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Investigation of the effects of increased levels of O-GlcNAc protein modification on protein kinase C and Akt

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Investigation of the effects of increased levels of O-GlcNAc protein modification on protein kinase C and Akt
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Matthews, Jason Aaron
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Translocation
Isoforms
Astroglial cells
Phosphorylation
Apoptosis
Dissertations, Academic -- Chemistry -- Doctoral -- USF
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ABSTRACT: O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and ubiquitous post-translational modification that has been shown to play a role in regulating a variety of intracellular processes. The pathway responsible for generating the O-GlcNAc modification, the hexosamine biosynthetic pathway (HBP), has also been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. To investigate if the effects of HBP flux on PKC translocation observed by others is related to the O-GlcNAc modification, O-GlcNAc levels in human astroglial cells were elevated using four separate O-GlcNAc modulating agents followed by analysis of cytosol and membrane concentrations of PKC-epsilon, -alpha, -betaII, and -iota. Of the four PKC isoforms analyzed, PKC-epsilon showed a significant reduction in its membrane associated levels in response to all agents tested whereas PKC-alpha showed reductions in response to only two agents.Investigation of the mechanism for thereductions in membrane associated PKC-epsilon and -alpha indicate that the increased O-GlcNAc levels did not disrupt the activation of these isoforms or their ability to translocate to the plasma membrane. Furthermore, results indicate that these reductions are not due to a disruption in the Hsp70 mediated recycling of the isoforms. It was found; however, that increased O-GlcNAc levels resulted in increased degradation of PKC-epsilon suggesting that the decreases in membrane associated PKC-epsilon may be a result of increased phosphatase or protease activity. Additional studies revealed that decreases in membrane bound PKC-epsilon and PKC-alpha, both of which act as anti-apoptotic enzymes, correlated with an increase in poly-(ADP-ribose) polymerase (PARP) cleavage --a well characterized hallmark of apoptosis. In addition to PKC, the effects of increased O-GlcNAc levels on a related kinase, Akt, were also examined. Initial investigation of the effects of increased O-GlcNAc modification of Akt activation using glucosamine or streptozotocin revealed a relatively large, short-term increase in Akt phosphorylation in response to these treatments. However, further analysis with other O-GlcNAc modulators indicated that this activation was not related to O-GlcNAc protein modification. Furthermore, this activation does not appear to be related to any hyperosmotic effects associated with the treatment conditions, nor does it appear to be related to oxidative stress. Therefore, further investigation is needed to characterize the novel pathway responsible for Akt activation following glucosamine or streptozotocin treatment.
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Dissertation (Ph.D.)--University of South Florida, 2006.
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by Jason Aaron Matthews.
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Investigation of the Effects of Increased Levels of O-Glc NAc Protein Modification on Protein Kinase C and Akt by Jason Aaron Matthews A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Co-Major Professor: Robert Potter, Ph.D. Co-Major Professor: Mildred Acevedo-Duncan, Ph.D. David J. Merkler, Ph.D. Larry P. Solomonson, Ph.D. Date of Approval: April 14, 2006 Keywords: translocation, isoforms, as troglial cells, phosphorylation, apoptosis Copyright 2006, Jason Aaron Matthews

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i Table of Contents List of Tables v List of Figures vi Abstract ix Chapter 1 O-GlcNAc protein modification 1 1.0 Introduction 1 1.1 O-GlcNAc Transferase 1 1.2 O-GlcNAcase 4 1.3 O-GlcNAc modification is reciprocal to O-phosphorylation 5 1.4 O-GlcNAc in cellular regulation 7 1.4.1 Protein regulation by O-GlcNAc 7 1.4.2 Regulation of nuclear transport 7 1.4.3 Regulation of protei n-protein interactions 8 1.4.4 Regulation of protein degradation 8 1.4.5 Regulation of stress response and cell cycle regulation 9 1.5 O-GlcNAc and disease 9 1.5.1 O-GlcNAc and Diabetes 9 1.5.2 O-GlcNAc and Neurodegenerative disease 10 1.5.3 O-GlcNAc and cancer 11 1.6 Modulation of O-GlcNAc levels 11 1.6.1 Glucosamine increases in tracellular O-GlcNAc levels 11 1.6.2 STZ increases intracellular O-GlcNAc levels 14 1.6.3 PUGNAc increases in tracellular O-GlcNAc levels 15 1.6.4 NAGBT increases intracellular O-GlcNAc levels 15 1.7 References Cited 17 Chapter 2 Protein kinase C 29 2.0 Protein kinase C isoforms 29 2.1 Protein kinase C structural domains 31 2.2 Protein kinase C activation 33 2.2.1 Activation by phosphorylation 33 2.2.2 Activation by cofactors 35 2.3 Protein kinase C regula tion by anchoring proteins 35 2.4 Protein kinase C deactivation 36 2.5 References Cited 38

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ii Chapter 3 Glial Cells 44 3.0 Introduction 44 3.1 Microglia 44 3.2 Schwann cells 47 3.3 Oligodendrocytes 47 3.4 Ependymal Glia 48 3.5 Astrocytes 50 3.6 References Cited 54 Chapter 4 Effects of increased O-Glc NAc protein modifi cation on Protein kinase C translocation 59 4.0 Introduction 59 4.1 Materials and methods 60 4.1.1 Materials 60 4.1.2 Cell culture 60 4.1.3 Fractionation of cytoso lic and membrane protein 61 4.1.4 Electrophoresis and Western blotting 62 4.1.5 Statistical Analysis 63 4.2 Results 63 4.2.1 Glucosamine, STZ, and PUGNAc increase O-GlcNAc modification on proteins 63 4.2.2 Effects of glucosamine on PKC translocation 67 4.2.3 Effects of streptozotocin on PKC translocation 68 4.2.4 Effects of PUGNAc on PKC translocation 71 4.2.5 Effects of NAGBT on PKC translocation 71 4.3 Discussion 71 4.4 References Cited 81 Chapter 5 Investigation of potential mechanisms for decreases in membrane associated PKCand PKC89 5.0 Introduction 89 5.1 Increased O-GlcNAc and PKCand translocation 90 5.2 Increases O-GlcNAc and Hsp70 mediated PKC recycling 91 5.3 Methods 91 5.3.1 Cell culturing and sample preparation for PMA treatment 92 5.3.2 Cell culturing and sample preparation for immunoprecipitation 92 5.4 Results 94 5.4.1 Effects of increased O-GlcNAc on PKC isozyme translocation 94 5.4.2 Effects of increased O-GlcNAc on PKC recycling 96 5.5 Discussion 104 5.6 References Cited 106 Chapter 6 PKCand PKCdownregulation and apoptosis induction 109

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iii 6.0 Introduction 109 6.1 Apoptosis 109 6.2 Protein Kinase C and apoptosis 112 6.3 Materials and methods 113 6.3.1 Cell culture 113 6.3.2 Cell fractionation 113 6.3.3 Electrophoresis and Western blotting 114 6.4 Results 114 6.4.1 Effects of PUGNAc and STZ treatment on PARP cleavage 114 6.4.2 Effects of glucosamine and NAGBT treatment on PARP cleavage 115 6.4.3 Caspase-7 activation 118 6.5 Discussion 118 6.6 References Cited 121 Chapter 7 Akt 128 7.0 Introduction 128 7.1 Akt structural domains 128 7.2 Akt activation 129 7.3 Akt regulation by anchoring proteins 132 7.4 References Cited 133 Chapter 8 Effects of O-GlcNAc increasing agents on Akt 137 8.0 Introduction 137 8.1 Materials and methods 137 8.1.1 Materials 138 8.1.2 Cell culture 138 8.1.3 Cell harvesting and fractionation 139 8.1.4 Electrophoresis and Western blotting 140 8.1.5 Statistical Analysis 141 8.2 Results 141 8.2.1 Effects of glucosamine on Akt phosphorylation and Akt distribution between cytosol and membrane 141 8.2.2 Effects of STZ on Akt phosphorylation and Akt distribution between cytosol and membrane 142 8.2.3 Effects of PUGNAc and NAGBT on Akt phosphorylation and Akt distribution between cytosol and membrane 145 8.2.4 Effects of galactosamine on Akt phosphorylation 148 8.2.5 Effects of N-acetyl-L-cysteine on Akt phosphorylation 148 8.2.6 Effects of Glucosamine or STZ on GRP 78 expression 152 8.3 Discussion 152 8.4 References Cited 161 Chapter 9 PKCand PKCassociated proteins 167 9.0 Introduction 167

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iv 9.1 Materials and Methods 167 9.2 Results 167 9.2.1 O-GlcNAc modified prot eins associated with PKCand 168 9.2.2 PKCand PKCassociation with Akt 168 9.3 Discussion 172 9.4 References Cited 174 About the Author End Page

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v List of Tables Table 1 Inhibition constants and sele ctivity of inhibitors for both O-GlcNAcase and -hexosaminidase 16 Table 2 Protein Kinase C Isoforms 30

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vi List of Figures Figure 1.1 Production of UDP-GlcNAc via the He xosamine Biosynthetic Pathway 2 Figure 1.2 O-GlcNAc Protein Modificat ion via the Hexosamine Biosynthetic Pathway 3 Figure 1.3 Hypothesized Yin-Yang Rela tionship Between O-GlcNAc and OPhosphate 6 Figure 1.4 Structures of OGlcNAc Increasing Agent 12 Figure 1.5 Points of Action for Glucosamine, STZ, PUGNAc, and NAGBT 13 Figure 2.1 Protein kinase C Is oforms' Structural Domains 32 Figure 2.2 Protein Kinase C Phosphorylation Sites 34 Figure 2.3 Protein Kina se C Life Cycle 37 Figure 3.1 Glial cells of th e central nervous system 45 Figure 3.2 Types of Glial Cells 46 Figure 3.3 Oligodendrocyte 49 Figure 3.4 Glutamate-glutamine cycli ng between central astrocytes and neurons 53 Figure 4.1 Effects of glucosamine, STZ, PUGNAc, and NAGBT treatments on O-GlcNAc modification of cytosolic proteins 65 Figure 4.2 Effects of glucosamine, STZ, PUGNAc, and NAGBT treatments on O-GlcNAc modification of membrane proteins 66 Figure 4.3 Effects of Glucosamine on PK C isoforms in membrane fractions 69 Figure 4.4 Effects of STZ on PKC is oforms in membrane fractions 70 Figure 4.5 Effects of PUGNAc on PKC isoforms in membrane fractions 72

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vii Figure 4.6 Effects of NAGBT on PKC is oforms in membrane fractions 73 Figure 5.1 Effects of 100nM PMA treatment on PKC isoform translocation 95 Figure 5.2 Effects of 5mM STZ treat ment on PMA induced PKC isoform translocation 97 Figure 5.3 Effects of 5mM STZ on PKCcleavage 99 Figure 5.4 PKCand Hsp70 Association 100 Figure 5.5 Hsp70 and PKCAssociation 101 Figure 5.6 Increased O-GlcNAc and PKC/Hsp70 Association 102 Figure 5.7 PKCand Hsp70 Association 103 Figure 6.1 Increased O-GlcNAc protei n modification and PARP cleavage 116 Figure 6.2 PUGNAc treatment and PARP cleavage 117 Figure 6.3 Increased O-GlcNAc and Caspase-7 activation 119 Figure 7.1 Akt Isoform Phosphorylation Sites 130 Figure 7.2 Akt life cycle 131 Figure 8.1 Effects of Glucosamine on phospho-Akt and Akt in cytosol and membrane fractions 143 Figure 8.2 Effects of STZ on phospho-Akt and Akt in cytosol and membrane fractions 144 Figure 8.3 Effects of PUGNAc on phospho-Akt and Akt in cytosol and membrane fractions 146 Figure 8.4 Effects of NAGBT on phos pho-Akt and Akt in cytosol and membrane fractions 147 Figure 8.5 Effects of decreasing NAGB T concentrations on phospho-Akt and Akt 149 Figure 8.6 Effects of decreasing P UGNAc concentrations on phospho-Akt and Akt 150

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viii Figure 8.7 Effects of Galactosamine on phospho-Akt and Akt 151 Figure 8.8 Effects of N-acetylcysteine and Glucosamine on phospho-Akt and Akt 153 Figure 8.9 Effects of N-acetylcysteine and STZ on phospho-Akt and Akt 154 Figure 8.10 Effects of Glucosamin e or STZ on GRP 78 expression 155 Figure 9.1 Association between PKCand and O-GlcNAc modified proteins 169 Figure 9.2 Association between PKCand Akt 170 Figure 9.3 Association between PKCand Akt 171

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ix Investigation of the Effects of Increased Levels of O-GlcNAc Protein Modification on Protein Kinase C and Akt Jason Aaron Matthews ABSTRACT O-linked N-acetylglucosamine (O-GlcNAc ) is an abundant and ubiquitous posttranslational modification that has been shown to play a role in regulating a variety of intracellular processes. The pathway responsible for generating the O-GlcNAc modification, the hexosamine biosynthetic pathway (HBP), has also been shown to affect the activity and translocation of certain protei n kinase C (PKC) isoforms. To investigate if the effects of HBP flux on PKC transloca tion observed by others is related to the OGlcNAc modification, O-GlcNAc levels in human astroglial cells were elevated using four separate O-GlcNAc modulating agen ts followed by analysis of cytosol and membrane concentrations of PKC, II, and Of the four PKC isoforms analyzed, PKCshowed a significant reductio n in its membrane associated levels in response to all agents tested whereas PKCshowed reductions in response to only two agents. Investigation of the mechanism for the reductions in membrane associated PKCand indicate that the increased O-GlcNAc leve ls did not disrupt th e activation of these isoforms or their ability to translocate to the plasma membrane. Furthermore, results indicate that these reductions are not due to a disruption in the Hsp70 mediated recycling of the isoforms. It was found; however, that increased O-GlcNAc levels resulted in increased degradation of PKCsuggesting that the decreases in membrane associated

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x PKCmay be a result of increased phosphatase or protease activity. Additional studies revealed that decreases in membrane bound PKCand PKC, both of which act as antiapoptotic enzymes, correlated with an increase in poly-(ADP-ribose) polymerase (PARP) cleavage a well characterized hallmark of apoptosis. In addition to PKC, the effects of incr eased O-GlcNAc levels on a related kinase, Akt, were also examined. Initial investig ation of the effects of increased O-GlcNAc modification of Akt activation us ing glucosamine or streptozot ocin revealed a relatively large, short-term increase in Akt phosphoryl ation in response to these treatments. However, further analysis with other O-GlcNAc modulators i ndicated that this activation was not related to O-GlcNAc protein modification. Furtherm ore, this acti vation does not appear to be related to any hyperosmotic effects associated with the treatment conditions, nor does it appear to be related to oxidative stress. Therefore, further investigation is needed to characterize th e novel pathway responsible for Akt activation following glucosamine or streptozotocin treatment.

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1 Chapter 1 O-GlcNAc protein modification 1.0 Introduction Protein monoglycosylation occurs when N-acetylglucosamine (GlcNAc) from uridine diphosphate-GlcNAc (UDP-GlcNAc) is enzymatically transferred to nuclear and cytoplasmic proteins on serine or threonine hydroxyl groups forming -O-linked Nacetylglucosamine moieties (O-GlcNAc). Unlike the more familiar multi-component glycosylation that occurs in the endoplasmic reticulum and Golgi, this single sugar addition occurs in the cytosol and is revers ible. The O-GlcNAc residue is added to proteins by a specific UD P-GlcNAc: polypeptide O-N-acetylglucosaminyl transferase (OGT) [1-4] and removed by a N-acetyl-D-glucosaminidase (O-GlcNAcase) [5-9]. The UDP-GlcNAc used to form O-Glc NAc is generated via the hexosamine biosynthetic pathway (HBP) (Figure 1.1). In the HBP, fructose-6-phosphate is converted to N-acetylglucosamine-6-phosphate by the ra te-limiting enzyme glutamine:fructose-6phosphate amidotransferase (GFAT) [10,11]. Subsequent steps ultimately produce UDPGlcNAc, the end product of the pathway (Figure 1.2). 1.1 O-GlcNAc Transferase OGT is a highly conserved enzyme found in all metazoans studied from Caenorhabditis elegans to humans, including plants. In liver, kidney, and muscle cells,

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Production of UDP-GlcNAc via the Hexosamine Biosynthetic Pathway Figure 1.1 Production of UDP-GlcNAc via the Hexosamine Biosynthetic Pathway [88] EC 2.3.1.4 glucosamine-phosphate N-acetyltransferase EC 2.3.1.157 glucosamine-1-phosphate N-acetyltransferase EC 2.6.1.16 glutaminefructose-6-phosphate transaminase (isomerizing) EC 2.7.7.23 UDP-N-acetylglucosamine diphosphorylase EC 3.5.1.25 N-acetylglucosamine-6-phosphate deacetylase EC 3.5.99.6 glucosamine-6-phosphate deaminase EC 5.4.2.3 phosphoacetylglucosamine mutase EC 5.4.2.10 phosphoglucosamine mutase 2

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O-GlcNAc Protein Modification via the Hexosamine Biosynthetic Pathway Figure 1.2 O-GlcNAc Protein Modification via the Hexosamine Biosynthetic Pathway [55] 3

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4 this enzyme exists as a heterotrimer consisting of two 110kDa subunits and one 78kDa subunit [12]. In all other ti ssue analyzed, the 78kDa subun it is not expressed and the enzyme is comprised of three 110kDa subunits [14]. The 110kD a OGT subunit has a K m for UDP-GlcNAc of 0.545 M [12] and is inhibited by the resulting UDP product [13]. The active site for OGT has been shown to be in the 110kDa subunit [14,15]. Southern blot analysis of the OGT gene has shown it to be highly conserved from nematodes to man [14]. The OGT gene is located on the X-chromosome and is necessary for embryonic stem cell viability [16,17] and mice with the OGT gene knocked out die early in embryonic development [16] suggesting an important role for OGT in development. The N-terminal domain of the 110kDa subunit c ontains 9-12 tetrat ricopeptide repeats (TPRs) [14,15]. The TPR domain is important for trimerization and stability of OGT [1]. OGT is both tyrosine phosphorylated and modified with O-GlcNAc [1,14]. No simple consensus motif on OGT substrates has been identified as the target for O-GlcNAc modification. Certain prot eins, such as Ataxin-10 (t he protein implicated in spinocerebellar ataxia) [ 18] and protein phosphatase-1 and have been shown to interact with OGT. OGT is ubiquitous but cy tosolic OGT activity is 10 times greater in brain tissue than in muscle, adipose, heart, and liver tissue [3]. 1.2 O-GlcNAcase O-GlcNAcase, originally called hexo saminidase C, was first purified and characterized from rat spleen [8]. Like OGT, O-GlcNAcase is ubiquitous but is more highly expressed in brain tissue, placenta, a nd pancreas than in other tissues [6]. OGlcNAcase is expressed in two forms, a 130kDa form and a splice variant with a molecular weight of 75kDa [6,19,20]. Eviden ce suggests that the 75kDa form is mostly

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5 localized in the nucleus [20] whereas the 130kD a form is predominately cytoplasmic [6]. Unlike the 130kDa form, the 75kDa variant has no O-GlcNAcase activity and its role is unknown [9]. Unlike other hexosaminidases, O-GlcNAcase is found in the cytosol and nucleus as opposed to the lysomsome, has a concomitantly more neutral optimal pH of 6.4 [6,8], and shows no other glycosidase ac tivity [6,8]. O-Glc NAcase selectively removes GlcNAc, but not GalNAc, from glycope ptides [6,9]. Very little is known about the regulation of this enzyme and is an area for future study. 1.3 O-GlcNAc modification is r eciprocal to O-phosphorylation Studies investigating the possible re lationship between O-GlcNAc and Ophosphate have demonstrated an inverse rela tionship (Figure 1.3). Treatment of cells with phosphatase inhibitors have led to decr eased levels of O-GlcNAc [21,22] whereas cellular treatment with kinase inhibitors increas es O-GlcNAc levels [ 21]. This reciprocal relationship has also been demonstrated on ce rtain individual protei ns. The C-terminal domain (CTD) of RNA polymerase II contains amino acid residues that are modified by both O-GlcNAc and O-phosphate [23,24]. Phosphor ylation of the CTD blocks the ability of OGT to modify this domain and O-GlcNAc modification of the CTD blocks the ability of the CTD kinase to phosphorylate the region [25]. Additionally, the estrogen receptor[26] and SV-40 large T-antigen [27] have also been shown to be reciprocally modified with O-GlcNAc and phosphate. While this yin-yang relationship has been demonstrated on several proteins, others, such as c-Myc are capabl e of being modified with both O-GlcNAc and O-phosphate simultaneously [28] indicating that the yin-yang hypothesis is overly simplistic. Further ev idence of this yinyang relationship was demonstrated when OGT and protein phosphatase 1and were shown to exist in

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Hypothesized Yin-Yang Relationship Between O-GlcNAc and O-Phosphate Figure 1.3 Hypothesized Yin-yang relationship between O-GlcNAc and Ophosphate [56] 6

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7 stable and active complex [29]. This complex was capable to dephosphorylating a synthetic peptide and then modifying the de phosphorylated residue wi th O-GlcNAc [29]. 1.4 O-GlcNAc in cellular regulation 1.4.1 Protein regulation by O-GlcNAc The O-GlcNAc protein modi fication has been demonstrated to function in the regulation of important signal tr ansduction enzymes. In endothelial cells, increases in OGlcNAc protein modification resu lted in both the direct modi fication and reduced activity of phosphoinositide 3'-OH-kinase (PI 3-K) [30]. Also, increases in cellular O-GlcNAc modification have also been demonstrated to increase p42/44 a nd p38 MAPK activities and these increases are associated with act ivation of upstream MAPK kinases [31]. The activation of endothelial nitric oxide synthase (eNOS) in re sponse to certain stimuli has also been shown to be impaired by increases in O-GlcNAc modificatio n. Federici et al. [30] showed that the insulin-stimulated activ ation of eNOS was impaired as well as its ability to undergo phosphorylation in endothe lial cells under conditi ons of increased OGlcNAc modification [30]. Also, the activati on of eNOS in the diabetic penis was shown to be impaired in response to fluid shear stress stimuli and vascul ar endothelial growth factor signaling and this im pairment was a result of the modification of Ser-1177 by OGlcNAc [32,32]. Additionally, the direct O-GlcNAc modifi cation of the transcription factor Sp1 has been shown to inhibit its transcriptional capability [33]. 1.4.2 Regulation of nuclear transport The O-GlcNAc modification has been implicated in the regulation of nuclear transport. The nuclear pore proteins that are responsible for the active transport of protein in and out of the nucleus are highly enriched in O-GlcNAc [34] [35,36]. Also, the nucleus

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8 contains a very high concentration of O-Glc NAc modified proteins [34]. Recently, the alpha4 phosphoprotein and Sp1, both of which contain O-GlcNAc moieties, showed a decreased translocation to the nucleus in res ponse to decreases cellular O-GlcNAc levels [37]. 1.4.3 Regulation of proteinprotein interactions The O-GlcNAc modification has been found on protein domains important for protein-protein interactions Mutation of the O-GlcNAc modification site on the transcription factor Sp1 blocked its interac tions with the Drosophila TAF110 protein and other Sp1 proteins [38]. The O-GlcNAc m odification has also been shown to mediate interactions between Sp1 and a p62 glycoprotein [39]. Certain cytoskeletal proteins such as keratins 8, 13, and 18 [40-42], neurofilament proteins [8,43] have been demonstrated to contain O-GlcNAc modifi cation sites in regions critical for protein-protein interactions. Other proteins such as the adenovirus fiber proteins 2 and 5 [44] and synapsin I [45] have sites shown to be m odified with O-GlcNAc that are believed to mediate its interactions with other proteins. 1.4.4 Regulation of protein degradation Proteins enriched with Pro, Glu, Ser, a nd Thr (PEST) sequences have been shown to be targeted for rapid degradati on [46,47] following phosphorylation of these sequences. Research has shown that proteins that have high PEST sequences are also modified with O-GlcNAc [26,48,49] and this finding has led to the hypothesis that OGlcNAc modification prevents phosphorylation of PEST sequences and thus prevents degradation of that protein. The cellula r glycoprotein p67 has been shown to be deglycosylated and rapidly degraded under conditions of serum starvation and heme

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9 depletion [50]. Also, the transcription factor Sp-1 has been shown to undergo proteosome dependent degradation when its level of O-GlcNAc modification is decreased [51]. Estrogen receptoris modified by O-GlcNAc on Ser19 [26] and when this residue was mutated to Ala16 in order to prevent it s modification, its tur nover rate was reduced; however, when Ser16 was mutated to Asp16 to mimic phosphorylati on, its turnover rate was increased [52]. 1.4.5 Regulation of stress response and cell cycle regulation A variety of cellular stress including UVB irradiation and thermal stress has been shown to increase intracellular O-GlcNAc le vels [53] and, conversely, decreases in OGlcNAc levels have been shown to reduce th e thermotolerance of mouse fibroblast cells [53]. Also, increases in O-GlcNAc have b een shown to elevate Hsp-70 expression [53], a protein known to stabilize proteins and thus protect cells from thermal stress. Increases in O-GlcNAc le vels disrupt the progression of HeLa cells through the cell cycle by delaying G 2 /M progression [54]. Also, ove rexpression of O-GlcNAcase or OGT has been shown to disrupt mitotic phosphorylation and the timed expression of cyclin proteins [54]. Finall y, increased OGT levels disrupted cytokinesis and resulted in aneuploidy [54]. 1.5 O-GlcNAc and disease 1.5.1 O-GlcNAc and Diabetes A large body of recent research has de monstrated a link between the O-GlcNAc modification and diabetes [55,56]. Increased levels of O-GlcNAc in adipocytes have been shown to result in d ecreased insulin stimulated glucose uptake [57]. Overexpression of OGT in mice resulted in insu lin [58], and insulin resistance in muscle

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10 cells due to the overexpression of Glut1 has b een linked to increased levels of O-GlcNAc [59]. Analysis of the insulin signaling pathway has reveal ed many proteins associated with this pathway to be modified with O-Gl cNAc suggesting an important regulatory role [30,60-62]. Phosphorylation of th e insulin receptor substrate 1 and 2 (IRS1 and IRS2) is the first step in the insulin-signaling pathwa y after activation of the receptor. Cellular treatment with glucosamine, a compound s hown to increase O-GlcNAc modification levels, results in a reducti on of insulin-stimulated IRS1 tyrosine phosphorylation and a subsequent reduction downstream signaling events and also in modification of IRS-1 and IRS-2 with O-GlcNAc [60]. Recently, mass sp ectrometry analysis of IRS-1 has reveal Ser-1036 at the carboxyl-terminus to be th e site of O-GlcNAc modification [62]. Pharmacological induced O-GlcN Ac increases have resulted in the disrupted activation of the insulin signaling kinase, Akt, in response to insulin stimulation [57,61,63]. Park et al. [61] showed that increased O-GlcNAc levels in adipocyt es reduced glucose uptake and GLUT4 translocation [61]. 1.5.2 O-GlcNAc and Neurodegenerative disease Many neuronal cytoskeletal proteins have been shown to be modified with OGlcNAc [64], in particular the Tau and -amyloid precursor protein. Tau, a microtubule binding protein associated in the pathology of Alzheimers disease, is both phosphorylated and extensively O-GlcNAc m odified in normal brain tissue [64], and hyperphosphorylated tau is found in the aggregat es of neurofibrillary tangles linked with Alzheimers. Together these discoveries have lead to th e hypothesis that decreasing OGlcNAc levels in the brain leads to the abnormal phosphorylation of tau [65]. Thus, OGlcNAc may have a protective effect in the brain. Beta amyloid precursor protein (APP),

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11 neurofilaments, and many synaptic vesicle pr oteins are also extensively modified with OGlcNAc [49,66,67]. Further evidence s uggesting a link between the O-GlcNAc modification and neurodegenerative diseases is the fact that bot h OGT and O-GlcNAcase map to loci linked to neurodegenerative diseas es; the locus for OGT is associated with Parkinsons disease, while the locus for O-GlcNAcase is linked with late onset Alzheimers disease. 1.5.3 O-GlcNAc and cancer Although not yet extensively investigat ed, preliminary data indicates a relationship between increases in O-GlcNAc modification and breast cancer. Slawson et al. [68] demonstrated a decrea se in total O-GlcNAc levels and a significant increase in OGlcNAcase activity with alterations in the patt ern of modified protei ns in primary human breast carcinomas [68]. This da ta suggests that there is a di sruption in the regulation of the O-GlcNAc modification as cells progress from normal to malignant possibly aiding the malignant phenotype [68]. 1.6 Pharmacological modulation of O-GlcNAc levels 1.6.1 Glucosamine increases intracellular O-GlcNAc levels Glucosamine is a compound known to enter the hexosamine biosynthetic pathway (HBP) downstream of the L-glutamine:Lfructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme in the pa thway (Figure 1.4 and 1.5). Since GFAT is regulated by negative feedback by its downstream product UDP-GlcNAc [11,69], the entry of glucosamine downstream of GFAT allows an increase in UDP-GlcNAc and ultimately increases in O-GlcNAc protein modi fication [70]. In addition to increasing

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12 Structures of O-GlcNAc Increasing Agents OHOHONHNOHCH3OOPhOGlucosamine ( GlcN)STZ)PUGNAc) (NAGBT) Streptozotocin (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (1,2-dideoxy-2'-propyl--D-glucopyranoso-[2,1-d]-2'thiazoline OHHOHHOHSNHHOH(CH2)2(CH)3 Figure 1.4 Structures of O-GlcNAc Increasing Agents

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13 Points of Action for Glucosamine, STZ, PUGNAc, and NAGBT Figure 1.5 Points of Action for Glucosamine, STZ, PUGNAc, and NAGBT [55]

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14 UDP-GlcNAc levels, glucosamine treatment has also been demonstrated to have other cellular effects such as altered endoplasmic re ticulum function [71] or calcium entry [72]. In addition to increasing O-GlcNAc le vels, glucosamine treatment has been shown to have a variety of other effects on cells. Glucosamine treatment has been shown to cause depletions of intracellular AT P content, possible through the enhanced conversion of glucosamine to glucosamin e-6-phosphate [73,74]. Also, treatment of macrophages with glucosamine was shown to inhibit Ca 2+ influx across the plasma membrane that was unrelated to ATP depl etion [72]. Additionally, the increased flux through the hexosamine biosynthetic pathway as a result of glucosamine treatment has been shown to lead to increased H 2 O 2 levels and increased oxidat ive stress in pancreatic -cell [75]. This increased oxidative stress wa s determined to not be related to increases in the O-GlcNAc modification [75]. Finall y, glucosamine has been shown to result in disruption of ER homeostasis that interferes with normal protein fo lding leading to an accumulation of misfolded or unfolded protei ns [71,76], a condition known as ER stress [77]. 1.6.2 STZ increases intracellular O-GlcNAc levels Streptozotocin (STZ) is an an tibiotic produced by the bacterium Streptomyces achromogenes This compound is derived from gl ucose and 1-methyl-1-nitrosouresa [61] forming an N-acetyl-glucosamine anal og with an N-methyl-N-nitrosourea group linked to the C-2 carbon on the s ugar (Figure 1.4). Over the last three decades, STZ has been used as a diabetic agent due to its ability to acutely induce pancreatic cell death [78]. STZ has the ability to release nitric oxide as well as donate methyl groups to nucleotides and DNA [79-81] although recently it s ability to release NO has been brought

