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Investigation of molecular signaling defects in PTP alpha-deficient mice : insulin and NMDA receptor… Le, Hoa Thi 2006

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INVESTIGATION OF M O L E C U L A R SIGNALING DEFECTS IN PTP ALPHA-DEFICIENT MICE: INSULIN AND NMDA RECEPTOR SIGNALING by HOA THI L E M . S c , Vietnam National University, Hanoi, 1998 B.Sc. , Vietnam National University, Hanoi, 1996 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Pathology and Laboratory Medicine) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A November 2006 © Hoa Thi Le, 2006 ABSTRACT The physiological roles of P T P a in insulin and N M D A receptor ( N M D A R ) signaling were investigated using gene-targeted mice deficient in PTPa . P T P a 7 " animals had normal body weights and circulating levels o f glucose and insulin. In glucose and insulin tolerance tests, their efficiency o f blood glucose clearance was comparable to wild-type mice. Kinetics and extents o f insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation were similar in wild-type and PTPa" 7" liver, muscle, and adipose tissue. However the association o f IRS-1 and PI3-K was altered in P T P a 7 " liver, with increased insulin-independent and reduced insulin-stimulated association compared to wi ld-type samples. This did not affect activation of the downstream signaling effector Akt . Thus, P T P a is not a negative regulator of insulin signaling and does not perform an essential role in mediating the physiological action of insulin. P T P a 7 " mice exhibit defects in NMDAR-associa ted processes. In vivo molecular effectors linking P T P a and the N M D A R were investigated in wild-type and P T P a 7 " mice. Tyrosine phosphorylation of the N M D A R N R 2 A and N R 2 B subunits was reduced upon P T P a ablation, indicating a positive effect of this phosphatase on N M D A R phosphorylation v ia intermediate molecules. The N M D A R is a substrate o f sre family tyrosine kinases (SFKs), and reduced activity of sre, fyn, yes, and lck, but not lyn, was detected in the absence o f PTPa . In addition, autophosphorylation of Pyk2, a tyrosine kinase linked to N M D A R signaling, was also reduced in P T P a 7 " samples. In an H E K 2 9 3 cell expression system, P T P a actions on N R 2 A / B phosphorylation mediated by sre and fyn were examined. The expression o f P T P a enhanced fyn- but reduced sre-mediated N R 2 A / B phosphorylation. This is partly i i due to the fact that P T P a complexes with src and fyn with strikingly distinct affinities and via different mechanisms of binding. Interestingly, P T P a Tyr789 phosphorylation was found to regulate P T P a action on fyn- and src-mediated N R 2 A / B phosphorylation. These studies eliminate P T P a as a candidate for the development of PTP-directed therapeutics for the treatment of diabetes and obesity. They demonstrate a key upstream regulatory role for P T P a in N M D A R signaling and function, and shed light on the involvement of specific SFKs and their potentially distinct targeting by P T P a in this process. i i i TABLE OF CONTENTS A B S T R A C T i i T A B L E O F C O N T E N T S iv LIST O F F I G U R E S ix L I S T O F A B B R E V I A T I O N S x i L I S T O F P U B L I C A T I O N S xiv A C K N O W L E D G E M E N T S xv 1 I N T R O D U C T I O N 1 1.1 Protein tyrosine phosphorylation 1 1.1.1 A general introduction to protein tyrosine phosphorylation 1 1.1.2 Protein tyrosine phosphatase (PTP) superfamily ; 1 1.2 Protein tyrosine phosphatase alpha (PTPa) 4 1.2.1 Structure o f P T P a 4 1.2.2 Regulation of P T P a 6 1.2.2.1 Dimerization 6 1.2.2.2 Phosphorylation 8 1.2.2.3 Proteolysis '. 9 1.2.3 Substrates of P T P a 10 1.2.3.1 Src family kinase (SFKs) 10 1.2.3.2 K v l . 2 potassium channel 13 1.2.3.3 p l 3 0 C a s 14 1.2.4 P T P a and cellular processes 14 1.2.4.1 Mitosis 14 1.2.4.2 Integrin signaling 16 1.2.4.3 Neuronal differentiation and outgrowth 17 1.2.4.4 T-cell signaling 18 1.2.4.5 Cancer 19 1.3 P T P a and insulin signaling 21 1.3.1 Insulin 21 1.3.2 The insulin receptor (IR) 22 1.3.3 Insulin signaling 22 iv 1.3.4 Insulin responses 27 1.3.4.1 Stimulation of glucose uptake 27 1.3.4.2 Stimulation of glycogen synthesis 27 1.3.4.3 Stimulation of protein synthesis 28 1.3.5 PTPs in insulin signaling 29 1.4 P T P a and N-methyl-D-aspartate receptor ( N M D A R ) signaling 34 1.4.1 L-Glutamate 34 1.4.2 The glutamate receptor 34 1.4.3 The N-methyl-D-aspartate receptor ( N M D A R ) 36 1.4.3.1 Properties 36 1.4.3.2 Subunit composition 37 1.4.3.3 Domain structure 39 1.4.3.4 Function 40 1.4.4 N M D A R signaling 41 1.4.5 PTPs i n N M D A R signaling 50 1.5 Hypothesis 53 2 M A T E R I A L S A N D M E T H O D S 56 2.1 Materials 56 2.1.1 Mice 56 2.1.2 Cultured cells 56 2.1.2.1 Human embryonic kidney ( H E K ) 293T cells 56 2.1.2.2 Primary cortical and hippocampal neurons 56 2.1.3 Antibodies 57 2.1.3.1 Primary antibodies 57 2.1.3.2 Secondary antibodies 58 2.1.4 Plasmids : 58 2.2 Methods 58 2.2.1 Physiological tests 58 2.2.1.1 Determination of blood glucose and plasma insulin concentrations 58 2.2.1.2 Glucose and insulin tolerance tests 59 2.2.1.3 Weight gain analysis 59 2.2.2 Insulin stimulation 59 2.2.3 Preparation of crude synaptosomal P2 fraction 60 2.2.4 Preparation o f l ipid rafts .....60 2.2.5 Protein extraction 61 2.2.5.1 Preparation of tissue lysates 61 2.2.5.2 Preparation o f cell lysates 61 2.2.5.3 Preparation of Triton X-100 soluble and insoluble fractions 62 v 2.2.6 Irnmunoprecipitation and immunoblotting 62 2.2.6.1 Irnmunoprecipitation 62 2.2.6.2 Immunoblotting 63 2.2.7 Kinase and phosphatase assays 64 2.2.7.1 Kinase assay 64 2.2.7.2 Phosphatase assay 65 2.2.8 Primary hippocampal and cortical neuron culture 65 2.2.9 Calcium phosphate transfection 66 2.2.10 Glutamate stimulation 67 2.2.11 Data analysis 67 3 I N S U L I N S I G N A L I N G A N D G L U C O S E H O M E O S T A S I S I N P T P a 7 " M I C E 68 3.1 Introduction and rationale 68 3.2 Results 69 3.2.1 P T P a expression in mouse tissues 69 3.2.2 Unaltered expression of insulin-related PTPs in PTPa" 7" mice 70 3.2.3 Normal weight gain in PTPa"'" mice 71 3.2.4 Glucose homeostasis is normal in PTPa" /" mice 71 3.2.5 Unaltered IR tyrosine phosphorylation in PTPa" 7" mice 73 3.2.6 IRS tyrosine phosphorylation and association with PI3-K in PTPa" 7" mice 77 3.2.6.1 IRS-1 tyrosine phosphorylation and association with PI3-K in PTPa" 7" muscle 77 3.2.6.2 IRS-1 and IRS-2 tyrosine phosphorylation and association with PI3-K in PTPa" 7" liver 79 3.2.7 Insulin-stimulated Ak t and M A P K activation in liver, muscle, and adipose tissues of PTPa" 7" mice 81 3.3 Discussion 86 3.3.1 Normal insulin sensitivity in PTPa" 7" mice 86 3.3.2 Altered IRS-1 phosphorylation and association with PI3-K in PTPa" 7" liver 88 3.3.3 Understanding the role of P T P a in insulin signaling in other systems 89 3.3.4 Summary 91 4 N M D A R S I G N A L I N G I N PTPa" 7" M I C E 92 4.1 Introduction and rationale 92 4.2 Results ...93 4.2.1 Localization of P T P a , SFKs , Pyk2, and N M D A R subunits in Triton X-100 solubilized synaptosomes 93 vi 4.2.2 Altered protein tyrosine phosphorylation in PTPa"'" synaptosomal fractions.... 96 4.2.3 Altered S F K phosphorylation in P T P a 7 ' synaptosomal fractions 97 4.2.3.1 S F K hyperphosphorylation at Tyr527 in P T P a 7 " synaptosomes 97 4.2.3.2 Four S F K s are hyperphosphorylated in P T P a 7 " synaptosomal fractions.. 99 4.2.4 N R 2 A and N R 2 B tyrosine phosphorylation is reduced in P T P a 7 " synaptosomes ....102 4.2.5 Pyk2 autophosphorylation is reduced in synaptosomes lacking P T P a 105 4.2.6 Localization of P T P a in lipid rafts 107 4.2.7 Glutamate-induced Akt , M A P K , and C R E B phosphorylation in P T P a 7 " hippocampal and cortical neurons 109 4.3 Discussion 112 4.3.1 P T P a is a positive regulator of N M D A R tyrosine phosphorylation 112 4.3.2 P T P a is a physiological regulator of four neuronal S F K s 114 4.3.3 P T P a is an upstream regulator of Pyk2 114 4.3.4 Summary 115 5 R E G U L A T I O N OF F Y N - A N D S R C - M E D I A T E D N R 2 A / B P H O S P H O R Y L A T I O N B Y P T P a 117 5.1 Introduction and rationale 117 5.2 Results 118 5.2.1 P T P a enhances fyn- but reduces src-mediated N R 2 A / B phosphorylation 118 5.2.2 P T P a dephosphorylates and activates both sre and fyn 121 5.2.3 P T P a dephosphorylates both fyn- and src-phosphorylated N R 2 A 124 5.2.4 Fyn and sre phosphorylate P T P a 125 5.2.5 F y n and sre have no effect on P T P a activity in vitro 129 5.2.6 P T P a complexes with fyn and sre with different affinities and via distinct mechanisms 131 5.2.7 Role of P T P a tyrosine phosphorylation in fyn- and src-mediated N R 2 A phosphorylation 134 5.2.8 Y789F P T P a and wild-type P T P a activate fyn and sre to similar extents 136 5.2.9 P T P a tyrosine phosphorylation is developmentally regulated 137 5.3 Discussion 138 5.3.1 Differential SFK-mediated N R 2 A / B phosphorylation is not due to differences in the intrinsic kinase activities of fyn or sre or in the phosphatase activity of P T P a 140 5.3.2 Potential functional and physical feedback signaling mechanisms between P T P a and fyn or sre 141 v i i 5.3.3 The significance of P T P a tyrosine phosphorylation 142 5.3.4 t he significance of the association of P T P a with fyn or src 144 5.3.5 The complexity o f the cell culture system 144 5.3.6 Summary 146 6 G E N E R A L D I S C U S S I O N 148 6.1 P T P a is not a major regulator of insulin signaling 150 6.2 P T P a regulates N M D A R tyrosine phosphorylation 153 6.3 Significance of the regulation of N M D A R tyrosine phosphorylation by P T P a in NMDAR-re la t ed processes 158 6.4 Future directions 160 6.5 Overall summary 161 7 R E F E R E N C E S . . . 164 v i i i LIST OF FIGURES Figure 1.1. Schematic structures and subgroups of representative PTPs 2 Figure 1.2. P T P a structure and regulatory features 5 Figure 1.3. P T P a dimerization 7 Figure 1.4. Structure and regulation of S F K s 11 Figure 1.5. Mechanism of src activation by P T P a serine phosphorylation during mitosis. 15 Figure 1.6. Insulin signaling 23 Figure 1.7. Ligand- and modulator-binding sites on the N M D A R 36 Figure 1.8. Domain structure of N M D A R subunits 39 Figure 1.9. N M D A R signaling at the postsynapse 42 Figure 1.10. Sites of tyrosine phosphorylation of N M D A R subunits by src and fyn 49 Figure 3.1. P T P a expression in mouse tissues 69 Figure 3.2. P T P a expression in insulin-responsive tissues of wild-type and PTPa" /" mice. 70 Figure 3.3. Body weights of wild-type and P T P a 7 " mice 72 Figure 3.4. Parameters o f glucose homeostasis are similar in wild-type and P T P a 7 " mice. 73 Figure 3.5. Insulin receptor tyrosine phosphorylation in liver, muscle, and adipose tissues of wild-type and P T P a 7 " mice ...75 Figure 3.6. Phosphosite-specific tyrosine phosphorylation o f insulin receptor in muscle and liver of wild-type and P T P a 7 " mice 76 Figure 3.7. Insulin-stimulated IRS-1 phosphorylation and association with PI3-K in muscle tissues from wild-type and P T P a 7 " mice 78 Figure 3.8. Insulin-stimulated IRS-1 phosphorylation and association with PI3-K in liver tissues from wild-type and P T P a 7 " mice 80 Figure 3.9. Insulin-stimulated IRS-2 phosphorylation and association with PI3-K in liver tissues from wild-type and P T P a 7 " mice 82 Figure 3.10. Insulin-stimulated A k t and M A P K activities in muscle tissues from wild-type and P T P a 7 " mice 83 Figure 3.11. Insulin-stimulated Ak t and M A P K activities in liver tissues from wild-type and P T P a 7 " mice 84 Figure 3.12. Insulin-stimulated Ak t and M A P K activities in adipose tissues from wild-type and P T P a 7 " mice 85 Figure 4.1. Developmental expression of P T P a , N M D A R , PSD-95, and tyrosine kinases in detergent-fractionated crude synaptosomes o f wild-type and P T P a 7 " mice.. 95 ix Figure 4.2. Phosphotyrosyl proteins and S F K tyrosine phosphorylation status in detergent-fractionated crude synaptosomes o f wild-type and PTPa ' 7 " mice 98 Figure 4.3. Altered tyrosine phosphorylation o f the synaptosomal S F K s sre and fyn in PTPa" 7" mice 100 Figure 4.4. Tyrosine phosphorylation of the synaptosomal S F K s yes, lck, and lyn in PTPa" 7" mice 101 Figure 4.5. Reduced tyrosine phosphorylation of N R 2 A and N R 2 B in synaptosomal fractions of PTPa" 7" mice 103 Figure 4.6. Specificity of ant i -NR2A and - N R 2 B antibodies 104 Figure 4.7. Pyk2 autophosphorylation and association with fyn are altered by ablated or increased P T P a expression 106 Figure 4.8. Localization of P T P a in lipid rafts 108 Figure 4.9. Glutamate-induced phosphorylation of Akt , C R E B , and M A P K in wild-type and PTPa" 7" hippocampal neurons 110 Figure 4.10. Glutamate-induced phosphorylation of Akt , C R E B , and M A P K in wild-type and PTPa" 7" cortical neurons I l l Figure 5.1. Schematic diagram illustrating the pathways through which P T P a potentially regulates N R 2 phosphorylation 119 Figure 5.2. Differential regulation of fyn- and src-mediated N R 2 A / B phosphorylation by P T P a 120 Figure 5.3. P T P a dephosphorylates and activates fyn and sre 123 Figure 5.4. P T P a dephosphorylates fyn- and src-phosphorylated N R 2 A 125 Figure 5.5. Schematic diagram depicting the potential ability of S F K s to phosphorylate P T P a and the possible actions of phospho-PTPa in regulating N R 2 phosphorylation 126 Figure 5.6. Phosphorylation of P T P a by fyn and sre 127 Figure 5.7. The intrinsic phosphatase activity of P T P a is not altered by co-expression with fyn or sre 130 Figure 5.8. Schematic diagram illustrating the potential association of P T P a with the SFKs fyn and sre, and the consequences of this on N R 2 phosphorylation 131 Figure 5.9. Differential association of P T P a with fyn and sre 133 Figure 5.10. Role of P T P a tyrosine phosphorylation in regulation of fyn- and src-mediated N R 2 A phosphorylation 135 Figure 5.11. Activation of fyn and sre by Y789F mutant P T P a 137 Figure 5.12. Phosphorylation of P T P a is developmentally regulated 138 LIST OF ABBREVIATIONS A k t / P K B protein kinase B A M P A R a-amino-3-hydroxy-5-methyl-4-isoxazone propionate receptor A T C C American Type Culture Collection A T P adenosine 5'-triphosphate B H K baby hamster kidney B S A bovine serum albumin C A D T K Ca 2 +-dependent tyrosine kinase C A K p cell adhesion kinase (3 C a M calmodulin C a M K I I calcium-CaM-dependent kinase II C N S central nervous system C R E c - A M P response element C R E B C R E binding protein Csk C-terminal src kinase Cys cystein D I membrane proximal domain D 2 membrane distal domain D M E M Dulbecco's modified Eagle's medium D S P dual specific phosphatase E C L enhanced chemiluminescence E D T A ethylenediaminetetraacetic acid eEF eukaryotic elongation factor E G F epidermal growth factor E G F R E G F receptor eEF eukaryotic initiation factor E r k l / 2 extracellular signal related kinase 1/2 F A K focal adhesion kinase F G F fibroblast growth factor G A B A y-amino butyric acid G A P guanine triphosphatase (GTPase)-activating protein G E F guanine nucleotide exchange factor G L U T glucose transporter G P C R G-protein-coupled receptor Grb2 adaptor protein growth factor receptor bound 2 G S K - 3 glycogen synthase kinase-3 h hour H B S Hepes-buffered saline H B S S Hank's balance salt solution H E K human embryonic kidney H R P horseradish peroxidase IGF-1 Insulin-like growth factor-1 IGF-1R IGF-1 receptor IR insulin receptor k D kiloDalton L A R leukocyte antigen-related P T P L M W low molecular weight L T D long term depression L T P long term potentiation M A M meprin-A5-mu M A P K mitogen-activated protein kinase M B S MES-buffered saline M E M minimum essential medium m E P S C miniature excitatory postsynaptic current min minute m T O R mammalian target of rapamycin N C A M neural cell adhesion molecule N M D A N-methyl-D-aspartate N M D A R N M D A receptor N R P T P intracellular/nonreceptor P T P O D optical density P B S phosphate-buffered saline P B S - T P B S containing 0.1% Tween 20 P D G F platelet-derived growth factor P D G F R P D G F receptor P D K phosphoinositide-dependent kinase x i i P D Z PSD-95, Dig , and ZO-1 Homology P H pleckstrin homology PI(3,4)P2 phosphatidylinositol 3,4-bisphosphate PI(3,4,5)P3 phosphatidylinositol 3,4,5-trisphosphate PI3-K phosphoinositide 3-kinase P K A protein kinase A P K C protein kinase C P M S F phenylmethylsulfonyl fluoride pNPP joara-nitrophenyl phosphate PP1 protein phosphatase 1 P P 1 G glycogen-bound form of protein phosphatase 1 PP2 4-amino-5-(4-chlorophenyl)-7-(?-butyl)pyrazolo[3,4-d]pyrimidine P P 2 B / C a N calcineurin, a calcium/CaM-regulated phosphatase P S D postsynaptic density PSD-95 postsynaptic density-95 scaffolding protein P T B phosphotyrosine binding P T K protein tyrosine kinase P T P protein tyrosine phosphatase P V D F polyvinylidene fluoride Pyk2 proline-rich kinase 2 R I P A radioimmunoprecipitation assay R P T P transmembrane/receptor-type PTP SDS sodium dodecyl sulfate S D S - P A G E SDS-polyacrymide gel electrophoresis Ser serine SH2 sre homology 2 SH3 sre homology 3 She sre homology collagen S T E P striatal enriched tyrosine phosphatase T C R T-cell receptor Tyr tyrosine W T wild-type x i i i LIST OF PUBLICATIONS Le H . T., Maksumova L . , Wang J., and Pallen C. J. (2006) Reduced N M D A receptor tyrosine phosphorylation in PTPa-deficient mouse synaptosomes is accompanied by inhibition of four src family kinases and Pyk2: an upstream role for P T P a in N M D A receptor regulation. J Neurochem. 98, 1798-809 Maksumova L . , Le H . T., Muratkhodjaev F., Davidson D . , Veillette A . , and Pallen C. J. (2005) Protein tyrosine phosphatase alpha regulates Fyn activity and C b p / P A G phosphorylation in thymocyte l ipid rafts. J Immunol. 175, 7947-56 Le H . T., Ponniah S., and Pallen C. J. (2004) Insulin signaling and glucose homeostasis in mice lacking protein tyrosine phosphatase alpha. Biochem Biophys Res Commun. 314, 321-9 Zeng L . , S i X . , Y u W . P., Le H . T., N g K . P., Teng R. M . , Ryan K . , Wang D . Z . , Ponniah S., and Pallen C. J. (2003) PTP alpha regulates integrin-stimulated F A K autophosphorylation and cytoskeletal rearrangement in cell spreading and migration. J Ce l l B i o l . 160, 137-46 x iv ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisor, D r Catherine J. Pallen for being an excellent supervisor. Her understanding, encouragement, patience, and support have provided me with an opportunity to complete my present thesis. I am deeply grateful to the chair o f my supervisory committee, Dr. E d Pryzdial, and my committee members Drs. Christopher Mcintosh and Poul Sorensen, for their knowledge and for their effort in reading my thesis and providing me with valuable comments. I also wish to thank all members in the lab (in both Canada and Singapore) for their assistance and collaboration. xv 1 INTRODUCTION 1.1 Protein tyrosine phosphorylation 1.1.1 A general introduction to protein tyrosine phosphorylation Protein tyrosine phosphorylation is a fundamental post-translational modification. Such phosphorylation can affect the target protein's conformation, interaction with other proteins, cellular localization, stability, and enzymatic activity. Tyrosine phosphorylation is involved in a wide variety of cellular processes in normal physiology, including cell growth (number and size), differentiation, metabolism, cel l -cel l communication, cell migration, ion channel activity, the immune response, and apoptosis/survival decisions (Hunter, 1998). Reversible protein tyrosine phosphorylation is controlled by the combined actions of two different classes of enzymes, protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Largely due to historical reasons, much more has been learned about P T K s than PTPs even though these two classes of enzymes equally contribute to the regulation o f protein tyrosine phosphorylation. Notably, similar to P T K s , deregulation o f PTP expression or activity can result in alterations of the cellular processes that underlie human diseases ( L i and Dixon, 2000). The first P T P was purified in 1988 (Tonks et al., 1988). To date, 107 P T P genes have been identified in the human genome (Alonso et al., 2004). 1.1.2 Protein tyrosine phosphatase (PTP) superfamily The P T P superfamily contains over 100 members, all possessing a conserved catalytic 1 motif C(X)sR, also known as the PTP signature motif (Fig. 1.1). Protein Tyrosine Phosphatase Superfamily 'Classical' PTPs DSPs cdc25 LMW PTPs PTP1B TCPTP L A R PTPa CD45 PTP|a PTP5 PTPs P T P K PTPa PJPX PTPH1 MEG1 Receptor-type PTPs V TI TT cdc2: T PRL1 MCE1 MPK1 MPK2 MPK3 LMW-PTP PTEN PRL2 PRL3 Intracellular PTPs P T P domain o F E R M domain MAM domain — P T P signature motif o P D Z binding domain C2 domain o E R anchor o F N Ill-like repeat • CH2 domain • SH2 domain Ig-like domain HI farnesylation site P E S T domain • extensive glycosylation / \ guanylyltransferase domain Figure 1.1. Schematic structures and subgroups of representative P T P s . PTP, protein tyrosine phosphatase; DSP , dual specific phosphatase; L M W , low molecular weight; E R , endoplasmic reticulum; SH2, src homology 2; PEST , polypeptide sequences enriched in proline (P), glutamate (E), serine (S), and threonine (T); F E R M , four-point one, ezrin, radixin, moesin; P D Z , PSD-95, Dig , and ZO-1 homology; F N , fibronectin; Ig, immunoglobulin; M A M , meprin- A5-mu; C H 2 , cdc25 homology. Modified from Wang et al. (2003). 2 These phosphatases are grouped into four subfamilies based on their structure and substrate specificity: the classical PTPs, the dual specific phosphatases (DSPs), the cdc25 phosphatases, and the low molecular weight ( L M W ) PTPs (Wang et al., 2003; Alonso et al., 2004). The DSPs are the most structurally and functionally diverse class o f PTPs. Many of the DSPs can remove phosphate groups from both tyrosine (Tyr) and serine/threonine (Ser/Thr) residues. The cdc25 (A, B , and C) phosphatases activate cyclin-dependent kinases by dephosphorylating inhibitory Thr l4 and Tyr 15 residues, indicating a role for these phosphatases i n cell cycle progression (Draetta and Eckstein, 1997). Although cdc25 phosphatases display dual specificity, they are classified separately because o f their distant relation to other PTPs in structure (Wang et al., 2003). The L M W PTPs are -18 k D proteins and are implicated in regulation of certain growth factor receptors such as the platelet-derived growth factor receptor (PDGFR) , the insulin receptor, and the ephrin receptor (Raugei et al., 2002). The active motif in the L M W PTPs is located near the N-terminus, whereas in all other PTPs it is found near the C-terminal end of the catalytic domain (Wang et al., 2003). The Tyr-specific P T P subfamily, termed the 'classical' PTPs, is comprised of - 40 members and is further divided into intracellular/nonreceptor PTPs (NRPTPs) and transmembrane/receptor-type PTPs (RPTPs) (Paul and Lombroso, 2003). The N R P T P s , such as P T P 1 B or SHP-1/2, reside in a variety of intracellular compartments including the cytosol, the plasma membrane, and the endoplasmic reticulum. They possess a single catalytic domain and various N - and C-terminal extensions. These extensions are usually important for N R P T P localization, protein-protein interactions, or substrate targeting (Feng, 1999; Tonks, 2003). The RPTPs contain an extracellular domain, a transmembrane domain, and one or two cytoplasmic P T P domains. The majority of RPTPs contain two catalytic domains and most, i f 3 not all , o f the phosphatase activity resides in the membrane proximal P T P domain ( D l ) . The function of the membrane distal domain (D2) is unclear, but it is speculated to play a regulatory role such as substrate targeting (Andersen et al., 2001). The extracellular motifs of the RPTPs include domains resembling immunoglobulin repeats, fibronectin type-Ill repeats, carbonic anhydrase-like, M A M (meprin-A5-mu) domains or cysteine-rich regions, suggesting a role for these domains in cell-cell or cell-matrix adhesion (Brady-Kalnay and Tonks, 1995; Beltran and Bixby, 2003). 1.2 Protein tyrosine phosphatase alpha (PTPa) 1.2.1 Structure of PTPa P T P a is a ubiquitously expressed transmembrane P T P (Fig 1.2) that is enriched in brain (Kaplan et al., 1990; Krueger et al., 1990; Matthews et al., 1990; Sap et al., 1990). Unlike other RPTPs , P T P a and the closely related P T P e lack cell adhesion motifs in the extracellular domain. Instead, P T P a and P T P e each have a remarkably short extracellular domain containing 123 residues or 27 residues, respectively. The extracellular region o f P T P a contains 8 putative sites for N-l inked glycosylation and many serine and threonine side chains for O-linked glycosylation (Daum et al., 1994). Extensive N - and O- glycosylation creates a mature 130 k D P T P a . N-glycosylation alone gives rise to a 100 k D protein, a precursor of the 130 k D P T P a . Unglycosylated P T P a is a -85 k D protein. L ike most other RPTPs , P T P a has two catalytic domains and the membrane proximal domain ( D l ) is responsible for the majority of the catalytic activity in vitro and all the activity in vivo (Wang and Pallen, 1991; den Hertog et al., 1993). Unlike the membrane distal 4 sre Figure 1.2. PTPa structure and regulatory features. P T P a c o n t a i n s a h i g h l y g l y c o s y l a t e d ( i n r e d ) e x t r a c e l l u l a r d o m a i n a n d t w o i n t r a c e l l u l a r c a t a l y t i c d o m a i n s ( D l a n d D 2 ) . A m i n o a c i d s t h a t a r e k n o w n t o u n d e r g o p h o s p h o r y l a t i o n ( P i n r e d c i r c l e ) a r e n u m b e r e d . R e g i o n s o r s i t e s w i t h i n P T P a t h a t c a n i n t e r a c t w i t h o t h e r p r o t e i n s a r e s h o w n ( a r r o w s ) . P S D - 9 5 , p o s t s y n a p t i c d e n s i t y - 9 5 ; N C A M , n e u r a l c e l l a d h e s i o n m o l e c u l e . ( D 2 ) d o m a i n s o f o t h e r R P T P s , P T P a - D 2 a l s o p o s s e s s e s s o m e in vitro c a t a l y t i c a c t i v i t y t o w a r d s t h e l o w m o l e c u l a r w e i g h t s y n t h e t i c s u b s t r a t e / ? a r a - n i t r o p h e n y l p h o s p h a t e ( p N P P ) ( a l t h o u g h t h i s i s s t i l l l o w r e l a t i v e t o t h a t o f P T P a - D l ) , b u t h a s n e g l i g i b l e a c t i v i t y t o w a r d s t h e p h o s p h o t y r o s i n e p e p t i d e s u b s t r a t e R R - s r c ( W a n g a n d P a l l e n , 1 9 9 1 ; L i m e t a l . , 1 9 9 7 ; W u e t a l . , 1 9 9 7 ) . T h e b a s i s o f t h e d i f f e r e n c e b e t w e e n P T P a - D l a n d - D 2 c a t a l y t i c a c t i v i t i e s t o w a r d s 5 the simple phenyl ring-based substrate pNPP lies in just two amino acids, valine 536 and glutamate 671 (L im et al., 1998; Buist et al., 1999). Mutation o f these two residues alters PTPa-D2 to an enzyme with catalytic activity towards pNPP comparable to that of P T P a - D l , however the mutations do not restore activity towards the more complex substrate RR-src (L im et al., 1998). This suggests that P T P a - D l and PTPa-D2 are distinct in substrate recognition/specificity, a difference that is evident with higher order peptide or protein substrates. The fact that PTPa-D2 has low intrinsic activity and that no physiological substrate of PTPa-D2 has been found so far suggests a regulatory role for PTPa-D2 rather than a catalytic one. PTPa-D2 may be important for protein-protein interactions that could bring P T P a - D l in close proximity to its substrates. In support o f this, PTPa-D2 has been reported to bind to the PDZ-2 domain of PSD-95 (postsynaptic density-95) (Lei et al., 2002) and to NCAJVI (neuronal cell adhesion molecule) (Bodrikov et al., 2005). Two cysteine residues (Cys433 in D l and Cys733 in D2) are essential for the enzymatic activity of P T P a (L im et al., 1997). In addition, three regulatory phosphorylation sites, Serl 80/204 in the juxtamembrane region and Tyr789 in the C-terminal tail, appear important for P T P a catalytic activity and substrate recognition. This is discussed further in section 1.2.2.2. 1.2.2 R e g u l a t i o n o f P T P a 1.2.2.1 D i m e r i z a t i o n Based on crystal structure and cell surface cross-linking data, P T P a - D l is capable o f forming a dimer via the interaction of a helix-turn-helix (at the N-terminal region of the P T P a - D l ) of one monomer with the catalytic cleft of another (Bilwes et al., 1996). Dimer formation is predicted to inhibit PTPa activity by interfering with substrate binding (Bilwes 6 et al., 1996; Majeti et al., 1998; Jiang et al., 2000). PTPa appears to constitutively form dimers on the cell surface (Jiang et al., 2000; Tertoolen et al., 2001). Overexpression of PTPa allows detection of homodimers by FRET (fluorescence resonance energy transfer) and on gel electrophoresis after chemical cross-linking (Jiang et al., 2000; Tertoolen et al., 2001; van der Wijk et al., 2004). However, dimers are not successfully detected in co-immunoprecipitation assays when using differently tagged forms of PTPa (Blanchetot and den Hertog, 2000), suggesting that PTPa dimers may not be stable. Oxidation upon H2O2, U V , or heat shock treatment rapidly inactivates the catalytic activity of P T P a - D l and induces conformational changes in PTPa-D2, thereby leading to a more stable PTPa dimer (Blanchetot et al., 2002; van der Wijk et al., 2004) (Fig 1.3). Nevertheless, it is unclear whether P T P a forms dimers in cells expressing a physiological level of PTPa , since all the above studies were carried out in cells overexpressing PTPa . Figure 1.3. PTPa dimerization. PTPa can be present as a dimer at the membrane. Oxidation of the D l and D2 catalytic sites can cause a conformational change and stabilize the dimer. 7 1.2.2.2 Phosphorylation About 20% of P T P a in NTH3T3 mouse fibroblasts is constitutively phosphorylated on Tyr789, a site located five residues from the C-terminus of P T P a (den Hertog et al., 1994). Tyr789 is phosphorylated by src as co-expression with src increases this phosphorylation (den Hertog et al., 1994). However, a recent study indicates that P T P a Tyr789 phosphorylation is regulated predominantly by a src/fyn/yes-independent tyrosine kinase (Hao et al., 2006). P T P a is also reported to autodephosphorylate itself at Tyr789 in vitro (den Hertog et al., 1994). Conflicting results arise when studying the effect of phosphorylation of Tyr789 on the intrinsic phosphatase activity of P T P a . Some groups report that this phosphorylation has no effect on the intrinsic activity of P T P a (Su et al., 1996; Zheng et al., 2000) while another group has observed a reduction in P T P a activity upon phosphorylation (den Hertog et al., 1994). Grb2 binds to phospho-Tyr789 o f P T P a v ia its SH2 (src homology 2) domain (den Hertog et al., 1994; Su et al., 1994). However the Grb2 binding partner, Sos, has not been found in the PTPa -Grb2 complex. It is possible that P T P a negatively regulates a Grb2-mediated pathway by competitively affecting Grb2 binding to other proteins (den Hertog et al., 1994; den Hertog and Hunter, 1996; Su et al., 1996). Grb2 binding protects the phospho-Tyr789 of P T P a from dephosphorylation and also blocks other SH2 containing proteins from associating with P T P a . Src-SH2 can also bind to phospho-Tyr789, but, its affinity is ~3-fold lower than that of Grb2-SH2 (Zheng et al., 2000). Thus, virtually all tyrosine phosphorylated P T P a is found in a complex with Grb2 (den Hertog et al., 1994; Su et al., 1994). In some situations, src activation is dependent on phospho-Tyr789 of P T P a , as P T P a phosphorylation at this site is required for transient PTPa-src association prior to P T P a action on src in mitosis (Zheng et al., 2000; Zheng and Shalloway, 2001). On the other hand, 8 this phospho-PTPa-dependent 'displacement model' (Zheng et al., 2000) of PTPa-mediated sre activation is clearly not required in other situations as a mutant form of P T P a lacking Tyr789 residue (Y789F PTPa), when expressed in P C 12 neuronal cells or embryonic fibroblasts, is capable o f activating sre (Yang et al., 2002; Chen et al., 2006). Thus, the role o f P T P a Tyr789 phosphorylation varies among different signaling pathways. This is discussed in more detail in section 1.2.4. It has been previously reported that PKC-catalyzed phosphorylation of P T P a (den Hertog et al., 1995; Tracy et al., 1995) on Serl80 and Ser204 in the juxtamembrane region increases the catalytic activity of P T P a by ~1.5- to 2-fold (Stetak et al., 2001; Zheng and Shalloway, 2001; Zheng et al., 2002). P K C 8 appears to be the only P K C isoform that is physically and functionally associated with PTPa (Stetak et al., 2001). Thus, P T P a may act as an intermediate effector in PKC-mediated sre activation as observed in smooth muscle cells (Brandt et al., 2003). It has been proposed that during mitosis, phosphorylation on these serine residues lead to conformational changes, resulting reduced Grb2-PTPa association, and consequently, in increased src-PTPa binding (Zheng and Shalloway, 2002). This may enable P T P a to activate sre. This is discussed in more detail in section 1.2.4.1. 1.2.2.3 Proteolysis The 130 k D form of P T P a can be cleaved by calpain at a site within the intracellular juxtamembrane region of P T P a to produce a 66 k D form (Tracy et al., 1995). The 66 k D form lacks an extracellular and transmembrane domain, suggesting that it has a cytoplasmic localization. In reality, it is found in both membrane and cytoplasmic cell fractions, indicating that some 66 k D proteins may associate with other membrane proteins to gain a membrane localization (Tracy et al., 1995). Notably, the proportion of the 66 k D form is 9 minimal relative to total PTPa . Further studies reveal the importance of membrane localization for its interaction with membrane substrates as truncated P T P a is unable to dephosphorylate src Tyr527 (Tracy et al., 1995) and has a very limited ability to dephosphorylate the Kv2.1 potassium channel (Gil-Henn et al., 2001). Treatment of NIH3T3 mouse embryonic fibroblasts expressing catalytically inactive P T P a (Cys433Ser) with pervanadate, a nonspecific inhibitor of PTPs, results in partial proteolysis o f P T P a at an unidentified site creating a 75 k D form of PTPa . This 75 k D form is no longer present at focal adhesions (Lammers et al., 2000). P T P a has been implicated in integrin signaling (see section 1.2.4.2 for detail), thus localization to focal adhesions may be required for its action in this particular event. These studies suggest that P T P a action on its substrates and cellular events is dependent on the proper localization of PTPa . 1 . 2 . 3 S u b s t r a t e s o f P T P a 1 . 2 . 3 . 