PAGE 26

15 into question [82]. STZ has also been demons trated to be an inhibitor or O-GlcNAcase [5,83-85] and is believed to lead to elevat ed intracellular O-Glc NAc levels via this inhibition (Figure 1.5). Recently, STZ has b een shown to inhibit O-GlcNAcase via the production of a transition state analog [86] O-GlcNAcase converts STZ to a compound that closely resembles the natural ligand transi tion state only more energetically stable. The resulting analog is catalyzed to comple tion very slowly thus out competing the normal GlcNAc substrate for the enzymes active site [86]. 1.6.3 PUGNAc increases intracellular O-GlcNAc levels PUGNAc (O-(2-acetamido-2-deoxy -D-glucopyranosylidene)amino-Nphenylcarbamate) was originally shown to be a potent -N-acetylglucosaminidase inhibitor of a variety of different -hexosaminidases [87] (Figure 1.4). It has also been demonstrated to be a highly potent inhibito r of O-GlcNAcase [8] and is effective at increasing intracellular O-GlcNAc levels wit hout the cytotoxic effects associated with prolonged STZ treatment [7] (Figure 1.5). Recent studies have demonstrated that, although it is a potent O-GlcNAcase i nhibitor, PUGNAc inhibits other hexosaminidases with similar K i values [82] (Table 1.1). 1.6.3 NAGBT increases intracellular O-GlcNAc levels NAGBT (1,2-dideoxy-2'-propyl-D-glucopyranoso-[2,1-d]2'-thiazoine) is a recently synthesized compound generated to function as an effective O-GlcNAcase inhibitor [82] (Figure 1.4 and 1.5). Although not inhibiting O-GlcNA case as potently as PUGNAc, it is much more select ive for O-GlcNAcase over other -hexosaminidases [82] (Table 1.1). It has also been shown to be effective at increasing intracellular O-GlcNAc modified proteins in African monkey ki dney cells [82]. Due to its recent

PAGE 27

characterization, no studies have been published to date using NAGBT to study O-GlcNAc effects and, therefore, any other effects that this compound may have in vitro are unknown. Inhibition constants and selectivity of inhibitors for both O-GlcNAcase and -hexosaminidase Compound O-GlcNAcase K I M -Hexosaminidase K I M Selectivity ratio ( -Hexosaminidase K I /O-GlcNAcase K I ) GlcNAc 1500 1200 0.8 STZ 1500 47,000 31 PUGNAc 0.046 0.036 0.8 NAGBT 0.23 340 1500 Table 1.1 Inhibition constants for various O-GlcNAcase inhibitors [82] 16

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17 1.7 References Cited 1. L.K. Kreppel, G.W. Hart, Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopept ide repeats, J. Biol. Chem. 274 (1999) 3201522. 2. W.A. Lubas, J.A. Hanover, Functional e xpression of O-linked GlcNAc transferase. Domain structure and substrate specificity, J. Biol Chem. 275 (2000) 10983-8. 3. R. Okuyama, S. Marshall, UDP-N-acetylglucosaminyl transferase (OGT) in brain tissue: temperature sensitivity and subcellu lar distribution of cytosolic and nuclear enzyme, J. Neurochem. 86 (2003) 1271-80. 4. R.J. Konrad, F. Zhang, J.E. Hale, M. D. Knierman, G.W. Becker, J.E. Kudlow, Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase, Biochem. Biophys. Res. Commun. 293 (2002) 207-12. 5. R.J. Konrad, I. Mikolaenko, J.F. Tolar, K. Liu, J.E. Kudlow, The potential mechanism of the diabetogeni c action of streptozotocin: inhibiti on of pancreatic beta-cell O-GlcNAc-selectiv e N-acetyl-beta-D-glucosaminidase, Biochem. J. 356 (2001) 31-41 6. Y. Gao, L. Wells, F.I. Comer, G.J. Parker, G.W. Hart, Dynamic O-glycosylation of nuclear and cytosolic pr oteins: cloning and charact erization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain, J. Biol. Chem. 276 (2001) 9838-45. 7. R.S. Haltiwanger, K. Grove, G.A. Philipsberg, Modulation of O-linked Nacetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-Nacetylglucosaminidase inhibitor O-(2-acetamido-2-

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18 deoxy-Dglucopyranosylidene)amino-Nphenylcarbamate, J. Biol. Chem. 273 (1998) 3611-7. 8. D.L. Dong, G.W. Hart, Puri fication and characterization of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosol, J. Biol. Chem. 269 (1994) 19321-30. 9. L. Wells, Y. Gao, J.A. Mahoney, K. Vosse ller, C. Chen, A. Rosen, G.W. Hart, Dynamic O-glycosylation of nuclear and cy tosolic proteins: furt her characterization of the nucleocytoplasmic beta-N-acetylglu cosaminidase, O-GlcNAcase, J. Biol. Chem. 277 (2002) 1755-61. 10. B.M. Pogell, R.M. Gryder, Enzymatic s ynthesis of glucosamine 6-phosphate in rat liver, J. Biol. Chem. 228 (1957) 701-12. 11. S. Kornfeld, R. Kornfeld, E.F. Neufeld, P.J. O'Brien, The feedback control of sugar nucleotide biosynthesis in liver Proc. Natl. Acad. Sci. U S A 52 (1964) 3719 12. R.S. Haltiwanger, M.A. Blomberg, G.W. Hart, Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-Nacetylglucosamine:polypeptide beta-N-acetyl glucosaminyltransferase, J. Biol. Chem. 267 (1992) 9005-13. 13. L. Wells, K. Vosseller, G.W. Hart, Glyc osylation of nucleocytoplasmic proteins: signal transduction and OGlc NAc, Science 291 (2001) 2376-8. 14. L.K. Kreppel, M.A. Blomberg, G.W. Ha rt, Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repe ats, J. Biol. Chem. 272 (1997) 9308-15.

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19 15. W.A. Lubas, D.W. Frank, M. Krause, J.A. Hanover, O-Linked GlcNAc transferase is a conserved nucleocytoplasmic protein co ntaining tetratricopeptide repeats, J. Biol. Chem. 272 (1997) 9316-24. 16. C. Slawson, S. Shafii, J. Amburgey, R. Potter, Characterization of the O-GlcNAc protein modification in Xenopus laevis oocyte during oogenesis and progesteronestimulated maturation, Biochi m. Biophys. Acta 1573 (2002) 121-9. 17. D. Nolte, U. Muller, Human O-GlcNAc transferase (OGT): genomic structure, analysis of splice variants, fine ma pping in Xq13.1, Mamm. Genome 13 (2002) 624. 18. S.S. Andrali, P. Marz, S. Ozcan, Ataxin-10 interacts with O-GlcNAc transferase OGT in pancreatic beta cells, Bioc hem. Biophys. Res. Commun. 337 (2005) 14953. 19. D. Heckel, N. Comtesse, N. Brass, N. Blin, K.D. Zang, E. Meese, Novel immunogenic antigen homologous to hyaluro nidase in meningioma, Hum. Mol. Genet. 7 (1998) 1859-72. 20. N. Comtesse, E. Maldener, E. Meese, Id entification of a nuclear variant of MGEA5, a cytoplasmic hyaluronidase and a beta -N-acetylglucosaminidase, Biochem. Biophys. Res. Comm un. 283 (2001) 634-40. 21. L.S. Griffith, B. Schmitz, O-linked N-acetylglucosamine levels in cerebellar neurons respond reciprocally to pertubati ons of phosphorylation, Eur. J. Biochem. 262 (1999) 824-31. 22. T. Lefebvre, C. Alonso, S. Mahboub, M.J. Dupire, J.P. Zanetta, M.L. CailletBoudin, J.C. Michalski, Effect of okad aic acid on O-linked N-acetylglucosamine

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20 levels in a neuroblastoma cell line, Biochim. Biophys. Acta 1472 (1999) 71-81. 23. W.G. Kelly, M.E. Dahmus, G.W. Hart RNA polymerase II is a glycoprotein. Modification of the COOH-terminal do main by O-GlcNAc, J. Biol. Chem. 268 (1993) 10416-24. 24. M.E. Dahmus, Reversible phosphorylation of the C-terminal domain of RNA polymerase II, J. Biol. Chem. 271 (1996) 19009-12. 25. F.I. Comer, K. Vosseller, L. Wells, M.A. Accavitti, G.W. Hart, Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine, Anal. Biochem. 293 (2001) 169-77. 26. X. Cheng, R.N. Cole, J. Zaia, G. W. Hart, Alternative O-glycosylation/Ophosphorylation of the murine estrogen receptor beta, Biochemistry 39 ( 2000) 11609-20. 27. L. Medina, K. Grove, R.S. Haltiwanger, SV40 large T antigen is modified with Olinked N-acetylglucosamine but not w ith other forms of glycosylation, Glycobiology 8 (1998) 383-91. 28. K. Kamemura, B.K. Hayes, F.I. Come r, G.W. Hart, Dynamic interplay between Oglycosylation and O-phosphorylation of nuc leocytoplasmic proteins: alternative glycosylation/phosphorylation of THR-58, a known mutational hot spot of c-Myc in lymphomas, is regulated by mitoge ns, J. Biol. Chem. 277 (2002) 19229-35. 29. L. Wells, L.K. Kreppel, F.I. Comer, B.E. Wadzinski, G.W. Hart, O-GlcNAc transferase is in a functional complex w ith protein phosphatase 1 catalytic subunits, J. Biol. Chem. 279 (2004) 38466-70. 30. M. Federici, R. Menghini, A. Mauriello, M.L. Hribal, F. Ferrelli, D. Lauro, P.

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21 Sbraccia, L.G. Spagnoli, G. Sesti, R. Lauro, Insulin-dependent activation of endothelial nitric oxide s ynthase is impaired by O-linke d glycosylation modification of signaling proteins in human coronary endothelial cells, Ci rculation 106 (2002) 466-72. 31. Z.T. Kneass,R.B. Marchase, Protein O-GlcNAc modulates motility-associated signaling intermediates in neutroph ils, J. Biol. Chem. 280 (2005) 14579-85. 32. B. Musicki, M.F. Kramer, R.E. Becker, A.L. Burnett, Inactivation of phosphorylated endothelial nitric oxide synthase (Ser-1177) by O-GlcNAc in diabetes-associated erectile dysfunction, Proc Natl. Acad. Sci. U. S. A. 102 (2005) 11870-5. 33. X. Yang, K. Su, M.D. Roos, Q. Chang, A.J. Paterson, J.E. Kudlow, O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transc riptional capability, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 6611-6. 34. G.D. Holt, C.M. Snow, A. Senior, R.S. Haltiwanger, L. Gerace, G.W. Hart, Nuclear pore complex glycoproteins contai n cytoplasmically disposed Olinked Nacetylglucosamine, J. Cell. Biol. 104 (1987) 1157-64. 35. J.A. Hanover, C.K. Cohen, M.C. Willingham, M.K. Park, O-linked Nacetylglucosamine is attached to protei ns of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycopro teins, J. Biol. Chem. 262 (1987) 9887-94. 36. L.I. Davis,G. Blobel, Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a prev iously unidentifie d cellular pathway, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 7552-6. 37. S.M. Dauphinee, M. Ma, C.K. Too, Ro le of O-linked beta-N -acetylglucosamine

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22 modification in the subcellular distributi on of alpha4 phosphoprotein and Sp1 in rat lymphoma cells, J. Cell. Biochem. 96 (2005) 579-588. 38. M.D. Roos, K. Su, J.R. Baker, J.E. Kudlow, O glycosylatio n of an Sp1-derived peptide blocks known Sp1 protein interac tions, Mol. Cell. Biol. 17 (1997) 6472-80. 39. I. Han, M.D. Roos, J.E. Kudlow, Interacti on of the transcription factor Sp1 with the nuclear pore protein p62 require s the C-terminal domain of p62, J. Cell. Biochem. 68 (1998) 50-61. 40. N.O. Ku, M.B. Omary, Expression, glyc osylation, and phosphorylation of human keratins 8 and 18 in insect cells Exp. Cell. Res. 211 (1994) 24-35. 41. J.A. King, R.L. Martino, J.A. Tucker, Anaplastic large-cel l lymphoma (Ki-1 lymphoma) and diffuse large-cell i mmunoblastic lymphoma: two diagnostic problem cases, Ultrastruct. Pathol. 22 (1998) 55-62. 42. N.O. Ku, M.B. Omary, Identification and mutational analysis of the glycosylation sites of human keratin 18, J. Biol. Chem. 270 (1995) 11820-7. 43. D.L. Dong, Z.S. Xu, M.R. Chevrier, R. J. Cotter, D.W. Cleveland, G.W. Hart, Glycosylation of mammalian neurofilamen ts. Localization of multiple O-linked Nacetylglucosamine moieties on neurofilament polypeptides L and M, J. Biol. Chem. 268 (1993) 16679-87. 44. M.L. Caillet-Boudin, G. Strecker, J.C. Mi chalski, O-linked GlcNAc in serotype-2 adenovirus fibre, Eur. J. Biochem. 184 (1989) 205-11. 45. R.N. Cole, G.W. Hart, Glycosylation sites flank phosphor ylation sites on synapsin I: O-linked N-acetylglucosamine residues ar e localized within domains mediating synapsin I interactions, J. Neurochem. 73 (1999) 418-28.

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23 46. S. Rogers, R. Wells, M. Rechsteiner Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis, Science 234 (1986) 364-8. 47. M. Rechsteiner, S.W. Rogers, PEST sequences and regula tion by proteolysis, Trends Biochem. Sci. 21 (1996) 267-71. 48. M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by Olinked N-acetylglucosamine, J. Biol. Chem. 272 (1997) 2421-8. 49. L.S. Griffith, M. Mathes, B. Schmitz, Be ta-amyloid precursor protein is modified with O-linked N-acetylglucosamine, J. Neurosci. Res. 41 (1995) 270-8. 50. M.K. Ray, B. Datta, A. Chakraborty, A. Chattopadhyay, S. Meza-Keuthen, N.K. Gupta, The eukaryotic initiation factor 2-associated 67-kDa polypeptide (p67) plays a critical role in regulation of protein synthesis initiation in animal cells, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 539-43. 51. I. Han,J.E. Kudlow, Reduced O glycosylation of Sp1 is associated with increased proteasome susceptibility, Mol. Cell. Biol. 17 (1997) 2550-8. 52. X. Cheng, G.W. Hart, Alternative O-glyc osylation/O-phosphoryl ation of serine-16 in murine estrogen receptor beta: post-tr anslational regulation of turnover and transactivation activity, J. Biol. Chem. 276 (2001) 10570-5. 53. N.E. Zachara, N. O'Donnell, W.D. Ch eung, J.J. Mercer, J.D. Marth, G.W. Hart, Dynamic O-GlcNAc modification of nucleoc ytoplasmic proteins in response to stress. A survival response of mamm alian cells, J. Biol. Chem. 279 (2004) 3013342. 54. C. Slawson, N.E. Zachara, K. Vosselle r, W.D. Cheung, M.D. Lane, G.W. Hart, Perturbations in O-linked beta-N-acetylg lucosamine protein modification cause

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24 severe defects in mitotic progression a nd cytokinesis, J. Biol. Chem. 280 (2005) 32944-56. 55. S.A. Whelan, G.W. Hart, Proteomi c approaches to analyze the dynamic relationships between nucleocytoplasmic protein glycosylation and phosphorylation, Circ. Res. 93 (2003) 1047-58. 56. N.E. Zachara, G.W. Hart, The emergi ng significance of O-GlcNAc in cellular regulation, Chem. Rev. 102 (2002) 431-8. 57. K. Vosseller, L. Wells, M.D. Lane, G.W. Hart, Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insuli n resistance associated with defects in Akt activation in 3T3-L1 adipocytes, Proc Natl. Acad. Sci. U. S. A. 99 (2002) 5313-8. 58. D.A. McClain, W.A. Lubas, R.C. Cookse y, M. Hazel, G.J. Parker, D.C. Love, J.A. Hanover, Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 10695-9. 59. M.G. Buse, K.A. Robinson, B.A. Marshall, R.C. Hresko, M.M. Mueckler, Enhanced O-GlcNAc protein modification is associated with insulin resistance in GLUT1-overexpressing muscles, Am. J. P hysiol. Endocrinol. Metab. 283 (2002) E241-50. 60. M.E. Patti, A. Virkamaki, E.J. Landaker, C.R. Kahn, H. Yki-Jarvinen, Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance of early postreceptor insulin signali ng events in skeletal musc le, Diabetes 48 (1999) 156271. 61. R.R. Herr, J.K. Jahnke, A.D. Argoudelis, The structure of stre ptozotocin, J. Am.

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25 Chem. Soc. 89 (1967) 4808-9. 62. L.E. Ball, M.N. Berkaw, M.G. Buse, Id entification of the Major Site of O-Linked {beta}-N-Acetylglucosamine Modification in the C Terminus of Insulin Receptor Substrate-1, Mol. Cell. Proteomics 5 (2006 ) 313-23. 63. G. Boehmelt, A. Wakeham, A. Elia, T. Sa saki, S. Plyte, J. Potter, Y. Yang, E. Tsang, J. Ruland, N.N. Iscove, J.W. Dennis, T.W. Mak, Decreased UDP-GlcNAc levels abrogate proliferation control in EMeg32-deficient cells, EMBO J. 19 (2000) 5092-104. 64. C.S. Arnold, G.V. Johnson, R.N. Cole, D.L. Dong, M. Lee, G.W. Hart, The microtubule-associated prot ein tau is extensively modified with O-linked Nacetylglucosamine, J. Biol. Chem. 271 (1996) 28741-4. 65. T. Lefebvre, S. Ferreira, L. Dupont-Wa llois, T. Bussiere, M.J. Dupire, A. Delacourte, J.C. Michalski, M.L. Caill et-Boudin, Evidence of a balance between phosphorylation and O-GlcNAc glycosylation of Tau proteins--a role in nuclear localization, Biochim. Biophys. Acta. 1619 (2003) 167-76. 66. R.N. Cole,G.W. Hart, Cytosolic O-glycosyl ation is abundant in nerve terminals, J Neurochem. 79 (2001) 1080-9. 67. P.J. Yao,P.D. Coleman, Reduction of O-linked N-acetylglucosamine-modified assembly protein-3 in Alzheimer's disease, J. Neurosci. 18 (1998) 2399-411. 68. C. Slawson, J. Pidala, R. Potter, Increased N-acetyl-beta-gl ucosaminidase activity in primary breast carcinomas corresponds to a decrease in N-acetylglucosamine containing proteins, Biochim. Biophys. Acta 1537 (2001) 147-57. 69. G.L. McKnight, S.L. Mudri, S.L. Mathew es, R.R. Traxinger, S. Marshall, P.O.

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26 Sheppard, P.J. O'Hara, Molecular cloning, cDNA sequence, and bacterial expression of human glutamine:fructose-6 -phosphate amidotransferase, J. Biol. Chem. 267 (1992) 25208-12. 70. M.D. Roos, I.O. Han, A.J. Paterson, J. E. Kudlow, Role of glucosamine synthesis in the stimulation of TGF-alpha gene tr anscription by glucose and EGF, Am. J. Physiol. 270 (1996) C803-11. 71. H.Y. Lin, P. Masso-Welch, Y.P. Di, J.W. Cai, J.W. Shen, J.R. Subjeck, The 170kDa glucose-regulated stress protein is an endoplasmic reticulum protein that binds immunoglobulin, Mol. Biol. Cell 4 (1993 ) 1109-19. 72. S. Vemuri,R.B. Marchase, The inhibition of capacitative calcium entry due to ATP depletion but not due to glucosamine is re versed by staurosporine, J. Biol. Chem. 274 (1999) 20165-70. 73. R.C. Hresko, H. Heimberg, M.M. Chi, M. Mueckler, Glucosamine-induced insulin resistance in 3T3-L1 adipocytes is caused by depletion of intracellular ATP, J. Biol. Chem. 273 (1998) 20658-68. 74. S. Marshall, O. Nadeau, K. Yamasaki Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels, J. Biol. Chem. 279 (2004) 35313-9. 75. H. Kaneto, G. Xu, K.H. Song, K. Suzu ma, S. Bonner-Weir, A. Sharma, G.C. Weir, Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress, J. Biol. Chem. 276 (2001) 31099-104.

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27 76. G.H. Werstuck, M.I. Khan, G. Femia, A.J. Kim, V. Tedesco, B. Trigatti, Y. Shi, Glucosamine-induced endoplasmic reticu lum dysfunction is associated with accelerated atherosclerosis in a hypergly cemic mouse model, Diabetes 55 (2006) 93-101. 77. R.J. Kaufman, Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene tran scriptional and translatio nal controls, Genes Dev. 13 (1999) 1211-33. 78. R.N. Arison, E.I. Ciaccio, M.S. Glitzer J.A. Cassaro, M.P. Pruss, Light and electron microscopy of lesions in rats rendered diabetic w ith streptozotocin, Diabetes 16 (1967) 51-6. 79. P. Golden, L. Baird, W.J. Malaisse, F. Malaisse-Lagae, M.M. Walker, Effect of streptozotocin on glucose-induced insulin secretion by isolated islets of Langerhans, Diabetes 20 (1971) 513-8. 80. P.S. Schein, 1-methyl-1-nitrosourea and dialkylnitrosamine depression of nicotinamide adenine dinucleotid e, Cancer Res. 29 (1969) 1226-32. 81. B. Rudas, [Streptozotocin], Arzneimittelforschung 22 (1972) 830-61. 82. M.S. Macauley, G.E. Whitworth, A.W. Debowski, D. Chin, D.J. Vocadlo, OGlcNAcase uses substrate-assisted catalysi s: kinetic analysis and development of highly selective mechanism-inspired i nhibitors, J. Biol. Chem. 280 (2005) 2531322. 83. J.A. Hanover, Z. Lai, G. Lee, W. A. Lubas, S.M. Sat o, Elevated O-linked Nacetylglucosamine metabolism in pancrea tic beta-cells, Arch. Biochem. Biophys. 362 (1999) 38-45.

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28 84. M.D. Roos, W. Xie, K. Su, J.A. Clar k, X. Yang, E. Chin, A.J. Paterson, J.E. Kudlow, Streptozotocin, an analog of N-acet ylglucosamine, blocks the removal of O-GlcNAc from intracellular proteins, Proc. Assoc. Am. Physicians 110 (1998) 422-32. 85. Y. Gao, G.J. Parker, G.W. Hart, Streptozotocin-induced beta-cell death is independent of its inhibition of O-Glc NAcase in pancreatic Min6 cells, Arch. Biochem. Biophys. 383 (2000) 296-302. 86. C. Toleman, A.J. Paterson, R. Shin, J.E. Kudlow, Streptoz otocin inhibits OGlcNAcase via the production of a transi tion state analog, Biochem. Biophys. Res. Commun. 340 (2006) 526-34. 87. M. Horsch, L. Hoesch, A. Vasella, D.M. Rast, N-acetylglucosaminono-1,5-lactone oxime and the corresponding (phenylcarbam oyl)oxime. Novel and potent inhibitors of beta-N-acetylglucosaminidase, Eur. J. Biochem. 197 (1991) 815-8. 88. UDP-N-Acetylglucosamine Biosynthe sis.http://www.chem.qmul.ac.uk/iubmb/ enzyme/reaction/polysacc/UDPGlcN.html (accessed March 2006).

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29 Chapter 2 Protein kinase C 2.0 Introduction Protein kinase C (PKC) is a family of serine-threonine kinases that play critical roles in the regulation of ce ll growth, differentiation, and a poptosis [1,2]. There are 12 known PKC isoforms that are divided into three groups based upon their means of activation. The conventional PKC is oforms (cPKC) consist of PKC, I, II, and These isoforms can be activated by Ca 2+ 1,2-diacylglycerol (DAG) or phorbol 12myristate 13-acetate (PMA), and phosphatid ylserine (PS). Th e novel PKC isoforms (nPKC) consist of PKC, , and These is forms lack a Ca 2+ binding domain but can still be activated by DAG, PS, and unsaturat ed fatty acids. Fi nally, the atypical PKC isoforms (aPKC) are PKCand (and its mouse homologue PKC). These PKCs are only activated by PS, phosphatidylinositides and unsaturated fatty acids. Also, the more recently discovered PKC(also called PKD) [3,4], is activated by phorbol esters [5] but, unlike other isoforms, contains a pleckstrin homology domain similar to Akt (Table 2.1). Whereas PKC, I, II, , and have been shown to be ubiquitous [6-8], other PKC isoforms demonstrate di stinct tissue localization. PKCis predominately found in the tissues of the central nervous system [7,9,10], whereas PKCis expressed

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30 Protein Kinase C Isoforms PKC isoform M w (kDa) Number of amino acids Activators Tissue distribution cPKC 76.8 672 PS,Ca 2+ ,DAG,FA,1,25D 3 ,PMA ubiquitous I 76.8 671 PS,Ca 2+ ,DAG,FA,PMA most tissues II 76.9 673 PS,Ca 2+ ,DAG,FA,PMA most tissues 77.9 697 PS,Ca 2+ ,DAG,FA,1,25D 3 ,PMA neural nPKC 77.5 674 PS,DAG,FA,PI,PMA ubiquitous 83.5 737 PS,DAG,PI,1,25D 3 ,PMA neural,immune,epithelium,heart 78.0 683 PI,PMA neural,epithelium 81.6 707 PI,PMA ovary,skeletal muscle,platelets aPKC 67.7 592 PS,FA,PIP3,ceramide,PA most tissues / 67.2 586 unknown ubiquitous PKC 115.0 912 unknown unknown Table 2.1 Protein kinase C isoform data PS phosphatidylserine; DAG diacylglycerol; FA fatty acid; 1,25-D 3 1,25-Dihydroxycholecalciferol; PMA phorbol 12-myristate 13-acetate; PI phosphatidy linositol; PA phospha tidic acid. [51]

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31 largely in skin and lung [11]. PKCis present in skeletal muscle and also in lung, spleen, skin, and brain [12] and PKCis largely found in thymus and lung [13]. 2.1 Protein kinase C structural domains All of the PKC groups are composed of an NH 2 -terminal regulatory region and a COOH-terminal catalytic region [14]. The cPKCs are composed of four conserved domains (C1-C4) separated by five variable domains (V1-V5). The C1, C2, V1, and V2 domains are located on the regulatory region wh ereas the C3, C4, V4, and V5 domain are located on the catalytic region. The V3 do main, also called the hinge region, connects the regulatory and catalytic regions (Figure 2.1) The C1 domain is a small globular stru cture approximately 8kDa consisting of several cysteine-rich repeats [15]. These cysteine-rich re peats resemble zinc finger domains found in many DNA-binding proteins, although there is no evidence that any PKC isoforms bind DNA [15]. Evidence suggests that two Zn 2+ atoms bind each Zn finger and coordinate with a histidine and three cysteine residues thus stabilizing a particular conformation [16]. This C1 do main is also essential for DAG and phorbol ester binding and also interacts with membra ne lipids [17,18]. The bi nding site for these hydrophobic molecules is formed by two pulled-apart sheets [15]. cPKCs and nPKCs have two C1 domains (C1A and C1B), but evidence suggests that only one of these participates in ligand binding [19]. aPKCs contain a nonfunctional C1 domain that lacks the key residues necessary for DAG or phorbol ester binding [19]. The C2 domain is a 12 kDa -strand rich region required for Ca 2+ binding. This domain contains several acidi c residues thought to participate in this binding [20,21].

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Protein kinase C Isoforms' Structural Domains Figure 2.1 Protein Kinase C isoforms' structural domains [14] 32

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33 nPKCs lack the key residues in the C2 domain involved in Ca 2+ binding [22], and aPKCs lack a C2 domain altogether [6]. The C3 domain contains the ATP binding si te and the C4 domain is the substrate recognition site [23]. All of the PKC isoforms contain C3, V4, C4, and V5 domains that make up the catalytic domain [24]. In addition to these domains, PKCs have a pseudosubstrate sequence (amino acids 13-30) that precedes the C1 domain [25]. This region contains a motif resembling the c onsensus sequence found in the phosphorylation sites of PKC substrates; howev er, it cannot be phosphorylated [26]. In PKCs inactive state, it interacts with the active site through Arg19 [27]. Th is arginine interacts with several acidic residues in the enzymes act ive site thus blocking the enzyme from becoming catalytically active [27]. 2.2 Protein kinase C activation 2.2.1 Activation by phosphorylation All PKC isoforms are initially tran scribed as a single unphosphorylated polypeptide chain. Studies of PKCII show that the enzyme must undergo three phosphorylations before it is capable of activation by DAG [28]. These phosphorylation sites are conserved among the PKC isoforms [2 4] (Figure 2.2). Ne wly transcribed PKCII initially has an apparent molecular wei ght of 76kDa and is found in the detergent insoluble fraction on cells [28]. It undergoes phosphorylation on Thr500 by 3phosphoinositide-dependent kinase-1 (PDK-1) at a segment near the entrance of the active site referred to as the activation loop [29-32]. This phosphorylation does not affect its electrophoretic mobility on SDS-PAGE. It then is autophosphorylated at Thr641at a region known as the turn motif altering its electrophoretic mobility and producing an

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Protein Kinase C Phosphorylation Sites Figure 2.2 Protein Kinase C Phosphorylation Sites [21] 34

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35 enzyme with an apparent molecular we ight of 78kDa [28]. Finally, PKC is autophosphorylated at Ser660 in a region called the hydrophobic motif giving it an apparent molecular weight of 80kDa [28]. After this final pho sphorylation, the enzyme is released from the cytoskeleton into the cytoso l [28] where it is capab le of being activated by various cofactors. 2.2.2 Activation by cofactors Once the fully phosphorylated PKC is releas ed into the cytosol and the enzyme remains inactive due to its active site being occupied by its NH 2 -terminal pseudosubstrate [33]. This occupation blocks the active site and prevents enzymatic activity. Upon increases in intracellular Ca 2+ the C2 domain of the phos phorylated cPKCs binds this free Ca 2+ As the cPKC diffuses through the ce ll and contacts the membrane, the bound Ca 2+ forms a low-affinity interaction with anionic phospholipids in the membrane [34,35]. Once at the membrane, PKC can th en interact with membrane embedded diacylglycerol via its C1 domain. This high-affinity interaction results in a conformational change that removes the pse udosubstrate from the s ubstrate-binding site [33]. The nPKCs lack a C2 domain and, therefore, do not bind Ca 2+ Consequentially, their translocation rate is an order of magnitude less than the cPKCs. The aPKCs do not bind Ca 2+ or diacylglycerol and therefore do not translocate in response to certain stimuli as do the cPKCs and nPKCs. 2.3 Protein kinase C regulati on by anchoring proteins The biological function and s ubstrate specificity of PKC is due in large part to its cellular localization [36]. Correct localizati on of active PKC positions it near the proper substrates and regulators such as phosphata ses. Proteins called RACKs (receptors for

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36 activated C-kinase) have been shown to bi nd active PKC in an isoform specific manner and localize them to specific cellular site s [37]. RACKS also bind other signaling enzymes such as phospholipase C (PLC ) [38], and PKC substrates such as dynamin-1 [39] linking them to the PKC isoform. Th e binding of RACK to active PKC has also been demonstrated to enhance PKC catalytic activity [40]. Thus far, two RACKs have been identified. RACK1 selectively binds PKCII and RACK (also called COP) binds PKC. RACK 1 and RACK contain seven repeat s of the WD40 motif [41,42] that is known to be involved in protein-pr otein interactions. Th e binding of RACK1 to PKCII occurs at the C2 domain of PKCII [43]. 2.4 Protein kinase C deactivation The activated conformation of PKC is highly sensitive to dephosphorylation [44] and is largely mediated by protein phospha tase-1 and -2A [45]. Once dephosphorylated, PKC localizes to the detergent-insoluble fractio n [46] of the cells where it ultimately is degraded by proteolysis [47,48] In addition to undergoing proteolysis, dephosphorylated PKC can also be recycled back to the memb rane [49]. The dephosphorylated turn motif of PKC provides a binding site for the mo lecular chaperone heat shock protein-70 (Hsp70) [49]. This binding stabilizes PKC a nd allows it to cycle back to the cytosolic PKC pool where it can undergo another activation cycle [49, 50] (Figure 2.3).