1 S r c f a m i l y k i n a s e ( S F K s ) S F K s comprise a group of nine intracellular protein tyrosine kinases including src, fyn, yes, fgr, lck, hck, blk, lyn, and yrk (Toyoshima et al., 1992; Courtneidge et al., 1993), which are essential for multiple cellular events such as cell growth, proliferation, differentiation, and migration (Thomas and Brugge, 1997; Bjorge et al., 2000; Irby and Yeatman, 2000; Frame, 2002). Src, fyn, and yes are ubiquitously expressed whereas the others have restricted localizations in various tissues such as brain and hematopoietic cells (Thomas and Brugge, 1997). In the case of fyn, a form called fynT that is distinct from the widely expressed form o f fyn (sometimes termed fynB due to its high expression in brain) can be generated by alternative splicing of exon 7 (Cooke and Perlmutter, 1989). FynT is 10 uniquely expressed in T lymphocytes (Appleby et al., 1992; Stein et al., 1992). SFKs have similar domain structures and contain a myristoylated/palmitoylated N-terminus, unique domain, SH3 domain, SH2 domain, kinase domain, and C-terminal tail (Brown and Cooper, 1996) (Fig. 1.4). Figure 1.4. Structure and regulation of S F K s . SFKs contain unique N-terminal sequences (that enable interaction with and docking at the inner leaflet of the plasma membrane), SH3, SH2, tyrosine kinase domains, and C-terminal regions (that play an important regulatory role). A) In the inactive conformation, the SH2 domain binds to the phosphorylated Tyr527 residue in the C-terminal tail, and the SH3 domain binds to the linker region located between the SH2 and the kinase domain. B) Activation of S F K s is mediated by dephosphorylation of the regulatory Tyr527 by PTPs (such as PTPa) or interaction with ligands (other proteins) via the SH2 and/or SH3 domains (red arrows), that disrupt intramolecular constraints and permit an 'open' activated conformation. Active S F K s undergo autophosphorylation of Tyr416 in the kinase domain, resulting in a stable, fully activated kinase. 11 The activity of S F K s is regulated by several mechanisms, including phosphorylation/dephosphorylation and protein-protein interactions. In the unstimulated state, S F K s exist in a closed conformation in which the SH3 domain interacts with the linker region between the SH2 domain and the kinase domain, and the SH2 domain interacts with the microsequence encompassing the phosphorylated tyrosine residue in the C-terminal tail (Sicheri and Kuriyan, 1997; Martin, 2001). To open up this conformation and become activated, S F K s have to either be dephosphorylated at the regulatory tyrosine (Tyr527) in the C-terminal tail or interact via their SH3 or SH2 domain with other proteins so as to abolish intramolecular inhibitory constraints. Following this, S F K s cz's-autophosphorylate at a tyrosine residue (Tyr416) in the catalytic domain to become fully active (Thomas and Brugge, 1997; Bjorge et al., 2000). Csk, a ubiquitously expressed cytosolic kinase (Okada et al., 1991), negatively regulates S F K s by phosphorylating the C-terminal regulatory tyrosine residue. PTPs, including P T P 1 B , SHP-1/2, CD45, PTPs, and PTPa , counteract the action of Csk to positively regulate S F K activity (Roskoski, 2005). The best-characterized positive regulator of S F K s is P T P a (Pallen, 2003). Various lines of evidence indicate the involvement of P T P a in S F K activation and SFK-dependent processes. Stable expression of P T P a in embryonic rat fibroblasts induces transformation as a result of dephosphorylation of Tyr527 in src and thereby its activation (Zheng et al., 1992). Similarly, expression of P T P a in embryonal carcinoma P19 cells promotes src-mediated neuronal differentiation (den Hertog et al., 1993). Activation of src in these cells is not accompanied by enhanced phosphorylation of src at Tyr416, indicating that activation o f src by P T P a is not always associated with an increase in autophosphorylation of src or that P T P a rapidly dephosphorylates this site as soon as it is phosphorylated. P T P a is 12 found to physically and functionally associate with fyn in brain and in PTPa-transfected cells, and to associate with sre and yes in PTPa-expressing A431 cells (Bhandari et al., 1998; Harder et al., 1998). Transfected P T P a is able to dephosphorylate endogenous fyn, sre, and yes, but not lyn, in A431 cells (Harder et al., 1998). Most importantly, sre and fyn in brain and embryonic fibroblasts from mice lacking P T P a exhibit a reduced activity to 30-50% of that in wild-type animals. This is in accord with an observed enhanced phosphorylation o f sre and fyn at Tyr527 (Ponniah et al., 1999; Su et al., 1999). Unlike brain and fibroblast sre and fyn, fynT of PTPa" 7" thymocytes is hyperphosphorylated at both tyrosine 527 and 416, suggesting that P T P a can act as either a positive or negative regulator of fynT (Maksumova et al., 2005). 1.2.3.2 Kvl.2 p o t a s s i u m c h a n n e l Signaling through the m l muscarinic acetylcholine receptor, a G protein-coupled receptor (GPCR) , promotes cellular kinase activity, leading to tyrosine phosphorylation and suppression of K v l . 2 potassium channel activity (Huang et al., 1993). Suppression of the K v l . 2 channel is involved in numerous events including m l receptor-mediated P K C -dependent activation of the epidermal growth factor (EGF) receptor, P K C - and C a 2 + -mediated Pyk2 activation and binding o f Pyk2 to sre and Grb2, and R h o A activity (Tsai et al., 1997; Cachero et al., 1998; Felsch et al., 1998). Phorbol 12-myristate 13-acetate ( P M A ) -induced activation of P K C also results in tyrosine phosphorylation and suppression of overexpressed K v l . 2 channel activity. Subsequent treatment with carbachol, an m l receptor stimulant, does not suppress channel activity further (Huang et al., 1993). Interestingly, in cells overexpressing PTPa , subsequent treatment with carbachol partially relieves P M A -induced suppression. This occurs together with an increase in the association o f P T P a with 13 K v l . 2 and tyrosine dephosphorylation of K v l . 2 (Tsai et al., 1999), suggesting that m l receptor stimulation initiates dephosphorylation of the K v l . 2 channel by P T P a in a direct and/or indirect manner. In a different system involving another G P C R , the serotonergic 5-HT2C receptor, 5 hydroxytryptamine (5-HT)-induced activation of 5-HT2C receptor in oocytes expressing 5-HT2C and either K v l . l or K v l . 2 leads to inhibition of these channel currents in a tyrosine phosphorylation dependent manner. Coexpression of P T P a slows down the time course of this inhibition (Imbrici et al., 2000). 1.2.3.3 pl30C a s A substrate trapping mutant of P T P a - D l (Cys433Ser), but not of PTP1B, is able to bind tyrosine phosphorylated p l 3 0 C a s (Buist et a l , 2000). In addition, tyrosine phosphorylation of p l 3 0 C a s , but not of the E G F receptor, is decreased when S K - N - M C neuroepithelioma cells are co-transfected with PTPa , suggesting that p l 3 0 C a s is a specific cellular substrate o f P T P a (Buist et al., 2000). 1.2.4 PTPa and cellular processes 1.2.4.1 Mitosis Src is transiently activated during mitosis and suppressed during interphase (Chackalaparampil and Shalloway, 1988). Activation of src during mitosis is associated with decreased Tyr527 phosphorylation (Bagrodia et a l , 1991), suggesting that PTPs including P T P a may be involved in src-mediated mitosis. It is now apparent that P T P a uniquely regulates this event as PTPa-nul l fibroblasts do not exhibit detectable src activation in mitosis (Zheng et al., 2002). During mitosis, P T P a is phosphorylated at Serl 80/204 in the 14 juxtamembrane region by P K C , resulting in enhanced PTPa activity and in decreased affinity of Grb2 for the phosphorylated Tyr789 residue in the PTPa tail (Zheng and Shalloway, 2001; Zheng et al., 2002) (Fig. 1.5). This permits the src-SH2 domain to bind to newly available phospho-Tyr789 of PTPa . Inactivated src exists in a closed conformation partly due to the intramolecular interaction between src-SH2 and phospho-Tyr527 in the src C-terminal tail (Sicheri and Kuriyan, 1997). However, phospho-Tyr789 of P T P a has a higher binding affinity for src-SH2 than does phospho-Tyr527 of src (Zheng et al., 2000), leading to the release of the src C-terminal tail. Phospho-Tyr527 of src is thereby exposed to and dephosphorylated by the src-associated PTPa (Zheng et al., 2002). Grb-2 Figure 1.5. Mechanism of src activation by P T P a serine phosphorylation dur ing mitosis. A) Src is inactive due to intramolecular interactions as depicted in Fig 1.4A and PTPa is complexed with the adaptor protein Grb2 via the interaction of the Grb2 SH2 domain with the phosphorylated (Y789-P) tail of PTPa. B) The PKC5-catalyzed phosphorylation of PTPa on two juxtamembrane serine residues (SI80 and S204) reduces the affinity of Grb2 for PTPa , allowing the src SH2 domain to bind to PTPa phospho-Tyr789 and thus the displaced phospho-Tyr527 site of src is exposed to and can be dephosphorylated by P T P a - D l . 15 1.2.4.2 Integrin signaling Integrin signaling is critical for multiple cellular events including cell growth, migration, and survival. Binding of extracellular matrix components to the integrin transmembrane receptors is accompanied by tyrosine phosphorylation of a series of molecules including focal adhesion kinase ( F A K ) . Autophosphorylation of F A K at Tyr397 creates a docking site for src and fyn via their SH2 domains. S F K - S H 2 binding to phospho-Tyr397 of F A K leads to the disruption of S F K intramolecular inhibitory constraint to promote S F K activation. Activated src and fyn, in turn, phosphorylate F A K at other tyrosine sites to promote full activation of F A K and FAK-associated proteins such as p l 3 0 C a s and paxill in, eventually resulting in focal adhesion formation and disassembly, actin stress fiber and cytoskeletal organization, and dynamic alterations in cell shape (Schlaepfer et al., 1999; Parsons et al., 2000; Schaller, 2001). A role for PTPa in integrin signaling has been revealed by studies of both PTPa overexpressing and deficient cells. A431 human epithelial carcinoma cells overexpressing PTPa exhibit an increase in src activation, F A K - s r c interaction, and tyrosine phosphorylation of the F A K / s r c substrate paxillin. This eventually leads to enhanced cell substratum attachment and resistance to EGF-induced cell rounding (Harder et al., 1998). In contrast, fibroblasts lacking PTPa exhibit defects in spreading, migration, and haptotaxis. This is in accord with reduced src and fyn activity, reduced tyrosine phosphorylation of F A K and p l 3 0 C a s , and diminished Erk activation upon integrin stimulation (Su et al., 1999; Zeng et al., 2003). Similar to PTPa_/" cells, cells treated with an S F K inhibitor (Salazar and Rozengurt, 2001) and cells lacking the three SFKs src, fyn, and yes (Klinghoffer et al., 1999) exhibit inhibited integrin-induced F A K tyrosine phosphorylation. Together, these findings indicate 16 the involvement of P T P a in promoting integrin signaling via its upstream actions on sre, fyn, and possibly yes. In support of this, P T P a is found physically associated with av/p3 subunits of integrins in cells spreading on the extracellular matrix proteins fibronectin and vitronectin (von Wichert et al., 2003). Furthermore, integrin-stimulated tyrosine phosphorylation of P T P a is essential for cytoskeletal reorganization and cell migration but is not required for early events such as activation of sre and F A K (Chen et al., 2006). It is possible that P T P a Tyr789 phosphorylation is required for P T P a to localize to focal adhesions (Lammers et al., 2000). 1.2.4.3 Neuronal differentiation and outgrowth P T P a expression is elevated during neuronal differentiation of embryonal carcinoma and neuroblastoma cells (den Hertog et al., 1993). Retinoic acid promotes P19 embryonal carcinoma cells to differentiate into endoderm/mesoderm while it induces cells with stably expressed P T P a to differentiate into neurons. Elevated P T P a expression in these cells is associated with enhanced sre activity as a result of reduced phosphorylation of sre at its regulatory Tyr527 site (den Hertog et al., 1993). This indicates that P T P a induces neuronal differentiation in a sre-dependent manner. Epidermal growth factor (EGF) alone is unable to mediate neurite outgrowth of P C 12 cells. However, overexpression of P T P a results in EGF-induced neurite outgrowth. This is most likely due to the ability o f P T P a to activate sre, resulting in tyrosine phosphorylation of the sre effectors Sin and p l 3 0 C a s , and their association with the adaptor proteins C r k L , C r k l l , and Nek that subsequently modulate downstream molecules (Yang et al., 2002). In contrast to E G F , fibroblast growth factor (FGF) alone can induce P C 12 neurite outgrowth. P T P a expression dramatically inhibits F G F action (Su et al., 1994; Yang et al., 2002), and this may 17 be due to the PTPa-mediated inhibition of Erk activation (Yang et al., 2002). This suggests that the effect of P T P a on F G F signaling diverges from its effect on the E G F response. Additionally, tyrosine phosphorylation of P T P a is not required for its inhibitory action, but EGF/PTPa-induced neuritogenesis is increased when Tyr789 is mutated (Yang et al., 2002). Neural cell adhesion molecules including N C A M and contactin are important for numerous morphogenetic events including neuronal migration and neurite outgrowth (Kiryushko et al., 2004). Fyn is present in a complex with contactin and N C A M and is activated by an unknown mechanism upon aggregation of these adhesion molecules (Zisch et al., 1995; Beggs et al., 1997). P T P a associates in cis with contactin through the extracellular region of P T P a , suggesting that P T P a l ikely acts as the transducing component of the receptor complex to link contactin to intracellular signaling molecules (including fyn) in response to contactin ligand binding (Zeng et al., 1999). Additionally, P T P a appears to interact with N C A M . P T P a binding links N C A M to fyn both physically and functionally, as mice lacking P T P a exhibit reduced N C A M and fyn association and abrogation of N C A M -induced neurite outgrowth (Bodrikov et al., 2005). These lines of evidence suggest that P T P a is involved in neuronal differentiation and neurite outgrowth, partly through activating src and fyn and/or interacting with neural adhesion molecules to couple these with downstream effectors. 1.2.4.4 T-cel l signaling Tyrosine phosphorylation is key to T-cell receptor (TCR) signaling and consequently to TCR-stimulated cell proliferation (Hermiston et al., 2002). T C R engagement induces activation of the S F K s fyn and lck, leading to phosphorylation of T C R / C D 3 subunits and formation of multicomponent complexes, to eventually activate several T-cell signaling 18 pathways. Thymocytes lacking P T P a develop normally but unstimulated PTPa"" cells exhibit enhanced tyrosine phosphorylation of specific proteins (Maksumova et al., 2005). Unlike the reduced fyn activity observed in P T P a 7 " fibroblasts and brain (Ponniah et al., 1999; Su et al., 1999) , fynT activity in unstimulated P T P a 7 " thymocytes is enhanced as a result of hyperphosphorylation of both tyrosine 528 and 417 (equivalent residues to sre Tyr527 and Tyr416) (Maksumova et al., 2005). This indicates that P T P a can both positively and negatively regulate fynT and that in P T P a 7 " thymocytes, enhanced Tyr417 phosphorylation probably predominates, to affect fynT activation. This suggests an important role for P T P a in controlling fyn activity prior to T C R activation. 1.2.4.5 Cancer P T P a m R N A was reported to be overexpressed in 70% (10 out of 14) of late stage colon tumors (Tahiti et al., 1995). In addition, enhanced P T P a protein and m R N A levels were found in 29% (15 out of 51) of primary breast carcinomas (Ardini et al., 2000). The causes and effects of this increased P T P a expression are not known. Although enhanced activation o f sre is documented in a number of human cancers including colon and breast cancers (Jacobs and Rubsamen, 1983; Bolen et al., 1987; Cartwright et al., 1989; Ottenhoff-K a l f f et al., 1992), a direct mechanistic link between elevated P T P a and sre activity in these tumors has not been demonstrated. Other studies indicate a potential tumor suppressive activity of P T P a (Ardini et al., 2000) . Overexpression of P T P a in breast cancers is associated with low tumor grade although also with positive estrogen receptor status. Expression of P T P a in M C F - 7 breast cancer cells activates sre and decreases cell growth. Furthermore, expression of P T P a in 19 mouse tumor cells transgenic for HER2/neu causes a reduction of tumor growth and delay in metastasis when the cells are injected into mice (Ardini et al., 2000). P T P a is also suggested to be a regulator of tumor cell growth inhibition and apoptosis (Stetak et al., 2001). The somatostatin analogue TT-232 inhibits proliferation and promotes apoptosis of many tumor cell lines (Keri et al., 1996). Due to its anti-tumorigenic and anti-metastasic effects, it is employed in the treatment of various cancer types. Stimulation of A431 cells with TT-232 leads to Erk activation, which is important for cell cycle arrest (Stetak et al., 2001). It is proposed that Erk activation is the result of a TT232-induced formation of a tricomplex of PI3-K, P K C S , and PTPa . In the complex, PI3-K-dependent activation of P K C 5 leads to serine phosphorylation and activation of PTPa , and eventually activation of sre. Activated sre transduces a signal leading to Erk activation. Stimulation of A431 cells with TT-232 also promotes the phosphorylation o f P T P a at Tyr789 (Stetak et al., 2001), most likely reflecting the activation of sre. The effect of P T P a on proliferation and transformation is cell-type dependent. P T P a overexpression in A431 carcinoma cells does not affect cell growth, but increases cell-substratum adhesion (Harder et al., 1998). Insulin receptor (IR)-expressing B H K (baby hamster kidney) cells exhibit insulin-induced growth inhibition and P T P a expression suppresses this inhibition (Moller et al., 1995). Overexpression of P T P a in rat embryo fibroblasts induces cell transformation (Zheng et al., 1992), which is associated with activation of Erk and c-Jun (Zheng and Pallen, 1994). Inducible expression of P T P a transforms NIH3T3 mouse embryo fibroblasts (Zheng et al., 2000). In contrast, Lammers et al. (2000) could not stably express wild-type P T P a in NIH3T3 cells, possibly because high levels o f the phosphatase are toxic. However, the stable expression of a catalytically active 20 P T P a mutant (Y781F PTPa) (less toxic than wild-type PTPa) does not induce focus formation in c-src expressing NIH3T3 cells, although the Y789F P T P a mutant does (Lammers et al., 2000). The above studies provide clear evidence that P T P a plays a role in tumorigenesis and metastasis in a cell-type dependent manner. However, it is unclear whether P T P a promotes or suppresses these phenomena. 1.3 PTPa and insulin signaling 1.3.1 Insulin Insulin (derived from the Latin word insula, "island") was discovered by Frederick Banting and Charles Best in 1921 (Banting and Best, 1990) and is a very well-studied polypeptide hormone. Insulin is produced by the p cells of the pancreatic islets o f Langerhans. It is initially synthesized as a long chain pro-insulin, then proteolytically cleaved to produce mature insulin and a connecting C peptide (Tager et a l , 1975). Mature insulin consists of a and p peptide chains linked by two disulfide bonds. It is secreted into the bloodstream in response to the elevation of glucose concentration after each meal. Insulin stimulates glucose uptake into cells of the body and the conversion o f glucose to glycogen in the liver (Saltiel and Kahn, 2001). Insulin initiates its action by binding to the insulin receptor (IR) on the cell surface (White and Kahn, 1994). A l l cell types are responsive to insulin. However, some tissues such as liver, muscle, and adipose are more sensitive to insulin. Glucagon has an opposing effect, causing a release, usually between meals, of glucose produced from glycogen breakdown (Unger, 1975). 21 1.3.2 The insulin receptor (IR) The IR belongs to a subfamily of ligand-activated receptor tyrosine kinases that includes insulin-like growth factor-1 receptor (IGF-1R) and FR-related receptor (J-RR) (Cheatham and Kahn, 1995; Patti and Kahn, 1998). The IR consists of two a and two p subunits, linked by disulfide bonds, that are derived from a single precursor by proteolytic cleavage (Massague et al., 1981; Kasuga et al., 1982). The 125-kD a subunits are located entirely outside the cell and contain the binding sites for insulin while the transmembrane 90-k D p subunits possess the kinase activity (Van Obberghen et al., 1981; Kasuga et al., 1982). In the basal state, the a subunits inhibit the kinase activity of the p subunits (Kahn, 1994). Insulin binding to the a subunits results in a conformational change, which enables the p subunit to bind A T P and undergo fra/w-autophosphorylation (Lee and Pilch, 1994; White and Kahn, 1994) on several tyrosine residues in the cytosolic region. These sites of phosphorylation include residues in the intracellular juxtamembrane region (Tyr960, a recognition site for IRS proteins and She), the kinase domain ( T y r l l 4 6 , T y r l l 5 0 , and T y r l l 5 1 ) , and the C-terminal region (Tyrl316 and Tyrl322) . Autophosphorylation in the kinase domain enhances IR activity 10-20 fold, enabling it to phosphorylate various substrates (White and Kahn, 1994). 1.3.3 Insulin signaling Activated IR binds to and phosphorylates several intermediate proteins including insulin receptor substrate (IRS) proteins and src-homology collagen (She), creating docking sites for effector proteins such as PI3-K, SHP-2, Grb2/Sos complex, Nek, and Crk, which in turn activate downstream signaling pathways (Saltiel and Kahn, 2001) (Fig. 1.6). 22 OUT P I 3 K / A k t pathway t S I G N A L S insulin © G r b 2 She Raf M E K rk1/2 - • Ras-MAPW pathway S I G N A L S glucose cell protein glycogen mitogen ic transport survival synthesis synthesis effects Figure 1.6. Insulin signaling. Insulin binding to its receptor (IR) stimulates IR autophosphorylation and tyrosine phosphorylation of the adaptor proteins insulin receptor substrates (IRSs) and She, which in turn interact with various SH2-containing proteins (such as PI3-K, Grb2, and SHP-2) to lead to activation of the R a s - M A P K pathway and the Ak t signaling pathway, with multiple effects. 23 Two major downstream pathways of insulin action are: i) the PI3-K/Akt pathway that regulates metabolic effects, including cell survival, glucose transport, and glycogen and protein synthesis and ii) the R a s / M A P K pathway that mainly regulates mitogenesis. Post-receptor defects in the insulin signaling pathway are associated with insulin resistance and subsequently non-insulin-dependent (Type 2) diabetes mellitus, a disease that accounts for 90% of diabetic patients (Saltiel and Kahn, 2001). Insulin receptor substrates (IRSs): Four IRS proteins have been identified. IRS-1 and IRS-2 are widely expressed, IRS-3 is present in mouse adipose tissues, and IRS-4 is found in thymus, brain, kidney, and possibly pancreatic P cells (Sesti et al., 2001; White, 2002). IRS proteins contain a highly conserved N-terminus, which includes a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, and a poorly conserved C-terminus. Unlike IRS-3 and IRS-4, IRS-1 and IRS-2 have a long C-terminal tail with approximately 20 potential tyrosine phosphorylation sites including six with a Y M X M motif that appears important for binding the SH2 domain o f PI3-K (Backer et al., 1992; Shoelson et al., 1992). Tyrosine phosphorylated IRS proteins bind to a variety of SH2-containing enzymes such as PI3-K, SHP-2, fyn, and adaptor proteins including Grb2, Nek, Crk, and others. IRS-1 and IRS-2 are intimately involved in carbohydrate metabolism but have tissue-specific roles in this, with IRS-1 being a primary effector of insulin action in skeletal muscle and IRS-2 primarily functioning in liver (Rother et al., 1998; Withers et al., 1998; K ido et al., 2000). Sre homology collagen (She): The adaptor protein She exists in three isoforms (46, 52, and 66 kD) that arise from alternative splicing o f a single transcript. A l l forms of She contain an 24 SH2 and a P T B domain (Kovacina and Roth, 1993). Both IRS proteins and She bind to phospho-Tyr960 of the IR (Kaburagi et al., 1995; Isakoff et al., 1996). The 52 k D isoform and, to a lesser extent, the 46 k D isoform are tyrosine phosphorylated and bind to the Grb2-Sos complex in response to insulin. Although IRSs also can bind Grb2, the She and Grb2 interaction is predominant upon insulin stimulation (Sasaoka and Kobayashi, 2000). Phosphoinositide 3-kinase (PI3-KV. The PI3-Ks are divided into three classes based on their in vitro l ipid substrate specificity, structure, and mechanism of regulation (Vanhaesebroeck et al., 1997). O f these, class l a PI3-K is the major effector of insulin signaling (Shepherd et al., 1998). Class l a PI3-K consists of an adaptor/regulatory subunit that contains an SH2 domain and a catalytic 110 k D subunit that exists in the three isoforms p i 10a, p l l O p , and p i 108. In the resting condition, the regulatory subunit maintains the catalytic subunit in a low activity state. Recruitment of the regulatory subunit of PI3-K to phosphorylated Y M X M motifs on IRSs leads to the activation o f the catalytic subunit (Myers et al., 1992). Activated PI3-K catalyzes the phosphorylation o f phosphoinositides on the 3 position of the inositol ring, leading to the production of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] and 3,4,5-trisphosphate [PI(3,4,5)P3]. PI(3,4,5)P3 binds to the P H domain of numerous signaling molecules thereby altering their activity or subcellular localization (Lietzke et al., 2000). Typically, PI(3,4,5)P3 recruits phosphoinositide-dependent kinase ( P D K ) 1 and 2, and protein kinase B (Ak t /PKB) to the plasma membrane. Once in proximity, P D K 1 and P D K 2 activate A k t / P K B by phosphorylating it on Thr308 and Ser473, respectively (Alessi and Cohen, 1998). Among three highly homologous isoforms of A k t / P K B , P K B p is the most critical intermediate in the regulation of insulin-mediated glucose homeostasis (Cho et al., 25 2001; Garofalo et al., 2003). Activated A k t / P K B translocates to the nucleus (Andjelkovic et al., 1997) and phosphorylates (among other substrates) glycogen synthase kinase-3 (GSK-3) , p70 ribosomal S6 kinase (p70 S6), forkhead transcription factors (FHTFs), and c A M P phosphodiesterase 3B, consequently leading to glucose metabolism, glycogen/lipid/protein synthesis, and cell survival (Hanada et al., 2004). The l ipid phosphatases P T E N and SHIP-2 have opposing effects to PI3-K as they respectively dephosphorylate the phosphate group at the 3 or the 5 position of the inositol ring, causing a decrease in PI(3,4,5)P3 levels (Maehama and Dixon, 1999; Clement et al., 2001). Adaptor protein growth factor receptor bound 2 (Grb2): Grb2 is composed of an SH3 domain lying between two SH2 domains. The SH3 domain constitutively associates with proline-rich regions in the guanine nucleotide exchange factor Sos. Grb2-SH2 binding to phosphorylated She and IRSs brings Sos to the plasma membrane (Skolnik et al., 1993). Here, Sos interacts with Ras and stimulates the formation of GTP-bound Ras. This activated form of Ras binds to the effector region of Raf, a serine kinase, and subsequently recruits Ra f to the membrane for further activation (Pronk and Bos, 1994) which in turn activates mitogen-extracellular signal-responsive kinase ( M E K ) by phosphorylation on two serine residues (Dent et al., 1992). M E K activates two isoforms of mitogen-activated protein kinase ( M A P K ) , Erk (extracellular signal related kinase) 1 and 2, by phosphorylation on Thr202 and Tyr204 (Crews and Erikson, 1992), leading to alterations in gene transcription to drive mitogenic responses (Nishida and Gotoh, 1993). 26 1.3.4 Insulin responses 1.3.4.1 Stimulation of glucose uptake Insulin stimulation results in the translocation o f glucose transporters (GLUTs) , mainly G L U T 4 , from intracellular pools to the plasma membrane with consequent increase in glucose transport into muscle and fat (Cushman and Wardzala, 1980; Suzuki and Kono, 1980; Slot et al., 1991). A key player that modulates glucose uptake into cells is PI3-K (Cheatham et al., 1994; Tsakiridis et al., 1995). However, PI3-K activation alone is not sufficient to stimulate glucose uptake as several growth factors activate PI3-K but fail to trigger glucose uptake (Isakoff et al., 1995; Wiese et al., 1995). Differential localization of activated PI3-K is produced in response to insulin and growth factors. While growth factors typically induce PI3-K localization to plasma membrane-situated growth factor receptors, insulin stimulation leads to increased content of PI3-K in intracellular compartments (Nave et al., 1996; Ricort et al., 1996) that contain G L U T 4 (Heller-Harrison et al., 1996). A k t / P K B and atypical P K C s are also implicated in insulin-stimulated glucose transport by acting downstream of PI3-K (Taha and K l i p , 1999). 1.3.4.2 Stimulation of glycogen synthesis Skeletal muscle is the major tissue that takes up most of the glucose in response to insulin. This glucose is deposited as glycogen through the action of glycogen synthase, an enzyme that is normally inhibited by phosphorylation at multiple sites by glycogen synthase kinase-3 (GSK-3) (Lawrence and Roach, 1997). Insulin causes phosphorylation and inactivation of G S K - 3 by PI3-K and its downstream targets A k t / P K B and mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70 S6 kinase), leading to glycogen synthesis (Cross et al., 1994; Welsh et a l , 1994; Shepherd et al., 1995; Uek i et al., 1998). Furthermore, 27 insulin also causes activation of the glycogen-bound form of protein phosphatase 1 (PP1G), by p90 ribosomal S6 kinase 2 (p90 rsk2), a downstream target of M A P K (Dent et al., 1990). P P 1 G is thought to enhance glycogen synthesis by dephosphorylating glycogen synthase, however, its precise contributions to insulin-stimulated glycogen synthesis are unclear as blocking the activation of M A P K and p90 rsk2 does not inhibit insulin-stimulated activation o f glycogen synthase (Lazar et al., 1995; Azpiazu et al., 1996). 1.3.4.3 Stimulation of protein synthesis A n early step in protein synthesis requires eukaryotic initiation factor (eIF)-4E binding to the m R N A cap structure to permit transcript translation. In the unstimulated state, 4E-BP1 (4E binding protein 1) binding prevents eIF-4E from recognizing the m R N A cap structure. Upon insulin stimulation, 4E-BP1 becomes phosphorylated and dissociates from eIF-4E, allowing translation to occur (Pause et al., 1994; L i n et al., 1995; Kimba l l et al., 1997). Insulin-induced phosphorylation of 4E-BP1 is dependent on PI3-K/Akt /mTOR signaling (Beretta et al., 1996; Mendez et al., 1996; Brunn et al., 1997; Gingras et al., 1998). Insulin stimulation also results in phosphorylation of eIF-4E itself by an additional unknown mechanism (Flynn and Proud, 1996). Phosphorylation of eIF-4E enhances its binding affinity to the m R N A cap structure (Minich et al., 1994). GTP-bound eIF-2 transfers the initiator M e t - t R N A to the 40S ribosomal subunits and this is required for initiation of translation (Proud and Denton, 1997). eIF-2B, a guanine nucleotide exchange factor for eIF2, is activated by insulin in a PI3-K/GSK-3-dependent manner (Welsh et al., 1997). Peptide elongation requires two elongation factors, eEF-1 and eEF-2 (Proud and Denton, 1997). Insulin-induced regulation o f eEF-1 (Chang and Traugh, 1997) and eEF-2 (Redpath et al., 28 1996) phosphorylation results in increased overall elongation rate. This effect of insulin appears to be through the P I3K/mTOR pathway (Redpath et al., 1996). 1.3.5 PTPs in insulin signaling A s insulin signaling is dependent on a series of tyrosine phosphorylation events, PTPs become potential regulators of insulin action. Early studies indicated that vanadate, a nonspecific inhibitor o f PTPs, acts as an insulin-mimetic agent (Brichard et al., 1992; Fantus et al., 1994; Carey et al., 1995). Several PTPs act as IR and/or IRS phosphatases in cell culture systems, including the receptors L A R , PTPa , PTPe, and CD45 , and the intracellular P T P 1 B , T C PTP , and SHP-2 (Cheng et a l , 2002; Asante-Appiah and Kennedy, 2003; Galic et al., 2005). However, heterologous P T P and/or substrate expression, possibly inappropriate cell type, and the non-physiological cell culture setting, are all factors that limit the ability o f these studies to evaluate the physiological role of a particular P T P in insulin action. Several studies of mice with genetically modified P T P expression have provided additional insight into the roles of various PTPs in insulin signaling and metabolic effects. P T P 1 B : Studies in cultured cells indicate the involvement of P T P I B in attenuation of various growth factor signaling pathways, including those initiated by platelet-derived growth factor (PDGF) , epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin (Roome et al., 1988; Milarski et al., 1993; Ahmad et a l , 1995; Flint et al., 1997; Lee et al., 1998; Buckley et al., 2002). Despite this, PTP1B _ / " mice appear normal, suggesting that PTP I B function is compensated for by other PTPs, at least during development. However, P T P IB-deficient mice exhibit an increase in insulin sensitivity, with enhanced insulin-29 induced tyrosine phosphorylation of the IR in liver and muscle but not in adipose tissue (Elchebly et al., 1999; Klaman et al., 2000), indicating a tissue-specific effect of PTP1B. These mice also exhibit a resistance to diet-induced obesity that is associated with higher metabolic rate and energy expenditure (Klaman et al., 2000). In another animal-based approach, a strain of diabetic (ob/ob) mice treated with P T P I B antisense oligonucleotide to reduce P T P I B expression showed decreased hyperglycemia and hyperinsulinemia, as well as increased insulin sensitivity (Zinker et al., 2002). Insulin signaling pathways were activated in the livers of these antisense-treated ob/ob mice, including increased IR, IRS-1, and IRS-2 tyrosine phosphorylation (Zinker et al., 2002; Gum et al., 2003). These studies strongly support a physiological role for P T P I B as an IR and IRS phosphatase in liver and muscle, and as a negative regulator of insulin signaling. L A R : Overexpression of L A R in cultured cells results in decreased insulin-induced IR and IRS-1 tyrosine phosphorylation, indicating a negative role for L A R in insulin signaling (L i et al., 1996; Zhang et al., 1996). Furthermore, wild-type or catalytically inactive 'substrate-trapping' mutants of L A R are found to be associated with IR/IRS (Ahmad and Goldstein, 1997). In addition, a causal link between increased L A R and insulin resistance noted in obese subjects (Ahmad and Goldstein, 1995) is supported by the finding that transgenic mice with muscle-specific overexpression of L A R have whole body insulin resistance, probably due to IRS dephosphorylation (Zabolotny et al., 2001). However, although the LAR-deficient mice have lower fasting glucose and insulin levels, and lower fasting hepatic glucose production, they exhibit a resistance to insulin-induced glucose disposal and inhibition of hepatic glucose output in euglycemic clamp studies (Ren et al., 1998). Due to differing phenotypes of L A R -30 deficient mice, the roles of L A R in normal insulin signaling remain unclearly defined (Schaapveld et al., 1997; Yeo et al., 1997; Ren et al., 1998). SHP-2: Stable expression of SHP-2 in 3T3-L1 mouse fibroblasts leads to impaired IRS-1 tyrosine phosphorylation and PI3-K activation, thus resulting in defective glycogen synthesis in response to insulin (Ouwens et al., 2001). There are several reports of a direct association between SHP-2 and IR and IRSs (Kuhne et al., 1993; Staubs et al., 1994; Rocchi et al., 1996). Transgenic mice expressing a dominant negative mutant lacking the P T P domain of SHP-2 exhibit an insulin-resistant phenotype (Maegawa et al., 1999). However, SHP-2-null mice die during embryonic development, SHP-2 hemizygous mice do not exhibit any alterations in insulin signaling and metabolism (Arrandale et al., 1996). A t this point, the function of SHP-2 in insulin signaling is unclear although SHP-2 has been shown to play important roles in other growth factor signaling pathways such as P D G F R , E G F R , IGF-1R signaling cascades (Stein-Gerlach et al., 1998) and in systems such as limb (Saxton et al., 2000), lymphoid, and hematopoietic cell (Qu et al., 2001) development. PTPe: A s observed upon P T P a overexpression, the overexpression of PTPs down-regulates insulin signaling and blocks insulin-induced cell rounding and detachment in IR overexpressing B H K (baby hamster kidney) cells (Moller et al., 1995; Andersen et al., 2001). N o defects in insulin signaling have been reported in PTPe 7 " mice although the lack of PTPe in these mice influences the activation of K v potassium channels and affects Schwann cell function in early development (Peretz et al., 2000). 