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Protein Kinase C Life Cycle Figure 2.3 Protein Kinase C life cycle [50] 37

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38 2.5 References Cited 1. E.C. Dempsey, A.C. Newton, D. Mochly-Rosen, A.P. Fields, M.E. Reyland, P.A. Insel, R.O. Messing, Protein kinase C isozymes and the regulation of diverse cell responses, Am. J. Physiol. Lung Ce ll. Mol. Physiol. 279 (2000) L429-38. 2. Y. Akita, Protein kinase Cepsilon (PKC-epsilon): its un ique structure and function, J. Biochem. (Tokyo) 132 (2002) 847-52. 3. F.J. Johannes, J. Prestle, S. Eis, P. Oberhagemann, K. Pfizenmaier, PKCu is a novel, atypical member of the protein ki nase C family, J. Biol. Chem. 269 (1994) 6140-8. 4. A.M. Valverde, J. Sinnett-Smith, J. Van Lint, E. Rozengurt, Molecular cloning and characterization of protein kinase D: a target for diacylg lycerol and phorbol esters with a distinctive catalytic domain, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 8572-6. 5. H. Abedi, E. Rozengurt, I. Zachary, Rapid activation of the n ovel serine/threonine protein kinase, protein kina se D by phorbol esters, angiotensin II and PDGF-BB in vascular smooth muscle cells, FEBS Lett. 427 (1998) 209-12. 6. H. Hug, T.F. Sarre, Protein kinase C isoenzymes: divergence in signal transduction?, Biochem. J. 291 ( Pt 2) (1993) 329-43. 7. W.C. Wetsel, W.A. Khan, I. Merchentha ler, H. Rivera, A.E. Halpern, H.M. Phung, A. Negro-Vilar, Y.A. Hannun, Tissue and cellular distribution of the extended family of protein kinase C isoenzymes, J. Cell. Biol. 117 (1992) 121-33. 8. D. Schaap, P.J. Parker, Expression, purif ication, and characte rization of protein kinase C-epsilon, J. Biol Chem. 265 (1990) 7301-7.

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39 9. Y. Nishizuka, The heterogeneity and differential expression of multiple species of the protein kinase C famil y, Biofactors 1 (1988) 17-20. 10. Y. Nishizuka, Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Science 258 (1992) 607-14. 11. N. Bacher, Y. Zisman, E. Berent, E. Livneh, Isolation and characterization of PKC-L, a new member of the protein kina se C-related gene family specifically expressed in lung, skin, and heart, Mol. Cell. Biol. 11 (1991) 126-33. 12. S. Osada, K. Mizuno, T.C. Saido, K. Suzuki, T. Kuroki, S. Ohno, A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle, Mol. Cell. Biol. 12 (1992) 3930-8. 13. J. Rennecke, F.J. Johannes, K.H. Richter, W. Kittstein, F. Marks, M. Gschwendt, Immunological demonstration of protein kinase C mu in murine tissues and various cell lines. Differential recognition of phosphorylated forms and lack of downregulation upon 12-O-tetradecanoylphorphol-13acetate treatment of cells, Eur. J. Biochem. 242 (1996) 428-32. 14. W.S. Liu, C.A. Heckman, The sevenfol d way of PKC regulation, Cell Signal. 10 (1998) 529-42. 15. G. Zhang, M.G. Kazanietz, P.M. Blumberg, J.H. Hurley, Crystal structure of the cys2 activator-binding domain of protein kinase C delta in complex with phorbol ester, Cell 81 (1995) 917-24. 16. S.R. Hubbard, W.R. Bishop, P. Kirschme ier, S.J. George, S.P. Cramer, W.A. Hendrickson, Identification and characteriza tion of zinc binding sites in protein kinase C, Science 254 (1991) 1776-9.

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40 17. Y. Nishizuka, Protein kinase C and lipid signaling for sustaine d cellular responses, FASEB J 9 (1995) 484-96. 18. D.J. Burns, R.M. Bell, Protein kina se C contains two phorbol ester binding domains, J. Biol. Chem. 266 (1991) 18330-8. 19. J.H. Hurley, A.C. Newton, P.J. Parker, P.M. Blumberg, Y. Nishizuka, Taxonomy and function of C1 protein kinase C ho mology domains, Protein Sci. 6 (1997) 47780. 20. R.M. Bell,D.J. Burns, Lipid activation of protein kinase C, J. Biol. Chem. 266 (1991) 4661-4. 21. M.H. Lee,R.M. Bell, Mechanism of protein kinase C activation by phosphatidylinositol 4,5-bisphosphate Biochemistry 30 (1991) 1041-9. 22. S. Ohno, Y. Akita, Y. Konno, S. Imajoh, K. Suzuki, A novel phorbol ester receptor/protein kinase, nPKC, distantly rela ted to the protein kinase C family, Cell 53 (1988) 731-41. 23. S.S. Taylor, J.A. Buechler, W. Y onemoto, cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes, Annu. Rev. Biochem. 59 (1990) 971-1005. 24. A.C. Newton, Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macrom olecular interactions, Chem. Rev. 101 (2001) 2353-64. 25. C. House,B.E. Kemp, Protei n kinase C contains a pse udosubstrate prototope in its regulatory domain, Science 238 (1987) 1726-8. 26. P.J. Parker, L. Coussens, N. Totty, L. Rhee, S. Young, E. Chen, S. Stabel, M.D.

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41 Waterfield, A. Ullrich, The complete prim ary structure of prot ein kinase C--the major phorbol ester receptor, Science 233 (1986) 853-9. 27. J.W. Orr, L.M. Keranen, A.C. Newton Reversible exposure of the pseudosubstrate domain of protein kinase C by phosphatidyl serine and diacylglycerol, J. Biol. Chem. 267 (1992) 15263-6. 28. L.M. Keranen, E.M. Dutil, A.C. Newton, Protein kinase C is regulated in vivo by three functionally distinct phosphoryl ations, Curr. Biol. 5 (1995) 1394-1403. 29. M.M. Chou, W. Hou, J. Johnson, L.K. Graham, M.H. Lee, C.S. Chen, A.C. Newton, B.S. Schaffhausen, A. Toker, Regul ation of protein kinase C zeta by PI 3kinase and PDK-1, Curr. Biol. 8 (1998) 1069-77. 30. J.A. Le Good, W.H. Ziegler, D.B. Pa rekh, D.R. Alessi, P. Cohen, P.J. Parker, Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1, Science 281 (1998) 2042-5. 31. E.M. Dutil, A. Toker, A.C. Newton, Re gulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1), Curr. Biol. 8 (1998) 1366-75. 32. V. Cenni, H. Doppler, E.D. Sonnenburg, N. Maraldi, A.C. Newton, A. Toker, Regulation of novel protein kinase C epsilon by phosphor ylation, Biochem. J. 363 (2002) 537-45. 33. J.E. Johnson, J. Giorgione, A.C. Newton, The C1 and C2 domains of protein kinase C are independent membrane targe ting modules, with specificity for phosphatidylserine conferred by the C1 domain, Biochemistry 39 (2000) 11360-9. 34. E.A. Nalefski,A.C. Newton, Membrane bindi ng kinetics of protei n kinase C betaII

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42 mediated by the C2 domain, Biochemistry 40 (2001) 13216-29. 35. M. Schaefer, N. Albrecht, T. Hofmann, T. Gudermann, G. Schultz, Diffusionlimited translocation mechanism of protei n kinase C isotypes, FASEB J 15 (2001) 1634-6. 36. D. Schechtman,D. Mochly-Rosen, Adaptor proteins in protein kinase C-mediated signal transduction, Onc ogene 20 (2001) 6339-47. 37. D. Mochly-Rosen, H. Khaner, J. Lop ez, B.L. Smith, Intra cellular receptors for activated protein kinase C. Identification of a binding site for the enzyme J. Biol. Chem. 266 (1991) 14866-8. 38. M.H. Disatnik, S.M. Hernandez-Sotomayor, G. Jones, G. Carpenter, D. MochlyRosen, Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C, Proc. Natl. Aca d. Sci. U. S. A. 91 (1994) 559-63. 39. M.M. Rodriguez, D. Ron, K. Touhara, C.H. Chen, D. Mochly-Rosen, RACK1, a protein kinase C anchoring protein, coor dinates the binding of activated protein kinase C and select pleckstrin homology do mains in vitro, Biochemistry 38 (1999) 13787-94. 40. D. Mochly-Rosen, A.S. Gordon, Anchoring proteins for protein kinase C: a means for isozyme selectivity, FASEB J. 12 (1998) 35-42. 41. M. Csukai, C.H. Chen, M.A. De Matteis D. Mochly-Rosen, The coatomer protein beta'-COP, a selective binding protein (RACK) for protein kinase Cepsilon, J. Biol. Chem. 272 (1997) 29200-6. 42. D. Ron, C.H. Chen, J. Caldwell, L. Jamieson, E. Orr, D. Mochly-Rosen, Cloning of an intracellular receptor for protein ki nase C: a homolog of the beta subunit of G

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43 proteins, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 839-43. 43. D. Ron, J. Luo, D. Mochly-Rosen, C2 region-derived peptides inhibit translocation and function of beta protein kinase C in vivo, J. Biol. Chem. 270 (1995) 24180-7. 44. E.M. Dutil, L.M. Keranen, A.A. DePa oli-Roach, A.C. Newton, In vivo regulation of protein kinase C by trans-phosphorylation followed by autophosphorylation, J. Biol. Chem. 269 (1994) 29359-62. 45. R. Ricciarelli,A. Azzi, Regulation of recombinant PKC alpha activity by protein phosphatase 1 and protein phosphatase 2A, Arch. Biochem. Biophys. 355 (1998) 197-200. 46. G. Hansra, P. Garcia-Paramio, C. Pre vostel, R.D. Whelan, F. Bornancin, P.J. Parker, Multisite dephosphoryl ation and desensitization of conventional protein kinase C isotypes, Biochem. J. 342 ( Pt 2) (1999) 337-44. 47. H.W. Lee, L. Smith, G.R. Pettit, J.B. Smith, Bryostatin 1 and phorbol ester downmodulate protein kinase C-alpha and -epsilon via the ubiquitin/proteasome pathway in human fibroblasts, Mol. Pharmacol. 51 (1997) 439-47. 48. Z. Lu, D. Liu, A. Hornia, W. Devoni sh, M. Pagano, D.A. Foster, Activation of protein kinase C triggers its ubiquitination and degrad ation, Mol. Cell. Biol. 18 (1998) 839-45. 49. T. Gao,A.C. Newton, The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C, J. Biol. Chem. 277 (2002) 31585-92. 50. A.C. Newton, Regulation of the ABC kina ses by phosphorylation: protein kinase C as a paradigm, Biochem. J. 370 (2003) 361-71. 51. J.P. Liu, Protein kinase C and its substr ates, Mol. Cell.Endocrinol. 116 (1996) 1-29.

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44 Chapter 3 Glial Cells 3.0 Introduction The central nervous system (CNS) is compos ed of both neurons and glial cells. In the brain, glial cells are up to fifty times more abundant than neurons [1] and are known as the "supporting cells" of the nervous system. The four main functi ons of glial cells are to surround neurons and hold them in place, to supply nutrients and oxygen to neurons, to insulate one neuron from another, and to re move dead or injured neuron via phagocytosis [2-4]. There are two types of glial cells: macroglial and microglial. Macroglial cells can be further divided into Schwann cells, locate d in the peripheral nervous system (PNS), and oligodendrocytes, ependymal glia, and astr ocytes of the CNS (Figure 3.1 and 3.2). 3.1 Microglia Microglia are the smallest of all the glial cells. They are the primary immune effectors in the CNS and have distinct mo rphologies and staining characteristics from other glia and neurons [5,6]. Microglia ha ve a comparable func tion to macrophages and serve as scavenger cells in the event of infection, inflammation, trauma, ischemia, and neurodegeneration in the CNS [ 6,7]. Microglia are believed to originate from monocytes that enter the brain du ring embryonic development and diffe rentiate into brain resident microglia [4]. They are found throughout the br ain but appear to be more densely located in the gray matter near blood vessels. In re sponse to brain injury, microglia undergo a

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Glial cells of the central nervous system Figure 3.1 Glial cells of the central nervous system. [39] 45

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Types of Glial Cells Figure 3.2 Types of glial cells [40] 46

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47 change in morphology to an ameboid shape and ar e then able to migrat e to the insult site where they can engulf foreign organisms or damaged cells by phagocytosis [8]. Activated microglia are also capable of releasing cytotoxi c substances such as oxygen radicals, proteases, and proi nflammatory cytokines [9,10]. 3.2 Schwann Cells Schwann cells ensheath and myelinate most of the surface of all axons in peripheral nerves. Schwann cells originate from cells in the neural crest and can be divided into two types based on their morphology: myelin-forming and nonmyelinforming [11]. Both types of Schwann cells ensheath the neuronal axons although by different means. A single nonmyelinating Schwann cells can ensheath several small diameter (<1 M) axons by forming small invaginati ons on its surface in which individual unmyelinated axons sit [12]. The roles of nonmyelinating Schwann cells have been poorly investigated but may play a role in th e maintenance of unmyelinated axons [12]. Alternatively, the more well studied myelinat ing Schwann cells associate with only one large diameter (>1 M) axon and wrap around it numerous times to form the multilaminated and highly compacted myelin sheaths that underlie fast, saltatory conduction [12]. Myelinating Schwann cells in fluence the structure of the axons they ensheath including axon diameter, axonal neurofilament spacing, and phosphorylation [13]. The gaps between the Schwann ce ll covered segments, called the Nodes of Ranvier, serve as important sites of ionic and other exchanges of the axon with the extracellular liquid. 3.3 Oligodendrocytes

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48 Similar to the Schwann cells of the PN S, oligodendrocytes ensheath neuronal axons in the CNS. One o ligodendrocyte may ensheath anywhere from 5 to 30 axons by forming either a loose wrapping around a group of unmyelinated axons or forming tight multilaminar myelin sheaths [5,14]. Simila r to astrocytes and ependymal glia, oligodendrocytes arise from the ectoderm of the developing neuroepithelium [14]. The principle function of o ligodendrocytes is to provide support to axons and to produce the myelin sheath that insulates axons. Myelin is 80% lipid and 20% protein and allows for the efficient conduction of acti on potentials down the axon [3]. Unlike Schwann cells, oligodendrocytes form segmen ts of myelin sheaths of numerous neurons at once. Each process from a given oligodend rocyte can wrap itself around portions of a nearby axon forming layers of myelin [3] and becoming a segment of the axon's myelin sheath (Figure 3.3). 3.4 Ependymal Glia Ependymal cells are epithelial cells that line the central cavities of the brain and the spinal cord [15]. They range in shape from squamous to columnar and in certain regions of the brain they po ssess cilia [15]. Ependymal cells form a relatively permeable barrier between the cerebrospinal fluid that fills those cavities and the tissue fluid that surrounds the cells of the CNS. In certain region of the brain ependymal cells possess cilia, the beating of which help to circulate the cerebrospinal fluid that cushions the brain [16]. Modified ependymal cells contribute to the formation of the choroids plexus which is a capillary knot that protrude s into a brain ventricle, and is involved in the synthesis of cerebrospinal fluid [17]. The cell bodies and nuclei of ependymal glia, however, are located primarily in the ependymal layer of the brain with their pr ocesses extending to

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Oligodendrocyte Figure 3.3 Oligodendrocyte [41] 49

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50 nearby blood vessels [18]. They can be di stinguished from astrocytes based on their apparent growth-promoting properties in adul t brain, their expressi on of both p75 and the estrogen receptor, and their abil ity to survive and proliferate in culture [19,20]. In adult mammals, the cerebral ventricles are nor mally lined by a layer of cuboidal and multiciliated ependymal cells [21,22]. These cel ls are at the interf ace between the brain parenchyma and the ventricular cavities and pl ay an essential role in the propulsion of CSF through the ventricular system [16,23]. The coordinated beating of cilia in ependymal cells creates a current of CSF along the walls of the lateral ventricle; ependymal malfunction leads to disturbances of CSF flow and hydro cephaly [24-26]. It has also been suggested that ependymal cells filter brain molecule s [27], insulate the brain from potentially harmful substances in the CSF [28], move cellular debris in the direction of bulk CSF flow, and optimize the dispersion of neural messengers in the CSF [29]. 3.5 Astrocytes Astrocytes, also called astroglial cells, are the most abundant types of glial cell. They are easily distinguishabl e from other types of glial ce lls during development due to their robust expression of the cytoskeletal prot eins GFAP and vimentin [5]. Astrocytes are generally star shaped cells with numerous projections th at anchor neurons to their blood supply and are found in both the white and gray matter of the brain. They ensheath regions of CNS neurons that are not covered by oligodendrocytes, su ch as the Nodes of Ranvier [2] and also encapsulat e synaptic regions between neur ons [2]. Both astrocytes and ependymal cells have processes that contact blood vessels and the pial surface of the CNS suggesting a role in trafficking metabo lites and eliminating waste products to and

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51 from neurons. Astrocytes are coupled toge ther with gap junctions that allow the movement of ions and small mo lecules between them [30]. Astrocytes carry out a large number of functions in the CNS including modulation of synaptic function [31]. Astrocytes actively take up the neurotransmitter glutamate via specific transporters, thereby shortening the synaptic current and protecting postsynaptic cells from potentially excitot oxic effects [31]. They then convert the glutamate back to glutamine via glutamine synthetase and shuttle it back to neurons where it is converted back to glutamate [31] (Figure 3.4). Astrocytes also regulate the neuron al microenvironment by controlling the extracellular pH and K + levels [32], and the supply of va rious metabolic substrates [2]. Astrocytes provide nourishment to neurons by receiving glucose from capillaries, breaking the glucose down into lactate, and re leasing the lactate into the extra cellular fluid surrounding the neurons [33] The neurons receive the l actate from the extracellular fluid and transport it to their mitochondria to use as an energy source [33]. In this process astrocytes store a small amount of glycogen, which stays on reserve for times when the metabolic rate of neurons in the ar ea is especially high [34]. In addition to regulating the neuronal microenvi ronment, astrocytes also affect neuronal development via the release of neurotrophic factors [35] and increase the nu mber of mature, functional synapses on CNS neurons [36]. Finally, astrocytes function as the primar y cell type responsible for the formation and maintenance of the blood brain barrier ( BBB), the structure that limits the entry of blood-borne elements in the CNS [2]. It has been demonstr ated that astrocyte ablation leads to the failure of the BBB to repair [37]. Additionally, astrocytes may regulate

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52 vasoconstriction and vasodila tion by producing substances such as arachidonic acid, whose metabolites are vasoactive.

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Glutamate-glutamine cycling between central astrocytes and neurons Figure 3.4 Glutamate-glutamine cycling between central astrocytes and neurons Abbreviations: -KG, -ketoglutarate; GS, glutamine synthetase; PAG, phosphate-activated glutaminase; SNAT1/2, system N/A amino acid transporter [38]. 53

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54 3.6 References Cited 1. A. Prat, K. Biernacki, K. Wosik, J. P. Antel, Glial cell influence on the human blood-brain barrier, Gl ia 36 (2001) 145-55. 2. Y. Dong,E.N. Benveniste, Immune functi on of astrocytes, Glia 36 (2001) 180-90. 3. N. Baumann,D. Pham-Dinh, Biology of oligodendrocyte and myelin in the mammalian central nervous system, Physiol. Rev. 81 (2001) 871-927. 4. S.U. Kim,J. de Vellis, Microglia in health and disease, J. Neurosci. Res. 81 (2005) 302-13. 5. A. Peters, K. Josephson, S.L. Vincent, Effects of aging on the neuroglial cells and pericytes within area 17 of the rhesus monkey cerebral cortex, Anat. Rec. 229 (1991) 384-98. 6. F. Gonzalez-Scarano, G. Baltuch, Microgl ia as mediators of inflammatory and degenerative diseases, Annu. Re v. Neurosci. 22 (1999) 219-40. 7. W.E. Thomas, Brain macrophages: evalua tion of microglia and their functions, Brain Res Brain Re s Rev 17 (1992) 61-74. 8. G. Raivich, Like cops on the beat: the active role of resting microglia, Trends Neurosci. 28 (2005) 571-3. 9. C.A. Colton,D.L. Gilbert, Production of superoxide anions by a CNS macrophage, the microglia, FEBS Lett. 223 (1987) 284-8. 10. R.B. Banati,G.W. Kreutzberg, Flow cyto metry: measurement of proteolytic and cytotoxic activity of microglia, Clin. Neuropathol. 12 (1993) 285-8. 11. G. Corfas, M.O. Velardez, C.P. Ko, N. Ratner, E. Peles, Mechanisms and roles of axon-Schwann cell interactions J. Neurosci. 24 (2004) 9250-60.

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55 12. J. Kamholz, R. Awatramani, D. Menichella, H. Jiang, W. Xu, M. Shy, Regulation of myelin-specific gene expression. Releva nce to CMT1, Ann. N. Y. Acad. Sci. 883 (1999) 91-108. 13. S.T. Hsieh, G.J. Kidd, T.O. Crawford, Z. Xu, W.M. Lin, B.D. Trapp, D.W. Cleveland, J.W. Griffin, Regional modulat ion of neurofilament organization by myelination in normal axons, J. Neurosci. 14 (1994) 6392-401. 14. J.C. Lee, M. Mayer-Proschel, M.S. Rao, Gliogenesis in the cen tral nervous system, Glia 30 (2000) 105-21. 15. J.B. Gabrion, S. Herbute, C. Bouille, D. Maurel, S. Kuchler-Bopp, A. Laabich, J.P. Delaunoy, Ependymal and choroidal cells in culture: ch aracterization and functional differentiation, Micr osc. Res. Tech. 41 (1998) 124-57. 16. W.C. Worthington Jr,R.S. Cathcart 3rd, Ep endymal cilia: distribution and activity in the adult human brain, Science 139 (1963) 221-2. 17. L.H. Strong, Early development of the epe ndyma and vascular patte rn of the fourth ventricular choroid plexus in the rabbit, Am. J. Anat. 29 (1956) 249-90. 18. G. Gudino-Cabrera,M. Nieto-Sampedr o, Estrogen recepto r immunoreactivity in Schwann-like brain macroglia, J. Neurobiol. 40 (1999) 458-70. 19. M.C. Langub Jr,R.E. Watson Jr, Estrogen receptor-immunoreactive glia, endothelia, and ependyma in guinea pig preoptic area and median eminence: electron microscopy, Endocrinology 130 (1992) 364-72. 20. S. Nadeau,S. Rivest, Effects of circulat ing tumor necrosis factor on the neuronal activity and expression of the genes encodi ng the tumor necrosis factor receptors (p55 and p75) in the rat brai n: a view from the blood-brai n barrier, Neuroscience 93

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56 (1999) 1449-64. 21. M.R. Del Bigio, The ependyma: a pr otective barrier be tween brain and cerebrospinal fluid, Glia 14 (1995) 1-13. 22. R. Bleier, The relations of ependyma to neurons and capillaries in the hypothalamus: a Golgi-Cox study, J. Comp. Neurol. 142 (1971) 439-63. 23. R.S. Cathcart 3rd,W.C. Worthington Jr, Cillary movement in the rat cerebral ventricles: clearing action and directions of currents, J. Neuropathol. Exp. Neurol. 23 (1964) 609-18. 24. D.L. Brody,D.T. Yue, Relief of G-protein inhibition of calcium channels and shortterm synaptic facilitation in cultured hippocampal neurons, J. Neurosci. 20 (2000) 889-98. 25. P.D. Taulman, C.J. Haycraft, D.F. Ba lkovetz, B.K. Yoder, Polaris, a protein involved in left-right axis pa tterning, localizes to basal bodies and cilia, Mol. Biol. Cell 12 (2001) 589-99. 26. Y. Kobayashi, M. Watanabe, Y. Okada, H. Sawa, H. Takai, M. Nakanishi, Y. Kawase, H. Suzuki, K. Nagashima, K. Ikeda, N. Motoyama, Hydrocephalus, situs inversus, chronic sinusitis, and male infertility in DNA polymerase lambdadeficient mice: possible implication fo r the pathogenesis of immotile cilia syndrome, Mol. Cell. Biol. 22 (2002) 2769-76. 27. J.E. Bruni, Ependymal development, proliferation, and f unctions: a review, Microsc. Res. Tech. 41 (1998) 2-13. 28. S. Kuchler, M.N. Graff, S. Gobaille, G. Vincendon, A.C. Roche, J.P. Delaunoy, M. Monsigny, J.P. Zanetta, Mannose depende nt tightening of th e rat ependymal cell

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57 barrier. In vivo and in vitro study using neoglycoproteins, Neurochem. Int. 24 (1994) 43-55. 29. Y. Roth, Y. Kimhi, H. Edery, E. Ah aronson, Z. Priel, Ciliary motility in brain ventricular system and trachea of hamsters, Brain Res. 330 (1985) 291-7. 30. E. Tani, M. Nishiura, N. Higashi, Freeze-fracture studies of gap junctions of normal and neoplastic astrocytes, Acta Neuropathol. (Ber l) 26 (1973) 127-38. 31. F.W. Pfrieger,B.A. Barres, New views on synapse-glia interactions, Curr. Opin. Neurobiol. 6 (1996) 615-21. 32. B.A. Barres, Glial ion channels, Curr Opin Neurobiol 1 (1991) 354-9. 33. G.A. Dienel,L. Hertz, Glucose and la ctate metabolism during brain activation, J Neurosci. Res. 66 (2001) 824-38. 34. J. Koizumi, H. Shiraishi, S. Minei, Ultrastructural appearance of glycogen in neuron and astrocyte of the human cerebral cortex adjacent to brain tumors, J. Electron. Microsc. (Tokyo) 19 (1970) 355-9 passim. 35. E.N. Benveniste, Cytokine actions in th e central nervous syst em, Cytokine Growth Factor Rev. 9 (1998) 259-75. 36. E.M. Ullian, S.K. Sapperstein, K.S. Christopherson, B.A. Barres, Control of synapse number by glia, Science 291 (2001) 657-61. 37. T.G. Bush, N. Puvanachandra, C.H. Horner, A. Polito, T. Ostenfeld, C.N. Svendsen, L. Mucke, M.H. Johnson, M.V. Sofroniew, Leukocyte infiltration, neuronal degeneration, and neurite outgr owth after ablati on of scar-forming, reactive astrocytes in adult transg enic mice, Neuron 23 (1999) 297-308. 38. B. Mackenzie,J.D. Erickson, Sodium-c oupled neutral amino acid (System N/A)

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58 transporters of the SLC38 gene fa mily, Pflugers Arch. 447 (2004) 784-95. 39. Herbrandson, C. Learning the Ne rvous System Chapters 14-19. http://academic.kellogg.edu/herbrandsonc/bio201_McKinley/Nervous%20System.h tm (accessed March 2006). 40. Patton, P. Cellular and Molecular Building Blocks I. http://soma.npa.uiuc.edu/courses/bio 303/Ch2.html (accessed March 2006). 41. Oligodendrocyte. http://members.tripod.com/blustein/O ligodendrocytes/oligodendrocytes.htm (accessed March 2006).

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59 Chapter 4 Effects of increased O-GlcNAc protein modification on Protein kinase C translocation 4.0 Introduction The O-GlcNAc protein modi fication has been demonstrated to function in the regulation of important signal tr ansduction enzymes. The activit y of certain kinases, such as PI 3-K [1] and p42/44 and p38 MAPK [2] have been shown to be affected by increases in cellular O-GlcNAc protein m odification (further discussed in section 1.5.1). In certain systems flux through the hexosamine bios ynthetic pathway (HBP), the pathway responsible for producing the substrate for th e O-GlcNAc transferase (OGT), has been shown to affect the activity of protein kinase C (PKC) [3-7]. Filippis [7] showed that increased flux through the HBP led to a 3-fold increase in overall PKC activity. Others have shown that flux through the HBP regul ates the translocation of specific PKC isozymes [3,6]. Although PKC regulation ha s been linked to flux through the HBP, a direct connection to O-GlcNAc levels has not been investigated. A good model system to investigate the relationship between PKC and O-GlcNAc is the brain. Brain tissue has been shown to express higher levels of PKC [8,9] and OGlcNAcase [10] and have 10 times greater OGT activity [11] than most other tissues. Therefore we examined this relationship in a SV-40 transformed human glial (SVG) cell line that has previously been used as a human astroglia l model system [12,13]. This

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60 chapter discusses experiments done in order to investigate the e ffects of increasing OGlcNAc protein modification on the translocation of severa l PKC isozymes. It also compares and contrasts the effects of four O-GlcNAc modulating agents on the rates and levels of O-GlcNAc increases in SVG cells. 4.1 Materials and methods 4.1.1 Materials SVG cell line, eagles minimum essentia l medium (EMEM) and fetal bovine serum (FBS) were obtained from American T ype Culture Collection (Rockville, MD). Penicillin/streptomycin was purchased from Fisher Scientific (Suwanee, GA). PKC(sc8393), PKCII (sc210), and PKC(sc214) specific antibodi es were purchased from Santa Cruz Biotechnology (S anta Cruz, CA) while PKC(P20520) was obtained from Transduction Laboratories (San Diego, CA ). CTD110.6 anti-O-GlcNAc antibody was a kind gift from Dr. Gerald Hart at Johns H opkins University (Baltimore, MD) and is now available from Covance Research Products (Berkeley, CA). Goat anti-mouse-HRP and goat anti-rabbit-HRP secondary antibodies were from BioRad (Hercules, CA). Dglucosamine and streptozotocin (STZ) were purchased from Sigma (St. Louis, MO) and O-(2-acetamido-2-deoxy-D-glucopyranosylid ene)amino-N-phenylcarbamate (PUGNAc) was from Carbogen (Aarau, Switzerland). 1,2-dideoxy-2'-propyl-D-glucopyranoso[2,1-d]2'-thiazoline (NAGBT) was a kind gift from Dr. David J. Vocadlo at Simon Fraser University (British Columbia, Canada). All other chemicals were purchased from Sigma and were of the purest grade available. 4.1.2 Cell culture

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61 SVG cells were grown at 37 C in a 95% air and 5% CO 2 environment. Cells were cultured in T-150cm 2 flask to 90% confluency in EMEM (5.4mM glucose) supplemented with 10% FBS and 10U/mL penicillin and 10g/mL streptomycin. Cell media was supplemented with 8mM glucosamine, 5mM STZ, 80 M PUGNAc, or 75 M NAGBT for the time periods indicated. 4.1.3 Fractionation of cytosoli c and membrane proteins To terminate experiments, flasks of cells were immediately placed on ice and washed with ice cold phosphate buffered saline (PBS). Cells were scraped into PBS and centrifuged a 2,000xg for 3 minutes to pellet cells PBS was removed and cell pellet was resuspended in 550 l of ice-cold homogenization buffer (20mM Tris, 1mM EDTA, 100mM NaCl, 1mM dithiothreitol, 1mM PMSF, 1mM Na 3 VO 4 4 g/ml aprotinin, pH 7.4). Cells were lysed by sonication (Fisher Scientific Sonic Dismembrator F60) on ice with 2, 10-second pulses at 7 watts. Any whole cells or debris was pelleted by centrifuging at 1000xg for 5 minutes at 4 C. Supernatants were then centrifuged at 100,000xg for 1 hour at 4 C. The resulting supernatants were removed and labeled the cytosol fraction. Pellets were resuspended in homogenization buffer with 1.0% Triton X100 by gentle agitation for 30 minutes followed by a brief 2-second sonication at 7 watts. Samples were centrifuged at 100,000xg for 30 minutes at 4 C. Supernatants were removed and labeled the membrane fractions. Protein concentrations of both fractions were determined using BioRad Protein A ssay Dye Reagent using bovine serum albumin (BSA) as a standard.

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62 4.1.4 Electrophoresis and Western blotting Proteins from cytosol and membrane frac tions were mixed with 0.3 volumes of 3x sample buffer (0.18M Tris-HCl pH 6.8, 6% s odium dodecyl sulfate (SDS), 30% glycerol, 0.025% Bromophenol Blue) and equal amounts of protein were loaded onto 8% SDSpolyacrylamide gels. Samples were electr ophoresed for 1 hour 30 minutes at 15mAmps and then transferred to nitrocellulose me mbranes by electroblotting in 50mM Tris, 77mM glycine, and 20% methanol transfer buffer for one hour at 12 volts. For western blot analysis with PKC antibodies, membranes we re blocked with 5% (w/v) non-fat dried milk in tris buffered saline (140mM NaCl, 2.7mM KCl, and 25mM Tris pH 8.0) with 0.05% Tween-20 (TBS-T). Membranes we re then incubated overnight at 4 C in TBS-T with 3% milk containing the PKC antibody. PKCantibody was diluted 1:15,000, PKCII antibody was diluted 1:1,500, PKCantibody was diluted 1:150, and PKCwas diluted 1:1000 for western blot analysis. Membranes were subsequently washed for 10 minutes (5 times) in TBS-T with 1% milk. Either goat anti rabbit-HRP (1:6000 dilution for PKCII and PKC) or goat anti mouse-HRP (1:20,000 dilution for PKCand 1:5000 dilution for PKC) in TBS-T 1% with milk was then incubated with the membranes. Membranes were again washed 10 minutes (6 times) in TBS-T with 1% milk and bands were detected by chemilumi nescence according to the manufactures instructions (Pierce, Rockford, IL). For analysis using CTD110.6 antibody, membranes were blocked in tris buffered saline with 0.3% Tween-20 (TBS-HT) for 1 hour then incubated overnight at 4 C in TBS-HT containing the CTD110.6 antibody (1:5 000 dilution) [14]. Membranes were washed for 10 minutes (2 times) in tris bu ffered saline with 1% Triton X-100, 0.1% SDS,

PAGE 74

63 0.25% deoxycholic acid (TBS-D) and (3 times) in TBS-HT. Goat anti rabbit Ig-M-HRP (1:15,000 dilution) in TBS-HT was then added to membranes. Membranes were washed as before and bands detected using chem iluminescence. Immunoblots were quantified using Scion Image 4.02 analysis prog ram (Scion Corp., Frederick, MD). 4.1.5 Statistical Analysis Data are given as standard error of the mean (S.E.M) for three to five experiments. Comparisons between means we re performed using tw o-tailed Students t test for unpaired data and graphed using SigmaPlot 8.0 Values with p<0.05 were considered significant. 4.2 Results 4.2.1 Glucosamine, STZ, PUGNAc, and NAGBT increase O-GlcNAc modification on proteins In order to examine the effect of increasing O-GlcNAc modification on PKC translocation to the plasma membrane, an SV -40 transformed human glial cell line (SVG) was treated independently with three compounds at levels known to increase O-GlcNAc in other systems [15-17]. S VG cells were exposed to either 8mM glucosamine, 5mM STZ, 80 M PUGNAc, or 75 M NAGBT for 1, 3, 5 and 9 hours. In conjunction with these time points, four untreated (control) samples were also examined in order to determine O-GlcNAc and PKC basal levels. Each experiment was repeated between three and five times as indicated. To confirm that these three compounds did indeed increase the intracellular OGlcNAc modification in this system and to compare each ones effect, we analyzed both the cytosolic and membrane fractions from treated and untreated SVG cells. The

PAGE 75

64 fractions were analyzed by SDS-PAGE followed by immunobloting with the CTD 110.6 antibody that specifically recognizes the OGlcNAc modification [ 18]. Western blot analysis of cytosol and membrane fractions sh owed, as expected, a significant increase in O-GlcNAc levels but with quantitative differences between the compounds. Both cytosol and membrane fractions showed numerous la beled proteins with the most prominent increases on proteins of 125kDa, 96.3 kD a, 89.0 kDa, 66.0 kDa, and 35.1 kDa in the cytosol (Figure 4.1). Membrane fractions s howed major increases in O-GlcNAc content on proteins between 134 kDa and 76.6 kDa and at 55.1 kDa (Figure 4.2). O-GlcNAc levels for all fractions were also quantifie d densitometrically by m easuring the intensity of banding between 200 and 50 kDa (data not shown). Glucosamine treated samples showed maximum O-GlcNAc levels in both th e cytosol and membrane fractions as early as one hour post treatment, increasing by ~43% in the cytosol and ~37% in the membrane fraction versus untreated samples. These levels remain nearly constant throughout the nine hour treatment period. STZ treated samples were at maximum O-GlcNAc levels as early as three hours afte r treatment in the cytosol (~51% increase) and five hours in the membrane fraction (~89% increase). Unlike glucosamine and STZ treatment, PUGNAc treatment continued to show increased O-GlcNAc throughout the time course. After nine hours of PUGNAc treatment, O-GlcNAc levels increased by ~121% in the cytosol and ~236% in the membrane. Densitometric anal ysis of these immunoblots revealed that 75 M NAGBT treatment increased overall O-GlcNAc levels on both cytosol and membrane associated proteins to approxima tely twice the levels seen following 8mM glucosamine or 5mM STZ treatment (Figur e 4.1 and 4.2). A comparison of O-GlcNAc levels following 75 M NAGBT and 80 M PUGNAc treatment rev ealed interesting

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Figure 4.1 Effects of glucosamine, STZ, PUGNAc, and NAGBT treatments on OGlcNAc modification of cytosolic proteins SVG cells were treated with either 8mM glucosamine, 5mM STZ, 75M NAGBT, or 80M PUGNAc for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), or 9 hours (T=9). Untreated (control) samples were also prepared. Cells were fractionated into cytosol and membrane fractions and western blotted for O-GlcNAc using the CTD110.6 anti-O-GlcNAc antibody. 65

PAGE 77

Figure 4.2 Effects of glucosamine, STZ, PUGNAc, and NAGBT treatments on OGlcNAc modification of membrane proteins SVG cells were treated with either 8mM glucosamine, 5mM STZ, 75M NAGBT, or 80M PUGNAc for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), or 9 hours (T=9). Untreated (control) samples were also prepared. Cells were fractionated into cytosol and membrane fractions and western blotted for O-GlcNAc using the CTD110.6 anti-O-GlcNAc antibody. 66

PAGE 78

67 similarities and differences. The effect of the compounds on cytosolic O-GlcNAc levels was very similar resulting in a 40% increase after one hour of treatment that increased to 147% after 9 hours of treatment. However, an alysis of membrane fractions from cells treated with 75 M NAGBT revealed a much slower increase than in cells treated with 80 M PUGNAc (Figure 4.2). After 3 and 5 hour s of NAGBT treatment, overall amounts of the protein modification on membrane proteins had increased by only about 15% whereas PUGNAc treatment over the same dura tion resulted in an ove rall 40% increase in the membrane fraction. By 9 hours of treatment, O-GlcNAc increases in membrane fractions produced by both NAGB T and PUGNAc were at near ly identical levels (50% and 54% respectively) (Figure 4.2). 4.2.2 Effects of glucosamine on PKC translocation In order to determine the effect of increased O-GlcNAc on active, membrane bound PKC, membrane fractions from SVG cells treated with glucosamine, STZ, or PUGNAc were separated by SDS-PAGE, i mmunoblotted, and probed with antibodies against PKC, PKCII, PKC, and PKC. In glucosamine treated samples, PKCII membrane concentrations increased by 41% % (p<0.015) compared to untreated control cells one hour after glucosamine treatment. PKCII continued to increase in the membrane fraction showing a maximal 73%% (p<0.00005) increase after three hours of treatment and 55%% (p<0.005) after fi ve hours (Figure 4.3). In contrast to increases in PKCII translocation, membrane associated PKClevels decreased after incubation with glucosamine. PKClevels began decreasi ng after five hours of treatment, and, after nine hours of incubation, PKClevels in the membrane fraction had

PAGE 79

68 decreased by 48%% (p<0.005) (Fi gure 4.3). In contrast to PKCII and PKC, membrane bound concentrations of PKCand PKCshowed no significant change compared to control cells (Figure 4.3). The concentration of these PKC isoforms was also measured in the cytosol fraction; however, no significant changes we re observed (data not shown). This is probably due to the fact that in unstimulated cells, the PKC concentration in the cytosol is much greater than in the membrane. Ther efore, upon activation, changes in membrane concentrations can be signifi cant while the corresponding cha nge in the cytosol may be much less observable [6]. 4.2.3 Effects of streptozot ocin on PKC translocation Cells treated with STZ were analyzed similar to those treated with glucosamine. In contrast to glucosamine treatment, STZ tr eatment resulted in a decrease in membrane bound PKC(Figure 4.4) while not increasing PKCII translocation (Figure 4.4). PKCunderwent a 78%% (p<0.005) decreas e after nine hours of incubation. Similar to glucosamine treatment, STZ treatme nt did not result in any significant change in PKC(Figure 4.4) while producing a decrease in membrane bound PKC. PKCshowed a 42%% (p<0.04) decrease after fi ve hours of incubati on and continued to decrease by an average of 87%% (p<0.0005) nine hours after addition of STZ (Figure 4.4). Also of note is that, although STZ has been shown to be toxic in pancreatic -cells [19,20], it has been effectively used to incr ease O-GlcNAc levels in other cell lines without a decrease in cell viab ility [21]. During our nine hour STZ treatment period, we

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69 Figure 4.3 Effects of Glucosamine on PKC isoforms in membrane fractions. SVG cells were treated with 8mM glucosamine for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), 9 hours (T=9). Untreated (control) samples were prepared for each experiment. Samples were separated into cytosol and membrane fractions as described in section 4.1.3. Equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Membrane fractions were treated with isoform specific anti PKC antibodies. Immunoblots were analyzed by densitometry and the results were graphed (A). Representative immunoblots for membrane fractions probed with anti PKC-II, PKC-, PKC-, and PKCantibodies are shown (B). Values are means S.E.M. for 5 determinations. represents p<0.05 and ** represents p<0.01

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70 Figure 4.4 Effects of STZ on PKC isoforms in membrane fractions. SVG cells were treated with 5mM STZ for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), 9 hours (T=9). Untreated (control) samples were prepared for each experiment. Samples were separated into cytosol and membrane fractions as described in section 4.1.3. Equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Membrane fractions were treated with isoform specific anti PKC antibodies. Immunoblots were analyzed by densitometry and the results were graphed (A). Representative immunoblots for membrane fractions probed with anti PKC-II, PKC-, PKC-, and PKCantibodies are shown (B). Values are means S.E.M. for 5 determinations. represents p<0.05 and ** represents p<0.01

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71 observed no changes in cell morphology or total protein levels or loss of cell viability (data not shown). 4.2.4 Effects of PUGNAc on PKC translocation The results obtained from PUGNAc treatmen t of SVG cells were similar to those obtained after STZ treatment. As with S TZ treatment, PUGNAc treatment reduced PKCand PKCin the membrane fractions and at a similar rate while not significantly changing PKCII and PKCmembrane concentrations (Figure 4.5). PKCwas reduced by 66%% (p<0.005) nine hours after the addition of PUGNAc (Figure 4.5). With PKC, a 40%% (p<0.02) reduction was dete cted five hours after treatment and a 73%% (p<0.0005) reduction after nine hours (Figure 4.5). 4.2.5 Effects of NAGBT on PKC translocation Unlike treatment with STZ or PUG NAc, NAGBT treatment resulted in no reduction in membrane associated PKCthroughout the treatment period and only a modest (13%.5%) after 9 hours of treatment (Figure 4.6). PKCII and PKCmembrane concentration did not deviate from control levels thr oughout the time course and cytosolic levels of all is oforms analyzed also did not change from control levels following NAGBT treatment (Figure 4.6). 4.3 Discussion Previous work in adipocytes and kidney cells has demonstrated that increased flux through the HBP can alter the ov erall activity and translocati on of certain PKC isoforms [3-7,22]. While the specific changes vary among the experi mental systems it has been hypothesized that these alterati ons in PKC may be the resu lt of increases in the O

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72 Figure 4.5 Effects of PUGNAc on PKC isoforms in membrane fractions. SVG cells were treated with 80M PUGNAc for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), 9 hours (T=9). Untreated (control) samples were prepared for each experiment. Samples were separated into cytosol and membrane fractions as described in section 4.1.3. Equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Membrane fractions were treated with isoform specific anti PKC antibodies. Immunoblots were analyzed by densitometry and the results were graphed (A). Representative immunoblots for membrane fractions probed with anti PKC-II, PKC-, PKC-, and PKCantibodies are shown (B). Values are means S.E.M. for 5 determinations. represents p<0.05 and ** represents p<0.01

PAGE 84

73 Figure 4.6 Effects of NAGBT on PKC isoforms in membrane fractions. SVG cells were treated with 70M NAGBT for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), 9 hours (T=9). Untreated (control) samples were prepared for each experiment. Samples were separated into cytosol and membrane fractions as described in section 4.1.3. Equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Membrane fractions were treated with isoform specific anti PKC antibodies. Immunoblots were analyzed by densitometry and the results were graphed (A). Representative immunoblots for membrane fractions probed with anti PKC-II, PKC-, PKC-, and PKCantibodies are shown (B). Values are means S.E.M. for 5 determinations. represents p<0.05 and ** represents p<0.01

PAGE 85

74 GlcNAc modification on certain intracellular proteins [ 5,22]. To investigate this hypothesis, we examined the effects of elevat ed O-GlcNAc levels on the translocation of specific PKC isoforms in an SV40-transforme d human astroglial cell line. Increases in cellular O-GlcNAc were achieved by increasing flux through the HBP with glucosamine [23] or inhibiting O-GlcNAcase with STZ, PUGNAc, and NAGBT [16,17,19,20,24,25]. A comparison of all four compounds demons trates their usefulness as tools for modulating O-GlcNAc levels while also revealing their quant itative and kinetic differences in O-GlcNAc protei n modification. These differen ces can, as discussed later, result in variations in prot ein regulation and possibly other intracellular functions related to this modification. While a link between O-GlcNAc and PK C isoform regulation has not been investigated the effects of glucosamine on these enzymes has been well documented. Studies demonstrating a connection between increased glucosamine and PKC regulation have, however, produced a wide variety of results. Therefore, after confirming that glucosamine increases O-GlcNAc in our syst em, we investigated the effect of this treatment on the translocation of four PKC is oforms to the cell membrane of SVG cells. We examined the translocation of two conventional isoforms (PKCand PKCII), one novel isoform (PKC), and one atypical isoform (PKC). In our 8 mM glucosamine treated samples, a relatively rapid in crease in membrane associated PKCII was observed (41%% one hour and 73%% three hours post treatment). A similar translocation of PKCwas observed by Kolm-Litty et al. [6] in kidney mesangial cells following treatment with 12mM glucosamine. In addition, we obser ved a relatively slow decrease of PKCfrom the membrane (48%% decrease nine hours post treatment)

PAGE 86

75 while PKCand PKClevels remained unchanged throughout the treatm ent period. Interestingly, these results are in contrast to Kolm-Littys findings that demonstrate an increased translocation of both PKCand PKCin response to glucosamine treatment [6]. In addition, other groups examining PKC observed both similar and contrasting results following glucosamine treatment. Expe riments by Goldberg et al. [5] also in kidney mesangial cells but with a much lowe r (2mM) glucosamine concentration did not find any changes in PKC isoform translocation; however, they did observe an increase in PKCI, PKC, and PKCenzyme activity. Furthermore, adipocytes treated with 3mM glucosamine [3] showed an in creased translocation of PKCbut no changes in PKCor PKC. While complex, it is widely recognized that the same PKC isoforms often have different or even opposing roles in different cell types [26]. These cell type differences most likely explain some of these variations in results while the varying concentrations of glucosamine used in these experiments likely contribute to other observed differences. All of this data, however, emphasizes a conn ection between increased glucosamine levels and PKC regulation and indicate that this connection occurs in a wide variety of cell types but with unique characteristics. To investigate if results obtained following glucosamine treatment were specifically due to elevated O-GlcNAc, intra cellular O-GlcNAc levels were increased by an alternative mechanism using the O-GlcNAcase inhibitors STZ, PUGNAc, and NAGBT. To our knowledge this study is the fi rst to examine the eff ects of all of these inhibitors on PKC isoform translocation. Similar to glucosamine treatment, cellular treatment with STZ and PUGNAc resulted in both increases in global O-GlcNAc levels and large, significant decreases in membrane associated PKClevels. The results

PAGE 87

76 obtained following treatment with NAGBT, however, were not consistent with this trend. Whereas NAGBT treatment produced large increases in total O-GlcNAc levels similar to those observed following PUGNAc treatme nt, it yielded a relatively small but reproducible decrease in membrane bound PKC(13%.5%). The reason for the divergent results obtained following NAGBT treatment is not known; however, there are two possibilities. The first possibility is th at the recently character ized NAGBT may have other unknown cellular effects that may c ounteract or delay a ny O-GlcNAc induced decreases in membrane bound PKC. This explanation is li kely because to date NAGBT has only been used in one published paper [24] and, other than its ability to inhibit OGlcNAcase and increase global O-GlcNAc leve ls in COS-7, its eff ects on other cellular pathways or systems have not been examined. A second, less likely, possibility for these results is that the large d ecreases in membrane bound PKCobserved following glucosamine, STZ, and PUGNAc treatments are the result of a shared pathway not related to the O-GlcNAc modification. This explanation seems unlikely because these compounds have been used extensively in a va riety of systems and, other than increasing O-GlcNAc modification levels, are not know n to affect another common pathway. Furthermore, although glucosamine and STZ have been demonstrated to produce oxidative stress in vitro [27-29], PUGNAc treatment has been shown to have an opposite, protective effect against various stressors in several cell ty pes [30]. PUGNAc has also been demonstrated not to mimic many of the cy totoxic effects of STZ in pancreatic cells [19]. Finally, unlike glucosamine and STZ, PUGNAc treatment does not alter phosphorylation of the serine/threonine kinase Akt providing further evidence against a shared pathway unrelated to O-GlcNAc (discussed further in chapter 8).

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77 Whereas treatment with all four O-GlcNAc modulating agents resulted in significant although varyi ng decreases in membrane associated PKC, only STZ and PUGNAc treatments produced significan t decreases in membrane bound PKCwithin the examined time period. Additionally, unlike membrane associated PKClevels that showed significant decrease s after 5 hours of STZ and PU GNAc treatment, significant decreases in PKCmembrane levels were not observed until 9 hours post treatment. This evidence suggests that the membrane association of PKCis less sensitive to increases in O-GlcNAc protei n modification and thus decreases more slowly than PKC. It is also possible that the decr eased levels of membrane bound PKCin response to STZ and PUGNAc treatments occur via a pathwa y not related to O-GlcNAc that is not affected by glucosamine or NAGBT. Furthe r investigation, including treatment periods longer than 9 hours, is necessary to more c onclusively determine a possible relationship between O-GlcNAc and PKC. Unlike glucosamine, neither STZ, PUGNAc, nor NAGBT resulted in increased translocation of PKCII. This result suggest that the effect of glucosamine on PKCII may not be linked to the increa ses in O-GlcNAc but due to alternate glucosamine effects such as certain oxidative stressors [27], calcium entry [31], altered endoplasmic reticulum function [32], or other yet unidentified path way and thus not affected by STZ, PUGNAc, or NAGBT (further discussed in Chapter 8). Also, none of the four treatment conditions had any significant effect on membrane associated levels of the atypical PKC, further underscoring the complex and varyi ng regulation of the PKC family.

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78 The use of a variety of O-GlcNAc m odulating agents in this study provides evidence that the O-GlcNAc modification may play a previously unknown role in regulating levels of PKCand possibly PKCat the cell membrane. Since PKC translocates to the cell membrane upon activ ation [33], the observed decreases in membrane bound PKCand PKCin response to increases in O-GlcNAc correspond to a decrease in their activity. Similarly, Akt, a serine-threonine kinase that, like PKC, depends on a series of ordered phosphorylati ons [34-38] and memb rane translocation [34] for activation, undergoes a decrease in activity in respon se to increased O-GlcNAc levels in adipocytes [39]. Additionally, evidence supporting a selective coordination in the regulation of PKCand PKChas been observed in other systems. In U251N glioma cells, PKCand PKCare the only isoforms to tran slocate to the cell membrane in response to phorbol ester stim ulation [40] and, in other cel l types, these two isoforms have been shown to act cooperatively in the regulation of c-Jun N-te rminal kinase (JNK) pathway [41] and in enhancing cell cycle progression [42]. The exact mechanism(s) by which O-GlcNAc may elicit changes in membrane associated PKC isoform levels remains to be determined. It is unlikely that these membrane associated decreases involve a disruption in the net synthesis of PKCor PKCbecause no alterations in the larger cytosolic pool of these isoforms were observed. Alternatively, prolonged activation of PKC isoforms has been shown to result in their down-regulation from the memb rane [40,43]; however, since increased translocation of PKCand PKCwas not observed prior to the observed decreases, it is unlikely that increases in ac tivators such as DAG or phospholipids account for our data. Since there is no evidence to suggest that PK C is directly modified by O-GlcNAc [5], the

PAGE 90

79 effects on these PKC isoforms may be mediat ed by O-GlcNAc modification of proteins that regulate their activat ion and/or degradation. There are several points at which incr eases in O-GlcNAc modifications could elicit the observed decreases in membrane associated PKCand PKC. First disrupting interactions with isoform-selective PKC bindi ng proteins, such as receptors for activated C kinase (RACKs) could reduce membrane a ssociation. For inst ance, disrupting the association between RACK1 and PKCII has been shown to block its translocation to the plasma membrane [44] and a RACK specific to PKC(RACK2) has been identified [45] and shown to play a critic al role in this isoforms tran slocation [46]. Korzick et al. [47] showed that an age-related de crease in the translocation of PKCand in rat myocardial cells following -adrenergic receptor stimulation was related to a decrease in RACK1 and RACK2 levels [47]. Second, alteri ng activation of upstream enzymes, such as phosphoinositide 3-OH-kinase (PI3-K), known to be involved in the PKC activation cascade could prevent translocation. While short (2 hour) glucosamine treatment has been shown to increase PI3-K activity in adipocytes [3], longer (> 4 hour) glucosamine treatment in skeletal muscle [48] and di rect PI3-K modificatio n with O-GlcNAc in endothelial cells [1] correla tes with reduced activity. Since PI3-K activates 3phosphoinositide-dependent kinase-1 (PDK-1) th at can in turn phosphorylate and activate conventional [49], novel [50,51], and atypical [52] PKC isoforms, an elevation of OGlcNAc affecting PI3-K could reduce PKC ac tivation. Third, increased O-GlcNAc could block the recycling of PKCand PKCback into the active PKC pool. Upon dephosphorylation PKC is deactivated and rem oved from the plasma membrane and is either degraded by proteolysis or stabilized by binding to Hsp70 [53] Once stabilized,

PAGE 91

80 PKC can be rephosphorylated, reactivated, and returned to the plasma membrane. Hsp70 has been shown to be modified by O-GlcNAc [54] and, therefore, increased O-GlcNAc may alter its ability to bind and prevent proteolysis of select PKC isoforms. In summary, our data in SVG cells de monstrates a novel link between increased levels of O-GlcNAc and decreases in active, membrane associated PKCand possibly PKC. Furthermore, glucosamine treatment resulted in the activation of PKCII via a pathway likely unrelated to the O-GlcNAc modifi cation while PKCwas unaffected by this modification.

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81 4.4 References Cited 1. M. Federici, R. Menghini, A. Mauriello, M.L. Hribal, F. Ferrelli, D. Lauro, P. Sbraccia, L.G. Spagnoli, G. Sesti, R. Lauro, Insulin-dependent activation of endothelial nitric oxide s ynthase is impaired by O-linke d glycosylation modification of signaling proteins in human coronary endothelial cells, Ci rculation 106 (2002) 466-72. 2. Z.T. Kneass,R.B. Marchase, Protein O-GlcNAc modulates motility-associated signaling intermediates in neutroph ils, J. Biol. Chem. 280 (2005) 14579-85. 3. C. Filippis, A. Filippis, S. Clark, J. Proiet to, Activation of PI 3-kinase by the hexosamine biosynthesis pathway, Mo l. Cell. Endocrinol. 194 (2002) 29. 4. L.P. Singh,E.D. Cook, Hexosamine regulat ion of glucose-mediated laminin synthesis in mesangial cells involves protein kinases A a nd C, Am. J. of Physiol. Renal Physiol. 279 (2000) F646-F654. 5. H.J. Goldberg, C.I. Whiteside, I.G. Fantus, The Hexosamine Pathway Regulates the Plasminogen Activator Inhibitor-1 Ge ne Promoter and Sp1 Transcriptional Activation through Protein Kinase C-beta I and -delta, J. Biol. Chem. 277 (2002) 33833-41. 6. V. Kolm-Litty, S. Tippmer, H.U. Hari ng, E. Schleicher, Glucosamine induces translocation of protein kinase C isoenzymes in mesangial cells, Exp. Clin. Endocrinol. Diabetes 106 (1998) 377-83. 7. A. Filippis, S. Clark, J. Proiet to, Increased flux through the hexosamine biosynthesis pathway inhibits glucose tr ansport acutely by activation of protein kinase C, Biochem. J. 324 ( Pt 3) (1997) 981-5.