31 CD45: CD45 is capable of dephosphorylating the IR in vitro (Tonks, 1990). Furthermore, expression of CD45 is associated with decreased insulin signaling in a human multiple myeloma cell line (Kulas et al., 1996). However, CD45 is uniquely expressed in B and T cells and is crucial for thymocyte development and B cell maturation (Hurley et a l , 1993; Byth et al., 1996). This suggests that results from cultured cells are artificial and that CD45 is unlikely to be a physiological regulator o f IR tyrosine phosphorylation. P T P a : There are no reported studies of insulin action in PTPa -nul l mice, despite the many studies that have been conducted in cultured cells, with mixed results, to investigate P T P a as a potential regulator of insulin signaling. Expression o f P T P a in B H K cells expressing a high level of the IR ( B H K - I R ) counteracts the detachment and growth inhibition of these cells that is induced by insulin (Moller et al., 1995). In contrast, the expression of P T P 1 B , known to negatively regulate insulin signaling in mice, does not abrogate the insulin-induced effects on B H K - I R cells (Moller et al., 1995). It may be that in this system P T P a counteracts insulin-induced cell detachment through actions independent of insulin signaling that promote cell attachment, akin to the increased substrate adhesion induced by P T P a expression in A431 carcinoma cells (Harder et al., 1998). Tyrosine phosphorylation o f the highly expressed IR is reduced by P T P a expression in B H K - I R cells, although this is similarly reduced by expression of the receptor PTP CD45 that has little ability to rescue insulin-stimulated cell detachment and growth inhibition (Moller et al., 1995). Ce l l type and heterologous protein expression levels may influence the ability of P T P a to dephosphorylate the IR, since in H E K 2 9 3 and B H K - I R cells, co-expression of P T P a and the IR results in lower insulin-stimulated IR and She phosphorylation (Lammers et al., 1998), but in IR-expressing G H 4 32 pituitary cells transient or stable expression of P T P a has no effect on insulin-induced IR, ERS-1 or She tyrosine phosphorylation or LR kinase activity (Jacob et al., 1998). However in the latter cells, P T P a inhibits insulin-stimulated expression o f a prolactin reporter plasmid. This inhibition was recently found to be through P T P a action on src (Vul in et al., 2005). Rat adipose cells transiently expressing P T P a have lower cell surface amounts of the glucose transporter G L U T 4 compared to parent cells and correspondingly reduced insulin-stimulated translocation o f G L U T 4 to the cell surface (Cong et al., 1999). Antisense oligonucleotide-mediated reduction of P T P a to undetectable levels in 3T3-L1 adipocytes had no effect on LR and LRS tyrosine phosphorylation upon insulin stimulation or on dephosphorylation following insulin withdrawal. Insulin-stimulated Erk2 activation and D N A synthesis were also unaffected by P T P a depletion (Arnott et al., 1999). In contrast, and in accordance with the action of P T P a as an upstream activator of src (Pallen, 2003), P T P a depletion greatly reduced the activity of this tyrosine kinase. In L 6 myoblasts, insulin-stimulated D N A synthesis was enhanced in cells overexpressing P T P a and reduced in cells in which PTPa expression was decreased using antisense c D N A (Lu et al., 2002). Elevated P T P a expression does not influence insulin-induced LR (or IGF-1R) tyrosine phosphorylation (Lu et al., 2002). A recent study using bioluminescence resonance energy transfer in H E K 2 9 3 cells expressing LR and 'substrate trapping' mutant of PTPa revealed the physical interaction of these proteins in the absence of insulin (Lacasa et al., 2005). In summary, enhanced or ablated expression o f P T P a in cultured cells exerts negative, positive, or no effects on insulin-stimulated cellular responses, and is sometimes accompanied by alterations in insulin-responsive LR tyrosine phosphorylation. P T P a has thus continually been proposed, but certainly not confirmed, to be 33 a regulator of LR phosphorylation and insulin action (Elchebly et al., 2000; Cheng et al., 2002; Asante-Appiah and Kennedy, 2003). 1.4 P T P a and N-methyl-D-aspartate receptor (NMDAR) signaling 1.4.1 L-Glutamate Communication between neurons takes place at synapses, specialized sites of contact between the presynaptic terminal of a neuron and a postsynaptic neuron. The space between the presynaptic and postsynaptic neuron endings is called the synaptic cleft (De Robertis and Bennett, 1954; Palade and Palay, 1954). The synaptic cleft contains a variety o f molecules at very high densities, including neurotransmitters that contribute to neuronal communication. Neurotransmitters are divided into three categories: amino acids (L-glutamate, y-amino butyric acid ( G A B A ) , aspartate, glycine), peptides (vasopressin, somatostatin, neurotensin), and monoamines (norepinephrine, dopamine, and serotonin) plus acetylcholine. They are synthesized in the neurons, stored in the presynaptic terminal, and released into the synaptic cleft in a calcium-dependent manner to act on the postsynaptic neurons or effector organs. L -glutamate and G A B A are major neurotransmitters in the mammalian C N S and have opposing effects (Lujan et al., 2005). L-glutamate is an excitatory neurotransmitter that acts through glutamate receptors, including N M D A R s , of the postsynaptic neuron (Riedel et al., 2003). 1.4.2 The glutamate receptor Glutamate receptors consist of ligand-gated ion channel (ionotropic) receptors and G -protein coupled (metabotropic) receptors (Riedel et al., 2003). Glutamate binding to 34 metabotropic receptors activates G protein coupling to second messenger enzymes, which eventually modulate cellular processes including regulation of ionotropic channels. On the other hand, activated ionotropic receptors are permeable to monovalent (Na + , K + ) and divalent (Ca 2 + ) cations. Ionotropic receptors are subdivided into N M D A R and n o n - N M D A R types. The latter group includes A M P A R (a-amino-3-hydroxy-5-methyl-4-isoxazone propionate receptor) and kainate receptor (Dingledine et al., 1999; Barnes and Slevin, 2003; Mayer and Armstrong, 2004). These names are derived from synthetic chemicals that are not naturally present in biological systems but that specifically bind to and activate the receptors. Although they all respond to glutamate, they possess numerous distinct characteristics including their subunit assembly, rates of appearance in the developing synapse, time courses of activation and deactivation, desensitization kinetics, ion permeability, and conductance properties (Barnes and Slevin, 2003). A M P A R s are composed of four subunit types, designated as GluR-1 , -2, -3, and -4, and are either homotetramers of GluR-1 and GluR-4 or heteromers o f GluR-2/3 and either GluR-1 or GluR-4. A M P A R s are responsible for the majority of fast excitatory central nervous system (CNS) synaptic transmission. L o w and high affinity kainate receptors are composed o f GluR-5 , -6, and -7, and K A - 1 and -2 subunits, respectively. Similar to A M P A R , kainate receptors have a fast electrophysiological response to glutamate. However, kainate receptors exhibit slower onset and decay kinetics, and smaller peak amplitude, compared to A M P A R s (Hollmann and Heinemann, 1994). The principal ions gated by A M P A R s and kainate receptors are N a + and K + (Hollmann and Heinemann, 1994). 35 1.4.3 The N-methyl-D-aspartate receptor (NMDAR) 1.4.3.1 Properties N M D A R s exhibit remarkable properties that distinguish them from other types of ligand-gated ionotropic receptors (Lynch and Guttmann, 2001) (Fig. 1.7). N M D A R s are the only receptor regulated by both ligand and voltage. In the basal state, the N M D A R is blocked by the physiological level of extracellular M g 2 + . Activation of the N M D A R requires the removal of M g 2 + , induced by membrane depolarization by other types of glutamate receptors. glutamate NMDA glycine C a 2 + polyamines 3 Y ® M g 2 + P C P MK-801 memantine redox Figure 1.7. Ligand- and modulator-binding sites on the NMDAR. The N M D A R contains binding sites for the agonists glutamate and N M D A , and the co-agonist glycine. The binding sites for M g 2 + or channel blockers, such as M K - 8 0 1 , phencyclidine (PCP), and memantine are within the ion channel pore region. The N M D A R also contains binding sites in the extracellular region for modulators such as zinc, polyamines, redox agents, and protons. The opening of the N M D A R channel leads to a calcium influx into cells, resulting in activation of various signaling molecules. 36 N M D A R activation also requires both glutamate and binding o f the co-agonist glycine. Glycine binding potentiates the glutamate response by reducing the magnitude o f desensitization of the N M D A R (Mayer et al., 1989). N M D A R s have higher affinity for glutamate than the other glutamate receptors (Patneau and Mayer, 1990). The activated N M D A R is highly permeable to C a 2 + . Excitatory postsynaptic currents resulting from N M D A R activation are exceptionally slow to rise and persist longer than those of A M P A R s (Hollmann and Heinemann, 1994). N M D A R s also contain various binding sites for endogenous modulators such as extracellular H + , Z n 2 + , and polyamines, suggesting roles for these factors in regulating N M D A R activity (McBain and Mayer, 1994). 1.4.3.2 Subunit composition There are seven N M D A R genes (NR1, N R 2 A - D , N R 3 A - B ) (Cull-Candy et al., 2001). Functional native N M D A R s are heteromeric complexes of two N R 1 and two N R 2 subunits. N R 1 is a ubiquitously expressed, indispensable subunit of the N M D A R . Eight isoforms of the N R 1 subunit can be generated from a single gene with three independent sites for alternative splicing (Dingledine et al., 1999). These isoforms are distinct in regional distribution and functional properties. The type of NR1 splice variant is important in modulation of the N M D A R s , for example, inhibition by protons and Z n 2 + , and potentiation by polyamines (Cull-Candy and Leszkiewicz, 2004). N R 2 subunits are made of four different but closely related genes. N R 2 subunits are differentially expressed according to developmental stages and C N S regions (Monyer et al., 1994; Standaert et al., 1996; Laurie et al., 1997). The N R 2 A subunit appears in most brain regions after birth. The N R 2 B subunit is restricted to the forebrain at postnatal stages but is present throughout the entire embryonic brain. The N R 2 C is postnatally enriched in cerebellum. The N R 2 D subunit is mainly found 37 in the diencephalon and brain stem at embryonic and neonatal stages. N R 2 subunits are critical in determining the biophysical and pharmacological properties of the receptor including its high affinity for glutamate and modulation by glycine, and sensitivity to M g , fractional C a 2 + current, and channel kinetics (Lynch and Guttmann, 2001; Cull-Candy and Leszkiewicz, 2004). The time course of N R 1 / N R 2 NMDAR-media ted current deactivation depends on the N R 2 subunit component following the sequence (from the fastest to the slowest): N R 2 A < N R 2 C = N R 2 B < N R 2 D (Cull-Candy et al., 2001). N R 2 A - and N R 2 B -containing N M D A R s generate 'high conductance' channels opening with high sensitivity to M g 2 + blockage, whereas N R 2 C - and NR2D-containing N M D A R s generate ' low conductance' channels with low sensitivity to extracellular M g 2 + (Cull-Candy et al., 2001). N M D A R subtypes also differ in their pharmacology as assessed by subunit-selective agonists and antagonists. Competitive antagonists such as A P 5 (2-amino-5-phosphonpentanoic acid), channel blockers such as MK-801 (dizocilpine), ketamine, phencyclidine, amantadine, and memantine, and novel non-competitive antagonists such as felbamate show moderate selectivity for certain subunit combinations. For instance, N R 2 A - and NR2B-containing N M D A R s are more sensitive to M K 8 0 1 than NR2C-containing N M D A R s (Lynch and Guttmann, 2002). Expression of the N R 3 subunits is associated with late prenatal and early postnatal development (Sun et al., 1998). The roles o f the N R 3 subunits are not well understood as they sometimes coassemble with N R 1 / N R 2 complexes, resulting in inhibition o f receptor activity (Matsuda et al., 2002). Moreover, N R 3 is also known to couple with N R 1 to form glycine receptors (Chatterton et al., 2002). 38 1.4.3.3 Domain structure NR1 and N R 2 subunits share a similar domain structure with an extracellular N -terminal ligand binding region, a middle region containing the structural components of the ion channel (four membrane regions), and an intracellular C-terminal region connecting the receptor to synaptic elements, second messengers, and intracellular events (Ziff, 1999) (Zheng etal., 2002) (Fig. 1.8). NH2 NH2 C 2 ^ C O O H C O O H Figure 1.8. Domain structure of NMDAR subunits. The ligand (glycine and glutamate) binding region is composed of residues from both the N -terminal domain and the extracellular loop that joins the M 3 and the M 4 regions. The M 2 region contributes to the channel pore. Eight isoforms of the N R 1 subunit (on the left) can be generated from a single gene with three independent sites for alternative splicing, one near N-terminus (NI) and two at the C-terminus ( C l and C2). The N R 2 subunit (on the right) contains a longer C-terminal region. 3 9 The N R 1 subunit has a shorter C-terminal region compared to the N R 2 subunit. Regions M - l , -3, and -4 are conventional membrane spanning domains. M - 2 is novel and is believed to have a membrane-reentrant hairpin structure that contributes to the receptor channel pore. The N-terminal domain is large, glycosylated, and contributes to form the agonist-binding site. The N R 1 subunit contains a binding site for glycine (Hirai et al., 1996) and the N R 2 subunit contains binding site for glutamate (Laube et al., 1997). Based on crystal structures o f bacterial amino acid binding proteins, each ligand-binding domain appears to be composed of residues from both the N-terminal domain and the extracellular loop that jo in M 3 and M 4 (Ziff, 1999). 1.4.3.4 Funct ion N M D A R s are important throughout life for synaptic formation, maintenance and plasticity (McBa in and Mayer, 1994). They also play a role i n higher brain functions such as learning and memory and certain behaviors (Riedel et al., 2003). Depending on their level of activation, N M D A R s may trigger two forms of mammalian experience-dependent synaptic plasticity, long term potentiation (LTP), that is a persistent increase in efficacy of synaptic strength induced by brief high frequency train of stimuli including tetanus, or long term depression (LTD) that is a persistent decrease in synaptic strength response to past patterns of electrical activity (Kullmann et al., 2000). Since the N M D A R is crucial for normal brain function, dysregulation of the N M D A R has numerous implications. Insufficient N M D A R -mediated transmission during development has been implicated in schizophrenia (Laruelle et al., 2003), whereas increased N M D A R activity promotes seizures and possibly epilepsy (Kohl and Dannhardt, 2001). Certain forms of pain and addiction that depend on aberrant synaptic plasticity may arise from inappropriate N M D A R stimulation (Laruelle et al., 2003; 40 Petrenko et al., 2003). Overactivation of the N M D A R leads to neuronal cell death (excitotoxicity) that is a cause o f hypoxia, brain and spinal cord injury, and chronic neurodegenerative diseases (Lynch and Guttmann, 2002). Although N M D A R antagonists prevent excitotoxicity in cellular and animal models, these drugs are not acceptable in clinical settings as they trigger various side effects such as psychosis, nausea, vomiting, memory impairment, and neuronal cell death caused by complete blockage of N M D A R , highlighting the crucial role o f the N M D A R in normal neuronal processes (Lipton and Rosenberg, 1994; Ikonomidou et al., 1999). 1.4.4 N M D A R signaling N M D A R s and other glutamate receptors are concentrated at the postsynaptic density (PSD), a dense structure visualized by electron microscopy that lies just below the postsynaptic membrane (Cotman et al., 1974; Cohen et al., 1977). One side of the P S D is connected to the cytoplasmic tails of ion channels, whereas the other face o f the P S D is associated with various cellular proteins. N M D A R s are found in a complex with numerous other proteins including signaling enzymes, scaffolding, cytoskeletal, and adaptor proteins, and cell signaling molecules (Husi et al., 2000), suggesting the involvement of the N M D A R in multiple signaling pathways. Activation of N M D A R s allows the influx of calcium into the postsynaptic spine, leading to the activation of various enzymes including kinases and phosphatases. These then regulate N M D A R s , A M P A R s , spine cytoskeletal changes, translation, transcription, and other events (Sheng and K i m , 2002) (Fig. 1.9). 41 NMDAR P K A +— sre -—-fr Pyk2 AMPAR e-... r C a 2 + _ ^ PKC c A M P / \ ^ CaMKII -R a s - G E F PP1 P P 2 B / C a N Ras I S y n - G A P I Raf I MEK1/2 I Erk1/2 Gene express ion Figure 1.9. N M D A R signaling at the postsynapse. N M D A R signaling is activated by calcium influx through the N M D A R . This eventually regulates the action of the N M D A R and the A M P A R . The feedback loop is highlighted in red. A C , adenylate cyclase; A M P A R , a-amino-3-hydroxy-5-methyl-4-isoxazone propionate receptor; C a M K I I , calcium-CaM-dependent protein kinase II; c A M P , cyclic A M P ; P K A , cAMP-dependent protein kinase; P K C , protein kinase C; Pyk2, proline-rich tyrosine kinase 2; PP2B/CaN, protein phosphatase 2B-calcineurin; PP1, protein phosphatase 1; Ras-GEF, Ras-guanine exchange factor; Syn-GAP, synaptic GTPase-activating protein. Modified from Sheng and K i m (2002). Postsynaptic density-95 (PSD-95): PSD-95 belongs to a family of structurally related proteins that includes chapsynl 10/PSD93, S A P 102, and SAP97. PSD-95 consists of three N -terminal P D Z (PSD-95, Dig , and ZO-1 homology) domains, an SH3 domain, and a C -42 terminal G K (guanylate kinase)-related domain ( K i m and Sheng, 2004). N M D A R s mainly interact with the PDZ-1 and PDZ-2 domains of PSD-95 via the C-terminal tails of N R 2 subunits (Kornau et al., 1995; Niethammer et al., 1996). PSD-95 links N M D A R s to components of the P S D , thus tethering the receptors at synapses and clustering them. Thus, PSD-95 binding to N M D A R is thought to be important for synaptic localization of the N M D A R , however, there has so far been no direct evidence to support this as N M D A R localization is unaffected in mice lacking PSD-95 (Migaud et al., 1998). Calcium-Calmodulin (CaM)-dependent protein kinase II (CaMKII) : C a M K I I is a serine/ threonine protein kinase that is abundant in the P S D (Kennedy, 2000), is activated upon N M D A R stimulation, and is important for NMDAR-re la ted L T P (Lisman et al., 2002). C a M K I I becomes activated in response to increased intracellular C a 2 + , this activated form of C a M K I I translocates to the synapses to interact with and to phosphorylate the N M D A R (Omkumar et al., 1996; Strack and Colbran, 1998; Leonard et al., 1999). A t synapses, N R 2 B and, to a lesser extent, N R 2 A binding to C a M K I I interfere with an autoinhibitory interaction within C a M K I I , thus stabilizing the activated state of C a M K I I (Bayer et al., 2001). C a M K I I can also bind cc-actinin (Walikonis et al., 2001), an actin binding protein, indicating a structural role for C a M K I I at synapses. The A M P A R - b i n d i n g proteins SAP97 and 4 . I N also interact with F-actin. In this close contact and by anchoring to N R 2 B , activated C a M K I I may phosphorylate A M P A R s and/or increase anchoring sites for newly delivered A M P A R s (Barria et al., 1997; Lee et al., 2000; Lisman et al., 2002), thereby enhancing synaptic transmission. 43 R a s / M A P K : The R a s / M A P K cascade is a major postsynaptic signaling pathway in synaptic plasticity (Adams and Sweatt, 2002). M A P K s E r k l and Erk2 are the predominant isoforms in post-mitotic neurons of the adult C N S (Boulton and Cobb, 1991; Fiore et al., 1993). Several studies report a differential regulation of E r k l and Erk2 in neurons (Bading and Greenberg, 1991; Fiore et al., 1993). This is most likely due to distinct localization and scaffolding interactions of these Erk isoforms, and/or to selective activation of E r k l and Erk2 by specific upstream kinases. It has been well documented that E r k l / 2 activation is induced by a Ras-dependent pathway (Cobb and Goldsmith, 1995). Increased calcium concentration activates Ras-guanine-nucleotide releasing factor (Ras-GRF), a guanine nucleotide exchange factor (GEF) for Ras, thereby stimulating Ras (Rosen et al., 1994). Several other Ca 2 +-regulated Ras-GEFs and the calcium- and PKC-activated Pyk2 may also be involved in R a s / M A P K activation (Lev et al., 1995; Orban et al., 1999). In addition, Ca 2 + - induced activation o f C a M K I I results in an inhibition of the synaptic Ras inhibitor, Syn-GAP, a guanine triphosphatase (GTPase)-activating protein (GAP) (Oh et al., 2004). Ras and its regulators (Ras-GEFs, Ras -GAP, and Pyk2) are prominent component of the N M D A R complex (Husi et al., 2000) and Ras activity is stimulated by N M D A R activation (Yun et al., 1998). Moreover, Ras is found critical for synaptic plasticity and learning and memory as mice deficient for R a s - G E F l exhibit loss of L T P in the amygdala (Brambilla et al., 1997). Altogether, these findings indicate a central role for Ras in N M D A R signaling. Activation of Ras eventually leads to activation of E r k l / 2 , which regulates various target proteins of three groups: cytoskeletal proteins, nuclear proteins, and signaling molecules (Graves et al., 1995). Inhibition of M E K , the upstream activator of M A P K , impairs L T P (Adams and Sweatt, 2002). C R E B (cAMP-response element binding protein) is a very important downstream 44 effector of R a s / M A P K in regulation of memory formation (Adams and Sweatt, 2002). Activation o f C R E B by phosphorylation on Ser 133 promotes its binding to the c A M P -response element (CRE) promoter element and a subsequent increase in the transcription o f target genes (Chrivia et al., 1993). PI3-K/Akt : P I3 -K activity is enhanced during hippocampal NMDAR-dependent L T P , which is essential for L T P expression but not induction (Sanna et al., 2002). Activation of PI3-K is also implicated in the amygdala for synaptic plasticity and memory consolidation (Lin et al., 2001). PI3-K is required for N M D A R - i n d u c e d trafficking of A M P A R s to the neuronal surface (Passafaro et al., 2001), thereby PI3-K may act in L T P to enhance the synaptic delivery of A M P A R s . A critical downstream effector of PI3-K is the serine/threonine protein kinase Ak t (Cantley, 2002). Protein kinase A ( P K A ) and protein kinase C (PKC) : N M D A R - i n d u c e d currents are elevated by activation of P K C (Gerber et al., 1989; Chen and Huang, 1992) and P K A (Raman et al., 1996). P K A and P K C can phosphorylate N R 1 and N R 2 subunits, resulting in an enhanced N M D A R response to agonists (Greengard et al., 1991; Kelso et al., 1992; Cerne et al., 1993). In addition, P K C can also indirectly mediate N M D A R function through enhancing src-induced N R 2 A and N R 2 B tyrosine phosphorylation. Inhibition of P K C does not alter the src-induced enhancement of N M D A R responses, whereas src inhibition depresses the potentiation activated by P K C (Lu et al., 1999). Furthermore, in src"/_ neurons, P K C -dependent activation of N M D A R is absent. This suggests an upstream role of P K C in src-45 mediated N M D A R phosphorylation and function. P K C has been repeatedly implicated in synaptic plasticity and L T P (MacDonald et al., 2001). Serine/threonine protein phosphatases: Inhibition of calcineurin (a calcium/CaM-regulated phosphatase, also termed PP2B) and PP1 (protein phosphatase 1) blocks hippocampal NMDAR-dependent L T D (Mulkey et a l , 1994). Expression of NMDAR-media ted L T D is associated with the internalization of A M P A R s as a result of dephosphorylation of A M P A R -G l u R l on Ser845, a P K A phosphorylation site (Ehlers, 2000; Lee et al., 2000; L i n et al., 2000) .The phosphatases PP1 and P P 2 B / C a N , and P K A bind to the same anchoring proteins (e.g. PP1 binds to yotiao, calcineurin binds to AKAP79 /150) that bind directly or indirectly to N M D A R s and A M P A R s (Westphal et al., 1999; Colledge et al., 2000). Therefore, both kinase and phosphatases are specifically targeted to glutamate receptors, facilitating their b i -directional regulation during synaptic plasticity (Westphal et al., 1999; Tavalin et al., 2002). Proline-rich kinase (Pyk2): Pyk2 (also known as Ca 2 +-dependent tyrosine kinase ( C A D T K ) or cell adhesion kinase p (CAKp)) is a nonreceptor protein tyrosine kinase. It is abundantly expressed in the C N S with high level in several brain regions including the hippocampus (Lev et al., 1995; Sasaki et al., 1995). Pyk2 is associated with the N M D A R complex and its activation is required for NMDAR-dependent hippocampal C A 1 L T P (Huang et al., 2001). In N M D A R regulation, Pyk2 is placed upstream o f sre (Huang et al., 2001) as the introduction o f recombinant Pyk2 into C A 1 neurons enhances N M D A R current, however, the application o f a sre-inhibitory peptide (src40-58) prevents this enhancement by Pyk2. Furthermore, activation of Pyk2 and its association with sre are increased by tetanus-induced L T P (Huang 46 et al., 2001). Pyk2 activation is initiated by a frans-autophosphorylation on Tyr402 and phosphorylation on Tyr579/580 in the catalytic domain (Girault et al., 1999), which is mediated by increased intracellular calcium and P K C activation (Lev et al., 1995) upon N M D A R activation. This leads to src binding to phospho-Tyr402 o f Pyk2 and src activation (Dikic et al., 1996). Thus, Pyk2 binding results in increased src activity, and hence N M D A R activation, thereby enhancing calcium influx during strong synaptic stimulation and promoting synaptic potentiation ( A l i and Salter, 2001; Huang et al., 2001). Src family kinases (SFKs): Five members of the S F K family, src, fyn, yes, lyn, and lck, are present in the mammalian C N S (Salter, 1998). Four o f these S F K s , the exception being lck, are physically associated with the N M D A R ( Y u et al., 1997; K a l i a and Salter, 2003). Y u et al., (1997) have investigated the effect of src on single N M D A R channel currents using inside-out patches (a small piece of membrane with cytosolic side facing the bath solution) from dorsal horn neurons. Application o f anti-cstl antibodies, which inhibit S F K s , to the cytoplasmic side of the inside-out patches results in a reduction o f basal N M D A R channel activity ( Y u et al., 1997). Similarly, introduction of anti-srcl antibody or src-inhibitory peptide [src(40-58)], that selectively block src but not other SFKs , causes inhibition of N M D A R activity and prevents L T P induction ( Y u et al., 1997). In contrast, applying a tyrosine phosphorylated peptide that has high binding affinity to S F K - S H 2 and thus activates SFKs , leads to enhanced N M D A R activity, whereas the non-phosphorylated form of the peptide exhibits no effect on N M D A R (Yu et al., 1997). In another experiment, whole cell currents from spinal dorsal horn neurons were recorded. Introduction o f recombinant src into neurons increases N M D A R current activity and induces long-lasting potentiation responses 47 (Wang and Salter, 1994). However, the mechanism of activation of recombinant N M D A R and native N M D A R appears to be distinct as src-induced potentiation of recombinant N M D A R is mediated by reducing zinc inhibition (Zheng et al., 1998), whereas in neurons, src-induced potentiation of native N M D A R current is clearly not (Xiong et al., 1999). Sre activity is increased upon tetanic stimulation that is sufficient for L T P induction (Lu et al., 1998), however, mice lacking sre exhibit similar L T P in the C A 1 region when compared with wild-type control animals (Grant et al., 1992). Fyn directly phosphorylates N M D A R s in vitro and can enhance glutamate-induced currents mediated by recombinant N M D A R (Suzuki and Okumura-Noji, 1995; Kohr and Seeburg, 1996), however, the role for fyn in regulation o f native N M D A R channel properties has not been tested. Although young fyn"7" mice appear normal, L T P from older fyn"7" mice is blunted but not abolished (Grant et al., 1992). Notably, the sre level is elevated in fyn"7" animals (Grant et al., 1995), suggesting a compensatory mechanism by sre. Introducing the wild-type fyn transgene into fyn"7" mice can restore the impairment of L T P in adult mice (Kojima et al., 1997). In summary, since administering specific blockers of sre causes inhibition of N M D A R activity and prevents L T P induction ( Y u et al., 1997), sre is, nonetheless, considered to be critical for L T P . Fyn may substitute for sre in sre"7" mice. The involvement of fyn in the molecular mechanism of L T P induction suggests that fyn may act upstream of sre. N R 2 A and N R 2 B contain multiple tyrosine residues in the C-terminal tail. The expression in human embryonic kidney ( H E K ) 293 cells of mutant forms of N R 2 A and N R 2 B , containing various mutations at these tyrosine residues, together with fyn or sre has identified Tyr l292 , Tyr l325 , and Tyr l387 in N R 2 A to be sites for sre phosphorylation (Yang and Leonard, 2001), and Tyr l252, Tyr l336, and Tyr l472 in N R 2 B to be sites for fyn 48 phosphorylation (Nakazawa et al., 2001) (Fig. 1.10). Also , Tyr l472 is the main fyn-catalyzed tyrosine phosphorylation site in recombinant N R 2 B (Nakazawa et al., 2001). Phosphorylation of the N M D A R by S F K s is not only associated with N M D A R activation but also with regulated synaptic localization and surface expression of the receptor (Grosshans et al., 2002; Thornton et al., 2003; Prybylowski et al., 2005; Suvarna et al., 2005). glycine glutamate ( ? ) Y1292 © Y1325 ® Y1387 NR2A Y 1 2 5 2 ( p ) Y 1 3 3 6 © H Y 1 4 7 2 © NR2B Figure 1.10. Sites of tyrosine phosphorylation of N M D A R subunits by sre and fyn. The N R 1 subunit (in blue) seems not to be phosphorylated on tyrosine. The C-terminal tails of N R 2 A and N R 2 B (in orange) contain 25 tyrosine residues. SFK-mediated phosphorylation sites in the C-terminal tails that have been verified in biochemical studies are shown (P in red): Y1292, Y1325, and Y1387 in N R 2 A , and Y1252, Y1336, and Y1472 in N R 2 B . 49 1 . 4 . 