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82 8. Y. Yoshida, F.L. Huang, H. Nakabayashi, K.P. Huang, Tissue distribution and developmental expression of protein kina se C isozymes, J. Biol. Chem. 263 (1988) 9868-73. 9. M. Goldberg,S.F. Steinberg, Tissue-speci fic developmental regulation of protein kinase C isoforms, Biochem. Pharmacol. 51 (1996) 1089-93. 10. Y. Gao, L. Wells, F.I. Comer, G.J. Parker, G.W. Hart, Dynamic O-glycosylation of nuclear and cytosolic pr oteins: cloning and charact erization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain, J. Biol. Chem. 276 (2001) 9838-45. 11. R. Okuyama,S. Marshall, UDP-N-acetylglucosaminyl transferase (OGT) in brain tissue: temperature sensitivity and subcellu lar distribution of cytosolic and nuclear enzyme, J. Neurochem. 86 (2003) 1271-80. 12. T.W. Corson, K.K. Woo, P.P. Li, J.J. Warsh, Cell-type specific regulation of calreticulin and Bcl-2 expression by mood stabilizer drugs, Eur. Neuropsychopharmacol. 14 (2004) 143-50. 13. E.O. Major, A.E. Miller, P. Mourrain, R.G. Traub, E. de Widt, J. Sever, Establishment of a line of human feta l glial cells that supports JC virus multiplication, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 1257-61. 14. C. Slawson, S. Shafii, J. Amburgey, R. Potter, Characterization of the O-GlcNAc protein modification in Xenopus laevis oocyte during oogenesis and progesteronestimulated maturation, Biochi m. Biophys. Acta 1573 (2002) 121-9. 15. I. Han, E.S. Oh, J.E. Kudlow, Responsiveness of the state of O-linked Nacetylglucosamine modification of nuclear pore protein p62 to the extracellular

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83 glucose concentration, Biochem. J. 350 Pt 1 (2000) 109-14. 16. M.D. Roos, W. Xie, K. Su, J.A. Clar k, X. Yang, E. Chin, A.J. Paterson, J.E. Kudlow, Streptozotocin, an analog of N-acet ylglucosamine, blocks the removal of O-GlcNAc from intracellular proteins, Proc. Assoc. Am. Physicians 110 (1998) 422-32. 17. R.S. Haltiwanger, K. Grove, G.A. Philipsberg, Modulation of O-linked Nacetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-Nacetylglucosaminidase inhibitor O-(2-acetamido-2deoxy-Dglucopyranosylidene)amino-Nphenylcarbamate, J. Biol. Chem. 273 (1998) 3611-7. 18. F.I. Comer, K. Vosseller, L. Wells, M.A. Accavitti, G.W. Hart, Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine, Anal. Biochem. 293 (2001) 169-77. 19. Y. Gao, G.J. Parker, G.W. Hart, St reptozotocin-induced beta-cell death is independent of its inhibition of O-Glc NAcase in pancreatic Min6 cells, Arch. Biochem. Biophys. 383 (2000) 296-302. 20. R.J. Konrad, I. Mikolaenko, J.F. Tolar, K. Liu, J.E. Kudlow, The potential mechanism of the diabetogeni c action of streptozotocin: inhibiti on of pancreatic beta-cell O-GlcNAc-selectiv e N-acetyl-beta-D-glucosaminidase, Biochem. J. 356 (2001) 31-41. 21. G. Majumdar, A. Harmon, R. Candelaria, A. Martinez-Hernandez, R. Raghow, S.S. Solomon, O-glycosylation of Sp1 and transcriptional regulation of the calmodulin gene by insulin and glucagon, Am. J. Physiol. Endocrinol. Metab. 285

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84 (2003) E584-91. 22. L.P. Singh,E.D. Crook, The effects of glucose and the hexosamine biosynthesis pathway on glycogen synthase kinase-3 a nd other protein kinases that regulate glycogen synthase activity, J. Investig. Med. 48 (2000) 251-8. 23. S. Marshall, V. Bacote, R.R. Traxi nger, Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance, J. Biol. Chem. 266 (1991) 4706-12. 24. M.S. Macauley, G.E. Whitworth, A.W. Debowski, D. Chin, D.J. Vocadlo, OGlcNAcase uses substrate-assisted catalysi s: kinetic analysis and development of highly selective mechanism-inspired i nhibitors, J. Biol. Chem. 280 (2005) 2531322. 25. J.A. Hanover, Z. Lai, G. Lee, W. A. Lubas, S.M. Sat o, Elevated O-linked Nacetylglucosamine metabolism in pancrea tic beta-cells, Arch. Biochem. Biophys. 362 (1999) 38-45. 26. J.D. Black, Protein kinase C-mediated regul ation of the cell cycl e, Front. Biosci. 5 (2000) D406-23. 27. H. Kaneto, G. Xu, K.H. Song, K. Suzuma, S. Bonner-Weir, A. Sharma, G.C. Weir, Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress, J. Biol. Chem. 276 (2001) 31099-104. 28. R. Mastrocola, F. Restivo, I. Vercelli natto, O. Danni, E. Brignardello, M. Aragno, G. Boccuzzi, Oxidative and nitrosative stress in brain mitochondria of

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85 diabetic rats, J. Endocrinol. 187 (2005) 37-44. 29. S.A. Wohaieb,D.V. Godin, Alterations in free radical tissue-defense mechanisms in streptozocin-induced diabetes in rat. Effects of insulin treatment, Diabetes 36 (1987) 1014-8. 30. N.E. Zachara, N. O'Donnell, W.D. Ch eung, J.J. Mercer, J.D. Marth, G.W. Hart, Dynamic O-GlcNAc modification of nucleoc ytoplasmic proteins in response to stress. A survival response of mamm alian cells, J. Biol. Chem. 279 (2004) 3013342. 31. S. Vemuri,R.B. Marchase, The inhibition of capacitative calcium entry due to ATP depletion but not due to glucosamine is re versed by staurosporine, J. Biol. Chem. 274 (1999) 20165-70. 32. H.Y. Lin, P. Masso-Welch, Y.P. Di, J.W. Cai, J.W. Shen, J.R. Subjeck, The 170kDa glucose-regulated stress protein is an endoplasmic reticulum protein that binds immunoglobulin, Mol. Biol. Cell 4 (1993 ) 1109-19. 33. A.S. Kraft,W.B. Anderson, Phorbol es ters increase the amount of Ca2+, phospholipid-dependent protein kinase asso ciated with plasma membrane, Nature 301 (1983) 621-3. 34. M. Andjelkovic, D.R. Alessi, R. Meier, A. Fernandez, N.J. Lamb, M. Frech, P. Cron, P. Cohen, J.M. Lucocq, B.A. He mmings, Role of translocation in the activation and function of protein kinase B, J. Bi ol. Chem. 272 (1997) 31515-24. 35. D.R. Alessi, M. Andjelkovic, B. Caudwell, P. Cron, N. Morrice, P. Cohen, B.A. Hemmings, Mechanism of activation of protein kinase B by insulin and IGF-1, EMBO J. 15 (1996) 6541-51.

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86 36. M. Andjelkovic, T. Jakubowicz, P. Cron, X.F. Ming, J.W. Han, B.A. Hemmings, Activation and phosphorylation of a pleckst rin homology domain containing protein kinase (RAC-PK/PKB) promoted by seru m and protein phosphatase inhibitors, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 5699-704. 37. A.D. Kohn, F. Takeuchi, R.A. Roth, Akt, a pleckstrin homology domain containing kinase, is activated primarily by phos phorylation, J Biol Chem 271 (1996) 21920-6. 38. A.C. Newton, Regulation of the ABC kina ses by phosphorylation: protein kinase C as a paradigm, Biochem. J. 370 (2003) 361-71. 39. K. Vosseller, L. Wells, M.D. Lane, G.W. Hart, Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insuli n resistance associated with defects in Akt activation in 3T3-L1 adipocytes, Proc Natl. Acad. Sci. U. S. A. 99 (2002) 5313-8. 40. A. Besson, T.L. Wilson, V.W. Yong, Th e anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains Requirements for adhesion and motility, J. Biol. Chem. 277 (2002) 22073-84. 41. W. Lang, H. Wang, L. Ding, L. Xiao Cooperation between PKC-alpha and PKCepsilon in the regulation of JNK activati on in human lung cancer cells, Cell Signal. 16 (2004) 457-67. 42. J.W. Soh,I.B. Weinstein, Roles of specifi c isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes, J. Biol. Chem. 278 (2003) 34709-16. 43. G. Hansra, P. Garcia-Paramio, C. Pre vostel, R.D. Whelan, F. Bornancin, P.J. Parker, Multisite dephosphoryl ation and desensitization of conventional protein

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87 kinase C isotypes, Biochem. J. 342 ( Pt 2) (1999) 337-44. 44. E.G. Stebbins,D. Mochly-Rosen, Binding specificity for RACK1 resides in the V5 region of beta II protein kinase C, J. Biol. Chem. 276 (2001) 29644-50. 45. M. Csukai, C.H. Chen, M.A. De Matte is, D. Mochly-Rosen, The coatomer protein beta'-COP, a selective binding protein (RACK) for protein kinase Cepsilon, J. Biol. Chem. 272 (1997) 29200-6. 46. D. Schechtman, M.L. Craske, V. Kheifets, T. Meyer, J. Schechtman, D. MochlyRosen, A critical intramolecular inte raction for protein kinase Cepsilon translocation, J. Biol. Chem. 279 (2004) 15831-40. 47. D.H. Korzick, D.A. Holiman, M.O. Boluyt, M.H. Laughlin, E.G. Lakatta, Diminished alpha1-adrenergic-mediated co ntraction and transl ocation of PKC in senescent rat heart, Am. J. Physiol. Heart Circ. Physiol. 281 (2001) H581-9. 48. M.E. Patti, A. Virkamaki, E.J. Landak er, C.R. Kahn, H. Yki-Jarvinen, Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance of early postreceptor insulin signaling events in skeletal muscle, Diabetes 48 (1999) 1562-71. 49. E.M. Dutil, A. Toker, A.C. Newton, Re gulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1), Curr. Biol. 8 (1998) 1366-75. 50. J.A. Le Good, W.H. Ziegler, D.B. Pa rekh, D.R. Alessi, P. Cohen, P.J. Parker, Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1, Science 281 (1998) 2042-5. 51. V. Cenni, H. Doppler, E.D. Sonnenburg, N. Maraldi, A.C. Newton, A. Toker,

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88 Regulation of novel protein kinase C epsilon by phosphor ylation, Biochem. J. 363 (2002) 537-45. 52. M.M. Chou, W. Hou, J. Johnson, L.K. Graham, M.H. Lee, C.S. Chen, A.C. Newton, B.S. Schaffhausen, A. Toker, Regul ation of protein kinase C zeta by PI 3kinase and PDK-1, Curr. Biol. 8 (1998) 1069-77. 53. T. Gao, A.C. Newton, The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C, J. Biol. Chem. 277 (2002) 31585-92. 54. J.L. Walgren, T.S. Vincent, K.L. Schey, M.G. Buse, High glucose and insulin promote O-GlcNAc modification of prot eins, including alpha-tubulin, Am. J. Physiol. Endocrinol. Metab. 284 (2003) E424-34.

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89 Chapter 5 Investigation of potential mechanisms fo r decreases in membrane associated PKCand PKC5.0 Introduction Treatment of SVG cells with the OGlcNAc modulating agents STZ and PUGNAc resulted in dramatic decreases in the levels of membra ne associated PKCand while not affecting membrane associated PKCII or (see chapter 4). These results suggest that this isoform-specific reduction is related to the O-Gl cNAc modification of certain proteins associated w ith the regulation of these PKC isoforms. As discussed in Chapter 4, there are several possible points at which O-GlcNAc prot ein modification may affect the levels of these isoforms at the cell membrane. Three of these possible interaction points are the inhibition of upstream PKCand PKCactivators, RACK mediated PKC isoform translocation, and the Hsp70 mediated recycling of these isoforms. First, PI 3-K, an upstream indir ect activator of PKC isoforms, has been shown to be directly modified by O-GlcNAc and th is modification correlates with a reduction in its activity [1]. Therefore, a decrease in th e activity of a PKC activ ator such as PI 3-K could result in decreased PK C isoform activation manifesting in a decrease in the translocation of these isofor ms to the plasma membrane. Second, although a relationship between RACKs and the O-GlcNAc modifi cation has not been investigated, the association between the PKCII and RACK1 and PKCand RACK2 has been

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90 demonstrated to facilitate PKC translocation to the plasma membrane [2-4]. Since the disruption of this interaction results in th e decreased translocati on of the PKC isoform [3], and the O-GlcNAc modification is known to affect protein-protein interactions (section 1.4.3), it is possible that a misregula tion of this step is responsible for the decreases in membrane associated PKCand observed in our system. Third, Hsp-70, an O-GlcNAc modified prot ein [5], has been demonstr ated to bind and stabilize dephosphorylated PKCII and [6]. This binding allows the isoforms to be rephosphorylated and recycled back to the plasma membrane [6]. Therefore, the sustained O-GlcNAc modificat ion of Hsp-70 may disrupt it s interactions with PKCand PKCresulting in decreased recycling a nd manifesting in decreased membrane associated levels and increased degradation of these isoforms. The focus of this chapter is to examine if increased levels of intracellular O-GlcNAc mo dification affects either the activation and/or tr anslocation of PKCand or their Hsp70 mediated recycling. 5.1 Increased O-GlcNAc and PKCand translocation In order to investigate if increased O-GlcNAc protein modification decreased membrane-associated levels of PKCand by either disrupting the phosphorylation of these isoforms or their interactions with RA CKs, SVG cells were pretreated for 5 hours with 5mM STZ and then treated with phorbol 12-myristate-13-acetate (PMA). Cells were treated for 5 hours with 5mM STZ because this treatment condition was demonstrated significantly increase global O-GlcNAc levels and also be sufficient to facilitate a 48% decrease in membrane associated PKC. Longer (9 hour) preincubation of cells with STZ prior to stimulation with PMA (up to 5 hour treatment) was avoided in

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91 order to reduce any toxic effects brought about by prolonged STZ exposure [7,8]. PMA was chosen to induce PKC translocation because it is a well characterized activator of both cPKC and nPKC isoforms. In order for PMA to induce translocation, PKC must first be fully phosphorylated [9] (Section 2.3.1). Additionally, bloc king the association between PKCII and RACK1 [3] or PKCand RACK1 [10] has been shown to prevent the PMA-induced translocation of these isoforms to the pl asma membrane or membrane associated focal adhesions respectively. Theref ore, if either the ac tivity of an upstream PKC activator or its ability to associate with a RACK is disrupted after pretreatment with 5mM STZ for 5 hours, a reduction in PMA-induc ed translocation of the isoforms should be observed. In order to de termine the normal translocation pattern of PKC isoforms in this cell type to PMA, cells were al so treated with 100nM PMA without STZ pretreatment. 5.2 Increases O-GlcNAc and Hsp70 mediated PKC recycling In addition to a disruption in PKC isof orm phosphorylation or RACK association, reduction in membrane PKCand levels could be caused by the disruption of Hsp70mediated PKC recycling. In order to inve stigate this hypothesis, immunoprecipitation was used to first analyze if either PKCor associated with Hsp70 in SVG cells and, if so, if this interaction was disrupted unde r conditions of increased O-GlcNAc protein modification. Additionally, since a disr uption in Hsp70 mediated PKC recycling correlates with increased PKC proteolysis and partitioning to the cyto skeletal fraction [6] (see section 2.4), cytoskeletal fractions from cells treated with or without O-GlcNAc modulating agents were analyzed for in creased PKC isoform degradation. 5.3 Materials and Methods

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92 Most materials and methods used in this chapter can be found in section 4.1. Materials and methods used solely in this chapter are described below. 5.3.1 Cell culturing and sample pr eparation for PMA treatment SVG cells were cultured as described prev iously. In order to determine the PKC isoform translocation pattern in response to PMA, SVG cells were treated with 100nM PMA in DMSO or in an equal volume of DM SO alone for 15 minutes, 1 hour, 5 hours, or 21 hours. To analyze any effects of in creased O-GlcNAc on PMA stimulated PKC translocation, cells were treate d with or without 5mM STZ fo r 5 hours. After the 5 hour preincubation period, cells were treated with 100nM PMA in DMSO or with an equal volume of DMSO alone for 15 minutes, 1 hour, or 5 hours without changing the medium. Longer time points (> 10 hours total STZ incubation) were not done because long incubations with STZ have been show n to induce DNA damage in pancreatic -cells [7,8]. After the appr opriate treatments, cells were fractionated into cytosol and membrane fractions and analyzed by SDSPAGE and western bl otting using isoform specific PKC antibodies as described in section 4.1.4. 5.3.2 Cell culturing and sample prep aration for immunoprecipitation SVG cells were cultured as previously de scribed. Samples were treated with or without 5mM STZ for 9 hours where appropria te. When cells reached approximately 90% confluency, cells were washed two times in cold PBS and scraped into 3ml of cold IP buffer (20mM Tris base (pH 7.4), 1mM EDTA, 1mM EGTA, 2mM MgCl 2 150mM NaCl, 1mM DTT, 1mM PMSF, 1mM Na 3 VO 4 4 g/ml aprotinin, 1.0% Triton X-100). Samples were incubated on a rocker at 4 C for 10 minutes followed by sonication two times for 3 seconds at 7 watts. All steps were performed at 4 C unless otherwise stated.

PAGE 104

93 Samples were centrifuged at 12000xg (12500rpm ) for 10 minutes and the supernatant was removed. The pellet was resuspended in 1x SDS-PAGE sample buffer, boiled for 5 minutes and analyzed as the cytoskeleton fract ion. Supernatant protein concentrations were determined by Bradford assay using BS A as a standard (section 4.1.3). Samples were then diluted to equal protein co ncentrations (1.5-1.0mg/ml) by adding an appropriate amount of IP buffer. Samples were precleared with immobilized Protein A agarose (Pierce Biotechnology, Rockfo rd, IL, catalog # 22811) by adding 25 l of beads to approximately 1.0ml of sample in a 1.5m l centrifuge tube. Samples were then incubated for 1 hour with gentle rocking. Samples were then centrifuged at 1000xg for 5 minutes and supernatant was removed. The pe llet, containing proteins nonspecifically bound to the Protein A agarose beads, was resu spended in 1x SDS-PAGE sample buffer, boiled for 5 minutes, and supernatant was removed for later analysis. Following preclearing, appropriate primar y or control antibody were added to each sample. Primary antibodies used for immunoprecipitation include anti-nPKC(Santa Cruz Biotechnol ogy, sc-214), anti-cPKC(Santa Cruz Biotechnology, sc-8393), and anti-Hsp70 (Stressgen, SPA-810) whereas an tibodies used as controls include normal rabbit IgG (Santa Cruz Biotec hnology, sc-2027), normal mouse IgG 1 (Santa Cruz Biotechnology, sc-3877), anti-p27 (Santa Cr uz Biotechnology, sc-1641), and anti-Mat1 (Santa Cruz Biotechnology, sc-13142). A variety of antibody amounts (5.0 g 0.5 g) were used for immunoprecipitation experiments with 1.0 g found to give the best signal to noise ratio following western blot analysis Samples with antibodies were incubated overnight with gentle rocking. The next morning, 10 l of immobilized Protein A agarose was added per 1 g of antibody and samples were incubated for 2 hours with gentle

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94 rocking. Samples were then centrifuged at 1000xg for 5 minutes, supernatants were removed and retained at -70 C, and beads were washed 4 times in IP buffer. Beads were then resuspended in 25 l of 3x SDS-PAGE sample buffer, boiled for 5 minutes, and supernatant was removed. In order to remove the small volume (~25 l) of sample from beads, a small hole was punctured in the botto m of the centrifuge tube with 6 beading 1 1 / 4 inch sewing needle. The punctured tube was then placed inside of another tube and briefly centrifuged. The liquid passed through the hole into the new tube leaving the beads in the punctured tube. Samples were th en analyzed by SDS-PAGE, as described in section 4.1.4, using specified antibodies. After initial immunoblot was complete the nitrocellulose membrane was subjected to a second immunoblot proce dure. The antibodies used for this immunodetection step were the same as th e antibody used for immunoprecipitation. This process was done to ensure that the immunopr ecipitation had effectiv ely precipitated the desired protein. First, the nitrocellulose membranes were washed 2 times for 15 minutes in TBST and then placed in stripping solution (200mM glycine pH 2.2) for 45 minutes. Membranes were then washed 1 time for 15 minutes in TBST, blocked in 5% nonfat dry milk in TBST, immunoblotted as described in section 4.1.4. 5.4 Results 5.4.1 Effects of increased O-GlcN Ac on PKC isoform translocation As expected, cellular treatment with 100nM PMA in DMSO had a dramatic effect of the translocation of PKC, and II but did not affect either cytosolic or membrane levels PKCwhen compared to DMSO treatme nt alone (Figure 5.1). Because PMA activates PKC by binding to the C1 do main (section 2.1), isoforms lacking a

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Effects of PMA Treatment on PKC Isoforms Figure 5.1 Effects of 100nM PMA treatment on PKC isoform translocation SVG cells treated with 100nM PMA in DMSO for 15min (T=0.25), 1 hour (T=1), 5 hours (T=5), or 21 hours (T=21). Additionally, cells were treated with an equivalent volume of DMSO alone for 1, 5, or 21 hours (control). Samples were then fractionated into membrane and cytosol fractions. 95

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96 functional C1 domain, such as the aPKCs are not affected by PMA treatment thus providing an explanation for the unresponsiveness of PKCto the treatment. Unlike PKC, after only 15 minutes of treatment, all detectable PKChad translocated from the cytosol to the membrane fraction. Also after 15 minutes of treatment, most PKCII and about half of PKChad translocated. After 5 hour s of PMA treatment, membrane associated levels of conventional PKCand II began to decrease and were not detectable after 21 hours of treatment. Memb rane associated levels of the novel PKCdid not begin to decline until 21 hours of treatment. These results are consistent with PMA-induced PKC isoform translocation patte rn observed by Besson et al. [10] in glioma cells. In SVG cells pretreated with 5mM STZ for 5 hours followed by treatment with 100nM PMA for 15min, 1 hour, or 5 hours, no si gnificant alterations in the PKC isoform translocation was observed (Figure 5.2). Tw enty-one hours of PMA treatment was not done because this would require a total of 26 hours of incubation with STZ. Such long exposures of cells to STZ has been shown to be toxic in pancreatic -cells [7,8]. 5.4.2 Effects of increased O-GlcNAc on PKC recycling In order to investigate th e effects of increased O-GlcNAc protein modification of PKC recycling, SVG cells were first treate d with or without 5mM STZ for 9 hours. These treatment conditions had been shown to decrease the level of membrane associated PKCand with a more dramatic effect on PKC. To determine if this treatment condition increased the degradation of PKC, a sign that PKC recycl ing is disrupted [6], cytoskeleton fractions prepared as descri bed in section 5.3.2 were analyzed by SDS

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Effects STZ treatment on PMA Induced on PKC Isoform Translocation Figure 5.2 Effects of 5mM STZ for 5 hours treatment on PMA induced PKC isoform translocation SVG cells were pretreated with 5mM STZ for 5 hours followed by stimulation with 100mM PMA in DMSO for 15 min (T=0.25), 1 hour (T=1), or 5 hours (T=5). Additionally, cells were pretreated with 5mM STZ for 5 hours followed by addition of an equivalent volume of DMSO for 15 min (control 15min), 1 hour (control 1hr), or 5 hours (control 5hr). An untreated sample was also prepared (untreated). Samples were then fractionated into cytosol and membrane fractions. Only membrane fractions are shown. 97

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98 PAGE and western blotting using an anti-PKCpolyclonal antibody (Santa Cruz Biotechnology, sc-214). Results showed increased levels of 45kDa and 30kDa degradation fragments in samples treated w ith STZ compared with untreated samples (Figure 5.3). Because Hsp70 has been shown to bind to PKC isoforms following their dephosphorylation thus preventi ng their degradation [6], the observed increase in PKCdegradation following STZ treatment suggests a possible disruption in its recycling in response to increased O-GlcNAc protein modi fication. To investigate if either PKCor PKCinteracts with Hsp70 in SVG cells and, if so, if this interaction was altered by increases in total cellular O-GlcNAc, associ ation between these isoforms and Hsp70 was investigated by immunopr ecipitation. Neither i mmunoprecipitation of PKC(Figure 5.7) nor PKC(Figures 5.4 and 5.5) showed an association with Hsp70 or immunoprecipitation of Hsp70 showed an asso ciation with the two PKC isoforms under experimental conditions in our cell system (Figures 5.4, 5.5, and 5.7). Additionally, no interaction between PKCand Hsp70 was observed after increasing O-GlcNAc levels by cellular treatment with 5mM STZ for 9 hour (Figure 5.6). For immunoprecipitation experiments performed using anti-PKC(mouse monoclonal IgG 1 ), negative control immunoprecipitations were done using a nonspecific mouse monoclonal IgG 1 and, because p27 and Hsp70 have been shown to inte ract in rat thyroid cells [11], anti-p27 was used as a positive control antibody for im munoprecipitations experiments (see section 5.3.2) (Figure 5.7). Additionally, an immunopreci pitation was done with an anti-mnage a trois 1 protein (Mat1) mouse monoclonal IgG 1 antibody. Interestin gly, a relatively

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99 Effects of 5mM STZ on PKCcleavage Figure 5.3 Effects of 5mM STZ on PKCdegradation SVG cells were treated with 5mM STZ for 9 hours (+STZ) or untreated (-STZ). Cells were then fractionated into cytoskeletal fractions (cytoskeleton) and soluble fractions (supernatant).

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PKCand Hsp70 Association Figure 5.4 PKCand Hsp70 Association SVG cells were lysed and immunoprecipitated using anti-PKCantibody (IP PKC-) or normal rabbit IgG (IP control IgG). Samples were then western blotted for Hsp70. The same membrane was then stripped and probed for PKCto ensure immunoprecipitation was effective. 100

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Hsp70 and PKCAssociation Figure 5.5 Hsp70 and PKCAssociation SVG cells were lysed and immunoprecipitated using anti-Hsp70 antibody (IP Hsp70) or normal mouse IgG (IP control IgG). Samples were then western blotted for PKC-. The same membrane was then stripped and probed for Hsp70 to ensure immunoprecipitation was effective. 101

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102 Increased O-GlcNAc and PKC-/Hsp70 Association Figure 5.6 Increased O-GlcNAc and PKC-/Hsp70 Association SVG cells treated with 5mM STZ for 9 hours (+STZ) or untreated (-STZ) followed by immunoprecipitaion using anti-PKCantibody (IP PKC) or normal rabbit IgG (IP IgG). Supernatants from immunoprecipitations treated with STZ and IPd for PKC(supernatant 1) or normal rabbit IgG (supernatant 2), or untreated and IPd for PKC(supernatant 3) or normal rabbit IgG (supernatant 4) were also analyzed. Samples were then western blotted for Hsp70.

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PKCand Hsp70 Association Figure 5.7 PKCand Hsp70 Association SVG cells were lysed and immunoprecipited using anti PKCantibodies (IP PKC-), mouse IgG 1 (IP mouse IgG 1 ), anti-p27 (IP p27), Mat1 (IP Mat1), or Protein A beads with no antibody (beads only). Samples were then western blotted for Hsp70. 103

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104 strong interaction betw een Mat1and Hsp70 was observed th at has not been previously documented in the literature (Figure 5.7). 5.5 Discussion Disruptions in PKC phosphoryla tion [9] or a disruption in the interaction between either PKC II [3] or PKC[10] and RACK1 have all been shown to decrease the translocation of PKC isoforms in response to PMA stimulation. Therefore, as indicated by the results, the decreases in membrane associated PKCand PKCunder conditions of increased O-GlcNAc did not appear to be due to either a disruption in the isoforms ability to translocate due to altered isoform phosphorylation or its ability to bind RACK. Results showing increased degradation of PKCfollowing pretreatment with STZ indicated that the previously observed decreases in membrane associated PKCand may be due to an increased rate of proteoly sis. The lack of detectable association between either PKCor PKCand Hsp70 suggest that this increased proteolysis in not a result of the Hsp70-mediated PKC recycling pathway. One alternate pathway that could be responsible for the increase in PKCdegradation under conditions of increased O-GlcNAc modifica tion is the increased action of specific phosphatases. It has been shown that dephosphorylation of conventional PKC isoforms precedes their degradations [12] and down regulation of PKCby dephosphorylation can be a protein phosphatase 1 (PP1) and 2A (PP2A) mediated event [13]. Additionally, OGT, the enzyme that adds O-GlcNAc to proteins exists in stable and active complexes with the se rine/threonine phosphatases PP1and PP1[14] suggesting that phosphatase activity may be linked to the regulation of the O-GlcNAc

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105 modification. Therefore, it is possible that in creases in global O-Gl cNAc levels result in increased phosphatase activity resulting in increased PKC isoform dephosphorylation and degradation. The isoform specificity may be due to specific co-com partmentalization of PKCand with the specific protein phosphatase [15] as both PP2A [16] and PP1and [17] have been shown to localize to specific and distinct cytoskeletal locations within neurons. Also, the decreases in membrane bound, and not cytosolic, PKCand could be explained by the f act that membrane bound PKC is two orders of magnitude more sensitive to dephosphorylation than the soluble form [18]. Investigations, using specific phosphatase inhibitors in conj unction with STZ and PUGNAc, could help determine if this mechanism is responsible for the observed decreases in membrane associated PKCand

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106 5.6 References Cited 1. M. Federici, R. Menghini, A. Mauriello, M.L. Hribal, F. Ferrelli, D. Lauro, P. Sbraccia, L.G. Spagnoli, G. Sesti, R. Lauro, Insulin-dependent activation of endothelial nitric oxide s ynthase is impaired by O-linke d glycosylation modification of signaling proteins in human coronary endothelial cells, Ci rculation 106 (2002) 466-72. 2. J.M. Pass, J. Gao, W.K. Jones, W.B. W ead, X. Wu, J. Zhang, C.P. Baines, R. Bolli, Y.T. Zheng, I.G. Joshua, P. Ping, Enhanced PKC beta II translocation and PKC beta II-RACK1 interactions in PKC ep silon-induced heart failure: a role for RACK1, Am. J. Physiol. Heart Ci rc. Physiol. 281 (2001) H2500-10. 3. E.G. Stebbins,D. Mochly-Rosen, Binding specificity for RACK1 resides in the V5 region of beta II protein kinase C, J. Biol. Chem. 276 (2001) 29644-50. 4. D.H. Korzick, D.A. Holiman, M.O. Boluyt, M.H. Laughlin, E.G. Lakatta, Diminished alpha1-adrenergic-mediated co ntraction and transl ocation of PKC in senescent rat heart, Am. J. Physiol. Heart. Circ. Physiol. 281 (2001) H581-9. 5. J.L. Walgren, T.S. Vincent, K.L. Schey, M.G. Buse, High glucose and insulin promote O-GlcNAc modification of prot eins, including alpha-tubulin, Am. J. Physiol. Endocrinol. Metab. 284 (2003) E424-34. 6. T. Gao,A.C. Newton, The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C, J. Biol. Chem. 277 (2002) 31585-92. 7. R.J. Konrad, I. Mikolaenko, J.F. Tolar, K. Liu, J.E. Kudlow, The potential mechanism of the diabetogeni c action of streptozotocin: inhibiti on of pancreatic beta-cell O-GlcNAc-selectiv e N-acetyl-beta-D-glucosaminidase, Biochem. J. 356

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107 (2001) 31-41 8. Y. Gao, G.J. Parker, G.W. Hart, St reptozotocin-induced beta-cell death is independent of its inhibition of O-Glc NAcase in pancreatic Min6 cells, Arch. Biochem. Biophys. 383 (2000) 296-302. 9. L.M. Keranen, E.M. Dutil, A.C. Newton, Protein kinase C is regulated in vivo by three functionally distinct phosphoryl ations, Curr. Biol. 5 (1995) 1394-1403. 10. A. Besson, T.L. Wilson, V.W. Yong, Th e anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains Requirements for adhesion and motility, J. Biol. Chem. 277 (2002) 22073-84. 11. S. Nakamura, I. Tatuno, Y. Noguchi, M. Kitagawa, L.D. Kohn, Y. Saito, A. Hirai, 73-kDa heat shock cogna te protein interacts direc tly with P27Kip1, a cyclindependent kinase inhibito r, during G1/S transition, Biochem. Biophys. Res. Commun. 257 (1999) 340-3. 12. G. Hansra, P. Garcia-Paramio, C. Pre vostel, R.D. Whelan, F. Bornancin, P.J. Parker, Multisite dephosphoryl ation and desensitization of conventional protein kinase C isotypes, Biochem. J. 342 ( Pt 2) (1999) 337-44. 13. R. Ricciarelli,A. Azzi, Regulation of recombinant PKC alpha activity by protein phosphatase 1 and protein phosphatase 2A, Arch. Biochem. Biophys. 355 (1998) 197-200. 14. L. Wells, L.K. Kreppel, F.I. Comer, B.E. Wadzinski, G.W. Hart, O-GlcNAc transferase is in a functional complex w ith protein phosphatase 1 catalytic subunits, J. Biol. Chem. 279 (2004) 38466-70. 15. A.T. Sim,J.D. Scott, Targeting of PKA, PKC and protein phosphatases to cellular

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108 microdomains, Cell. Calcium 26 (1999) 209-17. 16. S. Strack, J.A. Zaucha, F.F. Ebner, R.J. Colbran, B.E. Wadzinski, Brain protein phosphatase 2A: developmental regulation a nd distinct cellular and subcellular localization by B subunits, J. Comp. Neurol. 392 (1998) 515-27. 17. S. Strack, S. Kini, F.F. Ebner, B.E. Wadzinski, R.J. Colb ran, Differential cellular and subcellular localization of protein phosphatase 1 isoforms in brain J. Comp. Neurol. 413 (1999) 373-84. 18. E.M. Dutil, L.M. Keranen, A.A. DePa oli-Roach, A.C. Newton, In vivo regulation of protein kinase C by trans-phosphorylation followed by autophosphorylation, J. Biol. Chem. 269 (1994) 29359-62.

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109 Chapter 6 PKCand PKCdownregulation and apoptosis induction 6.0 Introduction Research discussed previously in chapter 4 demonstrated that increased levels of the O-GlcNAc protein modifica tion resulted in decreases in membrane associated PKCand possibly PKC. Research by others has indicat ed a link between these two PKC isoforms and promotion of apoptosis [1-3]. This chapter investigates whether decreases in the membrane associated, active forms of PKCand result in increased apoptosis in SVG cells. 6.1 Apoptosis Apoptosis is a programmed form of cell death with typical cell morphology including membrane blebbing, cell shrinkage, chromatin condensation, and DNA fragmentation [4]. Also, the activation of cysteine-dependent aspartate-directed proteases called caspases responsible for much of the apoptotic related proteolytic cleavage is another hallmark of apoptosis [5]. Unlike ce ll death by necrosis that typically occurs due to severe hypoxia, extreme temperatures, or mechanical trauma, apoptosis is a tightly regulated, energy requiring process that has been highly conserved throughout evolution [4]. Currently, there are two well studie d apoptotic pathways responsible for the activation of the caspases. One pathway is initiated by the binding of a ligand to its transmembrane death receptor that in turn recruits and activates certain caspases [6]. The

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110 other pathway involves the release of caspase -activating proteins from the mitochondria into the cytosol forming an apoptosome that can bind and activate se lect caspases [7]. The caspases are proteins synthesized in normal cells as proenzymes [8]. Following the appropriate signal, these pr oenzymes can be rapidly activated by autoproteolytic cleavage or clea vage by another caspase at speci fic aspartic acid residues [8]. There are currently 14 known members of the caspase family of which 7 mediate apoptosis [8]. In general, caspases with long pro-domains function as upstream, initiator, caspases and are capable of proteolytically activating downstream, effector, caspases which contain shorter pro-domains [8]. Th e effector caspases act on a variety of substrates resulting in proteoly sis of cellular proteins ultimat ely resulting in apoptotic cell death. All caspases specifically recogni ze and cleave a tetrapeptide sequence on their substrate with an absolute requirement for an Asp residue. The best characterized caspase substrate is poly-(ADP-ribose) polymerase (PARP), a nuclear protein implicated in DNA repair, which is cleaved into char acteristic 89kDa and 24kDa fragments [8]. PARP is one of the earliest proteins target ed for specific cleavage and is commonly used as a marker for the initiation of apoptosis [8]. Other caspase s ubstrates include ICAD (inhibitor of caspase-activated DNAse) that is cleaved and activated allowing CAD to translocate to the nucleus where it is re sponsible for internucleosmal DNA cleavage [9,10]. Also, caspase cleavage of lamins resu lts in nuclear shrinkage whereas cleavage of cytoskeletal proteins like fodrin and actin leads to cytosolic reorganization [11-13]. Furthermore, caspase-dependent cleavage of DNA-protein kinase, cell cycle regulators, transcription factors, and ce ll signaling proteins have also been reported [14-18]. The

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111 caspases are also responsible for cleaving pro-apoptotic proteins Bid and Bax and cell survival factors Bcl-2 and Bcl-Xl during apoptosis [19-21]. One well studied method for caspase activation is through ligand binding to plasma membrane receptors belonging to the tumor necrosis factor (TNF) receptor superfamily. This family includes Fas, TNF receptor-1, death receptor-3, TNF-related apoptosis inducing ligand receptor-1 (T RAIL-R1), TRAIL-R2, and DR6. The well studied Fas receptor is activated by the bindi ng of the Fas ligand (FasL) that induces trimerization and recruitment of specific adaptor proteins [22,23]. The Fas receptor contains a death domain (DD) in its cytoplasmic region that interacts with the adaptor protein Fas-associated deat h domain protein (FADD), formin g a death receptor-induced signaling complex [24,25]. FADD also contains a death effector domain (DED) that binds procaspase-8 (Medema, 1997). On ce bound, procaspase-8 is proteolytically activated to active caspase-8, which in turn can activate downstream effector caspases [8]. A second caspase-activating apoptotic pa thway involves the pa rticipation of the mitochondria. The process involves the fo rmation of the apoptosome in which cytochrome C released from the mitochondria interacts with apoptotic protease activating factor-1 (Apaf-1) and procaspase-9 in the presence of ATP [26]. The release of these proteins from the mitochondria is a resu lt of dramatic mitochondrial membrane depolarization. This depolari zation occurs following the fo rmation of the mitochondrial permeability transition pore (PTP) that is composed of adenine translocator at the inner membrane and voltage-dependent anion cha nnel (VDAC) at the outer membrane. The formation of this complex results in the activ ation of caspase-9 that in turn activates

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112 caspases-3, 6, and 7 [27]. One group of proteins that influence the opening of the PTP is the Bcl-2 family of proteins. The Bcl-2 family of proteins includes pro-apoptotic members, such as Bax. Bad, Bak, and Bim, and anti-apoptotic members such as Bcl-2 and Bcl-X L Proand anti-apoptotic Bcl-2 proteins are able to heterodimerize and suppress each others function; however, a single mechanism by which these proteins regulate apoptosis has not been completely de termined. Both proand anti-apoptitic Bcl2 proteins are subject to pos ttranslational modifications, such as phosphorylation, which affects their death or surviv al promoting functions [4]. 6.2 Protein Kinase C and apoptosis Early experiments investigating the potent ial role of PKC isoforms in apoptosis yielded contradictory results indicating PKC to be both pr o-and anti-apoptotic [28,29]. With the development of isoform-specific activ ators and inhibitors it became possible to investigate the role of individual isoform s roles in apoptotic regulation. Two PKC isoforms thus far implicated in cell survival are PKCand PKC. Cellular depletion of PKCusing antisense oligonucleotides or pho rbol ester-mediated downregulation has been shown to induce apoptosis in endotheli al cells [30] and glioma cells [31,32]. Inactivation of PKChas also increases apoptosis in liver cells [3]. Although the mechanism by which PKCprevents apoptosis is not fully understood, two potential mechanisms include its phosphorylation of Bcl-2 and/or phosphoryl ation of Raf-1. Phosphorylation of the anti-apoptotic Bcl-2 protei n stabilizes it and e nhances its ability to prevent apoptosis [33,34]. Additionally, PKCmediated phosphorylation of Raf-1 results in its localization to the mitochondrial membrane th rough an interaction with Bcl2 [35] where it can then phosphor ylate and inactivate the proapoptotic protein BAD [36].

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113 Similar to PKC, PKCis also widely accepte d as having anti-apoptitic properties. Overexpression of PKCin glioma cells inhibits TRAIL (tumor necrosis factorrelated apoptosis-inducing ligand) induced apoptosis [1]. PKCinhibits apoptosis in prostate can cer cells by interacting w ith Bax [2]. Although the mechanism(s) responsible for its an ti-apoptotic function is unknown, PKC, like PKC, has been shown to activate Raf-1 [37]. Conversely, unlike PKCand PKCis known to have pro-apoptotic functions. Many proapoptotic stimuli, such as signals initia ted by the death receptor [38], ultraviolet radiation [39], and etoposide [40], have been demonstrated to result in the activation of PKC. Additionally, it has been de monstrated that a loss of PKCis associated with tumor growth [ 41]. The down-regulation of PKCor overexpression of a kinase dead form of this enzyme provides a survival signal that prevents the induction of apoptosis in response to serum deprivation [42]. 6.3 Materials and methods 6.3.1 Cell culture SVG cells were cultured and treated with either 8mM glucosamine, 5mM STZ, 80 M PUGNAc, or 70 M NAGBT for 9 hours as described in section 4.1.2. 6.3.2 Cell fractionation For glucosamine, STZ, and PUGNAc treated samples, whole cell homogenates were generated by first washing cells 2 times in ice cold PBS. Cells were then scraped into 900 l of homogenization buffer with 1.0% Triton X-100 (section 4.1.3) and transferred to 1.5ml centrifuge tubes. Samples were then sonicated 2 times for 5 seconds

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114 at 7 watts using a Fisher Scientific Sonic Dismembrator F60. Unlysed cells and large cellular debris was pelleted out by ce ntrifuging at 1000xg for 5 minutes at 4 C. Supernatants were removed and labeled whole cell homogenates. Due to a limited supply of NAGBT, w hole cell homogenates treated with this inhibitor were not generated. Instead samples that had been previously fractionated into cytosol and membrane fractions as describe d in section 4.1.3 were analyzed for PARP cleavage. 6.3.3 Electrophoresis and Western blotting Samples were analyzed for PARP/PAR P cleavage fragment or procaspase7/caspase-7 as described in section 4.1.4 with a few modifications. Anti-PARP mouse monoclonal antibody (catalog # sc-8007) and an ti-caspase-7 goat pol yclonal (catalog # sc-8510) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and were both used at a 1:400 dilution in TBST with 3% nonfat dry milk. Goat anti mouse antibody conjugated with horseradish peroxida se (BioRad) or bovine anti-goat antibody conjugated with horseradish peroxidase (S anta Cruz Biotechnology catalog # sc-2350) were used in conjunction with the respective primary antibody at 1:10,000 dilutions also in TBST with 3% nonfat dry milk. 6.4 Results 6.4.1 Effects of PUGNAc and STZ treatment on PARP cleavage As discussed in Chapter 4, treatment of SVG cells for 9 hours with either PUGNAc or STZ resulted in a large (~80%) decreases in membrane associated PKCand whereas glucosamine and NAGBT treatments reduced PKCto a much less extent and produced no significant decrease in PKCover the same time frame. To

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115 determine if the large decreas e in the anti-apoptotic PKCand increased PARP cleavage, an early indicator of apoptosis, samples were treated with or without 5mM STZ or 80 M PUGNAc for 9 hours and then analyz ed by SDS-PAGE and immunoblotting with anti-PARP antibody. This antibody has been demonstrated to recognize both the 112kDa whole protein and 85kDa cleavage fragment [43]. Cellular trea tment with either STZ or PUGNAc for 9 hours resulted in an observable increases in the 85kDa PARP cleavage fragment with no observable change in uncleaved 112kDa PARP when compared to untreated control samples (Figure 6.1). As with previous experiments, there were no observable changes in cell morphology or total protein levels or loss of cell viability. In order to determine if PARP cleavage was detectable at time frames earlier than 9 hours, SVG cells were treated with 80 M PUGNAc or 5mM STZ for 1, 3, 5, and 9 hours, prepared as described in 4.1.3, and an alyzed by SDS-PAGE. When compared to untreated controls, increases in PARP cleav age were detectable after only 5 hours of treatment (Figure 6.2). 6.4.2 Effects of glucosamine and NAGBT treatment on PARP cleavage As with PUGNAc and STZ treatment, SVG cells were treated with either 8mM glucosamine or 70 M NAGBT for 9 hours and analyzed for PARP cleavage by SDSPAGE and western blot. Unlike PUGNAc a nd STZ treatment, no observable changes in PARP or PARP cleavage were observed following glucosamine or NAGBT treatment when compared to untreated control samples (Figure 6.1). Also, no changes in cell morphology or total protein levels or loss of cell viability were observed under these treatment conditions.

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Increased O-GlcNAc protein modification and PARP cleavage Figure 6.1 Increased O-GlcNAc protein modification and PARP cleavage SVG cells were treated with or without 80M PUGNAc (+PUGNAc and PUGNAc respectively), with or without 5mM STZ (+STZ and STZ respectively), with or without 8mM glucosamine (+GlcN and GlcN respectively), or with or without 75M NAGBT (+NAGBT and NAGBT respectively). Cells were treated with each compound for 9 hours before preparing samples as whole cell homogenates (WCH) or membrane fractions. Whole cell homogenates of NAGBT were not prepared due to limited supply of inhibitor (see section 6.3.2). Samples were western blotted with anti-PARP antibody that recognizes both full length PARP and the 85kDa apoptotic cleavage fragment. 116

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117 PUGNAc treatment and PARP cleavage Figure 6.2 PUGNAc treatment and PARP cleavage SVG cells were treated with 80M PUGNAc for 1 hour (T=1hr), 3 hours (T=3hr), 5 hours (T=5hr), or 9 hours (T=9hr) or untreated (Control). Cells were lysed and western blotted with anti-PARP antibody that recognizes both full length PARP and the 85kDa apoptotic cleavage fragment.

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118 6.4.2 Caspase-7 activation Caspase-7 has been shown to cleave PARP in HL-60 and MCF-7 cells during apoptosis [44]. In order to determine if the PARP cleav age observed following STZ and PUGNAc treatments in SVG cells was associat ed with increased caspase-7 activation, cells were first treated with either 5mM STZ or 80M PUGNAc or untreated for 9 hours. Samples were then analyzed by SDS-PAGE an d western blotting with an anti-caspase-7 antibody that recognizes both the inactive, pr ocaspase-7, form and the active, caspase-7, form of the enzyme [45]. Analysis revealed no detectable increase in active caspase-7 following STZ or PUGNAc treatment when comp ared to untreated controls (Figure 6.3) 6.5 Discussion The results obtained indicate a strong corre lation between decreases in membrane bound PKCand PKCresulting from treatment with select O-GlcNAc increasing agents and increased PARP cleavage. Cellular treatment with PUGNAc and STZ produced large decreases in PKCand and also corresponding increases in PARP cleavage whereas glucosamine and NAGBT prod uced smaller or no decreases in these isoforms and no observable increases in PARP cleavage. Examination of the time frames of PARP cleavage indicates that signi ficant increases begin around 5 hours after treatment with STZ or PUGNAc. The fact that significant decreases in membrane bound PKCbut not PKClevels are detectable after 5 hours of STZ and PUGNAc treatment suggests that decreases in active PKCmay be primarily responsible for the PARP cleavage. Although glucosamine and NAG BT treatments do produce deceases in membrane associated PKC, the level of decrease may not be large enough facilitate PARP cleavage. This data is the first to suggest a link between d ecreases in active PKC

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Increased O-GlcNAc and Caspase-7 activation Figure 6.3 Increased O-GlcNAc and Caspase-7 activation SVG cells were treated with or without 5mM STZ (+STZ and STZ respectively) or with or without 80M PUGNAc (+PUGNAc and PUGNAc respectively) for 9 hours. Cells were lysed and western blotted for Caspase-7 using an antibody that recognizes both the inactive Procaspase-7 and active Caspase-7 p20 cleavage product. 119

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120 as a result of increases in the O-Gl cNAc protein modifica tion and induction of apoptosis. Furthermore, Leverrier et al. [46] demonstr ated that in rat pituitary adenoma cells apoptosis induced from cis -platinum and UV irradiation, but not after serum deprivation resulted in the limited proteolysis of PKC, and Following cis -platinum treatment and UV irradiation, DNA fragmentatio n appeared after 9 hours and significant PARP cleavage was observed after 16 hours. The proteolytic cleavage of the PKC isoforms resulted in the formation of a catalytic fragment of between 48 and 42kDa localizing in the particulate fr action. This cleavage was al so shown to be calpain and caspase dependent [46]. As discussed in section 5.4.2, treatment of SVG cells for 9 hours with 5mM STZ resulted in increased fo rmation of a 45kDa catalytic and 30kDa regulatory fragment in the cytoskeletal (deter gent insoluble) fraction. Taken together with the results obtained from Leverrier et al. [46], it is possibl e that the increased cleavage of PKCobserved following increases in O-Gl cNAc is a result of increased calpain and/or caspase activity. This process does not appear to invol ve the activation of caspase-7, however, and further investigati on is needed to elucidate this pathway.

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121 6.6 References Cited 1. H. Okhrimenko, W. Lu, C. Xiang, N. Hamburger, G. Kazimirsky, C. Brodie, Protein kinase C-epsilon re gulates the apoptosis and su rvival of glioma cells, Cancer. Res. 65 (2005) 7301-9. 2. M.A. McJilton, C. Van Sikes, G.G. We scott, D. Wu, T.L. Foreman, C.W. Gregory, D.A. Weidner, O. Harris Ford, A. Morgan Lasater, J.L. Mohler, D.M. Terrian, Protein kinase Cepsil on interacts with Bax and pr omotes survival of human prostate cancer cells, Oncogene 22 (2003) 7958-68. 3. H.C. Jao, R.C. Yang, H.K. Hsu, C. Hsu, The decrease of PKCalpha is associated with hepatic apoptosis at early and late phases of polymicrobial sepsis, Shock 15 (2001) 130-4. 4. S.H. Kaufmann,M.O. Hengartner, Programme d cell death: alive and well in the new millennium, Trends Cell. Biol. 11 (2001) 526-34. 5. S.M. Harwood, M.M. Yaqoob, D.A. Allen, Caspase and calpai n function in cell death: bridging the gap between apoptosi s and necrosis, Ann. Clin. Biochem. 42 (2005) 415-31. 6. D.E. Bredesen, P. Mehlen, S. Rabizadeh, Receptors that mediate cellular dependence, Cell Death. Differ. 12 (2005) 1031-43. 7. L. Bouchier-Hayes, L. Lartigue, D.D. Newmeyer, Mitochondria: pharmacological manipulation of cell death, J. Clin. Invest. 115 (2005) 2640-7. 8. N.A. Thornberry,Y. Lazebnik, Caspases : enemies within, Science 281 (1998) 13126. 9. H. Sakahira, M. Enari, S. Nagata, Cleavage of CAD inhibitor in CAD activation

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122 and DNA degradation during apopto sis, Nature 391 (1998) 96-9. 10. X. Liu, H. Zou, C. Slaughter, X. Wang, DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis, Cell 89 (1997) 175-84. 11. K. Orth, A.M. Chinnaiyan, M. Garg, C.J. Froelich, V.M. Dixit, The CED-3/ICElike protease Mch2 is activated during a poptosis and cleaves the death substrate lamin A, J. Biol. Chem. 271 (1996) 16443-6. 12. T. Mashima, M. Naito, N. Fujita, K. Noguchi, T. Tsuruo, Identification of actin as a substrate of ICE and an ICE-like prot ease and involvement of an ICE-like protease but not ICE in VP -16-induced U937 apoptosis, Biochem. Biophys. Res. Commun. 217 (1995) 1185-92. 13. S.J. Martin, G.A. O'Brien, W.K. Ni shioka, A.J. McGahon, A. Mahboubi, T.C. Saido, D.R. Green, Proteolysis of fodrin (non-erythroid spectr in) during apoptosis, J. Biol. Chem. 270 (1995) 6425-8. 14. Q. Song, S.P. Lees-Miller, S. Kumar, Z. Zhang, D.W. Chan, G.C. Smith, S.P. Jackson, E.S. Alnemri, G. Litwack, K.K. Khanna, M.F. Lavin, DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis, EMBO J. 15 (1996) 3238-46. 15. C. Widmann, S. Gibson, G.L. Johnson Caspase-dependent cleavage of signaling proteins during apoptosis. A turn-off mechan ism for anti-apoptotic signals, J. Biol. Chem. 273 (1998) 7141-7. 16. M.O. Hengartner, The biochemistry of apoptosis, Nature 407 (2000) 770-6. 17. M. Barkett, D. Xue, H.R. Horvitz, T.D. Gilmore, Phosphorylation of IkappaB-

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123 alpha inhibits its cleavage by caspase C PP32 in vitro, J. Biol. Chem. 272 (1997) 29419-22. 18. X. Tan, S.J. Martin, D.R. Green, J.Y. Wang, Degradation of retinoblastoma protein in tumor necrosis factorand CD95-induced cell death, J. Biol. Chem. 272 (1997) 9613-6. 19. D. Grandgirard, E. Studer, L. Monney, T. Belser, I. Fellay, C. Borner, M.R. Michel, Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolyti c inactivation of Bcl-2, EMBO J. 17 (1998) 1268-78. 20. R.J. Clem, E.H. Cheng, C.L. Karp, D. G. Kirsch, K. Ueno, A. Takahashi, M.B. Kastan, D.E. Griffin, W.C. Earnsh aw, M.A. Veliuona, J.M. Hardwick, Modulation of cell death by Bcl-XL through caspase interaction, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 554-9. 21. H. Li, H. Zhu, C.J. Xu, J. Yuan, Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis, Cell 94 (1998) 491-501. 22. R.M. Pitti, S.A. Marsters, S. Ruppert, C.J. Donahue, A. Moore, A. Ashkenazi, Induction of apoptosis by Apo-2 ligand, a ne w member of the tumor necrosis factor cytokine family, J. Biol Chem. 271 (1996) 12687-90. 23. A. Ashkenazi,V.M. Dixit, Death recept ors: signaling and modulation, Science 281 (1998) 1305-8. 24. M.P. Boldin, I.L. Mett, E.E. Varfolom eev, I. Chumakov, Y. Shemer-Avni, J.H. Camonis, D. Wallach, Self -association of the "death domains" of the p55 tumor necrosis factor (TNF) receptor and Fas/APO1 prompts signaling for TNF and Fas/APO1 effects, J. Bi ol. Chem. 270 (1995) 387-91.

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124 25. A.M. Chinnaiyan, K. O'Rourke, M. Tewari, V.M. Dixit, FADD, a novel death domain-containing protein, in teracts with the death domain of Fas and initiates apoptosis, Cell 81 (1995) 505-12. 26. H. Zou, W.J. Henzel, X. Liu, A. Lutschg, X. Wang, Apaf-1, a human protein homologous to C. elegans CED-4, partic ipates in cytochrome c-dependent activation of caspase-3, Cell 90 (1997) 405-13. 27. P. Li, D. Nijhawan, I. Budihardjo, S. M. Srinivasula, M. Ahmad, E.S. Alnemri, X. Wang, Cytochrome c and dATP-depende nt formation of Apaf-1/caspase-9 complex initiates an apoptotic pr otease cascade, Cell 91 (1997) 479-89. 28. D.J. McConkey, P. Hartzell, M. Jo ndal, S. Orrenius, Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C, J. Bi ol. Chem. 264 (1989) 13399-402. 29. E.K. Azuma, S. Kitagawa, A. Yuo, H. Mizoguchi, K. Umezawa, F. Takaku, M. Saito, Activation of the respiratory burst a nd tyrosine phosphoryla tion of proteins in human neutrophils: no direct relationshi p and involvement of protein kinase Cdependent and -independent signaling pathways, Biochim. Biophys. Acta 1179 (1993) 213-23. 30. A. Haimovitz-Friedman, N. Balaban, M. Mc Loughlin, D. Ehleiter, J. Michaeli, I. Vlodavsky, Z. Fuks, Protein ki nase C mediates basic fi broblast growth factor protection of endothelial cells against radi ation-induced apoptosis, Cancer Res. 54 (1994) 2591-7. 31. S. Ahmad, T. Mineta, R.L. Martuza, R. I. Glazer, Antisense expression of protein kinase C alpha inhibits the growth and tumorigenicity of human glioblastoma cells,

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125 Neurosurgery 35 (1994) 904-8; discussion 908-9. 32. N.P. Dooley, G.H. Baltuch, N. Groome, J.G. Villemure, V.W. Yong, Apoptosis is induced in glioma cells by antisense oli gonucleotides to protein kinase C alpha and is enhanced by cycloheximide Neuroreport 9 (1998) 1727-33. 33. G. Meinhardt, J. Roth, G. Totok, Prot ein kinase C activati on modulates proand anti-apoptotic signaling pathways, Eur. J. Cell. Biol. 79 (2000) 824-33. 34. P.P. Ruvolo, X. Deng, B.K. Carr, W.S. May, A functional role for mitochondrial protein kinase Calpha in Bcl2 phosphorylation and suppression of apoptosis, J. Biol. Chem. 273 (1998) 25436-42. 35. J.J. Cheng, B.S. Wung, Y.J. Chao, D. L. Wang, Sequential activation of protein kinase C (PKC)-alpha and PKC-epsilon contributes to sustained Raf/ERK1/2 activation in endothelial cells under mechan ical strain, J. Biol. Chem. 276 (2001) 31368-75. 36. X. Fang, S. Yu, A. Eder, M. Mao, R.C. Bast Jr, D. Boyd, G.B. Mills, Regulation of BAD phosphorylation at serine 112 by th e Ras-mitogen-activated protein kinase pathway, Oncogene 18 (1999) 6635-40. 37. H. Cai, U. Smola, V. Wixler, I. Ei senmann-Tappe, M.T. Diaz-Meco, J. Moscat, U. Rapp, G.M. Cooper, Role of diacylglycerol-regulated protei n kinase C isotypes in growth factor activation of the Raf-1 protein kinase, Mol. Cell. Biol. 17 (1997) 732-41. 38. K. Mizuno, K. Noda, T. Araki, T. Imaoka, Y. Kobayashi, Y. Akita, M. Shimonaka, S. Kishi, S. Ohno, The proteolytic cleavage of protein kinase C isotypes, which generates kinase and re gulatory fragments, correlates with Fas-

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126 mediated and 12-O-tetradecanoyl-phorbol-1 3-acetate-induced a poptosis, Eur. J. Biochem. 250 (1997) 7-18. 39. M.F. Denning, Y. Wang, B.J. Nickoloff, T. Wrone-Smith, Protein kinase Cdelta is activated by caspase-dependent proteolysis during ultraviolet radiation-induced apoptosis of human ke ratinocytes, J. Biol Chem. 273 (1998) 29995-30002. 40. M.E. Reyland, S.M. Anderson, A.A. Ma tassa, K.A. Barzen, D.O. Quissell, Protein kinase C delta is e ssential for etoposide-induced a poptosis in salivary gland acinar cells, J. Biol. Chem. 274 (1999) 19115-23. 41. Z. Lu, A. Hornia, Y.W. Jiang, Q. Zang, S. Ohno, D.A. Foster, Tumor promotion by depleting cells of protein kinase C delta, Mol. Cell. Biol. 17 (1997) 3418-28. 42. M. Zhong, Z. Lu, D.A. Foster, Downre gulating PKC delta provides a PI3K/Aktindependent survival signal that overcom es apoptotic signals generated by c-Src overexpression, Oncogene 21 (2002) 1071-8. 43. M.E. Burow, C.B. Weldon, B.M. Collins-Burow, N. Ramsey, A. McKee, A. Klippel, J.A. McLachlan, S. Clej an, B.S. Beckman, Cross-talk between phosphatidylinositol 3-kinase and sphingom yelinase pathways as a mechanism for cell survival/death decisions, J. Biol. Chem. 275 (2000) 9628-35. 44. M. Germain, E.B. Affar, D. D'Amours, V.M. Dixit, G.S. Salvesen, G.G. Poirier, Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase -7, J. Biol. Chem. 274 (1999) 28379-84. 45. B. Del Bello, M.A. Valentini, P. Mangiavacchi, M. Comporti, E. Maellaro, Role of caspases-3 and -7 in Apaf-1 proteoly tic cleavage and degradation events during cisplatin-induced apoptosis in melano ma cells, Exp. Cell. Res. 293 (2004) 302-10.

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127 46. S. Leverrier, A. Vallentin, D. Joubert, Positive feedback of protein kinase C proteolytic activation during apopt osis, Biochem. J. 368 (2002) 905-13.