5 P T P s i n N M D A R s i g n a l i n g When the N M D A R is tyrosine phosphorylated, currents through endogenous N M D A R s are enhanced (Wang and Salter, 1994). Similar results were obtained with recombinant N M D A R s (Chen and Leonard, 1996; Kohr and Seeburg, 1996). Inhibition of endogenous P T K activity by penetrating neurons with genistein, a P T K inhibitor, or an increase in P T P activity effected by introducing intracellularly exogenous T-cell PTP truncate into neurons, result in a decline in N M D A R currents (Wang et al., 1996). In contrast, inhibiting endogenous P T P activity or enhancing P T K activity by the introduction of exogenous src causes an elevation in N M D A R currents (Wang and Salter, 1994; Wang et al., 1996). This suggests that N M D A R currents are controlled by a balance o f tyrosine phosphorylation and dephosphorylation mediated by the actions of P T K s , including SFKs , and PTPs present in the N M D A R complex. However, the identity o f these PTPs is still far from clear. CNS-expressed PTPs are implicated in multiple neuronal processes including morphogenesis, development, diffentiation, and axon outgrowth and guidance (Stoker, 2001; Paul and Lombroso, 2003). However, only very few PTPs, including STEP, SHP-2, P T P M E G , and PTPa , have been reported to play a role in N M D A R regulation (Lin et al., 1999; Hironaka et a l , 2000; L e i et al., 2002; Pelkey et al., 2002). STEP: STEP (striatal enriched tyrosine phosphatase) is a nonreceptor and brain-specific PTP. Originally, S T E P was identified as a P T P that was highly enriched in neurons of the striatum (Lombroso et al., 1993). Later studies revealed the presence of S T E P in other regions of the brain, including neurons of the hippocampus and cerebral cortex (Boulanger et al., 1995). Alternative splicing produces several STEP isoforms including STEP61, STEP46, STEP37, 50 and STEP33 (Boulanger et a l , 1995). STEP61, the only membrane-associated STEP, is present in the P S D (Oyama et al., 1995) and is associated with the N M D A R complex in the spinal cord and hippocampus (Pelkey et a l , 2002). Introducing recombinant STEP to the cytoplasmic face o f the inside-out patches extracted from spinal cord results in inhibition of N M D A R channel activity. In addition, microinjection of STEP into these neurons suppresses NMDAR-media ted synaptic currents measured by miniature excitatory postsynaptic currents (mEPSCs). Similarly, administering STEP to C A 1 hippocampal slices prevents tetanus-induced L T P . In contrast, application of anti-STEP antibodies leads to enhanced N M D A R synaptic currents, revealing a role of endogenous STEP in N M D A R regulation. N M D A R blockage by the antagonist M K - 8 0 1 or src inhibition by peptide src40-58 abolishes the effect of administration of anti-STEP antibodies, indicating that STEP functions by opposing src-mediated enhancement o f N M D A R currents (Pelkey et al., 2002). Recently, S T E P has been reported to directly associate with and to constitutively dephosphorylate the N M D A R (Braithwaite et al., 2006). Furthermore, STEP is shown to regulate N M D A R endocytosis by modulating the phosphorylation o f N R 2 B (Snyder et al., 2005). In addition, STEP61 can specifically bind to and dephosphorylate fyn at Tyr416 (Nguyen et al., 2002), in the same study, no interaction of STEP61 and src was detectable. Thus STEP can directly or indirectly down-regulate N M D A R phosphorylation and function. SHP-2: SHP-2 (also known as PTP1D, PTP2C, SHPTP2, SHPTP3, and Syp), a cytosolic PTP , is found in the P S D and functionally associated with N R 2 B (L in et al., 1999; Husi et al., 2000). Brain-derived neurotrophic factor ( B D N F ) stimulation leads to a transient increase in N R 2 B phosphorylation and SHP-2-NR2B association in the P S D . This indicates a 51 potential role of SHP-2 in decreasing and terminating BDNF-induced N R 2 B phosphorylation (Linet a l , 1999). P T P M E G : P T P M E G is a member of the band 4.1 domain-containing PTP subfamily and is prominently expressed in brain (Gu and Majerus, 1996). A detailed study reveals the enrichment of P T P M E G in mouse thalamus and Purkinje cells (Hironaka et al., 2000). In coimmunoprecipitation experiments, P T P M E G is found to associate with N R 2 A both in vitro and in vivo (Hironaka et al., 2000). In H E K 2 9 3 cells expressing N R 2 A and constitutively active fyn, P T P M E G further enhances fyn-mediated N R 2 A phosphorylation in a phosphatase^dependent manner (Hironaka et al., 2000). A s indicated in other studies, the band 4.1 domain appears to act as a binding domain for another transmembrane protein or as an adaptor domain involved in GTPase signaling cascade to regulate the organization of actin cytoskeleton (Tsukita et al., 1994; Takahashi et al., 1997; Takahashi et al., 1998). Thus, it is proposed that active P T P M E G rearranges the cytoskeleton around N R 2 A to make it easily accessible by fyn (Hironaka et al., 2000). PTPa : PTPa-nul l mice display defects in processes linked to N M D A R function such as learning and memory, hippocampal neuron migration, and C A 1 hippocampal L T P (Petrone et al., 2003; Skelton et al., 2003). Evidence of physical interactions between PTPa , src and fyn, and the N M D A R is provided by studies demonstrating that P T P a associates, as do fyn and src, with the N M D A R through the PSD-95 scaffolding protein (Lei et al., 2002). It has also been suggested that PTPa-D2 is important for the PSD-95-PTPa interaction (Lei et al., 2002). The introduction of P T P a into NRl /NR2A-express ing P T P a 7 " fibroblasts enhances NMDAR-media ted currents in a manner dependent on functional SFKs , while antibody 52 inhibition of P T P a in hippocampal neurons reduces NMDAR-media ted currents (Lei et al., 2002) . Intracellular application of G S T fusion protein containing the cytosolic region (D1+D2) of PTPa , but not the D l domain alone, into cultured hippocampal neurons potentiates NMDAR-media tes currents. Pretreatment of these neurons with the S F K inhibitor PP2 prevents this effect of P T P a (Lei et al., 2002). In contrast, introduction of anti-PTPa antibodies into postsynaptic neurons inhibits tetanic stimulation-induced long lasting potentiation of extracellular postsynaptic currents (EPSCs) of C A 1 hippocampal slices, indicating a role of endogenous P T P a in regulating synaptic plasticity (Lei et ah, 2002). These lines of evidence suggest a positive role of P T P a in N M D A R regulation in a S F K -dependent manner. 1.5 Hypothesis P T P a is ubiquitously expressed, indicating the potential for a universal cellular function and/or for a variety of tissue-specific roles of this PTP in cell signaling. The physiological function of PTPa in several signaling systems has been investigated using cells and mice deficient for PTPa . Several lines of evidence indicate a role for P T P a in a cell-type independent manner. For instance, previous studies have demonstrated the functional involvement of P T P a in mitosis and in integrin signaling (Zheng et a l , 2000; Zeng et al., 2003) , both of which are fundamental processes that operate in virtually all cell types. However, other studies suggest an involvement of P T P a in certain specialized pathways such as T cell signaling (Maksumova et al., 2005), and neuronal differentiation and outgrowth (Yang et al., 2002), indicating a tissue- and/or cell-type specific role for PTPa . Several 53 reports suggest that P T P a may also be involved in the LR- and NMDAR- in i t i a t ed signaling cascades. Thus, these two distinct signaling systems were selected to investigate the physiological functions of P T P a due to the following considerations. Firstly, N M D A R s are concentrated at the P S D of the C N S , whereas IRs are predominantly expressed in insulin-sensitive tissues (liver, muscle, and adipose tissues). Thus, these distinct signaling pathways provide opportunities to determine physiological tissue-specific, and neuronal versus non-neuronal, functions of PTPa . Secondly, P T P a is hypothesized to be involved in insulin signaling by affecting IR and/or LRS phosphorylation and activity. However in neuronal systems, including N M D A R signaling, P T P a appears to function via its upstream action on S F K s , tyrosine kinases that are well characterized signaling effectors of PTPa . Thus, elucidating the function of P T P a in the LR and N M D A R signaling pathways may provide additional insight into P T P a upstream action on distinct in vivo effectors (SFKs versus non-SFKs) , which may account for the basis of signaling system-specific functions of PTPa . Lastly, P T P a is capable of activating more than one S F K . Although these S F K s share a common domain structure and regulatory mechanism, previous studies have demonstrated both redundant and distinct functions of these S F K s (Stein et al., 1994). Thus it is important to determine whether P T P a functions in individual signaling pathway via its upstream action on specific SFKs . Therefore, this study not only addresses the physiological function of P T P a in the IR and N M D A R signaling pathways but also elucidates the mechanisms by which P T P a regulates distinct signaling pathways. To date, a role for P T P a in insulin signaling is controversial. Studies conducted with cultured cells indicate a negative, a positive, or no effect of P T P a in insulin signaling and insulin-dependent cellular processes (Moller et al., 1995; Jacob et al., 1998; Arnott et al., 54 1999; Cong et al., 1999). Factors such as heterologous P T P a and/or substrate expression, inappropriate cell types, and the non-physiological cell culture setting limit the abilities of these studies to determine the physiological role of P T P a in insulin action. N o studies have been conducted in animals to confirm or refute a role for P T P a in insulin action. Thus, the first aim of this thesis was to determine whether or not P T P a has a physiological function in the insulin signaling pathway. / hypothesize that PTPa functions as an IR phosphatase to down-regulate insulin action, with a consequent alteration in insulin signaling and glucose homeostasis. This was investigated in mice carrying a targeted deletion in the P T P a gene. M i c e lacking P T P a exhibit some defects in NMDAR-dependent L T P and other NMDAR-associa ted processes such as spatial learning and hippocampal neuron migration (Petrone et al., 2003; Skelton et al., 2003). However, in vivo effectors linking P T P a to the N M D A R have not been reported. Thus, the second aim of this thesis was to elucidate the mechanism underlying the action of P T P a on N M D A R function. N M D A R function can be regulated by tyrosine phosphorylation of the N R 2 A and N R 2 B subunits of the receptor by the S F K s sre and fyn (Nakazawa et al., 2001; Yang and Leonard, 2001). P T P a is a physiological regulator of S F K s (Ponniah et a l , 1999; Su et a l , 2001). However, whether P T P a functions to regulate N M D A R tyrosine phosphorylation, and i f so, how P T P a exerts its effect(s) on the N M D A R , is still unknown. / hypothesize that PTPa acts as a physiological regulator of NMDAR phosphorylation via its upstream action as an activator of sre and/or fyn. N M D A R phosphorylation and S F K activity were investigated in synaptic membranes of wild-type and PTPa-deficient mice. A heterologous expression system was also employed to examine the direct regulation of fyn- and src-mediated N R 2 A / B phosphorylation by PTPa . 55 2 MATERIALS AND METHODS 2.1 Materials 2.1.1 Mice M i c e deficient for P T P a (Ponniah et al., 1999) were maintained as an advanced intercross line (129Sv£v x Black Swiss, 50:50 mixed background). Animal care and use followed the guidelines of the Canadian Council on Animal Care and the University of British Columbia. Mouse genotyping was done by Dr. Jing Wang according to Ponniah et al. (1999). 2.1.2 Cultured cells A l l cells were cultured at 37°C with 5% C 0 2 . 2.1.2.1 Human embryonic kidney (HEK) 293T cells H E K 2 9 3 T cells (CRL-1573) were obtained from American Type Culture Collection ( A T C C ) . These cells were maintained in Dulbecco's modified Eagle's medium ( D M E M , Invitrogen) (4.5 g/L D-glucose with L-glutamine and 110 mg/L sodium pyruvate) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 100 L i g / m l streptomycin and 100 units/ml penicillin (Invitrogen). 2.1.2.2 Primary cortical and hippocampal neurons Primary cortical and hippocampal neurons were isolated from the pregnant mouse between day 18 (E18) and day 19 (E19) of gestation by Dr. Jing Wang (see section 2.2.8 for detail). 56 2.1.3 Antibodies 2.1.3.1 P r i m a r y antibodies Rabbit anti-insulin receptor p (C-19), anti-IRS-1 (C-20), anti-lyn (44), c-src (SRC 2), and anti-lck (2102) polyclonal antibodies were obtained from Santa Cruz Biotechnology (SantaCruz, C A , U S A ) . Mouse anti-phosphotyrosine clone 4G10 and rabbit polyclonal antibodies to PI3-kinase p85 (anti-serum), IRS-2, and SHP-2 were from Upstate Biotechnology (Lake Placid, N Y , U S A ) . Rabbit antibodies to phospho-Akt (Ser473 or Thr308), Akt , phospho-p44/42 M A P K (Thr202/Tyr204), and p44/42 M A P K , and mouse antibodies to phospho-Pyk2 (Tyr402) clone RR102 were from Cel l Signaling Technology (Mississauga, ON) . Mouse monoclonal antibodies to L A R , P T P 1 B , Grb2, yes (purified), Pyk2 (purified), and PSD-95, and rabbit anti-She polyclonal IgG were purchased from B D Transduction Laboratories (Mississauga, ON) . Phosphosite-specific antibodies raised against phospho-Tyr960 and T y r l 146-1150-1151 sites of the IR, and to the C-terminal phosphorylation site (pTyr527) and autophosphorylation site (pTyr416) of sre were obtained from Biosource International (Camarillo, C A , U S A ) . Purified mouse anti-fyn monoclonal IgG and goat anti-fyn (FYN-3G) polyclonal IgG were obtained from B D Transduction Laboratories and Santa Cruz Biotechnology, respectively. Anti-v-src (Ab-1) monoclonal mouse antibodies were from Oncogene (San Diego, C A , U S A ) . Rabbit a n t i - N M D A R 2 A / B and purified mouse a n t i - N M D A R l antibodies were purchased from Pharmingen (Mississauga, ON) . Goat a n t i - N M D A R 2 A (C-17) and a n t i - N M D A R 2 B (C-20), and rabbit a n t i - N M D A R 2 A and a n t i - N M D A R polyclonal antibodies were obtained from Santa Cruz Biotechnology and Chemicon (Temecula, C A , U S A ) , respectively. Antibodies to the transferrin receptor were from Zymed Laboratories, Inc. (South San Francisco, C A , U S A ) . 57 Mouse anti-tubulin clone GTU-88 and rabbit affinity-isolated anti-actin antibodies were from Sigma (St Louis, M O , U S A ) . Rabbit ant i -PTPa-Dl antiserum 2205 (raised against fusion protein containing P T P a - D l ) was produced as described by L i m et al. (1998). 2.1.3.2 Secondary antibodies Horseradish peroxidase (HRP)-conjugated Protein A was purchased from Biorad Laboratories. Goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP were from Sigma. Bovine anti-goat IgG-HRP was obtained from Santa Cruz Biotechnology. 2.1.4 Plasmids The plasmids pXJ41neo-wtPTPa encoding wild-type PTPa , pXJ41neo-Dl(C433S)D2(C733S)PTPa encoding inactive PTPa , and pXJ41neo-Y789FPTPa encoding P T P a lacking tyrosine phosphorylation site have been described (Zheng et al., 1992; L i m et al., 1997; Chen et al., 2006). The expression vector pXJ41neo-fyn and pXJ41neo-c-src have been described (Bhandari et al., 1998). The plasmids p R K 5 - N R 2 A and p R K 5 - N R 2 B were gifts from Dr. L . A . Raymond, University of British Columbia. Expression vector p G W l -EGFP-PSD95 was a gift from Dr. A . El-Husseini, University of British Columbia. The plasmid pcDNA-myc-Pyk2 was a gift from Dr. M . Gold, University of British Columbia. 2.2 Methods 2.2.1 Physiological tests 2.2.1.1 Determination of blood glucose and plasma insulin concentrations Blood samples were collected from the tail vein by cutting 1-2 mm from the tail tip with scissors. Blood glucose levels were measured with a One Touch Ultra Glucose meter 58 (Lifescan). A small drop (~5 ul) of blood was placed on the glucose meter test strip. After 5-seconds developing time, the blood glucose was recorded. Blood was also collected into heparin-coated capillary tubes (Sarstedt, Germany) by gently squeezing the tail from the base to the tip to obtain -50 ul of blood. The closed tubes were placed into a 1.5-ml eppendorf tube. Serum was then separated by centrifugation at 5000 rpm for 5 min and then transferred to a 500-ul eppendorf tube and stored at -20°C until use. Plasma insulin concentrations were determined using a Mercodia Mouse Insulin E L I S A kit (Alpco Diagnostics, U S A ) . 2.2.1.2 Glucose and insulin tolerance tests Animals were fasted overnight (15-16 hours) prior to each experiment but allowed free access to water. Prior to performing the tests, body weight and baseline glucose level were measured. Animals were then injected intraperitoneally with glucose (1.5 mg/g body weight) (Sigma) or human insulin (0.75 U/kg body weight) (El i L i l l y ) using a 1-riil syringe, then the animals were returned to the cage. Blood glucose was measured at 15, 30, 60, and 120 min after injection. 2.2.1.3 Weight gain analysis Animals were weighed on a weekly basis from 4 weeks to 10 weeks of age. Body weight of each mouse was recorded and then compared among groups of female or male animals. 2.2.2 Insulin stimulation M i c e (10-14 weeks old) were fasted overnight but allowed free access to water. Animals were anesthetized by intraperitoneal injection with tribromoethanol (250 mg/kg 59 bodyweight, Sigma). The abdominal cavity of the unconscious animal was opened, the portal vein was exposed, and insulin (5 U/mouse) or saline was injected into the vein using a 1-ml syringe. The needle was left untouched to prevent blood from coming out. A t selected times post-injection, pieces of liver, skeletal muscle, and abdominal fat pad were rapidly excised and immediately frozen in liquid nitrogen, and then stored at -80°C until use. 2.2.3 Preparation of crude synaptosomal P2 fraction M i c e were killed by cervical dislocation and whole brains were quickly collected and frozen in l iquid nitrogen, and stored at -80°C until use. Brain fractionation was carried out essentially as described by Huttner et al. (1983) and L i n et al. (1998). Frozen whole mouse brains were thawed on ice and then homogenized in ice-cold homogenizing buffer (320mM sucrose, 10 m M T r i s - H C l pH7.4, 1 m M N a H C 0 3 , 1 m M M g C l 2 , 1 m M N a 3 V 0 4 , 20 m M NaF, 10 u.g/ml aprotinin, 5 u.M leupeptin, 0.2 m M P M S F ) using a Dounce homogenizer. The homogenates were centrifuged at lOOOg for 10 min at 4°C to remove nuclei and large debris. Supernatants were collected and further centrifuged at lOOOOg for 15 min at 4°C. The resulting pellets (crude synaptosomal fraction, P2) were kept at -80°C for later studies. 2.2.4 Preparation of lipid rafts Crude synaptosomal fractions were lysed in M B S buffer (25 m M M E S - N a O H pH6.5, 150 m M N a C l , 2 m M E D T A , 1 m M N a V 0 4 , 20 m M NaF, 10 ixg/ml aprotinin, 5 u.M leupeptin, 0.2 m M P M S F ) containing 1% Triton X-100 for 1 h. One m l of 80% sucrose solution in M B S was added to 1 ml of the lysates with thorough mixing to obtain a final solution of 40% sucrose. These solutions were placed in the bottom of a clear tube (Beckman 60 Coulter) and overlaid with 4 ml of 30% sucrose and 2 m l of 5% sucrose solution in M B S . The sucrose gradients were centrifuged at 200000g for 16 h in an SW-40 rotor (Beckman). One-ml fractions were collected from the top for Western blot analysis. 2.2.5 Protein extraction 2.2.5.1 Preparation of tissue lysates Liver, muscle, and adipose tissues were homogenized in lysis buffer (20 m M Tris-HC1 p H 7.4, 150 m M N a C l , 2 m M E D T A , 1 m M N a V 0 4 , 50 m M NaF, 10 ng/ml aprotiniri, 10 nM leupeptin, 5 u M pepstatin, 0.2 m M P M S F , and 1% Triton X-100) using a Polytron P T 3100. The tissue homogenate was incubated on ice for 1 h and then centrifuged at 55000 rpm in a TLA100 .2 rotor (Beckman) for 30 min. The resulting supernatant was collected and assayed for protein concentration using Biorad reagent (Biorad). 2.2.5.2 Preparation of cell lysates Cells were washed with cold phosphate-buffered saline (PBS) and then lysed in modified R J P A (radioimmunoprecipitation assay) buffer (20 m M Tr i s -HCl pH7.5, 150 m M N a C l , 2 m M E D T A , 1 m M N a 3 V 0 4 , 20 m M NaF, 10 ng/ml aprotinin, 5 u M leupeptin, 0.2 m M P M S F , 1% Triton X-100, '0 .5% N a D O C , and 0.1% SDS). In experiments to determine the association of P T P a and fyn, the Triton X-100, N a D O C , and SDS were replaced with 1% Brij-98. In experiments to determine the phosphorylation of Pyk2 and its association with fyn, the Triton X-100, N a D O C , and SDS were replaced with 1% NP40. Cel l homogenates were incubated on ice for 30 min and then centrifuged at 16000g for 15 min. The resulting supernatants were collected and assayed for protein concentration using Biorad reagent. 61 2.2.5.3 Preparation of Triton X-100 soluble and insoluble fractions Crude synaptosomal fractions were lysed in Triton X-100 buffer (20 m M Tr i s -HCl pH7.5, 150 m M N a C l , 2 m M E D T A , 1 m M N a V 0 4 , 20 m M NaF, 10 ug/ml aprotinin, 5 u M leupeptin, 0.2 m M P M S F , and 1% Triton X-100) on ice for 1 h. The lysates were centrifuged at 16000g for 15 min. The resulting supernatants and pellets were designated as Triton X-100 soluble and insoluble fractions, respectively. Supernatants were assayed for protein concentration using Biorad reagent. Pellets from crude synaptosomes were dissolved in 1% SDS, boiled for 3 min, diluted 10-fold with Triton X-100 buffer, and centrifuged to obtain clear homogenates. 2.2.6 Immunoprecipitation and immunoblotting 2.2.6.1 Immunoprecipitation Immunoprecipitation was performed by incubating the appropriate amounts of precleared protein homogenates with 2 u.g of the indicated antibody on a rotator at 4°C for 2 h to overnight. Protein G Plus Protein A Agarose (Santa Cruz Biotechnology) was added to the samples and incubated with rotation for another 2 h at 4°C. The immunocomplexes were centrifuged at 5000 rpm for 3 min and the supernatant was carefully aspirated. The immunocomplexes were then washed 3 times with 1 m l lysis buffer. After the last wash, the remaining lysis buffer was carefully removed. The immunocomplexes were eluted with 20 ul 2 X SDS sample buffer (62.5 m M Tr i s -HCl pH6.8, 20% glycerol, 2% SDS, 572 m M (3-mercaptoethanol, 0.008% bromophenol blue), vortexed, boiled at 100°C for 5 min, and centrifuged at 16000 g for 1 min. The supernatant was loaded onto gels. 62 2.2.6.2 Immunoblott ing Immunoprecipitated proteins or total lysates in SDS sample buffer were resolved by S D S - P A G E in SDS-running buffer (192 m M glycine, 25 m M Tris-base, 0.1% SDS) at 150V. Prestained protein markers (Broad range, New England BioLabs) were also loaded onto the gels to enable the estimatation of sample protein molecular weights. Proteins were then transferred from the gel to a polyvinylidene fluoride ( P V D F ) membrane (Millipore) as follows. The P V D F membrane was pre-wet with 100% methanol for - 10 seconds, rinsed with distilled H 2 0 , and then immersed in transfer buffer (75 m M glycine, 10 m M Tris-base, 10% methanol). The transfer stack was assembled in transfer buffer. The transferring procedure was set up at 100V for 60 min (for most of the proteins) or 75 min (for high molecular weight proteins including N R 2 A , N R 2 B , LRS-1, and IRS-2). The membranes were then transferred to a square petri dish containing blocking buffer (2% B S A or 5% skim milk in P B S - T (PBS containing 0.1%Tween 20)) for 1 h at room temperature. Membranes prepared for phosphotyrosine detection were always blocked with 2% B S A . The blocked membranes were incubated with primary antibody diluted in 1% B S A in P B S - T overnight at 4°C with gentle agitation. The membranes were then washed 3 times (10 min each wash) with PBS-T , followed by incubation with secondary antibody in P B S - T for 1 h at room temperature on a shaker. After 3 further washes with PBS-T , proteins were detected using E C L detection reagent with exposure to X-ray film. In experiments to determine protein phosphorylation status, the membranes were subjected to anti-phosphotyrosine immunoblotting first, and then stripped and probed for other proteins of interest. 63 Membrane stripping;: Membranes were placed in stripping solution (62.5 m M Tr i s -HCl pH6.5, 2% SDS, 100 m M p-mercaptoethanol) in a ~55°C water bath for a minimum of 30 min with intervals of shaking. The membranes were then washed twice with P B S - T and were ready for re-immunoblotting. Antibody dilution: Most primary antibodies were diluted at 1:1000 (final concentration), except for anti-phosphotyrosine antibody (1:3000) and anti-tubulin antibody (1:5000). Secondary antibodies were used at the dilution of 1:3000 (Protein A H R P and goat anti-mouse IgG-HRP), 1:5000 (goat anti-rabbit IgG-HRP), and 1:10000 (bovine anti-goat IgG-H R P ) . 2.2.7 Kinase and phosphatase assays 2.2.7.1 Kinase assay Fyn and src immunoprecipitates were prepared from 300-400 p.g of lysate protein from transfected H E K 2 9 3 cells using anti-fyn antibody ( F Y N 3-G, Santa Cruz) or anti-v-src antibody (Oncogene). The kinase activity of these immunoprecipitates was determined in 20 ul o f kinase buffer containing 10 m M PIPES pH7, 5 m M M n C l 2 , 0.5 m M D T T , and 10 u C i [y - 3 2 P]ATP, and with or without 0.2 unit enolase (Sigma) at 30°C for 10 min (Bhandari et al., 1998). The enolase was treated with 10 m M N a A c pH3.5 at 37°C for 5 min prior adding to the kinase buffer. Reactions were terminated by adding 20 u.1 of 2 X SDS sample buffer, boiled for 5 min, and subjected to S D S - P A G E . The S D S - P A G E gels were dried and autoradiographed. Another set of immunoprecipitates was immunoblotted with anti-fyn antibody ( B D Transduction Laboratories) or anti-v-src antibody to determine the amounts of immunoprecipitated fyn or src proteins. 64 2.2.7.2 Phosphatase assay P T P a was immunoprecipitated from 500 p.g protein prepared from transfected H E K 293T cells lysed in modified R J P A buffer without tyrosine phosphatase inhibitor NaaVCXt. Dephosphorylation of /?ara-nitrophenyl phosphate (pNPP) was measured in 450 ul reactions containing 50 m M sodium acetate (pH5.5), 0.5 mg/ml B S A , and 0.5 m M D T T , and 2 m M pNPP (Sigma) (L im et al., 1998). Reactions were carried out at 30°C for 30 min and terminated by adding 50 ul of 13% K2HPO4. Released nitrophenol in the supernatant was quantified by measuring the O D at A ^ n m . The immunoprecipitates (pellets) were resolved by S D S - P A G E and immunoblotted with anti-PTPa antibody to determine the amount of PTPa protein. 2.2.8 Primary hippocampal and cortical neuron culture (done by Dr. Jing Wang) Pregnant mice were anesthetized with urethane and sacrificed by cervical dislocation. The abdominal cavity was opened and the embryos, which were still in their placental sacs, were collected. The sacs were then open, the head of the fetuses was cut off using sterilized scissors, and then brains were carefully transferred to 10-cm plate containing cold Hank' balance salt solution (HBSS) . The meninges were removed and the two hemispheres of the brain were separated. The cortices and hippocampi were dissected under the microscope and collected into 15-ml Falcon tubes containing H B S S . The hippocampi and cortices were triturated using 200-ul pipetman to obtain single cell suspension. The single cells were placed onto 6-cm dish (2 hippocampi or 1 cortex per dish) coated with 10 ng/ml of poly-D-lysine in plating medium (Neurobasal supplemented with 2% B27 supplement, 0.5 m M L -65 glutamine, and 5 p M L-glutamate). The plating medium was changed to maintenance medium (Neurobasal supplemented with 2% B27 supplement, and 0.5 m M L-glutamine) the next day. The medium was then replaced every 5 days. 2.2.9 Calcium phosphate transfection H E K 2 9 3 T cells were transfected with combinations of the following plasmids as indicated in the Figure Legends: 5 pg o f p R K 5 - N R 2 A or p R K 5 - N R 2 B ; 5 pg o f pXJ41neo-fyn or pXJ41neo-src; 1 pg of pXJ41neo containing c D N A encoding wild-type PTPa , inactive P T P a (C414S/C704S), or Tyr789 P T P a mutant (Y789F); 1 pg o f P S D 9 5 - w t - G F P - G W l ; 2 pg o f Pyk2-wt-pCDNA3-cmyc. H E K 2 9 3 cells were transfected using calcium phosphate transfection method. Cells were plated the day before transfection and were about 60-70% confluent on the day of transfection. The old medium was replaced with 9 m l of fresh medium - 2 h prior to transfection. In an eppendorf tube, 50 pi of 2.5 M CaCL. was added to 450 p i o f diluted D N A (appropriate amount o f D N A in sterile distilled H2O). The D N A / C a C k mixture was added, dropwise, to a 15-ml Falcon tube containing 500 p i of 2 X H B S (50 m M H E P E S , 280 m M N a C l , 1.5 m M N a 2 H P 0 4 , p H : 7.05-7.12). The bubbles were made in the Falcon tube while adding the D N A / C a C ^ mixture. The solution was incubated at room temperature for -20 minutes, mixed well before adding, dropwise, to the plate. The plate was gently swirled to distribute the precipitates evenly over the cells and returned to the 37°C incubator. After 5-h incubation, the precipitates were washed away by rinsing the cells twice with warm P B S . Fresh medium was added and returned to the incubator. The cells were harvested -36 h post-transfection. 66 2.2.10 Glutamate stimulation Cortical and hippocampal neurons were maintained in culture for 10 and 14 days, respectively, with the medium replaced every 5 days. In preparation for stimulation, the cells were washed once with minimum essential medium ( M E M ) and then 4 ml of fresh M E M containing 1.25 m M CaCl2, 0.5 m M M g C b , 0.5 m M glycine, and 1 p M tetrodotoxin ( T T X , Sigma) was added to each 6-cm dish of cells. The cells were returned to the incubator for 1 h, and then treated with 100 u,M of glutamate (Calbiochem) for the indicated time. 2.2.11 Data analysis Autoradiographs were scanned with a laser densitometer. The densities of the bands on the fi lm were quantified using the software program Quantity One (Biorad Laboratories). The amount o f protein phosphorylation or protein-protein association was determined by calculating the ratio of the intensity of the phosphorylated protein band or of the associated protein band to the intensity of the protein or protein partner band. This ratio was reported in arbitrary units. The ratio of the control sample was normalized to 1, and all other ratios are expressed relative to it. Normalized values from multiple experiments were averaged and graphed. The Y-axis was represented in arbitrary units (% of the control sample or fold-change over the control sample). Error bars in each of the figures represent the standard deviation (SD). The statistical significance was analyzed using two-tail paired or unpaired Student's t test. 67 3 INSULIN SIGNALING AND GLUCOSE HOMEOSTASIS IN PTPa 7 MICE 3.1 Introduction and rationale Insulin binding activates the insulin receptor (LR), resulting in tyrosine phosphorylation of the LR and of intermediate signaling molecules including the insulin receptor substrate (IRS) proteins. This eventually activates two major downstream pathways, i) the PI3-K/Akt pathway and ii) the Ras/MAPK pathway, and, consequently, regulates glucose metabolism, glycogen/protein/lipid synthesis, and mitogenic events (Saltiel and Kahn, 2001). Several PTPs including PTPa have been implicated as regulators of LR and/or LRS tyrosine phosphorylation, and insulin action (Elchebly et al., 2000; Cheng et al., 2002; Asante-Appiah and Kennedy, 2003). Elevation or ablation of PTPa expression in some cell culture systems results in altered IR tyrosine phosphorylation (Moller et al., 1995) and/or insulin signaling-dependent cellular responses (Moller et al., 1995; Cong et al., 1999; Lu et al., 2002), while in other systems, it does not (Jacob et al., 1998; Arnott et al., 1999). Thus, PTPa has been controversially proposed, although not confirmed, to be a candidate regulator of IR tyrosine phosphorylation and insulin action. No studies have so far been carried out in a physiological setting, such as in mice with genetically modified PTPa expression, to evaluate the role of PTPa in insulin action. Mice lacking PTPa have been generated in our laboratory (Ponniah et al., 1999). This has provided an opportunity to investigate the physiological function of PTPa in insulin 68 a c t i o n . I h y p o t h e s i z e t h a t P T P a p l a y s a r o l e i n i n s u l i n s i g n a l i n g a s a n e g a t i v e r e g u l a t o r o f I R a n d / o r I R S t y r o s i n e p h o s p h o r y l a t i o n . G l u c o s e h o m e o s t a s i s a n d t h e i n s u l i n s i g n a l i n g c a s c a d e s i n w i l d - t y p e ( W T ) a n d P T P a - d e f i c i e n t m i c e w e r e e x a m i n e d a n d c o m p a r e d . 3.2 Results 3.2.1 PTPa expression in mouse tissues P T P a i s r e p o r t e d t o b e w i d e l y e x p r e s s e d ( K a p l a n e t a l . , 1 9 9 0 ; S a p e t a l . , 1 9 9 0 ) . I m m u n o b l o t d e t e c t i o n o f P T P a w a s c a r r i e d o u t t o c o n f i r m t h e e x p r e s s i o n o f P T P a i n i n s u l i n -s e n s i t i v e t i s s u e s ( l i v e r , m u s c l e , a n d a d i p o s e ) a n d t o c o m p a r e i t s e x p r e s s i o n l e v e l b e t w e e n t h e s e a n d o t h e r m o u s e t i s s u e s ( F i g . 3 . 1 ) . 175 -4 47.5 H Brain Heart Intestine kidney Liver Lung Muscle Spleen Stomach Adipose +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/-*~ Hi - -_ ~ w a r mm*** P T P a Act in Figure 3.1. PTPa expression in mouse tissues. L y s a t e s o f t i s s u e s f r o m w i l d - t y p e ( + / + ) a n d PTPa" /_ ( - / - ) m i c e w e r e i m m u n o b l o t t e d f o r PTPa (upper panel) a n d a c t i n (lower panel). T h e m i g r a t i o n p o s i t i o n s o f m o l e c u l a r m a s s m a r k e r s ( k D ) a r e s h o w n . 6 9 Mouse brain, followed by kidney and stomach, expressed the highest levels of P T P a . P T P a expression in liver and adipose tissues was comparable to that in lung and spleen. L o w levels of P T P a were detected in muscle and heart, and it was almost undetectable in intestine. P T P a was confirmed to be absent in all tissues from P T P a 7 " mice. Among the three insulin-sensitive tissues, P T P a was expressed at higher levels in liver and adipose tissues than in muscle. 3.2.2 Unaltered expression of insulin-related PTPs in PTPa7" mice There are multiple PTPs present in insulin sensitive tissues. To address whether the loss o f P T P a was compensated for by alterations in the expression level of one or more other PTPs implicated in insulin signaling, such as PTP I B , L A R , and SHP-2, their expression in P T P a 7 " mice was evaluated by immunoblotting. The lack o f P T P a did not result in altered expression of any o f these PTPs in insulin-sensitive tissues (Fig. 3.2). Liver Muscle Adipose +/+ -/- +/+ -/- +/+ -/-175 62 47.5 83 62 ESS ». . A . ,d 47.5 -4 LAR PTP 1B SHP-2 Actin Figure 3.2. PTPa expression in insulin-responsive tissues of wild-type and PTPa7" mice. Lysates of liver, muscle, and adipose tissues from wild-type (+/+) and P T P a 7 " (-/-) mice were immunoblotted for L A R (top panel), PTP I B (upper middle panel), SHP-2 (lower middle panel) and actin (bottom panel). The migration positions of molecular mass markers (kD) are shown. 70 This indicates that i) any effects of ablating P T P a in these tissues that might be observed would not be due to upregulation o f these PTPs, or ii) a potential lack o f effect o f ablating P T P a in these tissues would not reflect compensation achieved by upregulated expression of these PTPs. 3.2.3 Normal weight gain in PTPa"'" mice Body weight is controlled by a balance o f energy intake and energy expenditure. Insulin is critical for the regulation of energy intake in the form of glucose metabolism (Saltiel and Kahn, 2001). Insulin increases glucose uptake into muscle and adipose tissues and inhibits glucose production by liver. Thus altered glucose metabolism can result in altered body weight. To investigate a role for P T P a in regulating body weight, wild-type and PTPa" 7" mice were weighed weekly over a period between 4 and 10 weeks o f age. Both male and female animals that were maintained on a standard chow diet were found to have similar initial body weights and rates of weight gain regardless o f the presence or absence of P T P a (Fig. 3.3). 