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128 Chapter 7 Akt 7.0 Introduction Akt (also known as protein kinase B) is a family of serine-threonine kinases that play a major role in signal tr ansduction activated by extracellu lar stimuli. There are three known Akt isoforms, Akt1, Akt2, and Akt3, each encoded by a separate gene. All three genes share greater than 85% homology. All three isforms are assumed to have similar substrate specificity although c onclusive testing has not b een done and all three share similar regulation (discussed be low). All three isoforms are ubiquitously expressed but the level of expression varies with tissue [1-3]. Akt1 is highly expressed in most tissues [4], Akt2 is largely expressed in insulin-re sponsive tissues [5], and Akt3 is most highly expressed in testis and brain [6,7]. 7.1 Akt structural domains All three Akt isoforms consist of an amino terminal pleckstrin homology (PH) domain, a central kinase doma in, and a carboxyl-terminal regu latory domain containing a hydrophobic motif. The PH domain is compri sed of approximately 100 amino acids and was originally found in pleckstrin, the ma jor phosphorylation substrate for PKC in platelets [8]. The PH domain binds the lipid products phosphatidylinositol (3,4,5) trisphosphate (PIP3) produced PI3-K and phos phatidylinositol (3,4) bisphosphate (PIP2) and allows Akt to interact with cell memb ranes through these lipid products. Akt has

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129 been shown to bind PIP3 and PIP2 with sim ilar affinity [9,10]. The PH domain of Akt shares similarity with the PH domains of other proteins that bind 3-phosphoinositides [11,12]. The kinase domain of Akt is located in the central region of the protein and exhibits high homology to other AGC kinases such as PKC, protein kinase A (PKA), and p70S6K. All three isoforms have a 40 ami no acid section at the carboxyl terminal end possessing an F-X-X-F/Y-S/T-Y/ F (X is any amino acid) hyd rophobic motif that is also share by most members of the AGC kinase family. 7.2 Akt activation Like PKC, Akt is initially transcribed as an unphosphorylated single polypeptide chain. All three Akt isoforms undergo two phosphorylations to produce the stable, active enzyme. The translocation of Akt to the pl asma membrane is a prerequisite for its phosphorylation [13]. Once at the membrane Akt1 is phosphorylated on Thr308 and Ser 473, Akt2 is phosphorylated on Thr309 and Ser474, and Akt3 is phosphorylated on Thr305 and Ser472 [14] (Figure 7.1). The threonine residue is located in the activation loop and the serine residue is located in the hydrophobic motif. The phosphorylation of Akt on Thr308 (or equivalent residue) has been shown to be mediated by PDK1 [15,16] (Figure 7.2). The kinase responsible fo r the phosphorylation of Ser473 (or equivalent residue) is controversial and PDK1 [17], integrin-linked protein kinase [3], Akt [18], DNA-PK [19] and mTOR [20] ar e all potential candidates. It is generally accepted that, once at the plasma membrane, Akt is fi rst phosphorylated on Ser473 [21-23]. The phosphorylation of Ser473 stab ilizes the Akt and allows ATP binding [22]. The phosphorylation of the hydrophobi c motif then promotes th e phosphorylation of Thr308

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130 Akt Isoform Phosphorylation Sites Figure 7.1 Akt Isofrom Phosphorylation Sites PHplecsktrin homology domain; catalytic catalytic domain; regul. C-terminus regulatory domain [14]

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131 Akt Life Cycle Figure 7.2 Akt life cycle PTEN Phosphatase and Tensin homolog deleted on chromosome Ten, PHLPP PH domain Leucine-rich repeat Protein Phosphatase [24]

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132 by PDK1. Following both phosphorylations, Akt detaches from the nucleus and translocates to the cytosol and nucleus [13]. The mechanism of Akt dephosphorylation and inactivation is largely unknown. Recently, a PH domain leucine-rich repeat protein phosphatase (PHLPP) was identified that specifically dephosphoryl ates the hydrophobic motif of Akt (Ser473 in Akt1) [24] (Figure 7.2). This PHLPP mediated dephosphorylation of Akt correlated with increased apoptosis and suppressing tumor grow th in glioblastoma cells [24]. 7.3 Akt regulation by anchoring proteins Several non-substrate proteins have been shown to bind Akt and regulate its activity. The carboxyl-terminal modulator protein (CTMP) in teracts with the carboxylterminal of Akt at the plasma membrane and reduces its phosphorylation on both Thr308 and Ser473 [25]. Another protein, Trb3, binds to the central kinase region of Akt and also reduces its phosphorylation [26]. The cytoskeletal protein Keratin K10 binds Akt and sequesters it to the cytoskel eton thus inhibiting its ability to translocate to the plasma membrane [27]. Other proteins, such as heat shock proteins 90 [28] and 27 [29] have been demonstrated to bind Akt and positively regulate its activity.

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133 7.4 References Cited 1. D. Brodbeck, M.M. Hill, B.A. Hemmings, Two splice variants of protein kinase B gamma have different regulatory capacity depending on the presence or absence of the regulatory phosphorylati on site serine 472 in the carboxyl-terminal hydrophobic domain, J. Biol. Chem. 276 (2001) 29550-8. 2. H. Konishi, S. Kuroda, M. Tanaka, H. Matsuzaki, Y. Ono, K. Kameyama, T. Haga, U. Kikkawa, Molecular cloning and ch aracterization of a new member of the RAC protein kinase family: association of the pleckstrin homology domain of three types of RAC protein kinase with protei n kinase C subspeci es and beta gamma subunits of G proteins, Biochem. Biophys. Res. Commun. 216 (1995) 526-34. 3. S. Persad, S. Attwell, V. Gray, N. Mawji, J.T. Deng, D. Leung, J. Yan, J. Sanghera, M.P. Walsh, S. Dedhar, Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: cr itical roles for kinase activity and amino acids arginine 211 and serine 343, J. Biol. Chem. 276 (2001) 27462-9. 4. D.A. Altomare, K. Guo, J.Q. Cheng, G. Sonoda, K. Walsh, J.R. Testa, Cloning, chromosomal localization and expression analysis of the mouse Akt2 oncogene, Oncogene 11 (1995) 1055-60. 5. D.A. Altomare, G.E. Lyons, Y. Mitsuuchi J.Q. Cheng, J.R. Testa, Akt2 mRNA is highly expressed in embryonic brown fat and the AKT2 kinase is activated by insulin, Oncogene 16 (1998) 2407-11. 6. D. Brodbeck, P. Cron, B.A. Hemmings, A human protein kinase Bgamma with regulatory phosphorylation sites in the activation loop and in the C-terminal hydrophobic domain, J. Biol Chem. 274 (1999) 9133-6.

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134 7. K. Nakatani, H. Sakaue, D.A. Thomps on, R.J. Weigel, R.A. Roth, Identification of a human Akt3 (protein kinase B gamma ) which contains the regulatory serine phosphorylation site, Biochem. Bi ophys. Res. Commun. 257 (1999) 906-10. 8. J.H. Brumell, K.L. Craig, D. Ferguson, M. Tyers, S. Grinstein, Phosphorylation and subcellular redistribution of pleckstr in in human neutrophils, J. Immunol. 158 (1997) 4862-71. 9. S.R. James, C.P. Downes, R. Gigg, S.J. Grove, A.B. Holmes, D.R. Alessi, Specific binding of the Akt-1 protein kinase to phospha tidylinositol 3,4,5trisphosphate without subse quent activation, Biochem. J. 315 ( Pt 3) (1996) 709-13. 10. M. Frech, M. Andjelkovic, E. Ingle y, K.K. Reddy, J.R. Falck, B.A. Hemmings, High affinity binding of inositol phospha tes and phosphoinositides to the pleckstrin homology domain of RAC/prot ein kinase B and their influence on kinase activity, J. Biol. Chem. 272 (1997) 8474-81. 11. S.E. Lietzke, S. Bose, T. Cronin, J. Klarlund, A. Chawla, M.P. Czech, D.G. Lambright, Structural basis of 3-phosphoinositide recognition by pleckstrin homology domains, Mol. Cell 6 (2000) 385-94. 12. K.M. Ferguson, J.M. Kavran, V.G. Sankara n, E. Fournier, S.J. Isakoff, E.Y. Skolnik, M.A. Lemmon, Structural basi s for discrimination of 3-phosphoinositides by pleckstrin homology domains, Mol. Cell 6 (2000) 373-84. 13. M. Andjelkovic, D.R. Alessi, R. Meier, A. Fernandez, N.J. Lamb, M. Frech, P. Cron, P. Cohen, J.M. Lucocq, B.A. He mmings, Role of translocation in the activation and function of protein kinase B, J. Bi ol. Chem. 272 (1997) 31515-24. 14. E.S. Kandel, N. Hay, The regulati on and activities of the multifunctional

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135 serine/threonine kinase Akt/PKB Exp. Cell. Res. 253 (1999) 210-29. 15. D.R. Alessi, S.R. James, C.P. Downes, A.B. Holmes, P.R. Gaffney, C.B. Reese, P. Cohen, Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha, Curr. Biol. 7 (1997) 261-9. 16. L. Stephens, K. Anderson, D. Stokoe, H. Erdjument-Bromage, G.F. Painter, A.B. Holmes, P.R. Gaffney, C.B. Reese, F. McCormick, P. Tempst, J. Coadwell, P.T. Hawkins, Protein kinase B kina ses that mediate phosphatidylinositol 3,4,5trisphosphate-dependent activation of pr otein kinase B, Science 279 (1998) 710-4. 17. A. Balendran, R. Currie, C.G. Armstrong, J. Avruch, D.R. Alessi, Evidence that 3-phosphoinositide-dependent protein kinase -1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252, J. Biol. Chem. 274 (1999) 37400-6. 18. A. Toker,A.C. Newton, Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site, J. Biol. Chem. 275 (2000) 8271-4. 19. J. Feng, J. Park, P. Cron, D. Hess, B.A. Hemmings, Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-de pendent protein kinase, J. Biol. Chem. 279 (2004) 41189-96. 20. D.D. Sarbassov, D.A. Guertin, S.M. Ali, D.M. Sabatini, Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex, Science 307 (2005) 1098-101. 21. M.P. Scheid, P.A. Marignani, J.R. Woodgett, Multiple phosphoinositide 3-kinasedependent steps in activation of protein kinase B, Mol. Cell. Biol. 22 (2002) 624760. 22. J. Yang, P. Cron, V. Thompson, V.M. Good, D. Hess, B.A. Hemmings, D. Barford, Molecular mechanism for the re gulation of protei n kinase B/Akt by

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136 hydrophobic motif phosphorylation, Mol. Cell 9 (2002) 1227-40. 23. D.R. Alessi, M. Andjelkovic, B. Caudwell, P. Cron, N. Morrice, P. Cohen, B.A. Hemmings, Mechanism of activation of protein kinase B by insulin and IGF-1, EMBO J. 15 (1996) 6541-51. 24. T. Gao, F. Furnari, A.C. Newton, PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis and suppresses tumor growth, Mol. Cell 18 (2005) 13-24 25. S.M. Maira, I. Galetic, D.P. Brazil, S. Kaech, E. Ingley, M. Thelen, B.A. Hemmings, Carboxyl-terminal modulator pr otein (CTMP), a nega tive regulator of PKB/Akt and v-Akt at the plasma membrane, Science 294 (2001) 374-80. 26. K. Du, S. Herzig, R.N. Kulkarni, M. Montminy, TRB3: a tribbles homolog that inhibits Akt/PKB activation by insuli n in liver, Science 300 (2003) 1574-7. 27. J.M. Paramio, C. Segrelles, S. Ruiz, J. L. Jorcano, Inhibition of protein kinase B (PKB) and PKCzeta mediates keratin K10-i nduced cell cycle arrest, Mol. Cell. Biol. 21 (2001) 7449-59. 28. D.B. Solit, A.D. Basso, A.B. Olshen, H.I. Scher, N. Rosen, Inhibition of heat shock protein 90 function dow n-regulates Akt kinase and sensitizes tumors to Taxol, Cancer. Res. 63 (2003) 2139-44. 29. H. Konishi, H. Matsuzaki, M. Tanaka, Y. Takemura, S. Kuroda, Y. Ono, U. Kikkawa, Activation of prot ein kinase B (Akt/RAC-pro tein kinase) by cellular stress and its association with heat shock protein Hsp27, FEBS Lett. 410 (1997) 493-8.

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137 Chapter 8 Effects of O-GlcNAc increasing agents on Akt 8.0 Introduction The ubiquitous serine threonine kinase Akt (protein kinase B) is a member of the ACG superfamily of kinases and plays an important role in mediating a variety of cellular functions, particularly in the brain. Akt has been shown to regulate such critical brain processes as the differentiation of neural stem cells into as trocytes [1], regulation of neuronal cell survival [2], and protection against ischemic inju ry [3]. The misregulation of Akt in the brain has been implicated in the progression of brain cancers from anaplastic astrocytoma to glioblastoma mu ltiforme [4] underscoring the need to fully understand the regulation of this enzyme. Recen tly, in adipocytes, increased intracellular levels of the O-GlcNAc posttranslational modi fication have been demonstrated to prevent the insulin-stimulated ac tivation of Akt [5,6]. These recen t studies suggest an important new regulatory mechanism for Akt, however, the effects of O-GlcNAc on Akt activation in the brain have yet to be investigated. Because the O-GlcNAc modification has b een shown to affect Akt activation in adipocytes [7,8] and the tran slocation of other members of the ACG superfamily of kinases in glial cells [9], we sought to dete rmine if increased O-GlcNAc levels affected Akt activity and/or subcelluar localization in an SV-40 transformed human glial cell line. 8.1 Materials and methods

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138 8.1.1 Materials SVG cell line, eagles minimum essential medium (EMEM) and fetal bovine serum (FBS) were obtained from American Type Culture Collection (Rockville, MD). Penicillin/streptomycin was purchased from Fisher Scientific (Suwanee, GA). Anti-Akt (catalog #9272) and anti-phospho-Akt (Serine 473) (catalog #4058) specific antibodies were purchased from Cell Signaling Technol ogy (Beverly, MA). Anti-GRP 78 (catalog # ac-13968) was purchased from Santa Cruz Bi otechnology (Santa Cruz, CA) whereas the CTD110.6 anti-O-GlcNAc antibody was a kind gi ft from Dr. Gerald Hart at Johns Hopkins University (Baltimor e, MD) and is also available from Covance Research Products (Berkeley, CA). Goat anti-mous e-HRP and goat anti-rabbit-HRP secondary antibodies were from BioRad (Hercules, CA). D-glucosamine, D-galactosamine, Nacetyl-L-cysteine, and streptozotocin (STZ) were purchased from Sigma (St. Louis, MO). O-(2-acetamido-2-deoxy-D-glucopyranosylid ene)amino-N-phenylcarbamate (PUGNAc) was from Carbogen (Aarau, Switzer land), and 1,2-dideoxy-2'-propyl--Dglucopyranoso-[2,1-d]2'-thiazoline (NAGBT) was kindly provided by Dr. David J. Vocadlo at Simon Fraser University in Brit ish Columbia, Canada. All other chemicals were purchased from Sigma and were of the purest grade available. 8.1.2 Cell culture SVG cells were grown at 37 C in a 95% air and 5% CO 2 environment. Cells were cultured in T-150cm 2 flask to 90% confluency in EM EM (5.4mM glucose) supplemented with 10% FBS and 10U/mL penicillin and 10g/mL stre ptomycin. Cell media was supplemented with 8mM glucosamine, 8mM galactosamine, 5mM STZ, 75 M NAGBT, or 80 M PUGNAc for the time periods indicated. For studies examining the possible

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139 effect of oxidative stress on glucosamine or STZ induced Akt phosphorylation, cells were treated with 6mM N-acetyl-L-cys teine (pHd to 7.0 in phosphate buffered saline) alone or in combination with 8mM glucosamine or 5mM STZ for 1, 3, or 5 hours. 8.1.3 Cell harvesting and fractionation At the indicated time points, experiments were terminated by immediately placing flasks of cells were on ice and washing with ice co ld phosphate buffered saline (PBS). Cells were scraped into PBS and centrifuged a 2,000xg for 3 minutes to pellet cells. For preparation of whole cell homogenates, PBS was removed and cells were resuspended in 600 l of ice-cold homogenization buffer (2 0mM Tris, 1mM EGTA, 1mM EDTA, 2mM MgCl 2 150mM NaCl, 1mM dithiothreitol, 1mM PMSF, 1mM Na 3 VO 4 4 g/ml aprotinin, and 1.0% Triton X-100). Cells we re lysed by sonication (Fisher Scientific Sonic Dismembrator F60) on ice with 2, 10-s econd pulses at 7 watts. Any whole cells or debris was pelleted by centrif uging at 1000xg for 5 minutes at 4 C. Protein concentrations of supernatants were de termined using BioRad Protein Assay Dye Reagent using bovine serum albumin as the standard according to the manufactures instructions. Fractionation of cells into cytosol and membrane fractions was done according to a method previously demonstrated to successf ully separate membrane associated Akt from cytosolic Akt [10] with few modificat ions. Briefly, SVG ce lls were harvest as described above except that cells were ini tially resuspend in ic e-cold homogenization buffer without Triton X-100. After lysing cells by sonication, debris was pelleted by centrifuging at 1000xg for 5 minutes at 4 C. Supernatants were then centrifuged at 100,000xg for 1 hour at 4 C. The resulting supernatants were removed and labeled as

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140 cytosol fractions. Pellets were resuspended in homogenization buffer with 1.0% Triton X-100 by gentle agitation for 30 minutes follo wed by a brief 2-second sonication at 7 watts. Samples were centrif uged at 100,000xg for 30 minutes at 4 C. Supernatants were removed and labeled as membrane fractions. Protein concentrations of both fractions were determined using BioRad Protein A ssay Dye Reagent using bovine serum albumin as the standard according to the manufactures instructions. 8.1.4 Electrophoresis and Western blotting Proteins from cytosol and membrane frac tions were mixed with 0.3 volumes of 3x sample buffer (0.18M Tris-HCl pH 6.8, 6% s odium dodecyl sulfate (SDS), 30% glycerol, 0.025% Bromophenol Blue) and equal amounts of protein were loaded onto 8% SDSpolyacrylamide gels. Samples were electr ophoresed for 1 hour 30 minutes at 15mAmps and then transferred to nitrocellulose me mbranes by electroblotting in 50mM Tris, 77mM glycine, and 20% methanol transfer buffer for one hour at 12 volts. For western blot analysis with Akt and phospho-Akt antibodies, membranes were blocked with 5% (w/v) non-fat dried milk in tris buffered saline (140mM NaCl, 2.7mM KCl, and 25mM Tris pH 8.0) with 0.05% Tween-20 (TBS-T). Membra nes were then incubated overnight at 4 C in TBS-T with 3% bovine serum albumin (BSA) containing the Akt antibody. For western blots with cytosol fractions, both Akt and phospho-Akt antibodies were diluted 1:2000 while for membrane fractions a dilu tion of 1:500 was used. Membranes were subsequently washed for 10 minutes (3 times) in TBS-T. Goat anti rabbit-HRP (1:6000 dilution) in TBS-T with 3% BSA was then incubated with the membranes. Membranes were again washed 10 minutes (4 times) in TBS-T and bands were detected by chemiluminescence according to the manufacture s instructions (Pierce, Rockford, IL).

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141 For analysis using CTD110.6 antibody, membranes were blocked in tris buffered saline with 0.3% Tween-20 (TBS-HT) for 1 hour then incubated overnight at 4 C in TBS-HT containing the CTD110.6 antibody (1:5 000 dilution) [11]. Membranes were washed for 10 minutes (2 times) in tris buffered saline with 1.0% Triton X-100, 0.1% SDS, 0.25% deoxycholic acid (TBS-D) and (3 ti mes) in TBS-HT. Goat anti rabbit Ig-MHRP (1:15,000 dilution) in TBS-HT was then added to membranes. Membranes were washed as before and bands detected using chemiluminescence. Immunoblots were quantified using Scion Image 4.02 analysis program (Scion Corp., Frederick, MD). 8.1.5 Statistical Analysis Data are given as standard error of the mean (S.E.M ) for three to five experiments. Comparisons between means were performed using two-tailed Students t test for unpaired data and graphed using SigmaPlot 8.0 Values with p<0.05 were considered significant. 8.2. Results 8.2.1 Effects of glucosamine on Akt phosphoryl ation and Akt distribution between cytosol and membrane In order to examine if increased glob al levels of the O-GlcNAc protein modification affected Akt activity or subcellu lar distribution, SVG cells were first treated with either 8mM glucosamine, 5mM STZ, 80 M PUGNAc or 75 M NAGBT. Treated cells were then fractionated into cytoso l and membrane fractions, separated by SDSPAGE, immunoblotted, and probed with anti bodies against anti-phospho-Akt (Serine 473) or for total Akt (see section 8.1).

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142 Following 8mM glucosamine treatment, phospho-Akt levels in both cytosol and membrane fractions were rapidly and significan tly increased when compared to untreated controls. After one hour of treatment, phospho-Akt levels had increased by 43.4% 23.6 in cytosol and by 31.3% 23.6 in membrane fractions (Figure 8.1). Phospho-Akt levels continued increasing th rough 3 hours of treatment with increases of 75.7% 31.9 in the cytosol and 49.5% 41.3 in membrane fractio ns (Figure 8.1) and were maximal after 5 hours, increasing by 96.8% 24.6 in the cytosol and 57.7% 38.1 in membrane fractions. After 9 hours of treatment, phospho-Akt levels began to decrease in the cytosol (67.8% 18.6) and membrane (33.8% 11.4) fractions (Figure 8.1). While phospho-Akt levels were significantly altered following glucosamine treatment, there were no significant changes in total Akt levels in eith er the cytosol or membrane fraction (Figure 8.1). 8.2.2 Effects of streptozotocin on Akt phos phorylation and Akt distribution between cytosol and membrane To further investigate th e potential role of the OGlcNAc modification on the activity and cellular distribution of Akt, S VG cells were treated with the O-GlcNAcase inhibitor STZ [12-14] and prepared as de scribe following glucosamine treatment. Analysis of Akt phosphorylation following 5m M STZ again showed a marked increase in phospho-Akt similar to that seen following gl ucosamine treatment. After one hour of treatment with 5mM STZ, phospho-Akt levels increased by 44.4% 12.3 in cytosol and by 32.3% 11.3 in membrane fractions (Figure 8.2). Phospho-Akt levels were maximal after 3 hours of treatment with increases of 96.8% 11.0 in the cytosol and 66.3% 18.7

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143 Figure 8.1 Effects of Glucosamine on phospho-Akt and Akt in cytosol and membrane fractions. SVG cells were treated with 8mM glucosamine for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), 9 hours (T=9). Untreated (control) samples were prepared for each experiment. Samples were separated into cytosol and membrane fractions as described in section 4.1.3. Equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Cytosol and membrane fractions were treated with either phospho-specific Akt antibodies (p-Akt) or antibodies against total Akt (Akt). Immunoblots were analyzed by densitometry and the results were graphed (A). Representative immunoblots for cytosol and membrane fractions probed with anti-phospho serine-473 Akt or total Akt (B). Values are means S.E.M. for 5 determinations. represents p<0.05 and ** represents p<0.01

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144 Figure 8.2 Effects of STZ on phospho-Akt and Akt in cytosol and membrane fractions. SVG cells were treated with 5mM STZ for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), 9 hours (T=9). Untreated (control) samples were prepared for each experiment. Samples were separated into cytosol and membrane fractions as described in section 4.1.3. Equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Cytosol and membrane fractions were treated with either phospho-specific Akt antibodies (p-Akt) or antibodies against total Akt (Akt). Immunoblots were analyzed by densitometry and the results were graphed (A). Representative immunoblots for cytosol and membrane fractions probed with anti-phospho serine-473 Akt or total Akt (B). Values are means S.E.M. for 5 determinations. represents p<0.05 and ** represents p<0.01

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145 in membrane fractions (Figure 8.2). Phospho-Akt levels returned to control levels after 5 hours of treatment in membrane fractions and 9 hours in cytosol fractions (Figure 8.2). Total Akt levels were also analyzed with and without STZ treatment and showed no significant changes from control levels (Figure 8.2). 8.2.3 Effects of PUGNAc and NAGBT on Akt phosphorylation and Akt distribution between cytosol and membrane In order to determine if the increas ed Akt phosphorylation observed following cellular treatment with glucosamine and STZ is a result on increased O-GlcNAc protein modification, SVG cells were treated with either 80 M PUGNAc or 75 M NAGBT for 1, 3, 5, or 9 hours and prepared as described fo r glucosamine treatment. Interestingly, the results obtained from PUGNAc and NAGBT treatment of SVG cells differed from both glucosamine treatment and STZ treatment. Neither 80 M PUGNAc nor 75 M NAGBT incubation resulted in no significant increas e in Akt phosphorylation in either the cytosol or membrane fractions at any of the time points examined when compared to untreated controls (Figure 8.3 and 8.4). To determine if exposure of SVG cells to NAGBT for time periods shorter than 1 hour had an effect on phospho-Akt levels, cells were treated for 15, 30, or 45 minutes. These exposures did not significantly alter Akt phosphorylation (data not shown). Also, total levels of Akt in ne ither the cytosol nor the membrane fractions were significantly altered by these treatments at any time points (Figure 8.3 and 8.4). Analysis of global O-GlcNAc levels from both cytosol and membrane fractions of SVG cells treated with either glucosamin e, STZ, PUGNAc, or NAGBT revealed that, whereas all compounds effectively increased O-GlcNAc levels, glucosamine and STZ treatment resulted in much lower levels of increase. It is therefore possible that the much

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146 Figure 8.3 Effects of PUGNAc on phospho-Akt and Akt in cytosol and membrane fractions. SVG cells were treated with 80M PUGNAc for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), 9 hours (T=9). Untreated (control) samples were prepared for each experiment. Samples were separated into cytosol and membrane fractions as described in section 4.1.3. Equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Cytosol and membrane fractions were treated with either phospho-specific Akt antibodies (p-Akt) or antibodies against total Akt (Akt). Immunoblots were analyzed by densitometry and the results were graphed (A). Representative immunoblots for cytosol and membrane fractions probed with anti-phospho serine-473 Akt or total Akt (B). Values are means S.E.M. for 5 determinations. represents p<0.05 and ** represents p<0.01

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147 Figure 8.4 Effects of NAGBT on phospho-Akt and Akt in cytosol and membrane fractions. SVG cells were treated with 75M NAGBT for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), 9 hours (T=9). Untreated (control) samples were prepared for each experiment. Samples were separated into cytosol and membrane fractions as described in section 4.1.3. Equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Cytosol and membrane fractions were treated with either phospho-specific Akt antibodies (p-Akt) or antibodies against total Akt (Akt). Immunoblots were analyzed by densitometry and the results were graphed (A). Representative immunoblots for cytosol and membrane fractions probed with anti-phospho serine-473 Akt or total Akt (B). Values are means S.E.M. for 5 determinations. represents p<0.05 and ** represents p<0.01

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148 larger O-GlcNAc increases may inhibit the pathway(s) responsible for the glucosamine and STZ induced increase in Akt phosphorylatio n. In order to determine if the global level of O-GlcNAc accounted for the differe nt effects of these O-GlcNAc modulating agents, cells were treated with reduced c oncentrations of PUGNAc and NAGBT. Cells were treated with either 40 M, 20 M, or 10 M NAGBT or PUGNAc for 1 or 3 hours; however, none of these treatment conditions re sulted in any detectable increase in Akt phosphorylation (Figure 8.5 and 8.6). 8.2.4 Effects of galactosamine on Akt phosphorylation Because Akt activation has been demonstrated to be affected by increases in osmotic pressure [15-17], SVG cells were treat ed with 8mM galactosamine to examine if the concentrations of glucosamine and STZ used in this study affected Akt phosphorylation via an osmotic pressure re lated pathway. Additionally, the results obtained following treatment with galactosam ine would determine if the effects of glucosamine and STZ treatment on Akt are mimick ed by other structurally related sugars. Unlike glucosamine and STZ treatment, however, cellular treatment with 8mM galactosamine for 1, 3, 5, or 9 hours did not a lter phospho-Akt levels or total Akt levels significantly from untreated control levels (Figure 8.7). 8.2.5 Effects of N-acetyl-L-cysteine on Akt phosphorylation In addition to osmotic stress, oxidative stress has also been shown to affect Akt activation and glucosamine and STZ have been demonstrated to increase oxidative stress [18]. To examine if the increases in Akt phosphorylation following glucosamine and STZ treatment are a result of oxidative stress, SVG cells were treated with either 8mM glucosamine and 6mM N-acetyl-L-cysteine, 5mM STZ and 6mM N-acetyl-L-cysteine,

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Effects of decreasing NAGBT concentrations on phospho-Akt and Akt Figure 8.5 Effects of decreasing NAGBT concentrations on phospho-Akt and Akt. SVG cells were treated with 10M NAGBT for 1 or 3 hours (10M T=1 and 10M T=3 respectively), 20M NAGBT for 1 or 3 hours (20M T=1 and 20M T=3 respectively), or 40M NAGBT for 1 or 3 hours (40M T=1 and 40M T=3 respectively). Additionally, two untreated (Control) samples were prepared. Whole cell homogenates were prepared and equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Immunoblots were treated with either phospho-specific Akt antibodies (p-Akt) or antibodies against total Akt (Akt). 149

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Effects of decreasing PUGNAc concentrations on phospho-Akt and Akt Figure 8.6 Effects of decreasing PUGNAc concentrations on phospho-Akt and Akt. SVG cells were treated with 10M PUGNAc for 1 or 3 hours (10M T=1 and 10M T=3 respectively), 20M PUGNAc for 1 or 3 hours (20M T=1 and 20M T=3 respectively), or 40M PUGNAc for 1 or 3 hours (40M T=1 and 40M T=3 respectively). Additionally, two untreated (Control) samples were prepared. Whole cell homogenates were prepared and equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Immunoblots were treated with either phospho-specific Akt antibodies (p-Akt) or antibodies against total Akt (Akt). 150

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Effects of Galactosamine on phospho-Akt and Akt Figure 8.7 Effects of Galactosamine on phospho-Akt and Akt SVG cells were treated with 8mM galactosamine for 1 hour (T=1), 3 hours (T=3), 5 hours (T=5), 9 hours (T=9). Untreated (control) samples were prepared for each experiment. Whole cell homogenates were prepared and equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Immunoblots were treated with either phospho-specific Akt antibodies (p-Akt) or antibodies against total Akt (Akt). 151

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152 6mM N-acetyl-L-cysteine alone, or untreated fo r 1, 3, or 5 hours. Cellular treatment with N-acetyl-L-cysteine has been well documented to attenuate the effects of oxidative stress in other systems [18]. In our system 6mM N-acetyl-L-cysteine treatment did not significantly alter the increased phos pho-Akt levels observed following 8mM glucosamine or 5mM STZ alone (Figure 8.8 and 8.9). Furthermore, 6mM N-acetyl-Lcysteine treatment did not alter phospho-Akt or total Akt levels from untreated control levels at any of the time point s analyzed (Figure 8.8 and 8.9). 8.2.6 Effects of Glucosamine or STZ on GRP 78 expression GRP 78 is a member of the heat shock protein family whose expression has been shown to increase in response to endoplasmic reticulum (ER) st ress [31, 34]. In order to determine if either glucosamine or STZ were enducing ER stress, thus giving a possible mechanism for the increases in phospho-Akt, cells were treated with either 8mM glucosamine or 5mM STZ and samples were western blotted for GRP 78. Results indicated an increase in GRP 78 expressi on after 5 hours of glucosamine treatment followed by a much larger increase 9 hours pos t treatment. Cells tr eated with 5mM STZ showed no increase in GRP 78 expression at any of the tim e points analyzed. 8.3 Discussion The O-GlcNAc modification has been show n to regulate the activity of key signal transduction enzymes including PI 3-K in e ndothelial cells [19], MAPK in neutrophils [20], eNOS in rat penis tissue [21] and Akt in adipocytes [7,8]. Previously, our research in human glial cells, suggests that increased levels of this modification also decreases the active, membrane bound forms of PKCand possibly PKC(chapter 4), two kinases that share a high degree of st ructural and sequence homology with Akt [22]. In order to

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Effects of N-Acetylcysteine and Glucosamine on Akt Figure 8.8 Effects of N-acetylcysteine and Glucosamine on phospho-Akt and Akt SVG cells were treated with either 8mM glucosamine and 6mM N-acetylcysteine for 1, 3, or 5 hours (GlcN+NAC T=1, GlcN+NAC T=3, GlcN+NAC T=5 respectively) or 6mM N-acetylcysteine alone for 1, 3, or 5 hours (NAC T=1, NAC T=3, NAC T=5 respectively). Additionally, two untreated (Control) samples were prepared. Whole cell homogenates were prepared and equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Immunoblots were treated with either phospho-specific Akt antibodies (p-Akt) or antibodies against total Akt (Akt). 153

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Effects of N-Acetylcysteine and STZ on Akt Figure 8.9 Effects of N-acetylcysteine and STZ on phospho-Akt and Akt SVG cells were treated with either 5mM STZ and 6mM N-acetylcysteine for 1, 3, or 5 hours (GlcN+NAC T=1, GlcN+NAC T=3, GlcN+NAC T=5 respectively) or 6mM N-acetylcysteine alone for 1, 3, or 5 hours (NAC T=1, NAC T=3, NAC T=5 respectively). Additionally, two untreated (Control) samples were prepared. Whole cell homogenates were prepared and equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Immunoblots were treated with either phospho-specific Akt antibodies (p-Akt) or antibodies against total Akt (Akt). 154

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155 Effects of Glucosamine or STZ on GRP 78 expression Figure 8.10 Effects of Glucosamine or STZ on GRP 78 expression SVG cells were treated with either 8mM glucosamine (top, GlcN) or 5mM STZ (bottom, STZ) for 1, 3, 5 or 9 hours (T=1, T=3, T=5, or T=9 respectively). Additionally, four untreated (Control) samples were prepared. Whole cell homogenates were prepared and equal amounts of protein (18g) were separated by SDS-PAGE, and transferred. Immunoblots were treated with antibodies against total GRP 78. STZ GlcN

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156 further investigate the effects of the O-GlcNAc protein m odification on signal transduction pathways in the brain, we sought to examine if Akt, like PKCand possibly PKC, either the activity or subcellular local ization affected by increases in this modification in glial cells. As indicated by our results, cellular treatment with four separate O-GlcNAc modulating agents pr oduced both different levels of global OGlcNAc increase as well as different e ffects on Akt phosphorylation. Glucosamine and STZ treatments produced low-level sustained increases in global O-GlcNAc (Figure 4.1 and 4.2) and also a rapid ri se in phospho-Akt levels in both cytosol and membrane fractions. These increases reached identic al maxima that were followed by a steady decline toward basal levels. The similarities between the glucosamine and STZ induced increases in Akt phosphorylation suggest that both compounds activate Akt via the same pathway. Alternatively, the more potent O-GlcNAcase inhibitors PUGNAc and NAGBT produced relatively large, rapidly rising OGlcNAc increases (Figure 4.1 and 4.2) but failed to significantly alter phospho-Akt levels from basal levels. One possible explanation for the data is th at the low level, global increases in OGlcNAc correspond to the modification of one or more specific proteins that then trigger an Akt activation pathway. As total levels of the O-GlcNAc modi fication continue to accumulate additional proteins are modified (o r the same proteins increasingly modified) reaching a level sufficient to stimulate a deac tivation pathway and/or block the activation pathway for Akt. Cellular treatment with lower concentrations of PUGNAc and NAGBT, however, failed to activate Akt suggest ing that the effects of glucosamine and STZ are not due to increases in the O-GlcNAc modification.

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157 Additionally, the increases in O-GlcNAc do not appear to affect Akt transcription, degradation, or translocati on since neither treatment altered total Akt levels or its intracellular distribution between the cytoso l and membrane (although a change in Akt transcription and a proportional, opposite change in Akt degrad ation cannot be ruled out). Similarly, incubation of adipocytes for 18 hours with 2.5mM glucosamine has been shown not to affect the translocation of Akt to the plasma membrane [23] suggesting that O-GlcNAc does not regulate Akt translocati on in insulin independe nt or dependent cell types. Another possible explanati on for the effects of glucosamine and STZ on Akt is that the experimental concentrations us ed (8mM and 5mM respectively) increased osmotic stress. Akt has been shown to be activated [17] by hype rosmolarity in renal tubular cells. Treatment of cells with 8m M galactosamine, an equimolar osmotic alteration that does not enha nce the O-GlcNAc modificatio n however, did not result in significant alterations in Akt phosphorylation. This data indi cates that the increased Akt phosphorylation following 8mM glucosamine and 5mM STZ treatments is a response to hyperosmotic conditions. Furthermore, the lack of effect of galactosamine on phosphoAkt levels also indicates that the effects of glucosamine and STZ are not mimicked by another structurally similar sugar. An alternate hypothesis explaining the e ffects of glucosamine and STZ on Akt phosphorylation, is Akt activation due to increas ed oxidative stress. Akt has been shown to become activated under conditions of oxidative stress [24,25]. Furthermore, in addition to modulating intr acellular O-GlcNAc levels cellular treatment with glucosamine [18] or STZ [26,27] is known to increase oxidative st ress. Increased flux

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158 through the hexosamine biosynthetic pathway as a result of glucosamine treatment has been shown to lead to pancreatic -cell deterioration presumably as a result of oxidative stress not related to increa ses in the O-GlcNAc modifica tion [18]. Although the exact mechanism by which glucosamine produces oxidative stress is unknown, it has been shown to increase H 2 O 2 levels [18]. Additionally, long term exposure to STZ has been shown to increase nitric oxide levels and suppress glutathion e peroxidase activity in STZ treated rat brain [26]. Many of the eff ects of glucosamine [18] and STZ induced oxidative stress, such as reduced arterial bl ood pressure [28], have been demonstrated to be reduced or reversed by N-acetylcysteine treatment. N-acetylcysteine is readily reduced to cysteine and can thus increase in tracellular levels of reduced glutathione [29,30]. The increases in phospho-Akt levels seen following exposure to glucosamine and STZ were not attenuated by treatment with N-acetylcysteine indicating that the mechanism of Akt activation does not likely involve oxidative stress. In addition to O-GlcNAc protein modi fication, oxidative stress, and osmotic stress, glucosamine has been shown to a ffect several other cellular functions. Glucosamine has been shown to induce endoplas mic reticulum (ER) stress in several cell systems [31,32]. The effects of STZ on ER stress, however, have not been examined. ER stress is described as a disruption of ER homeostasis that interferes with normal protein folding leading to an accumulation of misfolded or unfolded proteins [33]. Akt has been demonstrated to be activated in MCF-7 human breast cancer cells and H1299 human lung cancer cells in response to thapsi gargin and tunicamycin induced ER stress [34]. Similar to our pattern of Akt activa tion following glucosamine and STZ treatment, the pattern of Akt activation observed by Hu et al. [34] occurred rapidly with maximal

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159 levels occurring around 4 hours post treatment and then returning to basal levels between 8 and 12 hours post treatment. Analysis of GRP 78 expression, a well doc umented indicator of ER stress [31, 34], indicated that glucosamine, but not STZ, was inducing ER stress in our astroglial cell model. These results suggest that the increases in phosphoAkt induced glucosamine are due to increased ER stress whereas the increases in phospho-Akt observed after STZ treatment may be mediated via a different m echanism. STZ is a well known nitrosylating agent and may be inducing cellular stress via DNA damage. Although the mechanism by which glucosamine may induce ER stress in not known, these compounds may disrupt norma l protein N-glycosylation via the accumulation of lipid-linked oligosaccharide (LLO) such as Glc 3 Man 9 GlcNAc 2 -P-Pdolichol that have been shown to induce ER stress [35]. Additionally, glucosamine may disrupt the normal activity of N-acetylgluc osaminyltransferase V, an enzyme found predominately in intestine, lung and brain [36,37] that faci litates ER stress when its activity is blocked [38]. Although increased ER stress has been s hown to activate Akt, the mechanism by which this activation occurs is not known [34]. Two potential mechanisms by which ER stress may activate Akt are via a disruption on Ca 2+ homeostasis or the activation of stress activated protein kinases. Ca 2+ and calmodulin are known to activate PI 3-K, a well known upstream activator of Akt. Our ear lier findings show that whereas 8mM glucosamine treatment also result ed in an activation of the Ca 2+ sensitive PKCII similar to that of Akt, it failed to activate the Ca 2+ sensitive PKC[9]. Furthermore, Kohout et al. [39] demonstrated that PKCbinds to the plasma membrane longer and more

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160 efficiently at lower intracellular Ca 2+ concentrations than PKCII suggesting that increases in Ca 2+ likely do not account for our results. Another possible mechanism by which ER stress could activate Akt is by th e activation of the protein kinase R-like endoplasmic reticulum associated protein kina se (PERK) or the high inositol requiring-1 protein kinase (IRE1). Both of the ER transmembrane proteins are Ser/Thr kinases that are activated in response to ER stress [40]. Recently PERK has been demonstrated to be rapidly activated by 3mM glucosamine in retina l neuronal cells [40] and attributed this activation to ER stress. A lthough there is no evidence that PERK or IRE1 directly phosphorylate Akt, they are known to activate a variety of signal transduction pathways [41]. In conclusion, this study demonstrates a novel activation of Akt in response to glucosamine and STZ treatments. These treatments do not appear to affect the translation, degradation, or tran slocation of the enzyme. Investigation of the cause of this activation suggests that it is not relate d to increases in the O-GlcNAc protein modification, increased osmotic stress, or oxidative stress. It is likely that the increase in phospho-Akt brought on by glucosamine treatment is a result of incr eased ER stress and the increases observed after STZ treatment are mediated via an alternate mechanism.

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161 8.4 References Cited 1. O. Hermanson, K. Jepsen, M.G. Rosenfeld, N-CoR controls differentiation of neural stem cells into astr ocytes, Nature 419 (2002) 934-9. 2. H. Dudek, S.R. Datta, T.F. Franke, M.J. Birnbaum, R. Yao, G.M. Cooper, R.A. Segal, D.R. Kaplan, M.E. Greenberg Regulation of neuronal survival by the serine-threonine protein kina se Akt, Science 275 (1997) 661-5. 3. Z. Jiang, Y. Zhang, X.Q. Chen, P.Y. Lam, H. Yang, Q. Xu, A.C. Yu, Apoptosis and activation of Erkl/2 and Akt in astr ocytes postischemia, Neurochem. Res. 28 (2003) 831-7. 4. Y. Sonoda, T. Ozawa, K.D. Aldape, D.F. Deen, M.S. Berger, R.O. Pieper, Akt pathway activation converts anaplastic astrocytoma to glioblastoma multiforme in a human astrocyte model of glioma, Cancer Res. 61 (2001) 6674-8. 5. K. Vosseller, L. Wells, M.D. Lane, G.W. Hart, Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insuli n resistance associated with defects in Akt activation in 3T3-L1 adipocytes, Proc Natl. Acad. Sci. U. S. A. 99 (2002) 5313-8. 6. R.R. Herr, J.K. Jahnke, A.D. Argoudelis, The structure of stre ptozotocin, J. Am. Chem. Soc. 89 (1967) 4808-9. 7. K. Vosseller, K. Sakabe, L. Wells, G.W. Hart, Diverse re gulation of protein function by O-GlcNAc: a nuclear and cyt oplasmic carbohydrate post-translational modification, Curr. Opin. Chem. Biol. 6 (2002) 851-7. 8. S.Y. Park, J. Ryu, W. Lee, O-Gl cNAc modification on IRS-1 and Akt2 by

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162 PUGNAc inhibits their p hosphorylation and induces in sulin resistance in rat primary adipocytes, Exp. Mol. Med. 37 (2005) 220-9. 9. J.A. Matthews, M. Acevedo-Duncan, R.L. Potter, Selective decrease of membraneassociated PKC-alpha and PKC-epsilon in response to elevated intracellular OGlcNAc levels in transformed human glial cells, Biochim. Biophys. Acta 1743 (2005) 305-15. 10. M. Andjelkovic, D.R. Alessi, R. Meier, A. Fernandez, N.J. Lamb, M. Frech, P. Cron, P. Cohen, J.M. Lucocq, B.A. He mmings, Role of translocation in the activation and function of protein kinase B, J. Bi ol. Chem. 272 (1997) 31515-24. 11. C. Slawson, S. Shafii, J. Amburgey, R. Potter, Characterization of the O-GlcNAc protein modification in Xenopus laevis oocyte during oogenesis and progesteronestimulated maturation, Biochi m. Biophys. Acta 1573 (2002) 121-9. 12. R.J. Konrad, I. Mikolaenko, J.F. Tolar, K. Liu, J.E. Kudlow, The potential mechanism of the diabetogeni c action of streptozotocin: inhibiti on of pancreatic beta-cell O-GlcNAc-selectiv e N-acetyl-beta-D-glucosaminidase, Biochem. J. 356 (2001) 31-41. 13. J.A. Hanover, Z. Lai, G. Lee, W. A. Lubas, S.M. Sat o, Elevated O-linked Nacetylglucosamine metabolism in pancrea tic beta-cells, Arch. Biochem. Biophys. 362 (1999) 38-45. 14. M.D. Roos, W. Xie, K. Su, J.A. Clar k, X. Yang, E. Chin, A.J. Paterson, J.E. Kudlow, Streptozotocin, an analog of N-acet ylglucosamine, blocks the removal of O-GlcNAc from intracellular proteins, Proc. Assoc. Am. Physicians 110 (1998) 422-32.

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163 15. V. Pastukh, C. Ricci, V. Solodushko, M. Mozaffari, S.W. Schaffer, Contribution of the PI 3-kinase/Akt survival pathwa y toward osmotic preconditioning, Mol. Cell. Biochem. 269 (2005) 59-67. 16. A.S. Galvez, J.A. Ulloa, M. Chiong, A. Criollo, V. Eisner, L.F. Barros, S. Lavandero, Aldose reductase induced by hyperosmotic stress mediates cardiomyocyte apoptosis: differential effect s of sorbitol and mannitol, J. Biol. Chem. 278 (2003) 38484-94. 17. Y. Terada, S. Inoshita, S. Hanada, H. Shimamura, M. Kuwahara, W. Ogawa, M. Kasuga, S. Sasaki, F. Marumo, Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells Kidney Int. 60 (2001) 553-67. 18. H. Kaneto, G. Xu, K.H. Song, K. Suzuma, S. Bonner-Weir, A. Sharma, G.C. Weir, Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress, J. Biol. Chem. 276 (2001) 31099-104. 19. M. Federici, R. Menghini, A. Mauriello, M.L. Hribal, F. Ferrelli, D. Lauro, P. Sbraccia, L.G. Spagnoli, G. Sesti, R. Lauro, Insulin-dependent activation of endothelial nitric oxide s ynthase is impaired by O-linke d glycosylation modification of signaling proteins in human coronary endothelial cells, Ci rculation 106 (2002) 466-72. 20. Z.T. Kneass, R.B. Marchase, Protein O-GlcNAc modulates motility-associated signaling intermediates in neutroph ils, J. Biol. Chem .280 (2005) 14579-85. 21. B. Musicki, M.F. Kramer, R.E. Becker, A.L. Burnett, Inactivation of phosphorylated endothelial nitric oxide synthase (Ser-1177) by O-GlcNAc in

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164 diabetes-associated erectile dysfunction, Proc Natl. Acad. Sci. U. S. A. 102 (2005) 11870-5. 22. A.C. Newton, Regulation of the ABC kina ses by phosphorylation: protein kinase C as a paradigm, Biochem. J. 370 (2003) 361-71. 23. B.A. Nelson, K.A. Robinson, M.G. Buse, Defective Akt activation is associated with glucosebut not glucosamine-induced insulin resistance, Am. J. Physiol. Endocrinol. Metab. 282 (2002) E497-506. 24. A. Tapodi, B. Debreceni, K. Hanto, Z. Bognar, I. Wittmann, F. Gallyas Jr, G. Varbiro, B. Sumegi, Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADP-ribose)polymerase1 inhibition in oxida tive stress, J. Biol. Chem. 280 (2005) 35767-75. 25. M. Shaw, P. Cohen, D.R. Alessi, The act ivation of protein kinase B by H2O2 or heat shock is mediated by phosphoinositide 3-kinase and not by mitogen-activated protein kinase-activated pr otein kinase-2, Biochem. J. 336 ( Pt 1) (1998) 241-6. 26. R. Mastrocola, F. Restivo, I. Vercelli natto, O. Danni, E. Brignardello, M. Aragno, G. Boccuzzi, Oxidative and nitrosative stress in brain mitochondria of diabetic rats, J. Endocrinol. 187 (2005) 37-44. 27. S.A. Wohaieb,D.V. Godin, Alterations in free radical tissue-defense mechanisms in streptozocin-induced diabetes in rat. Effects of insulin treatment, Diabetes 36 (1987) 1014-8. 28. Z. Xia, P.R. Nagareddy, Z. Guo, W. Zhang, J.H. McNeill, Antioxidant Nacetylcysteine restores systemic nitric oxide availability and corrects depressions in arterial blood pressure and heart rate in diabetic rats, Free Radic. Res. 40 (2006)

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165 175-84. 29. P. Moldeus, I.A. Cotgreave, M. Ber ggren, Lung protection by a thiol-containing antioxidant: N-acetylcysteine, Re spiration 50 Suppl 1 (1986) 31-42. 30. S. De Flora, C.F. Cesarone, R.M. Ba lansky, A. Albini, F. D'Agostini, C. Bennicelli, M. Bagnasco, A. Camoirano, L. Scatolini, A. Rovida, a.l. et, Chemopreventive properties and mechanisms of N-Acetylcysteine. The experimental background, J. Cell. Biochem. Suppl. 22 (1995) 33-41. 31. H.Y. Lin, P. Masso-Welch, Y.P. Di, J.W. Cai, J.W. Shen, J.R. Subjeck, The 170kDa glucose-regulated stress protein is an endoplasmic reticulum protein that binds immunoglobulin, Mol. Biol. Cell 4 (1993 ) 1109-19. 32. G.H. Werstuck, M.I. Khan, G. Femia, A.J. Kim, V. Tedesco, B. Trigatti, Y. Shi, Glucosamine-induced endoplasmic reticu lum dysfunction is associated with accelerated atherosclerosis in a hypergly cemic mouse model, Diabetes 55 (2006) 93-101. 33. R.J. Kaufman, Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene tran scriptional and translatio nal controls, Genes Dev. 13 (1999) 1211-33. 34. P. Hu, Z. Han, A.D. Couvillon, J.H. Exton, Critical role of endogenous Akt/IAPs and MEK1/ERK pathways in counteracting endoplasmic reticulum stress-induced cell death, J. Biol. Chem. 279 (2004) 49420-9. 35. M.A. Lehrman, Oligosaccharide-based information in endoplasmic reticulum quality control and other biological syst ems, J. Biol. Chem. 276 (2001) 8623-6. 36. H. Saito, J. Gu, A. Nishikawa, Y. Ihara, J. Fujii, Y. Kohgo, N. Taniguchi,

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166 Organization of the human N-acetylglucos aminyltransferase V gene, Eur. J. Biochem. 233 (1995) 18-26. 37. A. Nishikawa, J. Gu, S. Fujii, N. Taniguchi, Determination of Nacetylglucosaminyltransferases III, IV and V in normal and hepatoma tissues of rats, Biochim. Biophys. Acta 1035 (1990) 313-8. 38. H. Fang, W. Huang, Y.Y. Xu, Z.H. Shen, C.Q. Wu, S.Y. Qiao, Y. Xu, L. Yu, H.L. Chen, Blocking of N-acetylglucosam inyltransferase V induces cellular endoplasmic reticulum stress in human hepatocarcinoma 7721 cells, Cell Res. 16 (2006) 82-92 39. S.C. Kohout, S. Corbalan-Garcia, A. Torrecillas, J.C. Gomez-Fernandez, J.J. Falke, C2 domains of protein kinase C isoforms alpha, beta, and gamma: activation parameters and calcium stoichiometries of the membrane-bound state, Biochemistry 41 (2002) 11411-24. 40. C.L. Kline, T.L. Schrufer, L.S. Jefferson, S.R. Kimball, Glucosamine-induced phosphorylation of the alpha-subuni t of eukaryotic initiation factor 2 is mediated by the protein kinase R-like endoplasmic-reticulu m associated kinase, Int. J. Biochem. Cell. Biol. 2005. 41. R. Kim, M. Emi, K. Tanabe, S. Murakami, Role of the unfolded protein response in cell death, Apoptosis 11 (2006) 5-13.

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167 Chapter 9 PKCand PKCassociated proteins 9.0 Introduction PKC associated proteins have been shown to play a role in PKC isoform activation, translocation, and lo calization near the proper subs trates and regulators [1-4] (discussed in section 2.3). Additionally, the O-GlcNAc protein modification has been shown to regulate protein-protein interacti on is certain instances [5-7] (discussed in section 1.4.3). It is therefor e possible that the decreases in membrane associated PKCand following treatment with O-GlcNAc in creasing compounds (discussed in chapter 4) is a result of a disruption between these PKC isoforms and certain O-GlcNAc modified protein(s) that pl ay a role in PKCand membrane association. The goal of this chapter is to determine if any O-GlcNAc m odified proteins specifi cally associate with either PKCor thus opening new avenues for the investigation of the possible link between PKC regulation and the O-GlcNAc modification. Additionally, we sought to examine if there is an associa tion between either of these isoforms and the related kinase Akt. 9.1 Materials and Methods Cell culturing, SDS-PAGE, and immunoblottin g were carried out as described in section 4.1. Immunoprecipitations were done as described in section 5.3.2. 9.2 Results

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168 9.2.1 O-GlcNAc modified proteins associated with PKCand Untreated SVG cells were immunopr ecipitated with either anti-PKCor anti PKCantibodies or the appropriate control antibody (see section 5.3.2). Additionally, the immunoprecipation process was carried out using Protei n A agarose beads with no antibody in order to be able to subtract the proteins binding nonspecifically to the beads from those specifically binding to the anti bodies. Samples were then immunoblotted using the CTD110.6 anti-O-GlcNAc antibody in order to detect O-GlcNAc modified proteins that were immunoprecipitated. Co mparison of anti-O-GlcNAc immunoblots of samples immunoprecipitated with either anti-PKCand antibodies, control antibodies, or beads only revealed three dis tinct protein bands sp ecifically precipitated with the anti-PKCand antibodies. One O-GlcNAc modified protein of ~120kDa was immunoprecipitated by the anti-PKCantibody and two O-GlcNAc modified proteins of ~90kDa and ~80kDa were brought down with PKC(Figure 9.1). 9.2.2 PKCand PKCassociation with Akt In order to determine if either PKCor associated with Akt, immunoprecipitations were pe rformed using either PKCor PKCspecific antibodies followed by immunoblotting for Akt. Some immunoprecipitations were performed using IP buffer (see section 5.3.2) with either 1.0% or 0.1% Triton X-100 as detergent concentration has been shown to affect the a ssociation of Akt with certain proteins in immunoprecipitation experime nts [8]. Immunoprecipi tation with anti-PKCantibodies followed by immunoblotting for Akt revealed no significant association between the two

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Association between PKCand and O-GlcNAc modified proteins Figure 9.1 Association between PKCand and O-GlcNAc modified proteins. Untreated SVG cells were lysed and immunoprecipitated with anti-PKCantibodies (IP PKC-), anti-PKCantibodies (IP PKC-), normal rabbit IgG antibodies (IP rabbit IgG), normal mouse IgG (IP mouse IgG), or Protein A agarose beads with no antibodies (beads). Samples were separated by SDS-PAGE and immunoblotted using the CTD110.6 anti-O-GlcNAc antibody. indicates bands unique to immunoprecipitations performed using PKCor specific antibodies and not found in control immunoprecipitations. 169

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Association between PKCAkt Figure 9.2 Association between PKCand Akt. Untreated SVG cells were lysed and immunoprecipitated with anti-PKCantibodies (IP PKC-) or normal rabbit IgG antibodies (IP control IgG) in IP buffer containing either 1.0% or 0.1% Triton X-100. Samples were separated by SDS-PAGE and immunoblotted using anti-Akt antibodies. Non-immunoprecipitated whole cell homogenate (homogenate) was also analyzed. 170

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Association between PKCand Akt Figure 9.3 Association between PKCand Akt. Untreated SVG cells were lysed and immunoprecipitated with anti-PKCantibodies (IP PKC-), normal mouse IgG antibodies (IP mouse IgG 1 ), anti-p27 antibodies, anti-Mat1 antibodies, or Protein A agarose beads with no antibodies. Samples were separated by SDS-PAGE and immunoblotted using anti-Akt antibodies. Non-immunoprecipitated whole cell homogenate (homogenate) was also analyzed. 171

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172 proteins when compared to immunoprecipita tion with control antibodies using either 1.0% or 0.1% Triton X-100 in the IP buffer (Figure 9.2). Investigation of a possible association between PKCand Akt was done by immunoprecipitating PKCfrom untreated SVG cells with an anti-PKCmouse monoclonal antibody (Santa Cruz Biotec hnology)(see section 5.4.2). Control immunoprecipitations were perf ormed using normal mouse IgG 1 anti-p27 and anti-Mat1 mouse monoclonal antibodies (all from Sant a Cruz Biotechnol ogy), and Protein A agarose beads (discussed further in section 5.4.2). Immunoprecipita tion with anti-PKCantibodies followed by immunoblotting for Ak t revealed a significant association between PKCand Akt when compare to control immunoprecipitations or using Protein A agarose beads only (Figure 9.3). 9.3 Discussion PKC associated proteins have been s hown to play a role in its subcellular localization and its ability to translocate to the cell me mbrane upon activation [1-4]. There is no record in the literature, however, of analysis of any of these proteins for modification with O-GlcNAc. The finding th at specific proteins associate with PKCand PKCin human astroglial cells raises the possi bility that the decreases in membrane associated PKCand observed following treatment with O-GlcNAc modulating agents may be mediated through their interac tion with these modified proteins. Further analysis, including purificati on, identification, and characte rization of these O-GlcNAc modified proteins, is required in order to determin e their role, if any, in the regulation of PKCand

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173 Additionally, the finding the PKCassociates with Akt in human astroglial cells raises some interesting possibilities. This association may indicate cross-talk between the PKCand Akt mediated signal transduction pathways in astroglial cells. Further investigation is needed to reveal the significance of this interaction.

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174 9.4 References Cited 1. D. Mochly-Rosen, H. Khaner, J. Lop ez, B.L. Smith, Intracellular receptors for activated protein kinase C. Identification of a binding site for the enzyme, J. Biol. Chem. 266 (1991) 14866-8. 2. M.H. Disatnik, S.M. Hernandez-Sotomayor, G. Jones, G. Carpenter, D. MochlyRosen, Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C, Proc. Natl. Aca d. Sci. U. S. A. 91 (1994) 559-63. 3. M.M. Rodriguez, D. Ron, K. Touhara, C.H. Chen, D. Mochly-Rosen, RACK1, a protein kinase C anchoring protein, coor dinates the binding of activated protein kinase C and select pleckstrin homology do mains in vitro, Biochemistry 38 (1999) 13787-94. 4. D. Mochly-Rosen, A.S. Gordon, Anchoring proteins for protein kinase C: a means for isozyme selectivity, FASEB J. 12 (1998) 35-42. 5. M.D. Roos, K. Su, J.R. Baker, J.E. Kudlow, O glycosylatio n of an Sp1-derived peptide blocks known Sp1 protein interac tions, Mol. Cell. Biol. 17 (1997) 6472-80. 6. I. Han, M.D. Roos, J.E. Kudlow, Interacti on of the transcription factor Sp1 with the nuclear pore protein p62 require s the C-terminal domain of p62, J. Cell. Biochem. 68 (1998) 50-61. 7. N.O. Ku, M.B. Omary, Expression, glyc osylation, and phosphorylation of human keratins 8 and 18 in insect cells Exp. Cell. Res. 211 (1994) 24-35. 8. D.D. Sarbassov, D.A. Guertin, S.M. Ali, D.M. Sabatini, Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex, Science 307 (2005) 1098-101.

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About the Author Jason Aaron Matthews was born on June 17, 1971 in Waycross, GA. Following elementary school he and his family move d to Birmingham, AL where he completed junior high and high school. After graduating salutatorian of his class, he moved to Jacksonville, FL and attended the University of North Florida. He graduated in 1994 with a major in Biology. A few years later, he then moved to Tampa, FL and entered the graduate program in the department of chemistr y at the University of South Florida. He joined the laboratory of Dr. R obert Potter, a biochemistry professor, where he worked until obtaining his Ph.D. degree.


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Investigation of the effects of increased levels of O-GlcNAc protein modification on protein kinase C and Akt
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ABSTRACT: O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and ubiquitous post-translational modification that has been shown to play a role in regulating a variety of intracellular processes. The pathway responsible for generating the O-GlcNAc modification, the hexosamine biosynthetic pathway (HBP), has also been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. To investigate if the effects of HBP flux on PKC translocation observed by others is related to the O-GlcNAc modification, O-GlcNAc levels in human astroglial cells were elevated using four separate O-GlcNAc modulating agents followed by analysis of cytosol and membrane concentrations of PKC-epsilon, -alpha, -betaII, and -iota. Of the four PKC isoforms analyzed, PKC-epsilon showed a significant reduction in its membrane associated levels in response to all agents tested whereas PKC-alpha showed reductions in response to only two agents.Investigation of the mechanism for thereductions in membrane associated PKC-epsilon and -alpha indicate that the increased O-GlcNAc levels did not disrupt the activation of these isoforms or their ability to translocate to the plasma membrane. Furthermore, results indicate that these reductions are not due to a disruption in the Hsp70 mediated recycling of the isoforms. It was found; however, that increased O-GlcNAc levels resulted in increased degradation of PKC-epsilon suggesting that the decreases in membrane associated PKC-epsilon may be a result of increased phosphatase or protease activity. Additional studies revealed that decreases in membrane bound PKC-epsilon and PKC-alpha, both of which act as anti-apoptotic enzymes, correlated with an increase in poly-(ADP-ribose) polymerase (PARP) cleavage --a well characterized hallmark of apoptosis. In addition to PKC, the effects of increased O-GlcNAc levels on a related kinase, Akt, were also examined. Initial investigation of the effects of increased O-GlcNAc modification of Akt activation using glucosamine or streptozotocin revealed a relatively large, short-term increase in Akt phosphorylation in response to these treatments. However, further analysis with other O-GlcNAc modulators indicated that this activation was not related to O-GlcNAc protein modification. Furthermore, this activation does not appear to be related to any hyperosmotic effects associated with the treatment conditions, nor does it appear to be related to oxidative stress. Therefore, further investigation is needed to characterize the novel pathway responsible for Akt activation following glucosamine or streptozotocin treatment.
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Translocation.
Isoforms.
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Phosphorylation.
Apoptosis.
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