3.2.4 Glucose homeostasis is normal in PTPa"7" mice To directly evaluate the effect of P T P a on glucose homeostasis in mice, blood glucose and insulin levels were measured. N o differences in fed and fasted blood glucose levels (Fig. 3.4A) or circulating insulin (Fig. 3.4B) were apparent between W T and PTPa-null animals. To further investigate the responses to elevations in glucose and insulin in PTPa" 7" mice, glucose and insulin tolerance tests were carried out. B lood glucose clearance following intraperitoneal injection of glucose occurred with normal efficiency in mice 71 lacking P T P a (Fig. 3.4C). Likewise, W T and PTPa" 7" mice demonstrated similar alterations in blood glucose in response to intraperitoneal insulin injection (Fig. 3.4D). In conjunction, the results indicate that the lack of P T P a does not render the mice more sensitive to insulin, nor does it alter insulin-regulated glucose homeostasis in animals maintained on a chow diet. M a l e A g e (weeks) B F e m a l e 14-1 , , , , , 1 4 5 6 7 8 9 10 A g e (weeks) Figure 3.3. Body weights of wild-type and PTPa" 7" mice. Wild-type (+/+) and PTPa" 7" (-/-) mice were used for measurements. A, body weights (± S.D.) of male mice (+/+, solid circles, n=14; -/-, open circles, n=14) that were weighed weekly from 4 to 10 weeks of age. B, body weights (± S.D.) of female mice (+/+, solid circles, n=16; -/-, open circles, n=9) that were weighed weekly from 4 to 10 weeks of age. 72 A B Figure 3.4. Parameters of glucose homeostasis are similar in wild-type and PTPa"'" mice. Wild-type (+/+) and PTPa- / - (-/-) mice were used for measurements. A, blood glucose of 8-to 12-week-old male mice fed ad libitum (+/+, n = l l ; -/-, n=12) or fasted overnight (+/+, n=12; -/-, n=15). B, serum insulin of 12- to 15-week-old male mice fed ad libitum (+/+, n=5; -/-, n=5) or fasted overnight (+/+, n=6; -/-, n=6). C, glucose tolerance tests were conducted on 8- to 10-week-old male mice (+/+, solid circles, n=12; -/-, open circles, n=12) that were fasted overnight and then injected i.p. with glucose (1.5 mg/g body weight). D, insulin tolerance tests were conducted on 10- to 12-week-old male mice (+/+, solid circles, n=l 1; -/-, open circles, n=9) that were fasted overnight and then injected i.p. with insulin (0.75 U/kg body weight). A l l error bars show ± S.D. 3.2.5 Unaltered IR tyrosine phosphorylation in PTPa"7" mice Since P T P a resides at the plasma membrane and is thus in proximity to the IR, it might act as an IR phosphatase. Insulin-stimulated tyrosine phosphorylation of the IR was examined in liver, muscle, and adipose tissues of W T and PTPa" 7" mice. To determine liver 73 and muscle LR tyrosine phosphorylation, equal amounts of tissue lysate protein were subjected to irnmunoprecipitation with anti-LRp subunit antibodies and then analyzed by immunoblotting with anti-phosphotyrosine antibody, followed by anti-LR antibody. The low amounts and l ip id contamination of adipose samples precluded the irnmunoprecipitation and analysis with these samples. Nevertheless, probing the total lysates prepared from adipose tissues with anti-phosphotyrosine antibodies revealed a band that comigrated with the LR that was highly phosphorylated in response to insulin injection. A similar sized phosphorylated band has been identified elsewhere as the LR ( L i et al., 2000). Therefore IR phosphorylation in adipose tissues was determined by immunoblotting tissue lysates, while that in liver and muscle was determined by immunoblotting IR immunoprecipitates. The IR tyrosine phosphorylation was low to undetectable in liver, muscle, and adipose tissues prior to insulin treatment. This was due to the fact that mice were fasted overnight. In W T liver, enhanced phosphorylation of the LR was detected by 1 min post-insulin injection, and sustained for at least 10 min. Liver LR phosphorylation was unaffected by the absence of P T P a (Fig. 3.5A). In W T muscle, LR phosphorylation increased up until at least 6 min following insulin stimulation and remained readily detectable to 11 min, with no alterations observed in PTPa" 7" muscle (Fig. 3.5C). In adipose tissues from W T and PTPa" 7" mice, comparable insulin-induced tyrosine phosphorylation o f the LR was observed up to at least 7 min post-injection and that decreased by 12 min post-injection (Fig. 3.5E). Furthermore, quantification of insulin-stimulated tyrosine phosphorylation of the LR from liver, muscle, and adipose tissues at 5, 6, and 7 min, respectively, demonstrated that ablation of P T P a did not result in any alterations in phosphorylation (Fig. 3.5B, D , and F). 74 +/+ -/- B Insulin (min) 0 1 5 10 0 ~ T T ~ H IR-P - 83 IR - 83 c +/+ -/-Insulin (min) 0 2 6 11 0 2 6 11 I R - P IR h 83 h 83 c •£ £ 9-If °-< 1.2 1 0.8 0.6 0.4 0.2 0 c S- <° Ii a: — 2-4 n 2 1.6 1.2 0.8 0.4 0 (3) JL, (3) +/+ -/-(2) (3) +•/+ -/-+/+ -/-Insulin (min) 0 3 7 12 0 3 7 12 I R - P IR 83 h 83 a* £"L> II <^ a: — 1.5 1.2 0.9 0.6 0.3 0 (2) (3) +/+ -/-Figure 3.5. Insulin receptor tyrosine phosphorylation in liver, muscle, and adipose tissues of wild-type and PTPa7" mice. A, C, and E, Wild-type (+/+) and P T P a (-/-) mice were fasted overnight, anesthetized, and insulin (5U) was injected into the portal vein. Tissue was removed from these mice at the indicated times following insulin treatment and from control untreated mice (0). The IR was immunoprecipitated from lysates of liver (A) and muscle (C) and probed with anti-phosphotyrosine antibody (upper panels), then reprobed with anti-IR antibody (lower panels). Lysates o f adipose tissue (E) were probed with anti-phosphotyrosine antibody (upper panel), then reprobed with anti-IR antibody (lower panel). The migration position o f the 83 k D molecular marker is shown. B, D, and F, The IR phosphorylation in liver at 5 (B), in muscle at 6 (D), and in adipose tissue at 7 (F) min post-injection was quantified by densitometry (from the experiments shown in A , C, and E , and other experiments) and are shown in the bar graphs. Bars (±S.D.) represent the amount of tyrosine phosphorylation per amount o f IR (arbitrary densitometric units). The number of individual mouse samples is shown in brackets above the bars. 75 Although no difference in the total tyrosine phosphorylation o f the IR was detected, this could be due to P T P a acting on only a small subset of the numerous phosphorylation sites on the LR, and such a difference might not be detectable against the overall LR phosphorylation signal. Therefore, phosphosite-specific antibodies were employed. Probing muscle and liver lysates with an antibody that specifically recognizes phosphoTyr-960 within the N P E Y sequence, a recognition site for IRSs and She, and an antibody that recognizes phospho-Tyrl 146/1150/1151 in the kinase domain o f the IR, demonstrated efficient insulin-stimulated phosphorylation at all these sites in P T P a 7 " liver and muscle (Fig. 3.6A and B) . +/+ -/-Insulin (min) 0 1 5 10 0 1 5 10 IR-3Y-P IR 83 h 83 B +/+ -/-Insulin (min) 0 2 6 11 0 2 6 11 IR-3Y-P 83 83 +/+ -/-Insulin (min) 0 1 5 10 0 1 5 10 IR-960-P IR I *Wal^.r 83 +/+ -/-Insulin (min) 0 2 6 11 0 2 6 11 83 IR-960-P IR 83 83 Figure 3.6. Phosphosite-specific tyrosine phosphorylation of insulin receptor in muscle and liver of wild-type and PTPa7" mice. Wild-type (+/+) and P T P a (-/-) mice were fasted overnight, anesthetized, and insulin (5U) was injected into the portal vein. Tissue was removed at the indicated times following insulin treatment and from control untreated mice (0). Lysates of liver (A, C) and muscle (B, D) were probed with phosphosite-specific antibodies that recognize phospho-T y r l 146/1150/1151 (3Y-P) (A, B, upper panels) or phospho-Tyr960 (960-P) (C, D, upper panels), then reprobed with anti-IR antibody (lower panels). The migration position of the 83 k D marker is shown. 76 Thus, insulin-dependent LR activation and its potential interaction with IRSs and/or She appeared to be normal in mice lacking P T P a . These findings suggest that PTPa is not a critical regulator of IR tyrosine phosphorylation in insulin-responsive tissues. 3.2.6 IRS tyrosine phosphorylation and association with PI3-K in PTPa"7" mice Although LR tyrosine phosphorylation appeared normal in PTPa-deficient settings, any undetected changes in LR activity could be reflected by alterations in downstream signaling events. Moreover, other phosphotyrosyl intermediates in the insulin signaling pathway could be direct substrates of PTPa , with consequent effects on protein-protein interactions. Therefore, the insulin-stimulated tyrosine phosphorylation of LRS proteins, that are early and central effectors of insulin signaling, and their association with PI3-K in muscle and liver lysates, were examined (low protein and l ipid contamination precluded such analysis in adipose samples). Briefly, IRS immunoprecipitates prepared from saline- or insulin-treated W T and PTPa" 7" mice were immunoblotted with anti-phosphotyrosine antibody, followed by immunoblotting with anti-PI3-K (to determine the associated PI3-K) and anti-LRS-1 antibodies. 3.2.6.1 IRS-1 tyrosine phosphorylation and association with PI3-K in PTPa"7" muscle LRS-1 is a primary effector of insulin signaling in skeletal muscle (Kido et al., 2000). Thus the phosphorylation status of LRS-1 and its association with PI3-K were investigated. Insulin induced similar extents of LRS-1 tyrosine phosphorylation in muscle of W T and PTPa" 7" mice (Fig. 3.7A-C). The absence of P T P a also did not affect insulin-stimulated LRS-1 77 6 min +/+ -/-IRS-1-P IRS-1 PI3-K S I I S S I I J - 175 \ - 175 h 83 B IRS-1-P IRS-1 PI3-K 11 min +/+ s I s s I I 175 175 83 | _ 800 ra ro £ 1 600 | 5 400 cx o T g.200 w Cc 0 6 11 6 11 1 600 * i— 500 rt $ CL C 400 •o 5 ra°S300 £ •§ 100 o CO 6 11 6 11 +/+ +/+ I -/- +/+ PI3-K 83 Figure 3.7. Insulin-stimulated IRS-1 phosphorylation and association with PI3-K in muscle tissues from wild-type and PTPa7" mice. Wild-type (+/+) and PTPa"" (-/-) mice were fasted overnight, anesthetized, and saline (S) or insulin (I, 5U) was injected into the portal vein. Muscle was removed at 6 (A), or 11 (B) min post-injection. LRS-1 immunoprecipitates from the tissue lysates were resolved by S D S -P A G E , transferred to membranes, and the upper portions of the membranes were probed for phosphotyrosine (A, B, upper panels) and reprobed for IRS-1 (A, B, middle panels). The bottom portions of the membranes were probed for PI3-K (A, B, bottom panels). Each lane represents an individual mouse. C, Bars (±S.D.) represent the amount of tyrosine phosphorylation per amount of immunoprecipitated IRS-1 (arbitrary densitometric units). D, Bars (±S.D.) represent the amount of co-immunoprecipitated PI3-K per amount of immunoprecipitated LRS-1 (arbitrary densitometric units). In C and D, the calculated ratio from saline-treated wild-type samples was taken as 100%, and all other ratios are expressed relative to this. The number of individual mouse samples is shown in brackets above the bars. Statistical analysis showed no significant differences between the values obtained from wi ld-type muscle and those from correspondingly treated PTPa" 7" muscle. E, Muscle lysates (from the 6 min saline- or insulin-treated samples) were probed for PI3-K expression. The migration positions o f molecular mass markers (kD) are shown. 78 association with PI3-K. In both W T and P T P a " muscle, an approximately 4- to 5-fold increase in protein association was detected at 6 min and declined to a ~3-fold increase over control by 11 min (Fig. 3.7A, B , and D). PI3-K expression in P T P a 7 " muscle was found comparable to that in W T muscle (Fig 3.7E). These results indicate that insulin signaling through LRS-1 in muscle is unaltered by the absence of P T P a . 3.2.6.2 IRS-1 and IRS-2 tyrosine phosphorylation and association with PI3-K in PTPa7" liver Similarly, phosphorylation of LRS-1 and its association with PI3-K were examined in liver. A comparison o f LRS-1 tyrosine phosphorylation levels between W T and P T P a 7 " control samples (saline-treated), and between W T and P T P a 7 " insulin-treated samples, revealed no statistically significant differences (p>0.05) (Fig. 3.8A-C). However, the slight increase in LRS-1 tyrosine phosphorylation in the P T P a 7 " saline-treated control liver (relative to saline-treated W T liver) resulted in a lower-fold stimulation of LRS-1 phosphorylation by insulin in the P T P a 7 " liver compared to the insulin-induced fold-stimulation in W T liver. Indeed by 10 min following insulin treatment, IRS-1 phosphorylation was returned to the control level in P T P a 7 " samples, while in W T samples it was still ~2-fold higher than the W T control. Analysis of the association of LRS-1 with PI3-K revealed a reduced insulin-stimulated interaction in P T P a 7 " liver (Fig. 3.8A, B , and D) . This was in part due to a significant 2-fold increase in insulin-independent LRS-1 and PI3-K association in control saline-treated P T P a 7 " liver versus W T liver. Thus insulin induced a more than 3-fold enhanced association over saline-treated control in W T liver at 5 min, and only a ~1.5-fold enhanced association over that in saline-treated P T P a 7 " liver. The difference was more pronounced after 10 min insulin treatment, with a 4-fold increase in LRS-1/PI3-K association 79 5 min +/+ S I 1 1 S 1 1 1 IRS-1-P • • • mm* tmm mm m» <mm «• MM IRS-1 PI3-K Mhatm -iT OMtM, — - • -vm* wv"" •ipw'W qumpp C c g 400 i losphorylal :  WT basal 300 -200 -IRS-1 pr (% oi 100 -0 -B 1 7 5 IRS-1-P 175 IRS-1 83 PI3-K D o 10 min 5 10 5 10 s s I I +/+ -/-+/+ -/-S I S I PI3-K 83 +/+ -/-S I I S I I I m— fr— k-«* «• •** i**"-* IpW '^ H?. ?** * 175 400 i co 0. -o ° 300 " ^ 2 0 0 £ | 100 o 1 0 CO 5 10 5 10 +/+ -/-Figure 3.8. Insulin-stimulated IRS-1 phosphorylation and association with PI3-K in liver tissues from wild-type and PTPa7" mice. Wild-type (+/+) and P T P a (-/-) mice were fasted overnight, anesthetized, and saline (S) or insulin (I, 5U) was injected into the portal vein. Liver was removed at 5 (^4), or 10 (B) min post-injection. IRS-1 immunoprecipitates from the tissue lysates were resolved by SDS-P A G E , transferred to membranes, and the upper portions o f the membranes were probed for phosphotyrosine (A, B, upper panels) and reprobed for IRS-1 (A, B, middle panels). The bottom portions of the membranes were probed for P I 3 - K (A, B, bottom panels). Each lane represents an individual mouse. C, Bars (±S.D.) represent the amount of tyrosine phosphorylation per amount of immunoprecipitated IRS-1 (arbitrary densitometric units). D, Bars (±S.D.) represent the amount of co-immunoprecipitated P I 3 - K per amount of immunoprecipitated IRS-1 (arbitrary densitometric units). In C and D, the values shown were calculated after densitometric scanning of the autoradiographs shown in A and B, and from the results of identical experiments that are not shown. The calculated ratio from saline-treated wild-type samples was taken as 100%, and all other ratios are expressed relative to this. The number o f individual mouse samples is shown in brackets above the bars. The asterisks indicate significant differences between the indicated PTPa" 7" samples and the corresponding sample from the wild-type liver (*, p<0.05). E. Liver lysates (from the 5 min saline- or insulin-treated samples) were probed for P I 3 - K expression. 80 in W T liver over the saline-treated W T samples, but a much lesser 1.3-fold increase in PTPa" 7" liver over saline-treated control PTPa" 7" samples. Nevertheless, independent o f the increased control IRS-1/PI3-K interaction in the PTPa-deficient liver, there was still a small but significant reduction in the level of the IRS-1/PI3-K complex in PTPa" 7" liver at the later time of 10 min when directly compared to that in W T liver. PI3-K expression was confirmed to be unaltered in the absence o f P T P a (Fig. 3.8E). These results indicate that the insulin-stimulated interaction of IRS-1 and PI3-K in liver either is reduced in extent or occurs over a shorter time in the absence of P T P a . IRS-2 plays an important role in regulating hepatic insulin action (Rother et al., 1998). Thus, features of the IRS-2 responses to insulin were investigated in PTPa' 7 " liver. A s observed in liver with IRS-1, insulin stimulation enhanced the phosphorylation of IRS-2 and its association with PI3-K. However, no significant differences were detected in insulin-induced IRS-2 tyrosine phosphorylation or interaction with PI3-K between W T and PTPa" 7" liver (Fig. 3.9). This indicates that P T P a ablation has a specific effect on insulin-induced IRS-1 responses that are not seen with IRS-2, albeit the latter is a closely related family member to IRS-1. 3.2.7 Insulin-stimulated Akt and MAPK activation in liver, muscle, and adipose tissues of PTPa"7" mice Insulin, acting via the IR and the IRS proteins, stimulates two major protein kinase-dependent signaling pathways, the PI3-K/Akt pathway and the R a s / M A P K (p42/44) pathway (Saltiel and Kahn, 2001). Activation of these signaling cascades is responsible for metabolic and mitogenic effects o f insulin action. Alterations in insulin signaling that occur upstream of 81 A 5 min B 10 min +/+ -/- +/+ -/-S 1 1 1 S 1 1 1 S I I S S I I I IRS-2-P < • « « « » H i - 175 IRS-2-P IRS-2 4 - 175 IRS-2 PI3-K m - 83 PI3-K «•»> mm mm **» **• h 175 83 c 400 o | | 300 | f &> 200 Q . O CN g? 100 rr CO w CNJ C (AS 5 10 5 10 300 -, 250 200 -I 150 100 50 0 5 10 5 10 +/+ +/+ Figure 3.9. Insulin-stimulated IRS-2 phosphorylation and association with PI3-K in liver tissues from wild-type and PTPa"'" mice. Wild-type (+/+) and PTPa (-/-) mice were fasted overnight, anesthetized, and saline (S) or insulin (I, 5U) was injected into the portal vein. Liver was removed at 5 (A), or 10 (B) min post-injection. IRS -2 immunoprecipitates from the tissue lysates were resolved by SDS-PAGE, transferred to membranes, and the upper portions of the membranes were probed for phosphotyrosine (A, B, upper panels) and reprobed for IRS -2 (A, B, middle panels). The bottom portions of the membranes were probed for P I 3 - K (A, B, bottom panels). Each lane represents an individual mouse. C, Bars (±S.D.) represent the amount of tyrosine phosphorylation per amount of immunoprecipitated IRS -2 (arbitrary densitometric units). D, Bars (±S.D.) represent the amount of co-immunoprecipitated P I 3 - K per amount of immunoprecipitated LRS-2 (arbitrary densitometric units). In C and D, the calculated ratio from saline-treated wild-type samples was taken as 100%, and all other ratios are expressed relative to this. The number of individual mouse samples is shown in brackets above the bars. Statistical analysis showed no significant differences between the values obtained from wild-type liver and those from correspondingly treated PTPa - /" liver. The migration positions of molecular mass markers (kD) are shown. 82 or at A k t or M A P K may be manifested by the altered activation o f these kinases, as is observed with the enhanced insulin-stimulated A k t activation observed in P T P IB-depleted mouse liver (Zinker et al., 2002). Insulin-dependent activation o f Ak t was measured by monitoring A k t Ser473 and Thr308 phosphorylation, whereas insulin-dependent phosphorylation o f M A P K was determined by monitoring the phosphorylation o f E r k l / 2 at Thr202 and Tyr204. This was carried out by using phosphosite-specific Ak t and E r k l / 2 antibodies (Figs 3.10-3.12). 6 min B 11 min +/+ -/- +/+ S I I S S I I S S I I I s Akt-P Akt 62 62 Akt-P Akt J - 62 T- 62 +/+ +/+ S I I I S I I I S I I I s MAPK-P MAPK h 47.5 h 47.5 MAPK-P MAPK j - 47.5 > 47.5 Figure 3.10. Insulin-stimulated Akt and MAPK activities in muscle tissues from wild-type and PTPa"7" mice. Wild-type (+/+) and P T P a (-/-) mice were fasted overnight, anesthetized, and saline (S) or insulin (I, 5U) was injected into the portal vein. Muscle was removed at 6 (A, Q and 11 (B, D) min post-injection. Lysates were prepared and probed for phospho-Ser473-Akt (A, B, upper panels), A k t {A, B, lower panels), phospho-Thr202/Tyr204-MAPK (p42/44) (C, D, upper panels) and M A P K (p42/44) (C, D, lower panels). The migration positions of molecular mass markers (kD) are shown. 83 Akt-P Akt 5 min +/+ s I I i s I I I 62 62 B Akt-P Akt-308P Akt +/+ 10 min s i i s s I h 62 h 62 r- 62 MAPK-P MAPK +/+ -/-S I I s s "•>—» h 47.5 47.5 MAPK-P MAPK +/+ S I I S S I h 47.5 h 47.5 Figure 3.11. Insulin-stimulated Akt and MAPK activities in liver tissues from wild-type and PTPa7" mice. Wild-type (+/+) and P T P a (-/-) mice were fasted overnight, anesthetized, and saline (S) or insulin (I, 5U) was injected into the portal vein. Liver was removed at 5 (A, C) and 10 (B, D) min post-injection. Lysates were prepared and probed for phospho-Ser473-Akt (A, B, upper panels), phospho-Thr308-Akt (B, middle panel), Akt (A, B, lower panels), phospho-Thr202/Tyr204-MAPK (p42/44) (C, D, upper panels) and M A P K (p42/44) (C, D, lower panels). The migration positions o f molecular mass markers (kD) are shown. Insulin treatment dramatically stimulated the phosphorylation o f A k t in liver, muscle, and adipose tissues o f W T mice (Figs. 3.10-3.12B). Furthermore, A k t phosphorylation was sustained for at least 10 to 12 min, indicating that the A k t response to insulin is robust and prolonged. The insulin-dependent activation of Akt , as measured bymonitoring Akt Ser473 phosphorylation, was not detectably different in P T P a 7 " liver, muscle, or adipose tissue at earlier (5, 6, and 7 min, respectively) (Figs. 3.10-3.12A) or later (10, 11, and 12 min, respectively) (Figs. 3.10-3.12B) times from that in the W T mice. In one set o f experiments in 8 4 liver at 10 min the phosphorylation of Akt at another site that is linked to its activation, Thr308 (Alessi and Cohen, 1998), was also examined. However, no differences were detected between W T and P T P a 7 " samples (Fig. 3.1 I B , middle panel). A k t phosphorylation per unit Ak t protein could not be reliably quantitated, because the initial probing of the membranes with anti-phospho-Akt antibody sometimes appeared to (especially in cases of high initial signal), even after its removal, partially block the efficient interaction o f the subsequently used anti-Akt antibody with A k t (as shown in Figs. 3.1 OA and 3.12B). 7 min B 12 min +/+ S I I I S I I +/+ S I I S I I I Akt-P Akt J— 62 r- 62 Akt-P Akt h 62 h 62 +/+ +/+ S I I I S I I I M A P K - P M A P K S I I S I I 47.5 h 47.5 M A P K - P M A P K j - 47.5 l - 47.5 Figure 3.12. Insulin-stimulated Akt and MAPK activities in adipose tissues from wild-type and PTPa7" mice. Wild-type (+/+) and P T P a (-/-) mice were fasted overnight, anesthetized, and saline (S) or insulin (I, 5U) was injected into the portal vein. Adipose tissues were removed at 7 (A, Q and 12 (B, D) min post-injection. Lysates were prepared and probed for phospho-Ser473-Akt (A, B, upper panels), Ak t (A, B, lower panels), phospho-Tnr202/Tyr204 M A P K (p42/44) (C, D, upper panels) and M A P K (p42/44) (C, D, lower panels). The migration positions of molecular mass markers (kD) are shown. 8 5 Insulin-induced M A P K activation was robust in muscle and adipose tissues. Insulin-dependent M A P K activation appeared similar in W T and PTPa" 7" muscle and adipose tissues at earlier (6 and 7 min, respectively) (Figs. 3.10 and 3.12C) or later (11 and 12 min, respectively) (Figs. 3.10 and 3.12D) times. In livers of both W T and PTPa" 7" mice, insulin-dependent M A P K activation was almost undetectable, apparently due to high basal M A P K phosphorylation in this tissue (Fig. 3.11C and D). Together, these findings reveal that mice lacking P T P a exhibit no obvious abnormalities in insulin-induced activation of the PI3-K/Akt and R a s / M A P K pathways. 3.3 Discussion 3.3.1 Normal insulin sensitivity in PTPa"7" mice The absence of P T P a did not result in altered insulin-induced tyrosine phosphorylation of IR or IRSs in any of the three insulin-sensitive tissues or in altered key parameters of glucose homeostasis. PTPa" 7" mice had normal body weight and normal circulating levels of insulin and glucose in random fed and fasted states, suggesting that insulin production, glucose metabolism, and food consumption and expenditure appears normal in these animals. Furthermore, these mice exhibited a blood glucose clearance efficiency in response to glucose load similar to that of W T animals. This suggests that glucose-induced insulin secretion and glucose uptake into muscle and adipose tissues are unaltered in PTPa" 7" mice. In response to insulin elevation, PTPa" 7" mice did not exhibit lower blood glucose nor did they remain hypoglycemic longer, indicating normal insulin sensitivity compared to W T control 86 animals. In contrast, the lack of PTP1B, a negative regulator of insulin signaling, results in increased insulin sensitivity as manifested by lower glucose and insulin levels in the fed state, enhanced blood glucose clearance, and a prolonged hypoglycemic state (Elchebly et al., 1999). Altogether, this clearly suggests that P T P a does not perform an essential role in the regulation of glucose homeostasis in mice. M i c e lacking P T P a also did not exhibit any fundamental molecular alterations in response to insulin. These mice had normal IR tyrosine phosphorylation in response to insulin as compared to W T animals. Furthermore, site-specific phosphorylation o f the IR, which is important for IRS and She binding as well as IR kinase activity, was investigated and was found to be unaltered in the absence o f P T P a . In addition, P T P a 7 " mice did not exhibit any alterations in IRS-1 or IRS-2 phosphorylation and the association of each with PI3-K in muscle and liver, respectively. High level of IRS-1 phosphorylation and association with PI3-K in the basal unstimulated state in P T P a 7 " liver partially contributed to the reduced insulin-stimulated association of these two proteins (this is discussed further in section 3.3.2). Nevertheless, this difference did not transduce into any alterations in activation of downstream signaling molecules including Ak t and M A P K in either the unstimulated or insulin-stimulated states. Altogether, these findings suggest that P T P a is neither a regulator o f IR tyrosine phosphorylation nor insulin signaling. Several cell culture studies also support this conclusion that P T P a is not a regulator of IR phosphorylation nor insulin signaling. Transient or stable expression of P T P a in IR-expressing G H 4 pituitary cells had no effect on insulin-induced IR, IRS-1 or She tyrosine phosphorylation, or IR kinase activity (Jacob et al., 1998). In another experiment, reduction o f P T P a expression to an undetectable level using antisense oligonucleotide in 3T3-L1 87 adipocytes did not result in any alterations in LR and LRS tyrosine phosphorylation upon insulin stimulation or dephosphorylation following insulin withdrawal. This was associated with unaltered Erk2 activation and D N A synthesis (Arnott et al., 1999). L u et al. (2002) revealed that elevation of PTPa expression did not influence insulin-induced LR (or IGF-1R) tyrosine phosphorylation in L6 myoblasts. The results described in this chapter clearly indicate that P T P a is not a negative regulator of insulin signaling and does not perform an essential role in mediating the physiological action of insulin. A potential role for P T P a in insulin signaling has been previously proposed in the scientific literature, however the results from the present study of the physiological action of P T P a in this key metabolic regulatory system have negatively resolved this question. 3.3.2 Altered IRS-1 phosphorylation and association with PI3-K in PTPa"7" liver The enhanced association of LRS-1 and PI3-K in control non-insulin-treated PTPa"7" liver partially contributed to the reduced effect o f insulin in stimulating association o f these two proteins (this was not observed in non-insulin treated muscle nor with LRS-2/PI3-K association in control PTPa" 7" liver). A similar but converse insulin-independent effect was reported in adipose cells upon overexpression of PTPa , whereupon the cell surface level o f the glucose transporter G L U T 4 was reduced when compared to control transfected cells (Cong et al., 1999). This was attributed to the possible attenuation of minor signaling by unoccupied insulin receptors. Likewise, it is possible that P T P a performs such a role in liver by maintaining basal phosphorylation of LRS-1. This is consistent with the observation o f a small but not significant increase in insulin-independent LRS-1 tyrosine phosphorylation in 88 PTPa"'" liver compared to W T liver. Similar to this role of P T P a in controlling phosphorylation of IRS-1 prior to insulin stimulation, it has been recently reported that P T P a also functions in regulating the tyrosine phosphorylation of various cellular proteins in resting thymocytes prior to T C R activation in response to antigen (Maksumova et al., 2005). In addition, mice lacking P T P a had a lesser or more rapidly attenuated insulin-stimulated interaction between IRS-1 and PI3-K in PTPa" 7" liver. Normal insulin-stimulated IRS-1 phosphorylation but reduced IRS-1/PI3-K association and PI3-K activity was previously observed in LAR-deficient liver. This is proposed to account for a complex phenotype o f secondary insulin resistance that arises from compensation for the initial insulin sensitivity (Ren et al., 1998). Subsequently, muscle-specific overexpression of L A R was also reported to reduce insulin-dependent association of PI3-K with IRS-1 without altering IRS-1 tyrosine phosphorylation (Zabolotny et al., 2001) and was linked to insulin resistance. This suggests that P T P a and L A R exhibit similar effects on IRS-1 and PI3-K association. However, the PTPa-regulated subpopulation of PI3-K is not critical for insulin action, as discussed above in section 3.3.1. 3.3.3 Understanding the role of PTPa in insulin signaling in other systems This mouse study and other cell culture studies (Jacob et al., 1998; Arnott et al., 1999) indicate that P T P a is not a critical regulator of insulin signaling (see section 3.3.1). However, some other studies in cultured cells suggest a potential role for P T P a in the regulation of insulin signaling. Expression of P T P a in B H K - I R cells (baby hamster kidney cells engineered to stably express the IR) reduced insulin-induced growth inhibition and attachment of these cells (Moller et al., 1995), whereas expression of P T P I B , a physiological 89 negative regulator of insulin signaling in mice, did not. This suggests that in this system P T P a counteracts insulin-induced cell detachment through actions independent of insulin signaling that promote cell attachment, similar to the increased substrate adhesion induced by P T P a expression i n A431 cells (Harder et al., 1998). P T P a expression was associated with not only reduced tyrosine phosphorylation of LR but also of other cellular proteins in B H K - L R cells (Lammers et al., 1998), indicating what is l ikely a non-specific action of overexpressed P T P a in this system. This likelihood is supported by the parallel finding that the expression of CD45 , a receptor PTP that is uniquely expressed in haematopoietic cells, in B H K - L R cells also caused a reduction in LR phosphorylation. Thus, this study system likely permits artificial effects of non-physiological protein overexpression. Transient expression of P T P a in rat adipose cells, cells that are naturally responsive to insulin, caused a reduction in cell surface G L U T 4 and in insulin-mediated translocation o f G L U T 4 to the cell surface (Cong et al., 1999). Notably, only 5% of the cells were transfected, thus the P T P a level in these few cells far exceeded a physiological level. In addition, the authors were unable to show an effect of P T P a overexpression on LR and LRS-1 tyrosine phosphorylation. Furthermore, P T P a overexpression in L6-myoblasts enhanced insulin-induced D N A synthesis and enhanced differentiation of these cells (Lu et al., 2002). This effect of P T P a appeared to be exerted through a signaling pathway, independent of LR signaling, since insulin-induced LR tyrosine phosphorylation in these cells was normal (Lu et al., 2002). Inconsistent results arise when studying the function o f P T P a in cultured cells. Thus heterologous P T P and/or substrate expression, inappropriate cell type, and the culture setting are all factors that limit the ability of the above studies to determine the physiological function of P T P a in insulin action. In view of the lack of function of P T P a in insulin 90 signaling demonstrated by studies of PTPa-nul l mice, the results of the above cell culture-based studies of heterologous P T P a that suggest a role of this P T P in modulating insulin action likely represent artifactual effects of non-physiological protein overexpression or extraneous effects o f P T P a via an intermediate molecule(s) that are independent o f insulin signaling. 3.3.4 Summary While it cannot be ruled out that other PTPs may compensate for the absence o f P T P a to maintain normal insulin action, this study indicates that P T P a does not play an essential role in the physiological regulation of insulin action. M i c e lacking P T P a appear normal and do not exhibit any significant changes in insulin signaling molecules and glucose homeostasis. This suggests that the effect of P T P a elevation or ablation in cultured cells is not a direct effect o f P T P a in insulin signaling but a secondary effect, for instance an effect o f P T P a exerted through it's insulin-independent action as an upstream activator of sre (Pallen, 2003). P T P a has been proposed to be a regulator o f insulin signaling, despite controversial results from cell culture studies. M y study provides clear evidence that P T P a is not a major regulator of insulin signaling and does not perform an essential role in controlling glucose homeostasis in mice. 91 4 NMDAR SIGNALING IN PTPa MICE 4.1 Introduction and rationale The N M D A R , a ligand-gated ion glutamate receptor, plays an important role in a wide variety of neuronal phenomena (McBain and Mayer, 1994; Riedel et al., 2003). Functional N M D A R s mainly consist of N R 1 and N R 2 (A-D) subunits (Cull-Candy et al., 2001) . One mechanism by which N R 2 subunits modulate N M D A R activity is through changes in their tyrosine phosphorylation state. Four of five neuronal S F K s ; src, fyn, yes, and lyn, but not lck, are associated with the N M D A R (Kalia and Salter, 2003). The SFKs src and fyn have been shown to regulate the phosphorylation of the N R 2 A and N R 2 B subunits (Suzuki and Okumura-Noji, 1995; Kohr and Seeburg, 1996; Nakazawa et al., 2001; Yang and Leonard, 2001). P T P a activates src and fyn by dephosphorylating their inhibitory C-terminal tyrosine residue (Ponniah et al., 1999; Su et al., 1999) and associates with the N M D A R (Lei et al., 2002) . This suggests that P T P a may regulate N M D A R tyrosine phosphorylation directly as a phosphatase or indirectly via its upstream action on src and fyn. A positive role for P T P a in regulating NMDAR-media ted currents in a SFK-dependent manner is suggested from cell culture studies (Lei et al., 2002). More importantly, mice lacking P T P a display defects in processes linked to N M D A R function (Petrone et al., 2003; Skelton et al., 2003). These lines o f evidence suggest that P T P a plays a physiological role in mediating N M D A R tyrosine phosphorylation and function, most likely via its upstream action on SFKs . 92 To address the molecular mechanisms that account for the defects in PTPa"'" mice, N M D A R tyrosine phosphorylation status and S F K activities in synaptosomal fractions (enriched with N M D A R ) o f wild-type (WT) and PTPa-deficient mice were evaluated and compared. Furthermore, N M D A R signaling molecules that are affected by P T P a ablation were also identified. 4.2 Results 4.2.1 Localization of PTPa, SFKs, Pyk2, and NMDAR subunits in Triton X-100 solubilized synaptosomes The N M D A R and multiple associated signaling proteins are highly enriched in detergent-insoluble post-synaptic densities (Moon et al., 1994; Husi et al., 2000). A s an initial step in determining the functional effects of P T P a upon N M D A R tyrosine phosphorylation, the physical localization of P T P a , N M D A R subunits, and non-receptor tyrosine kinases that can regulate N M D A R phosphorylation, was investigated in detergent fractionated synaptosomes. In addition, the effect of P T P a ablation on the detergent solubility and developmental expression of synaptosomal N M D A R subunits and non-receptor tyrosine kinases was determined. Crude synaptosomes (P2 fraction) were prepared from whole brains of wild-type (WT) and PTPa - / " mice, solubilized in 1% Triton X-100 as described in Materials and Methods, section 2.2.5.3, and probed for the expression of the proteins of interest (Fig. 4.1 A ) . P T P a and N R 1 , N R 2 A , and N R 2 B subunits were detectable in both Triton X-100-soluble and -insoluble fractions prepared from animals at postnatal day 7 (P7) to day 60 (P60) ages. 93 Triton X-100 soluble P7 P14 P30 P60 Triton X-100 insoluble P7 P14 P30 P60 +/+ -/- +/+ -/- +/+ -/- +/+ -/-P T P a NR2A NR2B NR1 PSD-95 fyn sre yes lyn Pyk2 Actin +/+ -/- +/+ -/- +/+ -/- +/+ -/-- 175 - 175 \ - 175 175 83 175 83 62 62 62 175 83 h 47.5 Soluble ® _a o CM O CO o o "3" 05 • • c 175 - P T P a B P60 P T P a NR2A NR2B NR1 PSD-95 fyn sre yes lyn Pyk2 Actin +/+ -/- +/+ -/-175 175 83 175 h 83 h 62 1- 62 175 - 83 47.5 Figure 4 .1 . Developmental expression of PTPa, NMDAR, PSD-95, and tyrosine kinases in detergent-fractionated crude synaptosomes of wild-type and PTPa"7" mice. A, Triton X - 1 0 0 soluble and insoluble fractions prepared from synaptosomes of W T (+/+) and PTPa" 7" (-/-) mice at postnatal days 7, 14, 30, and 60 (P7-60) were immunoprobed for developmental expression of the indicated proteins. The relative protein expression within either the detergent-soluble and insoluble samples can be compared, but not between detergent-soluble and insoluble fractions due to unequal sample and loading amounts. B, Relative protein expression between Triton X - 1 0 0 soluble (S) and insoluble (I) fractions was determined in P 6 0 samples after equal sample fractionation and loading. C, To more accurately compare the amount of P T P a present in the Triton X - 1 0 0 soluble and insoluble synaptosomal fractions, the PTPa-enriched detergent soluble fraction was diluted 1:10, 1:20, 1:30, and 1:40 with lysis buffer (vol/vol) and equal volumes of the diluted samples and the undiluted insoluble fraction were probed for P T P a . The results indicate that - 2 . 5 % of total P T P a is present in the Triton X - 1 0 0 insoluble fraction. In A-C, the migration positions of molecular mass markers (kD) are shown. P T P a was expressed in the Triton X-100-soluble and -insoluble fractions at all ages. Increased expression of N R 2 A , N R 2 B , N R 1 , and PSD-95 in the Triton X-100-insoluble fractions was observed post-P7, consistent with other reports (Sheng et al., 1994; Petralia et al., 2005). Four (fyn, src, yes, and lyn) of five S F K s that are reported to be expressed in the C N S (Thomas and Brugge, 1997) were present in Triton X-100-soluble and -insoluble fractions of the synaptosomal preparations. The fifth C N S S F K , lck, was not reliably detected in these fractions; although as described below, it could be immunoprecipitated from Triton X-100-insoluble synaptosomal fractions. Another tyrosine kinase, Pyk2, was present in both detergent-soluble and -insoluble fractions, with increasing expression detected in the latter fractions with age as reported (Menegon et al., 1999). N o differences were observed in N M D A R subunit, S F K , or Pyk2 expression levels or distribution between W T and PTPa" 7" mice of any age examined. The protein expression shown in Fig . 4.1 A does not reflect the relative protein amounts in Triton X-100-soluble versus -insoluble fractions, as these two types of fractions 95 were analyzed in amounts that enabled protein visualization rather than equivalency. To examine the relative expression levels in these fractions, P60 synaptosome preparations were fractionated into equal volumes of detergent-soluble and -insoluble material and equal volumes of these fractions were assessed by immunoprobing (Fig. 4 . IB) . Consistent with previous reports (Blahos and Wenthold, 1996; Perez and Bredt, 1998), the majorities of N R 1 , N R 2 A , N R 2 B , and PSD-95 were found in the insoluble fraction in mature animals. More fyn and yes were found to be Triton X-100 insoluble, whereas more sre, lyn, and Pyk2 were localized in the soluble fraction. To determine the amount of P T P a in the insoluble fraction relative to that in the soluble fraction, equal volumes o f undiluted insoluble fraction and soluble fraction diluted 1:10, 1:20, 1:30, and 1:40 with lysis buffer (vol/vol) were analyzed for P T P a level. Comparatively low amounts of PTPa were present in the insoluble fraction, this was estimated to be - 2-5% o f the total synaptosomal P T P a (Fig 4.1C). In summary, a small portion of P T P a is associated with the N M D AR-enriched Triton X-100-insoluble fraction of synaptosomes. The absence of P T P a did not result in any changes in expression or in distribution of the N M D A R , SFKs , and the cytosolic P T K Pyk2 at any stage of mouse development examined. 4.2.2 Altered protein tyrosine phosphorylation in PTPa"'" synaptosomal fractions A s a phosphatase, P T P a plays a role in controlling protein tyrosine phosphorylation status. Thus, P T P a ablation may lead to changes in protein phosphorylation which could be detected by probing the crude synaptosomal fractions prepared from W T and P T P a 7 " mice with anti-phosphotyrosine antibodies. The absence of P T P a resulted in reduced phosphorylation o f Triton X-100-insoluble proteins that migrated at -180 k D and -120 k D , 96 with the former detectable from P14 onwards, and the latter from P30 onwards (Fig. 4.2, top right panel). In contrast, in the same detergent-insoluble fractions the absence o f P T P a also resulted in the enhanced tyrosine phosphorylation, readily observed from P14 onwards, of a protein(s) that migrated at - 60 k D (Fig. 4.2, top right panel), about the same size as SFKs . Enhanced phosphorylation of a - 60 k D protein(s) was also detectable in Triton X-100-soluble fractions (Fig. 4.2, top left panel), although no other PTPa-dependent changes in tyrosine phosphorylation were observed in these fractions. Equivalent actin amounts were present in the paired W T and P T P a 7 " samples (Fig. 4.2), indicating that the observed changes in protein tyrosine phosphorylation were not due to unequal sample loading. The observations suggest that P T P a dephosphorylates, directly or indirectly, a protein(s) o f -60 k D that is present in both Triton X-100-soluble and -insoluble synaptosomal fractions, and mediates the phosphorylation o f a protein(s) o f - 1 8 0 k D that is localized to or predominates in detergent-insoluble synaptosomal fractions. These initial findings are consistent with the working hypothesis that P T P a acts in synaptosomes to dephosphorylate and activate SFKs ( M w -60 kD) that in turn phosphorylate tyrosine residues in the N M D A R 2 A and/or 2B subunits ( M w -180 kD). In addition, an unknown protein(s) of -120 k D appears to be a target o f the putative P T P a - S F K signaling pathway. 4.2.3 Altered SFK phosphorylation in PTPa 7' synaptosomal fractions 4.2.3.1 SFK hyperphosphorylation at Tyr527 in PTPa7" synaptosomes Previous studies have demonstrated that brain src/fyn kinase activities in P T P a 7 " mice are reduced to about half of those of W T animals (Ponniah et al., 1999; Su et al., 1999). This is concomitant with elevated phosphorylation of the regulatory C-terminal tyrosine residue of 97 Triton X-100 soluble Triton X-100 insoluble P7 P14 P30 P60 P7 P14 P30 P60 +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/-175 - fm^m^ f •4-83 -62 -47.5 -^MHM^ AjBJiHR^  WMMMM M i ta-4 -4MMNP MM •4-pTyr 4 -62 -^ _ src-pTyr416 62 - mMHtBmmmmmm src-pTyr527 47.5 - Actin Figure 4.2. Phosphotyrosyl proteins and SFK tyrosine phosphorylation status in detergent-fractionated crude synaptosomes of wild-type and PTPa"7" mice. Triton X-100 soluble and insoluble fractions prepared from synaptosomes of wild-type (+/+) and PTPa" 7" (-/-) mice at postnatal days 7, 14, 30, and 60 (P7-60) were immunoprobed for protein tyrosine phosphorylation using anti-phosphotyrosine antibody (upper panels). The arrows indicate differences detected between wild-type and PTPa" 7" samples. These fractions were also probed with phosphosite-specific antibodies to the autophosphorylation site of S F K s (anti-src pTyr416) and to the inhibitory C-terminal phosphorylation site of S F K s (anti-src pTyr527) (middle panels), and with anti-actin antibody (bottom panels). The migration positions of molecular mass markers (kD) are shown on the left. src (Tyr527) and fyn. Fu l l S F K activation requires autophosphorylation at another tyrosine residue (Tyr416 in src) in the activation loop region (Brown and Cooper, 1996; X u et al., 1999). However, the phosphorylation status of brain src and fyn at this site in PTPa" 7" brain has not been reported. Phosphosite-specific antibodies towards src phosphoTyr527 and phosphoTyr416 were used to determine i f the -60 k D protein(s) observed exhibits PTPa-dependent alterations in phosphorylation indeed co-migrates with S F K s , and i f so, which 98 tyrosine residue o f S F K s was hyperphosphorylated in the synaptosomal fractions. These antibodies recognize equivalent phosphotyrosyl residues in other S F K s such as fyn and yes (Reinehr et al., 2004). Both antibodies recognized a protein(s) that co-migrated with the anti-phosphotyrosine-reactive ~60 k D protein(s). Phosphorylation at Tyr416 (or the equivalent residue in other SFKs) did not differ in the Triton X-100-soluble and -insoluble fractions between W T and PTPa" 7" animals at any age (Fig. 4.2, upper middle panels). However, phosphorylation at Tyr527 (or its equivalent tyrosine residue in other SFKs) was enhanced in the detergent-soluble and -insoluble fractions of PTPa" 7" animals at the same ages as was the enhanced phosphorylation of the -60 k D band(s) (Fig. 4.2, lower middle panels). This indicates that the phosphotyrosyl 60 k D band is likely to represent one or more SFKs , and that enhanced phosphorylation of one or more S F K at the C-terminal tyrosine site is observed in the absence of PTPa . 4.2.3.2 Four SFKs are hyperphosphorylated in PTPa"7" synaptosomal fractions There are five S F K s expressed in the C N S and the phosphosite-specific antibodies recognize phosphotyrosyl residues in not only src but also other S F K s such as fyn and yes. To identify which S F K s exhibited enhanced C-terminal tyrosine phosphorylation in PTPa" 7" synaptosomes, individual SFKs were immunoprecipitated from adult animals (P60) and immunoprobed for phosphotyrosine. Fyn, src, and yes displayed significantly increased tyrosine phosphorylation in Triton X-100-soluble (2.3-, 1.6-, and 2.5-fold increases, respectively) and -insoluble (1.9-, 1.9-, and 2.6-fold increases, respectively) fractions from PTPa" 7" samples compared to W T samples (Figs. 4.3A and C, and 4.4A). In accord with enhanced overall S F K tyrosine phosphorylation detected in the synaptosomal fractions, this was confirmed to be due to enhanced phosphorylation at the C-terminal regulatory residue, as 99 A s s I Figure 4.3. Altered tyrosine phosphorylation of the synaptosomal SFKs sre and fyn in PTPa"7" mice. Sre and fyn were individually immunoprecipitated from Triton X-100 soluble (S) and insoluble (I) fractions prepared from synaptosomes of wild-type (+/+) and PTPa"7" (-/-) mice at postnatal day 60. A, B, Fyn and C, D, sre immunoprecipitates were probed for phosphotyrosine content (A, C, upper panels) or with a phosphosite-specific antibody that recognizes sre phospho-Tyr416 or the equivalent autophosphorylation site in fyn (Y416P) (B, D, upper panels), and with antibody to the specific kinase that was immunoprecipitated (lower panels). The bar graphs to the right show the amount of phosphotyrosine per amount of immunoprecipitated kinase (±S.D.), as determined by densitometric scanning o f the results of several independent experiments (n is shown in brackets above each graph). Gray bars represent wild-type (+/+) samples and are set at 1 unit, and the black bars represent PTPa" 7" (-/-) samples with the units expressed relative to those o f the W T samples. Asterisks note a significant difference (p< 0.005) between wild-type and PTPa" 7" samples. 100 A s s C s i +/+ -/- +/+ -/- _ (3) 1 l « i Figure 4.4. Tyrosine phosphorylation of the synaptosomal SFKs yes, lck, and lyn in PTPa"'" mice. Yes, lck, and lyn were individually immunoprecipitated from Triton X-100 soluble (S) and insoluble (I) fractions prepared from synaptosomes o f wild-type (+/+) and PTPa"'" (-/-) mice at postnatal day 60. A, Yes, B, lck, and C, lyn immunoprecipitates were probed for phosphotyrosine content (upper panels) and with antibody to the specific kinase that was immunoprecipitated (lower panels). The bar graphs to the right show the amount of phosphotyrosine per amount of immunoprecipitated kinase (±S.D.), as determined by densitometric scanning («=3). Gray bars represent wild-type (+/+) samples and are set at 1 unit, and the black bars represent PTPa"'" (-/-) samples with the units expressed relative to those of the wild-type samples. Asterisks note significant differences (*, p< 0.005; **, p< 0.05) between wild-type and PTPa"'" samples. The migration positions of molecular mass markers (kD) are shown. 101 probing sre or fyn immunoprecipitates with anti-src phosphoTyr416 antibody demonstrated no detectable differences in activation loop tyrosine phosphorylation o f these kinases in the P T P a 7 " samples relative to that of the kinases from W T samples (Fig. 4.3B and D). Although lck could not be detected in synaptosomal fractions by probing with anti-lck antibody, low amounts of lck could be immunoprecipitated from Triton X-100 insoluble fractions, and the phosphotyrosine content of lck from P T P a 7 " samples was 2-fold higher than that of lck from W T samples (Fig. 4.4B). In contrast to the altered tyrosine phosphorylation of fyn, sre, and yes, the lack of P T P a did not affect lyn tyrosine phosphorylation in either the Triton X-100 -soluble or -insoluble fractions (Fig. 4.4C). In section 4.2.3.1, enhanced tyrosine phosphorylation of S F K s in P T P a 7 " mice was confirmed to be specific for the tyrosine 527 residue in the C-terminal tail. Together, these results indicate that four S F K s (fyn, sre, yes, and lck) are hyperphosphorylated at the regulatory C-terminal residue in P T P a 7 " synaptosomal fractions, and the tyrosine phosphorylation status of a fifth S F K , lyn, remains unaltered upon P T P a ablation. 4.2.4 NR2A and NR2B tyrosine phosphorylation is reduced in PTPa"7" synaptosomes The N M D A R 2 A and 2B subunits are S F K substrates (Suzuki and Okumura-Noji, 1995; Tezuka et al., 1999; Cheung and Gurd, 2001; Nakazawa et al., 2001; Yang and Leonard, 2001). The -180 k D phosphotyrosyl protein(s) detected in Triton X-100 insoluble synaptosomal fractions co-migrated with the N M D A R 2 A and 2B subunits (Fig 4.2). Furthermore, the reduced phosphorylation of the 180 k D protein(s) that was apparent in P T P a 7 " Triton X-100-insoluble fractions correlates with the reduced S F K activity expected upon the observed increased C-terminal tyrosine phosphorylation of SFKs . To ascertain i f the 102 tyrosine phosphorylation status of these subunits was indeed altered by the ablation o f PTPa , N R 2 A and N R 2 B were immunoprecipitated from the PI4 , P30, and P60 fractions and probed with anti-phosphotyrosine antibody. The low amount o f the N M D A R in the P7 fractions precluded such analysis in these samples. N R 2 A tyrosine phosphorylation was significantly reduced in all PTPa" 7" samples compared to W T samples (Fig. 4.5A). Similar reduced levels o f N R 2 B tyrosine phosphorylation were detected in PTPa" 7" samples (Fig. 4.5B). P14 P30 P60 B +/+ ./- +/+ -/- +/+ -/-175 - — — 175 - i t i « M , i M — c P14 P30 P60 +/+ -/- +/+ -/- +/+ -/-100 NR2A-P < > 60 CM 0i ° 40 -\ NR2A 175 -A 175 - NR2B ~ 20 0 NR2B-P °r t CO > (4) (4) P14 P30 P60 (5) (6) P14 P30 P60 Figure 4.5. Reduced tyrosine phosphorylation of NR2A and NR2B in synaptosomal fractions of PTPa"7" mice. A, N R 2 A and C, N R 2 B were immunoprecipitated from Triton X-100 insoluble synaptosomal fractions of wild-type (+/+) and PTPa"7" (-/-) mice at postnatal days 14, 30, and 60 (PI4-60), and probed for phosphotyrosine (top panels) and for N R 2 A or N R 2 B as shown (bottom panels). The results o f several such experiments were quantified by densitometry and are shown in the bar graphs in B, N R 2 A ; and D, N R 2 B . The bars represent the amount of phosphotyrosine per amount of N R 2 A or N R 2 B (± S.D.). This ratio from wild-type samples was set at 100% (gray bars) at each age, and the ratio from PTPa" 7" samples (black bars) is expressed as a relative %. The number o f paired samples analyzed is shown in brackets above the bars. Asterisks depict significant differences between the wild-type and PTPa"7" samples (*, p<0.05; **, p<0.005; ***, p<0.0005). 103 To establish that the anti-NR2B antibodies did not cross-react with N R 2 A , the N R 2 A - and the NR2B-specific immunoprecipitates were probed with NR2A-specif ic and NR2B-specific antibodies. Min ima l crossing-reactivity between these antibodies was detected (Fig. 4.6A). Furthermore, the N R 2 A - and the NR2B-specific immunoprecipitates were probed with anti-phosphotyrosine antibody and with another antibody that recognizes both N R 2 A and N R 2 B to allow the comparison of relative N R 2 A and N R 2 B phosphorylation levels. Although equal amounts o f N R 2 A and N R 2 B were immunoprecipitated, a significantly higher phosphorylation of N R 2 B was observed when compared with that o f N R 2 A (Fig. 4.6B), this is consistent with a previous report (Moon et al., 1994) and further confirmed the specificity for the appropriate subunit o f the antibodies used in this study. These results demonstrate that mice lacking P T P a exhibit a reduction in tyrosine phosphorylation o f both the N R 2 A and the N R 2 B subunits o f the N M D A R . IP: NR2A NR2B IB: NR2A IB: NR2B 175 B IP: NR2A NR2B IB: pTyr 175 IB: NR2A/B h 175 h 175 Figure 4.6. Specificity of anti-NR2A and -NR2B antibodies. A, N R 2 A - and NR2B-specific antibodies were used to prepare immunoprecipitates from Triton X-100 insoluble fractions of adult wild-type animals (postnatal day 60). The N R 2 A (left side) and the N R 2 B (right side) immunoprecipitates were probed with NR2A-specific (upper panel) and NR2B-specific (lower panel) antibodies as shown. Min imal crossing-reactivity between these antibodies was detected. B, N R 2 A - and NR2B-specific immunoprecipitates as in A were probed with anti-phosphotyrosine antibody (upper panel) and with another antibody that recognizes both N R 2 A and N R 2 B (lower panel) to allow the comparison of relative N R 2 A and N R 2 B phosphorylation levels. The N R 2 B subunit has ~2-fold higher phosphotyrosine content (per unit protein) than does N R 2 A . 104 4.2.5 Pyk2 autophosphorylation is reduced in synaptosomes lacking PTPa Pyk2 is a component of the N M D A R complex, and can function upstream of src to up-regulate N M D A R function (Husi et al., 2000; Huang et al., 2001; L i u et al., 2001; Seabold et al., 2003). The autophosphorylation of Pyk2 at Tyr402, and src or fyn binding to this site, correlate with Pyk2 activation (Dikic et al., 1996; Sieg et al., 1998; L i et a l , 1999; Huang et al., 2001). Pyk2 Tyr402 phosphorylation was found reduced in Triton X-100 soluble synaptosomal fractions prepared from P7-P60 P T P a 7 " mice (Fig. 4.7A). Quantification of phospho-Tyr402 of immunoprecipitated Pyk2 from adult (P60) mouse synaptosomes demonstrated that ablation of P T P a resulted in about a 40% reduction in phosphorylation (Fig. 4.7B). Tyr402 phosphorylation status of Pyk2 in Triton X-100-insoluble fractions was not determined as there is a low level o f Pyk2 in these fractions. To determine i f the converse situation of increased P T P a activity accordingly promoted Pyk2 activation, H E K 2 9 3 cells were transfected with Pyk2 alone, with fyn, or with fyn and PTPa . Co-expressed P T P a and/or fyn did not increase the phosphorylation of transfected Pyk2 at Tyr402, and this is perhaps due to endogenous cellular factors or conditions promoting maximal or near maximal autophosphorylation of Pyk2 at this site (Fig. 4.7C). However, co-transfected fyn was found to be in association with Pyk2, and the co-expression o f P T P a further enhanced the association of fyn with Pyk2 by about 2-fold (Fig. 4.7D). The above results indicate that P T P a may positively regulate properties of Pyk2, specifically Tyr402 autophosphorylation and S F K binding, that are associated with Pyk2 activation. 105 175 -83 -175 83 -I 175 ^ 83 175 ^ 83 62 4 62 - | 175 P 7 P14 P 3 0 P60 +/+ - / - +/+ - / - +/+ . / - +/+ - / -+ + Pyk2-402P Pyk2 + + + P T P a fyn Pyk2 Pyk2-402P Pyk2 fyn fyn P T P a IP: Pyk2 TL B 100 a. r r CM H 7 c o «> ' ° CN'S 50 J ^ 2 5 o 3 i * i 1 CM $ 0.5 0 (6) I 1 +/+ fyn -/-fyn P T P a Figure 4.7. Pyk2 autophosphorylation and association with fyn are altered by ablated or increased PTPa expression. Pyk2 was immunoprecipitated from Triton X-100 soluble fractions of synaptosomes prepared from wild-type (+/+) and P T P a 7 " ( - / - ) mice at postnatal days 7, 14, 30, and 60 (P7-60). A, The immunoprecipitates were probed for phospho-Pyk2-Tyr402 (top panel) and for Pyk2 amount (bottom panel). B, The results of six such immunoprecipitations from paired P60 samples were quantified by densitometry. The bars represent the amount of autophosphorylation per amount of Pyk2 (± S.D.). This ratio is expressed as 100% for the wild-type samples and the ratio from P T P a 7 " samples is expressed as a relative %. C, H E K 2 9 3 cells were transiently co-transfected with plasmids encoding Pyk2, fyn, and/or P T P a as shown. Pyk2 immunoprecipitates were probed for Pyk2 phosphorylation at Tyr402, for Pyk2 amount, and for associated fyn (top three panels). Ce l l lysates were also probed for fyn and P T P a amounts (bottom two panels). D, The amount of fyn associated with Pyk2 in the absence or presence o f co-expressed P T P a (shown in C) (n=4) was quantified by densitometry. The bars represent the amount of fyn per unit of Pyk2 in arbitrary units (± S.D.). Asterisks depict significant differences between samples with or without P T P a ( * , p<0.005; **, p<0.0005). The migration positions o f molecular mass markers (kD) are shown. I P , immunoprecipitates; T L , total lysates. 106 4 . 2 . 6 L o c a l i z a t i o n o f P T P a i n l i p i d r a f t s Several synaptic proteins, including N M D A R , PSD-95, and A M P A R , have been shown to associate with l ipid rafts (Perez and Bredt, 1998; Hering et al., 2003). L ip id rafts have been implicated in numerous cellular processes including trafficking and sorting of membrane proteins, secretory and endocytosis pathways, and signal tranduction (Brown and London, 1998; Simons and Toomre, 2000; Lkonen, 2001). More importantly, l ipid rafts have been found to be essential in synaptic maintenance and function (Hering et al., 2003). Thus, we examined whether P T P a is also present in synaptosomal l ipid rafts, where it would be ideally located to regulate raft-associated N M D A R . L ip id rafts are microdomains on the cellular membrane that are enriched with cholesterol and sphingolipids. L ip id rafts are not soluble in detergents such as Triton X-100 at 4°C and therefore are present in the low-density fraction after density gradient centrifugation (Simons and Toomre, 2000) as described in Materials and methods, section 2.2.4. The fractions with 40% sucrose contain cytosolic and Triton X-100-soluble proteins. The pellets contain Triton X-100-insoluble protein complexes, probably nuclear and cytoskeletal proteins, and were not analyzed. Nine fractions of 1-ml each were collected from the top of the tubes and probed for the presence of PTPa , N M D A R , PSD-95, and SFKs (Fig. 4.8). The presence of transferrin receptor, as it is excluded from lipid rafts (Giurisato et al., 2003), was employed as a marker for the purity of the isolated l ipid rafts. Consistent with previous reports (Perez and Bredt, 1998; Hering et al., 2003), the N M D A R (NR1, N R 2 A , and N R 2 B ) and the scaffolding protein PSD-95 predominantly resided in the l ipid raft fraction. The S F K s fyn and yes were enriched in lipid rafts, whereas much of the SFKs src and lyn were present in non-raft fractions. More importantly, a small portion of PTPa was also 107 detected in l ipid rafts. The presence of P T P a in l ipid rafts suggests a potential role for P T P a in synaptic maintenance and function by regulating the action o f N M D A R , and, possibly, of A M P A R . Lipid raft Non raft Fraction number: 1 2 3 4 5 6 7 8 9 P T P a -fyn yes src lyn : NR1 NR2A • NR2B • PSD-95 • Transferrin-R • t 11 h 175 62 62 62 - 62 175 83 « » • • » h 175 h 175 175 h 83 1- 83 Figure 4.8. Localization of PTPa in lipid rafts. Synaptosomal fractions from P60 wild-type (+/+) and PTPa" 7" (-/-) mouse brains (P60) were prepared in Triton X-100 buffer and resolved by sucrose gradient centrifugation (Materials and Methods, section 2.2.4). Nine fractions (1 m l each) were collected starting from the top of the gradient and aliquots (30pl) of each fraction were immunoprobed to detect expression o f the indicated proteins. The migration positions o f molecular mass markers (kD) are shown on the right. Transferrin-R, transferrin receptor. 108 4.2.7 Glutamate-induced Akt, MAPK, and CREB phosphorylation in PTPa"" hippocampal and cortical neurons M i c e lacking P T P a exhibit impaired NMDAR-re l a t ed processes that are controlled mainly by hippocampal neurons (Petrone et al., 2003; Skelton et al., 2003). Results from studies in synaptosomes did not specify which subsets of neurons are affected in the absence of PTPa . Moreover, these studies were carried out under unstimulated conditions, thus whether P T P a also plays a role in N M D A R signaling in response to stimuli is unclear. To address these points, neurons from the hippocampus and cerebral cortex of mouse embryos were isolated and cultured. Glutamate responses were investigated and compared between W T and PTPa" 7" neurons that had been in culture for 10-14 days. L o w protein amounts in the samples precluded analyses using irnmunoprecipitation assays (such as N R 2 A / B tyrosine phosphorylation). Thus, the phosphorylation/activation o f downstream molecules o f N M D A R signaling including Akt , M A P K , and C R E B was determined, as the phosphosite-specific antibodies that detect the activated forms of these proteins are commercially available. It has been shown that the N M D A R channel blocker M K - 8 0 1 can inhibit glutamate-induced activation of Akt , M A P K , and C R E B (Mabuchi et a l , 2001; Sutton and Chandler, 2002), suggesting an essential role of N M D A R in glutamate-induced activation of these molecules. Glutamate-induced activation of Ak t and C R E B was observed in W T hippocampal neurons (Fig. 4 .9A and B , left panels). In contrast, PTPa" 7" hippocampal neurons exhibited impaired activation of Ak t and C R E B in response to glutamate stimulation (Fig. 4 .9A and B , right panels). Activation of M A P K in PTPa" 7" hippocampal neurons was robust and appeared similar to that in W T neurons (Fig. 4.9C). Sutton et al. (2002) reported that maximal activation of M A P K was obtained when stimulating the neurons with 25 p M , 109 but not with 100 u M , N M D A . Therefore, the high concentration o f glutamate (100 u,M) used in these experiments may attenuate the activation o f M A P K . A lower concentration of glutamate (25 u M ) may be required to precisely evaluate M A P K activation in PTPa"7" and W T hippocampal neurons. Similar results were obtained with cortical neurons (Fig.4.10A-C), +/+ -/-Glutamate (min) 0 2 5 1 0 0 2 5 1 0 Akt-P Akt B +/+ Glutamate (min) 0 2 5 10 . i.. . nwiiHm - — 5 10 h 62 h 62 47.5 47.5 +/+ -/-Glutamate (min) 0 2 5 1 0 0 2 5 1 0 M A P K - P h 47.5 M A P K h 47.5 Figure 4.9. Glutamate-induced phosphorylation of Akt, CREB, and MAPK in wild-type and PTPa"7" hippocampal neurons. Hippocampal neurons that had been cultured for 14 days were untreated (0) or treated with 100 u,M glutamate for the indicated times. Lysates from wild-type (+/+) and PTPa" 7" (-/-) hippocampal neurons were prepared and immunoprobed for phospho-Ser473-Akt, phospho-S e r l 3 3 - C R E B , and phospho-Thr202/Tyr204-MAPK (A-C, upper panels) and for Akt , C R E B , and M A P K protein amount {A-C, lower panels). The migration positions of molecular mass markers (kD) are shown. 110 although M A P K activation in these PTPa" 7" neurons was somewhat less efficient than in W T neurons. To confirm these observations, more investigations are required. These preliminary results suggest that P T P a may play a role in two or more types of neurons and that P T P a is involved in the regulation of N M D A R signaling in response to stimuli such as glutamate. +/+ Akt B +/+ Glutamate (min) 0 2 5 10 C R E B - P C R E B -/-Glutamate (min) 0 2 5 1 0 0 2 5 10 Akt-P -/-10 h 62 h 62 J- 47.5 \ - 47.5 +/+ -/-Glutamate (min) 0 2 5 1 0 0 2 5 1 0 M A P K - P M A P K h 47.5 h 47.5 Figure 4.10. Glutamate-induced phosphorylation of Akt, CREB, and MAPK in wild-type and PTPa"7" cortical neurons. Cortical neurons that had been cultured for 10 days were untreated (0) or treated with I O O L I M glutamate for the indicated times. Lysates from wild-type (+/+) and PTPa' 7 " (-/-) cortical neurons were prepared and immunoprobed for phospho-Ser473-Akt, phospho-Serl33-CREB, and phospho-Thr202/Tyr204-MAPK (A-C, upper panels) or Akt , C R E B , and M A P K protein amount (A-C, lower panels). The migration positions of molecular mass markers (kD) are shown. I l l 4.3 Discussion 4.3.1 PTPa is a positive regulator of NMDAR tyrosine phosphorylation In the present study, a small portion of PTPa was determined to be in the Triton X -100-insoluble fraction and to be associated with l ipid rafts o f synaptosomes, suggesting a potential role o f P T P a in signaling pathways focused at the P S D complex. This is consistent with the findings that a small portion of P T P a is associated with l ipid rafts of hippocampal neurons and thymocytes (Bodrikov et al., 2005; Maksumova et al., 2005). Furthermore, a previous study has also detected P T P a in the PSD-localized N M D A R complex (Lei et al., 2002). Most importantly, the tyrosine phosphorylation of the N R 2 A and N R 2 B subunits of the N M D A R is reduced in detergent-resistant synaptosomal fractions from PTPa" 7" mice, and this is detectable from two weeks of age into adulthood. This is in accord with the reduced phosphorylation of N R 2 B at Tyr l472 that has been reported in hippocampi of adult PTPa" 7" mice (Petrone et al., 2003). Furthermore, preliminary results from the neuron culture study suggest a potential role for P T P a in mediating N M D A R - i n d u c e d activation of downstream signaling molecules in response to stimuli such as glutamate. These findings indicate that P T P a is a physiological upstream activator of N M D A R phosphorylation and that P T P a exerts its effect on N M D A R phosphorylation via intermediate molecules, events that may be localized to or conveyed to the P S D where most N M D A R resides, to eventually modulate N M D A R downstream signaling. The S F K s sre and fyn can phosphorylate N M D A R (Cheung and Gurd, 2001; Nakazawa et a l , 2001; Yang and Leonard, 2001). In synaptosomal fractions from PTPa" 7" mice, the SFKs , sre, fyn, yes, and lck, exhibit reduced activity (see discussion section 4.3.2 for detail). Although only sre and fyn have been shown to phosphorylate the N M D A R , this 112 finding suggests that not only these S F K s but also yes and lck are candidate kinase intermediates in the observed PTPa-dependent regulation of N M D A R tyrosine phosphorylation in mouse synaptosomes. A l l these SFKs and P T P a are present in the Triton X-100 insoluble synaptosomal fraction where most of the N M D A R is found. Interestingly, P T P a and the SFKs src, fyn, and yes can physically associate with the N M D A R via their interactions with the scaffolding protein PSD-95 (Tezuka et al., 1999; Ka l i a and Salter, 2003). PSD-95 binding could thus promote phosphatase-kinase-NMDAR proximity to enhance N M D A R phosphorylation upon the PTPa-mediated dephosphorylation and activation of these S F K s . Since fyn is not the only target of PTPa , this may explain the partial but not complete overlap between P T P a 7 " (Petrone et al., 2003) and fyn 7" (Grant et al., 1992) phenotypes. The tyrosine kinase Pyk2 acts upstream o f S F K s to mediate N M D A R tyrosine phosphorylation and N M D A R - i n d u c e d L T P (Huang et al., 2001). Pyk2 activation was significantly reduced in P T P a 7 " synaptosomes as indicated by its reduced phosphorylation at a key activation site (see discussion in section 4.3.3 for detail), suggesting that P T P a may also act upstream o f Pyk2 to modulate N M D A R tyrosine phosphorylation. The possibility that P T P a can also act as a N M D A R phosphatase cannot be ruled out and could perhaps play a role in returning N M D A R phosphorylation to basal levels following stimulation. However, a positive effect of PTPa on N M D A R phosphorylation, v ia its upstream action on S F K s and, possibly, on Pyk2, is apparently predominant since an overall significant decrease in N M D A R 2 A and 2B subunit tyrosine phosphorylation is observed in PTPa-nul l synaptosomes. 113 4.3.2 PTPa is a physiological regulator of four neuronal SFKs The investigation o f the activity o f five neuronal S F K s as determined by their tyrosine phosphorylation status reveals that, in synaptosomal fractions from mice lacking PTPa , the S F K s sre, fyn, yes, and lck exhibit reduced activity as indicated by their enhanced phosphorylation at the C-terminal inhibitory tyrosine residue. This is consistent with the earlier reports that sre and fyn can be regulated by P T P a (Ponniah et al., 1999; Su et al., 1999). Interestingly, this is the first report indicating the regulation o f yes and lck by P T P a in a physiological setting by PTPa . The phosphorylation/activation of a fifth synaptosomal S F K , lyn, remained unchanged in the absence of PTPa . Similarly, transfected P T P a is capable of dephosphorylating endogenous sre, fyn, and yes, but not lyn, in A431 carcinoma cells (Harder et al., 1998). This demonstrates that P T P a has a broad range of action towards several SFKs . The similar structures and regulation of S F K family members suggest it is unlikely that lyn cannot be dephosphorylated by P T P a per se. L y n may be sublocalized such that it is not in proximity to P T P a and/or specific conditions are required to stimulate a functional interaction. Moreover, synaptosomal sre and fyn phosphorylation at the autophosphorylation site is unaltered in PTPa" 7" mice. This suggests that activation of sre and fyn is not always associated with an increase in autophosphorylation of sre, as has also been reported by Zheng et al. (1992), and that this may reflect an ability of P T P a to dephosphorylate this site as soon as it is phosphorylated. 4.3.3 PTPa is an upstream regulator of Pyk2 The tyrosine kinase Pyk2 (also known as C A K p / C A D T K ) acts downstream o f G -protein coupled receptors and integrins, and upstream of S F K s to mediate N M D A R tyrosine 114 phosphorylation and N M D A R - i n d u c e d L T P (Huang et al., 2001; Heidinger et al., 2002; Bemard-Trifilo et al., 2005). In PTPa" 7" synaptosomes, Pyk2 phosphorylation at its key activation site, Tyr402, is significantly reduced. In the converse situation in transfected H E K 2 9 3 cells, increased P T P a expression promoted an enhanced association of fyn and Pyk2. PTPa-dependent modulation of Pyk2 activation by regulation o f Pyk2 autophosphorylation and binding to SFKs has not been previously reported. Pyk2 Tyr402 phosphorylation occurs by an autocatalytic mechanism ( L i et al., 1999). In synaptosomes, the S F K inhibitor PP2 inhibits integrin-stimulated N M D A R tyrosine phosphorylation, but not Pyk2 Tyr402 phosphorylation (Bernard-Trifilo et al., 2005). This and other observations (Huang et al., 2001) place the SFKs downstream rather than upstream of Pyk2. However, Pyk2 tyrosine phosphorylation, including that at Tyr402, is dramatically decreased in hippocampi of mice lacking fyn (Corvol et a l , 2005). Pyk2 is closely related to focal adhesion kinase ( F A K ) . In integrin signaling, the PTPa-catalyzed activation of src and fyn promotes F A K autophosphorylation and association with these SFKs at a site analogous to Pyk2 Tyr402 (Zeng et al., 2003). P T P a could potentially regulate Pyk2 by a similar mechanism, although this, and the nature o f the upstream signals that engage P T P a to lead to Pyk2 activation, require further investigation. Pyk2 has been implicated in L T P (Huang et al., 2001), and impaired L T P in mice lacking P T P a (Petrone et a l , 2003) could involve altered Pyk2 activity. 4 . 3 . 4 Summary Biochemical studies indicate that mice lacking P T P a display a reduction in the phosphorylation of two subunits ( N R 2 A and N R 2 B ) of the synaptosomal N M D A R . Reduced 115 N M D A R tyrosine phosphorylation is in accord with the attenuation of activity of SFKs src, fyn, yes, and lck. The loss of PTPa also results in decreased Pyk2 autophosphorylation and binding to SFKs. This study provides evidence that aberrant NMDAR-associated functions reported in PTPa-null mice may be linked to impaired N M D A R tyrosine phosphorylation. The key molecular mechanism by which PTPa regulates neuronal NMDAR-mediated processes, such as learning and memory, hippocampal neuron migration, and LTP, is likely to be through controlling N M D A R tyrosine phosphorylation via its action as an upstream regulator of the SFKs src, fyn, yes, and/or lck and the PTK Pyk2. 116 5 REGULATION OF FYN-AND SRC-MEDIATED NR2A/B PHOSPHORYLATION BY PTPa 5.1 Introduction and rationale Although both fyn and sre can phosphorylate N M D A R (Kohr and Seeburg, 1996; Yang and Leonard, 2001; Nakazawa, 2001), they are quite distinct in their regulation of NMDAR-dependent processes. Sre has been implicated as a fundamental regulator of N M D A R signaling since application of anti-srcl antibody or sre-inhibitory peptide [src(40-58)] that selectively block sre but not other SFKs , leads to inhibition of N M D A R activity and prevents L T P ( Y u et al., 1997). However, mice lacking sre exhibit normal L T P in the hippocampal C A 1 region (Grant et al., 1992). It has been suggested that function of sre in these animals is substituted for by fyn. Similar to sre7" mice, young fyn 7" mice appear normal, however, L T P is blunted in older fyn 7" mice, indicating a unique role of fyn (Grant et al., 1992). In further support of fyn and sre having distinct NMDAR-re l a t ed functions is their specific interaction with different proteins linked to N M D A R regulation and signaling. For example, the scaffolding protein R A C K - 1 associates with fyn and N R 2 B to form a tricomplex (Yaka et al., 2002). The formation of this complex prevents fyn from phosphorylating N R 2 B , thus resulting in the down-regulation of NMDAR-media ted mEPSCs in the C A 1 region of the hippocampus (Yaka et al., 2002). This event is specific for fyn since R A C K - 1 is unable to bind sre. O n the other hand, H-ras, a small G T P binding protein, binds sre, but not fyn, both in vitro and in brain via the sre kinase domain, thus inhibiting sre activity (Thornton et al., 2003). This eventually causes reduced N R 2 A phosphorylation and 117 decreased N M D A R level at the membrane (Thornton et al., 2003). Altogether, these studies suggest distinct involvements of src and fyn in signaling pathways that regulate N M D A R action and/or localization. P T P a activates both src and fyn (Ponniah et al., 1999; Su et al., 1999) and positively regulates NMDAR-dependent processes (Lei et al., 2002; Petrone et al., 2003). However, it is not clear how the signal is transduced from P T P a to N M D A R via src and fyn. The aim of this study was to investigate and compare the regulation of fyn- and src-mediated N M D A R phosphorylation by P T P a in particular to determine i f this phosphatase exerted differential upstream regulation on the N M D A R via specific actions on fyn and src. A model is shown in Fig . 5.1 .that depicts some direct and indirect actions of P T P a that potentially regulate N M D A R N R 2 subunit tyrosine phosphorylation. Experiments were carried out with H E K 2 9 3 cells transfected with N R 2 A / B subunits of the N M D A R , fyn or src, and with or without PTPa . 5.2 Results 5.2.1 PTPa enhances fyn- but reduces src-mediated NR2A/B phosphorylation. H E K 2 9 3 cells, as they contain low levels of src and fyn and no N M D A R , have been extensively used as a transfectable model system to investigate the regulation of N M D A R phosphorylation by src and fyn (Tezuka et al., 1999; Nakazawa et al., 2001; Yang and Leonard, 2001). In addition, they are readily transfectable using the calcium phosphate method, and permit a high transfection efficiency to be achieved. Thus this cell line was employed to address the regulation of fyn- and src-mediated N R 2 A and N R 2 B phosphorylation by PTPa . 118 P T P a Figure 5.1. Schematic diagram illustrating the pathways through which PTPa potentially regulates NR2 phosphorylation. Step (A), PTPa dephosphorylates SFKs (such as fyn or sre) at their regulatory C-terminal tyrosine residue (depicted as Y527), thereby activating them. Step (B), PTPa-activated SFKs (such as fyn or sre) can then phosphorylate N R 2 A / B . Step (C), PTPa can dephosphorylate N R 2 A / B that was phosphorylated by SFKs (such as fyn or sre). N R 2 A or N R 2 B , fyn or sre, and wild-type or catalytically inactive PTPa were transiently transfected into HEK293 cells. N R 2 A or N R 2 B were immunoprecipitated from the cell lysates and analyzed for their tyrosine phosphorylation status. Virtually no tyrosine phosphorylation of N R 2 A was detected in cells expressing N R 2 A alone or with PTPa . The co-expression of fyn with N R 2 A induced N R 2 A phosphorylation, and this was further enhanced by the expression of P T P a (Fig. 5.2A and C) . Co-expression of sre with N R 2 A also induced phosphorylation of N R 2 A . In contrast, the additional co-expression of PTPa with 119 A PTPa fyn N R 2 A N R 2 A - P N R 2 A B wt - wt dm + + + 175 N R 2 A " P * * PTPa - wt dm src + + + N R 2 A + + + h 175 N R 2 A h 175 175 _ 3.5 ~ 2.8 4: | 2.1 CM s or 2 1.4 —y -4—1 1 0.7 fyn fyn wt fyn dm ^ 1.2 CL I 0.9 -e 0.3 < 0 src src src wt dm PTPa fyn N R 2 B N R 2 B - P N R 2 B wt dm + + + 175 r- 175 PTPa src wt dm + + + N R 2 B N R 2 B - P N R 2 B 175 175 Figure 5.2. Differential regulation of fyn- and src-mediated NR2A/B phosphorylation by PTPa. H E K 2 9 3 cells were transiently transfected with N R 2 A or N R 2 B , fyn or src, and without (-) or with wild-type (wt) or catalytically inactive (dm) P T P a . N R 2 A (A, B) and N R 2 B (E, F) were immunoprecipitated from the cell lysates and probed for phosphotyrosine content (upper panels), and N R 2 A or N R 2 B amount (lower panels). C, D, The results of 3 independent experiments as in A and B were quantified by densitometric scanning. The bars represent the amount of tyrosine phosphorylation per amount of immunoprecipitated N R 2 A (arbitrary densitometric units ±S.D.) . This ratio from cells expressing N R 2 A and fyn or src is set at 1 unit, and the ratio from cells expressing N R 2 A , fyn or src, and wild-type (wt) or catalytically inactive (dm) PTPa is expressed relative to this. 120 sre and N R 2 A caused a reduction in src-phosphorylated N R 2 A (Fig. 5.2B and D). This indicates that P T P a differentially regulates N R 2 A phosphorylation i n a sre- and fyn-dependent manner. To verify that these effects observed upon P T P a expression involved the catalytic activity of PTPa , sre- and fyn-dependent N R 2 A phosphorylation was also examined in cells co-expressing a catalytically inactive mutant of P T P a (Fig. 5.2A-D). Notably, expression of catalytically inactive P T P a did not result in readily apparent changes in src-phosphorylated N R 2 A but moderately increased fyn-phosphorylated N R 2 A . Similar results were obtained when cells were transfected with N R 2 B , fyn or sre, and P T P a (Fig. 5.2E and F). The co-expression experiments demonstrate that P T P a enhances fyn-mediated N R 2 A / B phosphorylation but reduces N R 2 A / B phosphorylation by sre. In addition to the SFK-dependent effects of wild-type P T P a expression, expression of inactive P T P a also differentially affected SFK-mediated N R 2 A / B phosphorylation. 5.2.2 PTPa dephosphorylates and activates both sre and fyn The simplest explanation of the above results is that P T P a enhances fyn activity but reduces sre activity towards N R 2 A / B . However, this seems very unlikely as fyn and sre are wel l documented to be activated by P T P a (Ponniah et al., 1999; Su et al., 1999). Nevertheless, to rule out this possibility, fyn and sre were immunoprecipitated from the H E K 2 9 3 cell expression system and assayed for their phosphorylation status and kinase activity. Expression of P T P a resulted in reduced tyrosine phosphorylation of sre and fyn (Fig. 5.3A and B , top panels). While P T P a appeared to dephosphorylate fyn more efficiently than it did sre, these findings demonstrate that both kinases are indeed P T P a substrates. The 121 dephosphorylation of sre and fyn was fully dependent on P T P a catalytic activity since catalytically inactive P T P a was unable to affect sre and fyn phosphorylation. In addition, P T P a action on sre and fyn was directly measured in an auto-kinase and enolase-kinase assay. Consistent with previous reports (Ponniah et al., 1999; Su et al., 1999), P T P a promoted autophosphorylation of fyn and sre, and enhanced phosphorylation of enolase by fyn and sre (Fig. 5.3A and B , upper middle panels). Catalytically inactive mutant P T P a was unable to dephosphorylate or activate sre (Fig. 5.3B, top and upper middle panels). However, fyn activity was increased in the presence of inactive P T P a (Fig. 5.3 A , upper middle panel) despite the lack o f effect of mutant P T P a on fyn phosphorylation (Fig. 5.3 A , top panel). This is in accord with the increased fyn-mediated N R 2 A / B phosphorylation observed upon co-transfection with inactive mutant P T P a (Fig. 5.3A and E). Together, these findings suggest that the activation o f fyn is partially independent o f P T P a catalytic activity. Activation o f fyn or sre in the in vitro kinase assay was also reflected by the increased phosphorylation of co-immunoprecipitated proteins that likely represent fyn- or sre-associated molecules (Fig. 5.3, lower middle panels). Notably, fyn and sre appeared to participate in some similar (pi 15 and p70) and some distinct (ie. fyn: p l45 , p85, p65; sre: p>250, p l05) protein associations, indicating that fyn and sre may have either distinct cellular substrates or different catalytic activities toward similar substrates. These results demonstrate that, in the presence o f N R 2 A , both sre and fyn are dephosphorylated and activated by PTPa , thus confirming step A in the model depicted in F ig . 5.1. Thus, the differential outcomes on N R 2 A / B tyrosine phosphorylation that are mediated by PTPa/fyn or PTPa/src signaling are not due to intrinsic differences in the ability o f P T P a to stimulate S F K activities in this system. 122 f >> CO CO CO CO 1 P T P a - wt dm fyn + + + NR2A + + + fyn-P Auto-P B Enolase-P 62 h 62 CO c 'CD CD o o CO CO < V 250 -* 150 - m m * p145 100 -*•» * - p115 * • * * p85 75 -p70 62 — * * p65 fyn - 62 f CO CO CO CO Auto-P 62 f- 62 Enolase-P 250 - \ c S 150 2 CL I 100 o o CO CO < V 75 62 sre p>250 « - p115 * p105 p70 ^ "mit t „„,„Mmm, 62 Figure 5.3. P T P a dephosphorylates and activates fyn and sre. H E K 2 9 3 cells were transiently transfected with N R 2 A , fyn or sre, and without (-) or with wild-type (wt) or catalytically inactive (dm) P T P a . Fyn (A) and sre (B) were immunoprecipitated from the cell lysates and analyzed for phosphotyrosine content (top panels) and kinase activity in an in vitro assay. In the in vitro assay, the autophosphorylation of fyn and sre and the phosphorylation o f the exogenous substrate enolase are shown in the upper middle panels, and the phosphorylation o f associated (co-immunoprecipitated) proteins ( M w -62 k D to -250 kD) are shown in the lower middle panels. The arrows to the right of the lower middle panels indicate proteins that co-immunoprecipitate with and are phosphorylated by both fyn and sre. The asterisks indicate proteins that uniquely co-immunoprecipitate with and/or are distinctly phosphorylated by fyn and sre. Portions of the immunoprecipitates were probed for fyn or sre (bottom panels). The migration positions of molecular mass markers (kD) are shown. 123 5.2.3 PTPa dephosphorylates both fyn- and src-phosphorylated NR2A P T P a activates both fyn and src but exerts differential, indeed opposite, effects on fyn and src-mediated N R 2 A / B phosphorylation. N R 2 A and N R 2 B contain multiple tyrosine residues that are potential sites of fyn and src phosphorylation, and some residues are preferentially phosphorylated by either fyn or src (Nakazawa et al., 2001; Yang and Leonard, 2001). This raises the possibility that P T P a acts as an N M D A R phosphatase (Fig. 5.1, step C) and that P T P a dephosphorylates src-specific N R 2 A / B phosphorylation sites but not fyn-specific sites of N R 2 A / B phosphorylation. To test this hypothesis, cells transfected with N R 2 A , src or fyn, and with or without P T P a were left untreated or treated with PP2, an S F K inhibitor. Under these conditions, PTPa/src/fyn-mediated phosphorylation of N R 2 A proceeded as described in section 5.2.1, with subsequent PP2-induced src/fyn inhibition allowing any phosphatase activity of P T P a towards N R 2 A to be detected in the absence of continued kinase-mediated phosphorylation. The outcome of this treatment on the phosphorylation status of N R 2 A was determined by probing N R 2 A immunoprecipitates with anti-phosphotyrosine antibody. In the absence o f exogenous P T P a , PP2-mediated inhibition of fyn and src activity caused significant reductions in N R 2 A phosphorylation, suggesting that dephosphorylation o f fyn- and src-mediated N R 2 A by endogenous PTPs is very rapid (Fig 5.4A and B , left panels). This dephosphorylation of N R 2 A was further enhanced in the presence o f exogenous P T P a (compare right and left panels within F ig 5.4A and B) , indicating that P T P a can dephosphorylate both fyn- and src-mediated N R 2 A . Furthermore, no major differences in the ability o f P T P a to dephosphorylate fyn- and src-phosphorylated N R 2 A were detected (18% vs. 22% dephosphorylation, respectively). 124 B fyn src PTPa PP2 NR2A-P NR2A h 175 f- 175 PTPa P P 2 NR2A-P NR2A h 175 175 Arbitrary Units: 1 0.25 1 0.07 1 0.26 1 0.04 Figure 5.4. PTPa dephosphorylates fyn- and src-phosphorylated NR2A. H E K 2 9 3 cells were transiently transfected with N R 2 A , fyn (A) or src (B), and without (-) or with (+) wild-type P T P a . Prior harvesting, the transfected cells were treated with D M S O (-) or 5 p M PP2 (+) for 5 min. N R 2 A was immunoprecipitated from the cell lysates and analyzed for phosphotyrosine content (top panels) and for N R 2 A (bottom panels). The results were quantified by densitometric scanning. The number shown at the bottom of each lane represents N R 2 A tyrosine phosphorylation per N R 2 A amount. The ratio from D M S O -treated samples was taken as 1 unit and the ratio from PP2-treated samples is expressed relative to this. 5.2.4 Fyn and src phosphorylate PTPa P T P a activates both fyn and src. In addition, P T P a also exhibits indistinguishable phosphatase activity towards fyn- and src-phosphorylated N R 2 A . Thus the similar characteristics o f P T P a discussed above cannot account for the differential effects of PTPa on fyn- and src-mediated phosphorylation of N R 2 A / B . However, the possibility remains that PTPa-activated src and fyn exhibit some feedback on P T P a that results in altered and distinct P T P a phosphatase activities towards N R 2 A / B . Thus the regulation of P T P a by fyn and src was investigated. Phosphorylation of PTPa by SFKs is a l ikely feedback mechanism that could potentially regulate PTPa actions (Fig. 5.5). 125 Figure 5.5. Schematic diagram depicting the potential abili ty of S F K s to phosphorylate P T P a and the possible actions of phospho-PTPa in regulating N R 2 phosphorylation. SFKs (such as fyn and src) phosphorylate PTPa as shown in step (D), potentially altering the ability of PTPa to activate SFKs as depicted in step (E). This may also alter the activity of PTPa as an N R 2 phosphatase, as shown in step (F). Indeed, it has been reported that src can phosphorylate PTPa at Tyr789, the major phosphorylatable tyrosine residue of PTPa (den Hertog et al., 1994). The ability of fyn to phosphorylate PTPa has not been reported. To examine whether src or fyn effected Tyr789 phosphorylation of PTPa in H E K 2 9 3 cells, PTPa was co-transfected with each kinase. PTPa immunoprecipitates from the cell lysates were analyzed for PTPa tyrosine phosphorylation status. PTPa tyrosine phosphorylation was low to undetectable in cells expressing PTPa alone. The co-expression of fyn or src with PTPa increased PTPa tyrosine phosphorylation dramatically (Fig. 5.6A, top panel). Notably, probing PTPa immunoprecipitates with anti-P T P a antibodies revealed two major forms of PTPa (Fig. 5.6A, bottom panel), the mature 126 PTPa-P i- 175 h- 175 B PTPa fyn ( L t g ) 2.5 5 10 PTPa-P PTPa r~ 175 i- 175 src ( n g ) 2.5 5 10 PTPa-P PTPa \ - 175 \ - 175 PTPa - wt d m 789 fyn + + + + NR2A + + + + PTPa-P PTPa \ - 175 175 PTPa - wt d m 789 sre + + + + NR2A + + + + PTPa-P PTPa \ - 175 175 Figure 5.6. Phosphorylation of PTPa by fyn and sre. A, H E K 2 9 3 cells were transiently transfected without (-) or with (+) wild-type P T P a and without (-) or with fyn or sre. B, C, HEK293 cells were transiently transfected with increasing amount o f fyn or sre plasmids as indicated and with a plasmid expressing wi ld-type PTPa . D, F, H E K 2 9 3 cells were transiently transfected with N R 2 A , fyn or sre, and without (-) or with wild-type (wt), catalytically inactive (dm), or Y 7 8 9 F (789) PTPa . P T P a was immunoprecipitated from the cell lysates and analyzed for phosphotyrosine content (top panels) and P T P a amount (bottom panels). The migration position o f the 175 k D marker is shown on the right. 1 2 7 glycosylated (-130 kD) and the incompletely glycosylated (-100 kD) P T P a (Daum et al., 1994); however, only the mature 130 k D P T P a was phosphorylated when co-tranfected with fyn or src. Furthermore, when P T P a was co-transfected with increasing amounts of fyn or src expression plasmids, a correspondingly increased phosphorylation o f P T P a was observed (Fig. 5.6C and D , top panels). To confirm that P T P a phosphorylation occurred at Tyr789, a mutant form of P T P a lacking this tyrosine residue (Y789F PTPa) was co-expressed with fyn and src. N o phosphorylation o f Y 7 8 9 F P T P a was detected (Fig 5.6D and F , top panels), indicating that Tyr789 is the sole site of phosphorylation by fyn or src on PTPa . Furthermore, the involvement of the catalytic activity of P T P a in its phosphorylation status was examined by comparing the tyrosine phosphorylation status of wild-type P T P a with that of inactive PTPa . Similar to wild-type PTPa , only the mature form of mutant P T P a was phosphorylated by fyn. Tyr789 phosphorylation of mutant P T P a was lower than that of wild-type P T P a (Fig. 5.6C, top panel). This is probably due to lower activation of fyn by mutant P T P a than by wild-type P T P a (as shown in Fig . 5.3A) and/or may simply indicate that wild-type P T P a is a preferred substrate. In contrast, in src co-expressing cells, Tyr789 phosphorylation of the mature form of mutant P T P a was higher than that of wild-type P T P a (Fig. 5.6D, top panel). Interestingly, the incompletely glycosylated (-100 kD) form of mutant PTPa , but not o f wild-type PTPa , was also phosphorylated by src (Fig. 5.6D, top panel). Although src activity is enhanced when co-transfected with wild-type but not mutant P T P a (as shown in Fig . 5.3B), src-mediated phosphorylation of mutant PTPa is significantly higher than that of wild-type PTPa . This result may arise from one or more of the following possibilities. Firstly, src may preferentially phosphorylate mutant PTPa , perhaps due to conformational changes in the 128 mutant protein. Secondly, mutant P T P a may have a distinct subcellular distribution (ie. mutant P T P a is in closer proximity to sre than is wild-type PTPa) such that it is more accessible to sre. This is supported by the observation that sre is able to phosphorylate the incompletely glycosylated (-100 kD) form of mutant P T P a but not that of wild-type PTPa . Lastly, PTPa tyrosine phosphorylation may also be regulated by other enzymes including PTPs, and mutant PTPa may complex with sre in such a way that phospho-Tyr789 is protected from dephosphorylation by cellular PTPs. P T P a has been shown to have autodephosphorylation activity in vitro (den Hertog et al., 1994). P T P a may exhibit this activity in vivo and thus contribute to its own dephosphorylation. The above results indicate that both fyn and sre phosphorylate P T P a at Tyr789 (Fig. 5.5, step D). However, P T P a tyrosine phosphorylation seems to be differentially regulated by these kinases. This is in accord with the finding described in section 5.2.1 that N R 2 A / B tyrosine phosphorylation is distinctly modulated by P T P a in a fyn- and sre-dependent manner. These observations clearly demonstrate the distinct characteristics of PTPa/fyn and PTPa/src, however, the mechanism underlying these differences is still unclear. 5.2.5 Fyn and sre have no effect on PTPa activity in vitro Some groups report that P T P a Tyr789 phosphorylation has no effect on the intrinsic activity of P T P a (Su et al., 1996; Zheng et al., 2000) while other groups have observed a reduction or an increase in P T P a activity upon phosphorylation (den Hertog et al., 1994; Hao et al., 2006). Thus, phosphorylation by fyn and sre may alter P T P a phosphatase activity towards substrates including N R 2 A / B (Fig. 5.5, step E and F). To assess the intrinsic phosphatase activity of P T P a from fyn- and sre-expressing cells, P T P a immunoprecipitates 129 from these cells were incubated with the synthetic substrate pNPP in an in vitro phosphatase assay as described in section 2.2.7.2. A low amount of endogenous PTPa was precipitated from control untransfected HEK293 cells and was assayed to determine baseline phosphatase activity (Fig 5.7). The expression of heterologous PTPa in the cells resulted in about 10-fold increase in immunoprecipitated PTPa phosphatase activity (Fig. 5.7). Notably, no difference in immunoprecipitated PTPa activity was readily detected from cells expressing PTPa alone or from cells co-expressing PTPa with either fyn or src. These results suggest that fyn and src phosphorylate PTPa but that neither kinase alters the intrinsic activity of PTPa towards pNPP. Nevertheless, this finding cannot rule out the possibility that the phosphatase activity of PTPa towards cellular substrates may be altered in vivo, where it could be influenced by interactions with multiple cellular molecules. ~ 0.6 | c 0.5 o o 0.4 CO a K 0.3 I— Q 0.2 °- s o.i 0 PTPa fyn src I I + + Figure 5.7. The intrinsic phosphatase activity of PTPa is not altered by co-expression with fyn or src. HEK293 cells were untransfected or were transiently transfected with PTPa alone or with fyn or src. PTPa was immunoprecipitated from the cell lysates and analyzed for PTPa activity in an in vitro assay with the substrate pNPP as described in section 2.2.7.2. At the end of the assay, the reactions were stopped and the reaction mixtures were briefly centrifuged, the released product (nitrophenol) in the supernatant was measured by its absorbance at A^snm. The actual OD shown in the graph represents the activity of PTPa measured in 3 independent experiments (±S.D.). The reaction pellets containing the PTPa immunoprecipitate were resolved by SDS-PAGE and probed for PTPa amount as shown in the panel above the graph. 130 5.2.6 P T P a complexes wi th fyn and sre wi th different affinities and v ia distinct mechanisms Protein-protein interactions are fundamental to protein function. Protein binding may alter the catalytic activity of a protein and/or its accessibility to its substrates. Thus, to investigate the basis of the differential P T P a - d e p e n d e n t regulation of N R 2 A / B phosphorylation by P T P a / f y n and P T P a / s r c , the association of P T P a with fyn and sre was examined (see model depicted in Fig . 5.8). P T P a can associate with fyn in transfected cells and in brain (Bhandari et al., 1998; Harder et al., 1998), and with sre in transfected cells (Harder et al., 1998; Zheng et al., 2000). However, whether P T P a preferentially or more strongly associates with fyn or with sre, and whether these interactions occur though similar mechanisms/protein domains, is far from clear. Figure 5.8. Schematic diagram il lustrating the potential association of P T P a wi th the S F K s fyn and sre, and the consequences of this on N R 2 phosphorylation. P T P a may associate with fyn and/or sre. The formation of P T P a / f y n and P T P a / s r c complexes may alter the ability of the SFKs to act on N R 2 as shown in steps (G) and (H) and/or the phosphatase activity of P T P a towards N R 2 as shown in steps ( I ) and ( J ) . 131 To address this, the association of these kinases with P T P a was assessed and compared by co-immunoprecipitation assays from H E K 2 9 3 cells expressing P T P a and fyn or sre (Fig. 5.9A). Fyn and sre were immunoprecipitated from the cell lysates and the immunoprecipitates were probed with anti-PTPa antibodies and with anti-src2 antibodies that recognize both fyn and sre (Harder et al., 1998; Kuo et al., 2005). Exogenous PTPa , but not endogenous PTPa , was detected in fyn and sre immunoprecipitates (Fig. 5.9A, top panel). Interestingly, much more P T P a was present in the fyn immunoprecipitate than in the sre immunoprecipitate despite the lower amount of fyn that was immunoprecipitated compared to sre (Fig. 5.9A, middle panel). The amount of P T P a in the fyn immunoprecipitate was calculated to be about 4-fold higher (per unit of kinase) than in the sre immunoprecipitate (Fig. 5.9B, graph [a]). This suggests that more PTPa directly and/or indirectly interacts with fyn than with sre. Probing the cell lysates for P T P a revealed two major forms o f P T P a (Fig. 5.9A, bottom panel), the mature glycosylated and the incompletely glycosylated P T P a (Damn et al., 1994). Only the mature 130 k D form of exogenous P T P a co-immunoprecipitated with fyn or sre (Fig. 5.9A, top panel). This is consistent with previous studies (Bhandari et al., 1998; Zheng et al., 2000) and is in accord with the finding (section 4.2.4) that only the mature 130 k D P T P a is phosphorylated by fyn and sre. It is possible that the immature 100 k D P T P a is incompletely processed and is trapped in the endoplasmic reticulum and/or Golgi apparatus (Bhandari et al., 1998). Thus, due to non-membrane localization, this form of P T P a may be unable to complex with membrane-bound fyn and sre, and therefore is not phosphorylated by fyn and sre. 132 fyn src TL PTPa - wt 789 - wt 789 NR2A + + + + + + IP: fyn/src PTPa S R C - 2 PTPa S R C - 2 \ - 175 62 - Ig heavy chain 175 62 wt 789 fyn src 1.2 1 0.8 0.6 0.4 0.2 0 (b) wt 789 src Figure 5.9. Differential association of PTPa with fyn and src. HEK293 cells were transiently transfected with NR2A, fyn or src, and without (-) or with wild-type (wt) or Y789F (789) PTPa. Fyn and src immunoprecipitates from cells lysed in 1% Brji98 buffer were resolved by SDS-PAGE and transferred to membranes. The upper portion of the membranes was probed for PTPa (A, top panel) and the bottom portion of the membrane was probed for fyn and src using anti-SRC-2 antibody (A, upper middle panel). Portions of the cell lysates were resolved by SDS-PAGE and probed for PTPa (A, lower middle panel), and for fyn and src using SRC-2 antibody (A, bottom panel). B, Bars represent the amount of co-immunoprecipitated PTPa per amount of immunoprecipitated fyn or src (arbitrary densitometric units) from 2 independent experiments. In B(a), the wild-type PTPa and fyn association was taken as 1 and other amounts of association (±S.D.) are expressed relative to this. In B(b), the wild-type PTPa and src interaction was taken as 1 unit, and the amount of associated Y789F PTPa (±S.D.) and src is expressed relative to this. The gray bars represent the amount of associated wild-type PTPa and the black bars represent the amount of associated Y789F PTPa. IP, immunoprecipitates; TL: total lysates. 133 Phosphorylation of P T P a at Tyr789 has been demonstrated to be essential for src binding to P T P a and src activation by P T P a in mitosis (Zheng et al., 2002). N o studies have examined the role of phospho-Tyr789 in PTPa-fyn binding. To investigate this, P T P a with a Tyr789 to Phe mutation (Y789F PTPa) was transfected into H E K 2 9 3 cells. The association of Y789F P T P a with fyn and src was analyzed and compared with wild-type PTPa . The Y789F mutation did not affect PTPa-fyn association (Fig. 5.9A, top panel and 5.9B, graph [a]), indicating that phospho-Tyr789 is not required for the PTPa-fyn interaction. In contrast, PTPa-src association was reduced by about 50% when Tyr789 is mutated, but not completely abolished (Fig. 5.9A, top panel and 5.9B, graph [b]) as observed by another group (Zheng et al., 2002). This suggests that phospho-Tyr789 is partially required for PTPa-src association but is not necessary for PTPa-fyn interaction. This also indicates that other binding sites exist within P T P a for src and fyn. The above results demonstrate that P T P a associates with fyn with higher binding affinity than src. The distinct binding affinity of fyn and src to P T P a may alter P T P a phosphatase activity towards N R 2 A / B (as depicted in F ig . 5.8, steps I and J). Alternatively, or in addition, the differential association of fyn and src with P T P a may alter the activities of these kinases towards N R 2 A / B (as depicted in Fig . 5.8, steps G and H) . 5.2.7 Role of PTPa tyrosine phosphorylation in fyn- and src-mediated NR2A phosphorylation Tyrosine phosphorylation of P T P a is critical for P T P a action in certain signaling pathways (Yang et al., 2002; Chen et al., 2006), and may regulate P T P a action in signaling events that determine N R 2 phosphorylation (as depicted in F ig . 5.5). To determine whether 134 B IP: N R 2 A T L P T P a fyn N R 2 A N R 2 A - P N R 2 A P T P a - wt 789 + + + + + + mm 175 - 175 175 IP: N R 2 A T L P T P a sre N R 2 A N R 2 A - P N R 2 A P T P a - wt 789 + + + + + + h - 175 \ - 175 h - 175 _ 5 CO c 4 < i 2 i l 0 fyn fyn fyn wt 789 1.2 CO -.—' 0. '% 0.9 w CN c r\ a or S •2 0.3 sre sre sre wt 789 Figure 5.10. Role of PTPa tyrosine phosphorylation in regulation of fyn- and src-mediated NR2A phosphorylation. H E K 2 9 3 cells were transiently transfected with N R 2 A , fyn or sre, and without (-) or with wild-type (wt) or Y789F (789) PTPa . A, B, N R 2 A was immunoprecipitated from the cell lysates and probed for phosphotyrosine content (top panels), and N R 2 A amount (middle panels). Portions of the cell lysates were resolved by S D S - P A G E and probed for P T P a (bottom panels). C, D, the results o f 3 independent experiments as in A and B were quantified by densitometric scanning. The bars represent N R 2 A tyrosine phosphorylation per N R 2 A amount (arbitrary units ±S.D.). The ratio from cells expressing N R 2 A and fyn or sre is set at 1 unit and the ratio from cells expressing N R 2 A , fyn or sre, and wild-type (wt) or Y789F (789) P T P a is expressed relative to this. Asterisks note significant differences (*, p<0.05; **, p< 0.005). IP, immunoprecipitates; T L , total lysates. 135 P T P a Tyr789 phosphorylation affected fyn- or src-mediated N R 2 A phosphorylation, the Y789F mutant P T P a was expressed in the H E K 2 9 3 cell system. The co-expression o f Y789F P T P a with fyn enhanced fyn-mediated N R 2 A phosphorylation better than did co-expression o f wild-type P T P a with fyn (Fig. 5.1 OA and C). Similarly, the co-expression o f Y789F P T P a with src also enhanced N R 2 A phosphorylation over that observed with wild-type P T P a (Fig. 5.1 OB and D). When also considered in relation to the N R 2 A phosphorylation that occurs in the absence of any form of PTPa , it is apparent that the lack of this phosphorylation of P T P a actually exerts opposite effects on N R 2 A phosphorylation that are dependent on the nature o f the co-expressed kinase. Namely, the unphosphorylatable Y789F P T P a promotes the positive effect of P T P a on fyn-mediated N R 2 A phosphorylation while inhibiting the negative effect o f P T P a on src-mediated N R 2 A phosphorylation. 5.2.8 Y789F PTPa and wild-type PTPa activate fyn and src to similar extents Fyn- and src-mediated N R 2 A phosphorylation is higher when these kinases are co-expressed with Y789F P T P a than with wild-type PTPa , suggesting that altered P T P a Tyr789 phosphorylation may influence P T P a activity on src and fyn (Fig. 5.5, step E) . Thus, activation of fyn and src by the mutant P T P a was examined. Src and fyn were immunoprecipitated from cell lysates as described in section 2.2.7 and assayed for their in vitro autophosphorylation activity. Fyn and src activities in Y789F and wild-type P T P a -expressing cells were similar, indicating that P T P a tyrosine phosphorylation is not necessary for PTPa-mediated src and fyn activation (Fig. 5.11 A and B) . The results suggest that P T P a promotes fyn and src activity through a P T P a tyrosine phosphorylation-independent mechanism. Nevertheless, the effects of P T P a on fyn- and src-mediated N R 2 A 136 phosphorylation are dependent on the tyrosine phosphorylation o f PTPa. However, overall, these results suggest that tyrosine phosphorylation o f PTPa, l ikely carried out by fyn inhibits the activity o f PTPa in either promoting fyn-mediated N R 2 A / B phosphorylation inhibiting src-mediated N R 2 A phosphorylation. or src, or P T P a - wt 789 fyn + + + IP: fyn TL Auto-P fyn P T P a 62 h- 62 175 B P T P a - wt 789 src + + + IP: src TL Auto-P src P T P a r- 62 r- 62 \ - 175 Figure 5.11. Activation of fyn and src by Y789F mutant PTPa. H E K 2 9 3 cells were transiently transfected with N R 2 A , fyn or src, and without (-) or with wild-type (wt) or Y789F (789) PTPa . Fyn (A) and src (B) were immunoprecipitated from the cell lysates and analyzed for kinase activity i n an autophosphorylation assay (top panels). Portions o f the immunoprecipitates were probed for fyn or src (middle panels). Portions of the cell lysates were resolved by S D S - P A G E and probed for P T P a (bottom panels) 5.2.9 PTPa tyrosine phosphorylation is development ally regulated A s P T P a tyrosine phosphorylation plays a role in the regulation o f src- and fyn-mediated N R 2 A phosphorylation in a cell culture-based heterologous expression system, the phosphorylation status of PTPa in mouse synaptosomes was examined to see whether tyrosine phosphorylation of PTPa is regulated in vivo. Synaptosomes were prepared from 137 whole brains o f wild-type mice (section 2.2.5.3) at age intervals between day 7 (P7) and day 60 (P60), and, to provide a negative control sample, from P T P a 7 " mice at day 7 (P7). P T P a immunoprecipitates from these samples were immunoblotted with anti-phosphotyrosine antibody. Tyrosine phosphorylation of PTPa was readily detectable at P7 and P14 and dramatically reduced to an almost undetectable level at P30 and P60 (Fig. 5.12, top panel). Reprobing the P T P a immunoprecipitates with anti-PTPa antibodies revealed similar amounts of P T P a in the immunoprecipitates and PTPa was confirmed to be absent in PTPa-null mice (Fig. 5.12, bottom panel). This indicates that P T P a tyrosine phosphorylation is tightly regulated during development, suggesting a role of this phosphorylation in PTPa function in vivo. -I- +/+ Postnatal day: 7 7 14 30 60 PTPa-P PTPa •Jan Figure 5.12. Phosphorylation of PTPa is developmentally regulated. P T P a was immunoprecipated from synaptosomes prepared from wild-type (+/+) mice at postnatal day 7, 14, 30, and 60 and from a P T P a 7 " (-/-) mouse at postnatal day 7. The immunoprecipitates were probed with anti-phosphotyrosine antibody (upper panel), and reprobed with anti-PTPa antibody (lower panel). 5.3 Discussion The studies described in Chapter 4 demonstrated that the ablation o f P T P a in mice resulted in reduced phosphorylation o f the inhibitory C-terminal tyrosine residue o f several 138 synaptosomal SFKs , among them sre and fyn, and the decreased tyrosine phosphorylation of the N R 2 A / B subunits of the N M D A R . Together, these findings suggest that, through dephosphorylating and activating certain SFKs , P T P a functions as a physiological upstream positive regulator of N M D A R tyrosine phosphorylation. In conjunction with other studies that have revealed defective NMDAR-dependent L T P and other NMDAR-associa ted process such as learning and memory in PTPa-null mice (Petrone et al., 2003; Skelton et al., 2003), this suggests that, through its action on SFKs , P T P a regulates N M D A R function and/or localization. In this Chapter, experiments are described that were designed to validate these actions of P T P a on sre and fyn, and on the consequent N R 2 A / B tyrosine phosphorylation, by employing a cell culture system as a model environment in which to examine the controlled interactions and regulation of these molecules. A s predicted, the co-expression of PTPa with fyn further enhanced N R 2 A / B tyrosine phosphorylation in this H E K 2 9 3 cell system, effecting a 2- to 3-fold increase in N R 2 A phosphorylation over that stimulated by the expression of fyn alone. However, in contrast to this result and contrary to expectation, the co-expression of P T P a with sre and NR2A/2J3 resulted in the inhibition of src-mediated N M D A R subunit tyrosine phosphorylation, to a level about 50% of that stimulated by sre in the absence of PTPa . These results were consistent and reproducible in multiple experiments and over a variety o f tested ranges of P T P a and fyn/src expression levels. Reasoning that differential regulation of fyn- or src-mediated N M D A R phosphorylation might represent an important mechanism underlying the distinct src-null and fyn-null phenotypes o f N M D A R -related processes (such as learning and memory) that are observed in mice (Grant et al., 1992), or at least might represent significant cellular mechanisms o f P T P a - S F K interaction and/or regulation, further experiments were carried out to attempt to elucidate the molecular 139 basis for the opposite fyn/src-dependent outcomes effected by P T P a on N R 2 A / 2 B phosphorylation. 5.3.1 Differential SFK-mediated NR2A/B phosphorylation is not due to differences in the intrinsic kinase activities of fyn or src or in the phosphatase activity of PTPa The obvious possibilities that P T P a differentially affected the kinase activities of fyn and src towards N R 2 (Fig 5.1, steps A and B) , or that P T P a itself displayed very different fyn- or src- dependent phosphatase activities towards phospho-NR2 (Fig. 5.1, step C), were initially investigated. In vitro assays of the intrinsic kinase activities of fyn and src demonstrated that both kinases were activated upon co-expression with P T P a (Fig 5.3), suggesting that the failure of P T P a to activate src did not underlie the reduced N R 2 A / B phosphorylation observed when P T P a was introduced into the src/NR2 expression system. Furthermore, the assays demonstrated the ability of P T P a to dephosphorylate both fyn and src, and revealed no obvious SFK-dependent differences in these phosphatase activities of P T P a (Fig 5.3). Likewise, the intrinsic phosphatase activity o f PTPa , determined in an in vitro assay with the artificial substrate pNPP, was not altered upon co-expression with fyn or src (Fig. 5.7). The activity of P T P a as an N R 2 phosphatase has not been previously reported. This was examined in the H E K 2 9 3 cells, however this cellular assay system was limited by the considerable endogenous N R 2 A phosphatase activity that was apparent in the absence o f P T P a in H E K 2 9 3 cells (upon PP2-induced inhibition of further SFK-mediated N R 2 A phosphorylation). Thus, only a very small additional extent o f N R 2 A dephosphorylation could be detected when P T P a was added to the cells, such that the PTPa-specific N R 2 A dephosphorylation that was monitored most likely represented phosphatase activity outside 140 the linear range o f the reaction kinetics. Despite this limitation, P T P a dephosphorylated both fyn-phosphorylated N R 2 A and src-phosphorylated N R 2 A in H E K 2 9 3 cells, and appeared to do so at approximately similar rates and to similar extents (Fig. 5.4). Overall, the above results did not reveal differential actions or abilities of P T P a to regulate the intrinsic kinase activities of fyn and sre, nor did they uncover fyn- and src-dependent differences in the intrinsic phosphatase activity o f PTPa . Although carried out with the limitations described above, in vivo H E K 2 9 3 cell assays indicated that fyn and sre did not have distinct effects on the N R 2 phosphatase activity of PTPa , either through these kinases exerting effects on P T P a or through their distinct specificities for various tyrosine residues in the N R 2 subunits to create potentially differing N R 2 substrates for P T P a . 5.3.2 Potential functional and physical feedback signaling mechanisms between PTPa and fyn or sre P T P a and the SFKs , together with N R 2 phosphorylation as an endpoint, represent a signaling network with multiple layers of complexity. A phosphatase (PTPa) activates a kinase (fyn or sre) (Fig. 5.1, step A ) that phosphorylates NR2A/J3 (NR2) (Fig. 5.1, step B) . The phosphatase can also dephosphorylate N R 2 (Fig. 5.1, step C) . Furthermore, additional physical and functional interactions between these signaling components (Figs. 5.5 and 5.8) have the potential to up- or down-regulate kinase and/or phosphatase activities and thus determine N R 2 phosphorylation status. For example, sre can phosphorylate P T P a (den Hertog et al., 1994). This has been reported to affect the phosphatase activity of P T P a (den Hertog et al., 1994) and/or to regulate PTPa-mediated activation of sre (Zheng et al., 2000) or the SFK-dependent and -independent actions of P T P a in certain signaling systems (Yang 141 et al., 2002; Chen et al., 2006). P T P a can also associate with src (Harder et al., 1998) and fyn (Bhandari et al., 1998; Harder et al., 1998), with effects on S F K and P T P a activities. Thus, the phosphorylation of P T P a by fyn and src (Fig. 5.5, step D) , the physical interaction of P T P a with fyn and src (Fig. 5.8), and the relationship of these events and their consequences on N R 2 phosphorylation, were investigated in the H E K 2 9 3 cell co-expression system. Here, both fyn and src phosphorylated P T P a at its C-terminal tail residue, Tyr789, in a dose-dependent manner (Fig. 5.6). In addition, both kinases associated with PTPa , although clearly more P T P a directly and/or indirectly associated with fyn than with src (Fig. 5.9), providing the first evidence of S F K specificity. While the association of P T P a with fyn was independent of P T P a phosphorylation, about 50% of the association of P T P a with src was dependent upon P T P a phosphorylation (Fig. 5.9), a further indication that distinct modes of P T P a - S F K interaction occur. 5.3.3 The significance of PTPa tyrosine phosphorylation The phosphorylation of PTPa affected N R 2 phosphorylation status, as significantly different N R 2 phosphorylation was observed, in the presence of fyn and src (Fig. 5.10), when wild-type P T P a or a non-phosphorylatable mutant form of P T P a (Y789F) was expressed. However, irrespective o f which kinase was co-expressed with PTPa , the higher N R 2 A phosphorylation observed in the presence of non-phosphorylatable P T P a compared to wi ld-type PTPa , suggested that P T P a phosphorylation had an inhibitory effect on N R 2 phosphorylation. Despite this similar effect with fyn and src, it was apparent that N R 2 A phosphorylation was stimulated in the presence o f fyn/PTPa regardless o f P T P a phosphorylation status, while N R 2 A phosphorylation was significantly reduced (albeit to 142 varying extents) in the presence of src/PTPa regardless of P T P a phosphorylation status. Notably, this key difference was specific for N R 2 phosphorylation outcome, as both wi ld-type and mutant Y789F P T P a were confirmed to stimulate fyn and sre kinase activities in an in vitro assay (Fig. 5.11). Thus, although P T P a phosphorylation modulates the degree o f SFK-regulated N R 2 A phosphorylation, it is not responsible for the basic PTPa-dependent difference in N R 2 A phosphorylation that is observed in the presence o f fyn and sre (i.e. enhanced with fyn or reduced with sre). Phosphorylation of P T P a by fyn or sre cannot account for the opposite effects exerted by PTPa/fyn and PTPa/src on N R 2 phosphorylation. Nevertheless, the results of these studies, namely that post-translational modification of P T P a can negatively modulate both fyn- and src-mediated N R 2 phosphorylation, infer that P T P a can exert a level of regulation on SFK-mediated N R 2 phosphorylation that is in addition to its role as an enzymatic activator of SFKs . A t least in the case of fyn, this regulation is independent of PTPa-fyn association, as wild-type and mutant Y789F P T P a bind to fyn with similar affinities. Intriguingly, the dramatic down-regulation of P T P a phosphorylation detected in mouse synaptosomes after postnatal day 14 indicates that P T P a modification could represent a physiologically relevant mechanism to enhance P T P a action during the later stages of maturation towards adulthood (Fig. 5.12). Another finding made using the H E K 2 9 3 cell expression system that supports the concept of a non-enzymatic mechanism of PTPa-directed regulation is that catalytically inactive P T P a (dm) can stimulate fyn activity and fyn-mediated N R 2 phosphorylation. Together, the above results suggest that protein-protein interactions may be an important regulatory feature of this signaling micro-network. 143 5.3.4 The significance of the association of PTPa with fyn or src A major difference observed in the P T P a / f y n and the PTPa/s rc co-expression systems is the differential extents of association o f P T P a with each kinase, and this difference was most marked when the parameter o f P T P a phosphorylation was eliminated through use o f the Y789F mutant P T P a to measure association. Approximately 10 times more Y789F P T P a (and ~4 times more wild-type P T P a ) directly and/or indirectly associated with fyn than with src (Fig 5.9). The region(s) of fyn and of P T P a that are involved in this interaction are undefined. It is also unclear whether this fyn/src-based specificity accounts for the distinct outcomes of PTPa/kinase co-expression on N R 2 phosphorylation. Certainly there are precedents for fyn- and src-specific protein interactions (with R A C K - 1 and H-ras, respectively) that regulate kinase activity towards the N M D A R subunits (Yaka et al., 2002; Thornton et al., 2003). It would be informative to carry out structure/function studies to systematically replace domains o f fyn with the equivalent portions of src to determine these P T P a binding requirements, and to investigate the role o f this interaction in N R 2 phosphorylation. 5.3.5 The complexity of the cell culture system The phosphorylation of any given protein is directly regulated by the opposing actions of protein kinases and phosphatases. In the present system examining N R 2 phosphorylation, the S F K s src and fyn act as N R 2 kinases and, in addition to unidentified cellular phosphatases, P T P a is demonstrated to have activity as an N R 2 phosphatase. Further complicating the analysis of these NR2-directed kinase and phosphatase activities are the additional actions of P T P a as an S F K phosphatase (and activator) and of fyn/src as P T P a 144 kinases (and potential modulators). To explain the PTPa-enhanced N R 2 phosphorylation in the presence of fyn and the contrasting PTPa-inhibited N R 2 phosphorylation in the presence of src, it is necessary to postulate either that PTPa inhibits src activity while having no effect on or enhancing fyn activity towards N R 2 and/or that src enhances while fyn inhibits or does not affect P T P a phosphatase activity towards N R 2 . These possibilities all necessitate kinase-phosphatase inter-regulation, through catalysis and/or physical interactions, especially between P T P a and src. However as described and discussed above, in vitro phosphatase and kinase assays did not reveal SFK-mediated effects on the phosphatase activity o f PTPa , and confirmed the expected action of P T P a as an activator not only of fyn but also of src. This may indicate that factors in the cellular environment, such as other proteins or even S F K / P T P a cellular localization, are required for S F K / P T P a inter-regulation. However, the phosphatase activities of P T P a towards fyn and src, and towards N R 2 , were indeed measured in the cellular setting and did not exhibit any detectable SFK-specific alterations. Despite the lack of detectably distinct modulating effects of fyn and src upon P T P a catalytic activity, it is clear that the phosphatase activity per se o f P T P a is required for its surprising inhibitory effect on src-mediated N R 2 tyrosine phosphorylation. This is evidenced by the lack o f inhibition exerted by co-expression of catalytically inactive mutant P T P a with src and N R 2 . In addition, the src-catalyzed phosphorylation of P T P a can modulate the inhibitory effect of PTPa , as the PTPa-dependent inhibition of src-mediated N R 2 phosphorylation was partially mitigated by the co-expression of non-phosphorylatable mutant (Y789F) P T P a with src and N R 2 (Fig. 5.10). 145 5.3.6 Summary The studies described in this Chapter have revealed an unexpected effect of P T P a as a negative modulator of src-mediated N R 2 subunit tyrosine phosphorylation that is distinct from the stimulatory effect o f P T P a upon fyn-mediated N R 2 phosphorylation. The basis for the inhibitory effect of P T P a in the former instance was investigated by determining whether P T P a exerted differential effects on the kinase activities o f sre and fyn, and whether sre and fyn exerted differential effects on the phosphatase activities of P T P a . N o readily apparent differences were observed in the variety of in vitro and in vivo assays employed. The potential regulatory mechanisms of protein-protein interaction (between P T P a and fyn/src) and of protein phosphorylation (SFK-catalyzed phosphorylation of PTPa) were also investigated. Both SFKs formed complexes with P T P a , and both S F K s phosphorylated P T P a at its C-terminal tail tyrosine residue, Tyr789. Clear and novel differences were demonstrated in (i) the much higher extent of PTPa - fyn association compared to PTPa-src association, and (ii) the independence of PTPa-fyn association on P T P a phosphorylation compared to the partial dependence of PTPa-src association on P T P a phosphorylation. However, determination of the role of P T P a - S F K association in N R 2 phosphorylation requires future definition o f the specific P T P a and S F K structures/regions involved in each phosphatase-kinase interaction. SFK-catalyzed phosphorylation of P T P a was demonstrated to modulate the extent o f SFK-dependent N R 2 phosphorylation, but not to represent the basis of the observed PTPa-dependent inhibition of src-mediated N R 2 phosphorylation. Finally, it can be concluded that P T P a catalytic activity is required for the inhibitory effect o f P T P a on sre-dependent N R 2 phosphorylation. 146 The finding that fyn and sre phosphorylated P T P a and that this could regulate N R 2 phosphorylation status led to the very interesting finding that P T P a tyrosine phosphorylation in mouse synaptosomes is tightly regulated during postnatal development. Synaptosomal phospho-PTPa was present during at least the first two weeks of age but had virtually disappeared in adult mice (by one month of age). In conjunction with results from the H E K 2 9 3 cell studies, this suggests that P T P a phosphorylation might represent a physiological mechanism to limit or prevent over-stimulation of SFK-mediated N R 2 phosphorylation and thus of N M D A R activity in the maturing animal at a time of intense synaptic development and formation of neural pathways involved in learning and memory circuits. 147 6 GENERAL DISCUSSION I have investigated the role of P T P a in two different systems, the insulin and the N M D A R signaling pathways. This was carried out using mice with a targeted disruption of the P T P a gene that were previously generated by our lab (Ponniah et al., 1999). P T P a 7 " mice are viable, have normal life span, and appear indistinguishable from W T mice. However, biochemical, cell biological and histological studies reveal some alterations in P T P a 7 " mice. Several findings previously made using P T P a 7 " mice, or cells derived from these mice, indicate that S F K s are key substrates and targets of PTPa . Brain src and fyn activities in P T P a 7 " mice are reduced to less than half of those in W T mice (Ponniah et al., 1999). This is concomitant with elevated phosphorylation of src and fyn at the inhibitory C-terminal tyrosine residue (Ponniah et a l , 1999). Similarly, embryonic fibroblasts from P T P a 7 " mice exhibit reduced src and fyn activities (Ponniah et al., 1999; Su et al., 1999). These findings indicate that P T P a is a physiological positive regulator of src and fyn. In contrast to brain and fibroblast src and fyn, fyn activity in P T P a 7 " thymocytes (fynT) is enhanced as a result of the increased phosphorylation of both inhibitory and autophosphorylation sites, suggesting that P T P a can both positively and negatively modulate fynT in this specialized cell type, and that increased autophosphorylation predominates to affect fynT activation (Maksumova et al., 2005). These results suggest that the S F K s src and fyn are in vivo effectors of PTPa . Defects associated with several SFK-mediated signaling pathways occur in the absence o f PTPa . PTPa-deficient embryonic fibroblasts exhibit defective spreading, migration, and haptotaxis likely due to the reduced src and fyn activity, indicating a role for 148 P T P a in integrin signaling (Su et al., 1999; Zeng et al., 2003). PTPa-deficient thymocytes develop normally but the unstimulated thymocytes exhibit enhanced tyrosine phosphorylation of specific proteins and reduced thymocyte proliferation is observed in response to T cell receptor stimulation (Maksumova et al., 2005). This could be due to the enhanced kinase activity o f fynT and/or the absence o f phosphatase activity o f P T P a , indicating a role for P T P a in controlling cellular tyrosine phosphorylation. Alterations in neuronal signaling pathways of PTPa-null animals have also been described. N C A M (neural cell adhesion molecule)-induced fyn activation and neurite outgrowth are abolished in PTPa" 7" hippocampal neurons (Bodrikov et al., 2005), indicating the involvement of P T P a in NCAM-media ted signaling. M i c e lacking P T P a exhibit some defects in NMDAR-re la ted processes such as C A 1 hippocampal neuron migration and learning and memory (Petrone et al., 2003; Skelton et al., 2003). However, in vivo molecular effectors linking P T P a and the N M D A R remain to be identified. N o defects in glucose homeostasis in PTPa" 7" mice have been reported although several studies in cultured cells suggest a role for P T P a in insulin signaling (Moller et al., 1995; Lammer et al., 1998). Thus the physiological functions of P T P a in insulin signaling and the precise molecular role of P T P a in N M D A R signaling remain undefined. Several considerations guided me to investigate the actions of P T P a in these signaling pathways. N M D A R signaling is a neuronal system, whereas insulin signaling is a non-neuronal system, this providing an opportunity to determine physiological tissue-specific functions o f PTPa . In addition, P T P a action in N M D A R signaling is most likely mediated through its action on SFKs , whereas P T P a is reported to operate in insulin signaling via its action on IR/IRSs in an S F K -independent manner. Thus whether the upstream action of P T P a on distinct in vivo effectors, 149 in particular the S F K s as opposed to non-SFK targets, is responsible for the tissue-specific functions of PTPa , is an important question to resolve. Interestingly, a recent finding suggests that the neuronal deficiency o f PTP1B, a key regulator o f LR, affects body weight, glucose homeostasis, and insulin sensitivity (Bence et al., 2006). This uncovers new connections between the neuronal system and insulin-related processes. Accordingly, this study also addresses the possibility that neuronal P T P a could play a role in the regulation of peripheral insulin sensitivity. 6.1 PTPa is not a major regulator of insulin signaling A potential role for P T P a in insulin signaling has been proposed but not confirmed due to the mixed results from cell culture systems with enhanced or ablated expression of P T P a (Moller et al., 1995; Lammers et al., 1998; Jacob et al., 1998). Thus, the involvement of P T P a in insulin signaling was investigated using mice with a targeted disruption of the P T P a gene (Ponniah et al., 1999). Overall, these mice did not exhibit any major alterations in insulin signaling cascades (in three insulin-sensitive tissues) or in glucose homeostasis. P T P a 7 " mice had unaltered circulating blood insulin and glucose levels in random fed and fasted states. Moreover, P T P a 7 " mice performed normally in both intraperitoneal glucose and insulin tolerance tests, with similar blood glucose clearance efficiency and insulin sensitivity, when compared to wild-type mice. Many other PTPs have been investigated for regulation of insulin signaling including L A R , SHP2, PTPe, and PTP IB (Elchebly et al., 2000; Cheng et al., 2002; Asante-Appiah and Kennedy, 2003). O f these, P T P 1 B appears to be the only physiological negative regulator of insulin signaling. In contrast to P T P a 7 " mice, 150 mice lacking P T P I B are able to maintain glucose homeostasis with about half of the circulating insulin and display enhanced glucose clearance efficiency and insulin sensitivity in response to exogenous insulin and glucose (Elchebly et al., 1999). This genetic evidence suggests that, unlike P T P I B , P T P a does not perform any role in the regulation of glucose homeostasis in mice. Ablation of P T P a expression in mice did not result in any changes in insulin-induced IR tyrosine phosphorylation or IR activity in liver, muscle, and adipose tissues. In contrast, when insulin is injected into the portal vein of PTP1B" 7" mice, a significant elevation in IR phosphorylation is observed in muscle and liver when compared to wild-type mice, indicating that P T P 1 B is an IR-specific P T P (Elchebly et al., 1999). In addition to normal IR tyrosine phosphorylation, IRS-1 or IRS-2 tyrosine phosphorylation and the association of each with PI3-K in muscle and liver, respectively, were unaltered in P T P a 7 " mice. However, IRS-1 phosphorylation and its association with PI3-K was found to be higher at the basal unstimulated state in P T P a 7 " liver when compared to W T liver, suggesting that P T P a may function as a phosphatase maintaining basal phosphorylation of IRS-1 in liver prior to insulin stimulation. Nevertheless, this alteration in P T P a 7 " liver did not translate into any differences in the activities of downstream signaling molecules, including Ak t and M A P K , in both unstimulated and insulin-stimulated states. While it cannot be ruled out that the loss of P T P a is compensated for by other PTPs, including PTP I B , that are expressed in insulin-sensitive tissues, these findings clearly indicate that P T P a is not a major regulator of either IR tyrosine phosphorylation or insulin signaling. P T P a has been reported to function as an IR phosphatase as coexpression of PTPa and the IR results in lower insulin-induced IR phosphorylation in B H K and H E K 2 9 3 cells 151 (Moller et al., 1995; Lammers et a l , 1998). However, CD45 , a PTP that is uniquely expressed in T and B cells, is also able to dephosphorylate LR when expressed in this system (Moller et al., 1995). Thus this study system likely represents an artificial effect o f non-physiological protein expression. In contrast, the ablation or overexpression of P T P a in other systems such as G H 4 pituitary cells or 3T3L1 adipocytes does not affect IR tyrosine phosphorylation (Jacob et al., 1998; Arnott et al., 1999). Altogether, this indicates that heterologous P T P a and/or LR expression levels, possibly inappropriate cell types, and non-physiological settings may limit the ability of these studies to evaluate the physiological role of PTPa . Several lines of evidence suggest the involvement of P T P a in insulin-dependent processes. For instance, P T P a counteracts insulin-induced detachment and growth inhibition in B H K - L R cells (Moller et al., 1995), inhibits insulin-stimulated expression of a prolactin reporter plasmid in G H 4 pituitary cells (Jacob et al., 1998), or enhances insulin-stimulated D N A synthesis in myoblasts (Lu et al., 2002). M y present data clearly indicate that P T P a does not play any essential role in insulin signaling. Thus the effect o f P T P a in these insulin-mediated events may be via a mechanism that is independent of insulin signaling. For instance, P T P a may inhibit insulin-induced detachment (Moller et al., 1995) via its action on integrin signaling, resulting in enhanced substrate adhesion as observed in PTPa-expressing A431 cells (Harder et al., 1998). Furthermore, a study has revealed that the inhibitory effect of P T P a on insulin-stimulated expression of a prolactin reporter (Jacob et al., 1995) is through PTPa ' s action on src (Vul in et al., 2005). It is proposed that P T P a activates src/Rho/PI3K signaling and induces cytoskeletal changes, resulting in blockage of activation of the prolactin gene. Studies from our lab and others have reported that the role of P T P a in 152 various cellular processes is mainly dependent on its upstream action on the SFKs sre and fyn (Pallen, 2003). Therefore, it is not surprising that IR signaling and glucose homeostasis in PTPa V"mice are unaltered since S F K s are not required for these events. Thus, in some cases, P T P a may act on S F K s to produce an effect that is additive to the effect of insulin. In summary, the studies described in Chapter 3 have revealed that P T P a is neither a major regulator of insulin signaling nor a major player in controlling glucose homeostasis in mice. This finding eliminates P T P a as a potential target for PTP-directed therapeutics for the treatment of metabolic disorders such as diabetes and obesity. 6.2 P T P a regulates N M D A R tyrosine phosphorylation Previous studies have revealed defects in NMDAR-dependent L T P and other NMDAR-associa ted processes such as spatial learning and C A 1 hippocampal neuron migration in PTPa-deficient mice (Petrone et al., 2003; Skelton et al., 2003). Furthermore, application o f the P T P a intracellular region containing both catalytic domains (D1+D2) into hippocampal neurons enhances NMDAR-media ted currents, whereas blockage of endogenous P T P a causes a reduction in NMDAR-dependent currents (Lei et al., 2002). These findings indicate a positive role of PTPa in modulating N M D A R activity and, consequently, in neuron migration and learning and memory. However, the mechanisms underlying the function of P T P a in NMDAR-media ted processes have not been characterized. The studies in Chapter 4 and 5 in this thesis have identified some in vivo molecular effectors and mechanisms that functionally link P T P a and the N M D A R . 153 I have observed that tyrosine phosphorylation of the N R 2 A and N R 2 B subunits of the N M D A R is reduced in detergent-resistant synaptosomal fractions from P T P a 7 " mice. Tyrosine phosphorylation of N R 2 subunits is reduced by about 25-35% in the absence o f PTPa . This suggests that P T P a functions as a physiological upstream positive regulator of N M D A R tyrosine phosphorylation though an intermediate molecule(s). The S F K s src and fyn regulate N M D A R function by phosphorylating the N R 2 A and N R 2 B subunits of the N M D A R (Cheng and Gurd, 2001; Nakazawa et al., 2001; Yang and Leonard, 2001). Synaptosomal S F K s src, fyn, yes, and lck, but not lyn, from P T P a 7 " mice had reduced activity as indicated by their enhanced phosphorylation at the inhibitory tyrosine residue. This is in accord with a report of a 30-50% reduction in brain src and fyn activity in PTPa-nul l mice when compared to wild-type littermates (Ponniah et al., 1999; Su et al., 1999). N o previous studies have implicated yes and lck in the regulation of the N M D A R . However, yes has been shown to be a component of the N M D A R complex (Kalia and Salter, 2003). Altogether, these findings indicate that src, fyn, and possibly yes and lck are candidate kinase intermediates for the observed PTPa-dependent regulation of N M D A R tyrosine phosphorylation, and consequently N M D A R function in mouse synaptosomes. In support of this, constitutive regulation of N M D A R function by S F K s is reported to be dependent on PTPa , as inhibition of P T P a activity abolishes the effect o f the S F K inhibitor PP2 on NMDAR-media ted currents in hippocampal neurons (Lei et al., 2002). Direct evidence supporting a mechanism of P T P a action on N M D A R through its ability to activate fyn was obtained from experiments utilizing a heterologous co-expression system. A s expected, co-expression of PTPa with fyn activated fyn and further enhanced N R 2 A / B tyrosine phosphorylation. This is consistent with the reduced N R 2 B 154 phosphorylation at Tyr l472 , a major fyn target site, that has been reported in hippocampi of adult P T P a 7 " mice (Petrone et al., 2003). Furthermore, fyn 7" mice and P T P a 7 " mice share some phenotypes, including aberrant hippocampal development, impaired L T P , and impaired spatial learning (Grant et al., 1992). Altogether, these results indicate that fyn is an effector linking P T P a to the N M D A R . Surprisingly and unexpectedly, co-expression of P T P a with sre in the same system, the H E K 2 9 3 cell expression system, resulted in the inhibition of src-mediated N R 2 A / B phosphorylation (although sre was also activated by PTPa) . Various studies indicate an essential role of sre in N M D A R function (Wang and Salter, 1994; Y u et al., 1997). However, unlike fyn 7" adult mice, sre7" mice exhibit normal L T P , suggesting that fyn may compensate for the lack of sre in sre7" mice (Grant et al., 1992). Neuroanatomical or behaviorial abnormalities in sre7" mice have not been reported, therefore whether sre is also physiological intermediate molecule of P T P a in the regulation of N M D A R tyrosine phosphorylation and function is unclear. Efforts have been made to determine the basis of the differential regulation of N R 2 A / B tyrosine phosphorylation by PTPa/src and PTPa/fyn. N o differences were observed in a variety o f in vitro assays employed to determine the intrinsic activities o f sre/fyn and PTPa . Notably, P T P a was found capable of dephosphorylating both fyn- and src-phosphorylated N R 2 . Clear and novel differences in protein-protein interactions (between P T P a and fyn or P T P a and sre) and protein phosphorylation (fyn- or src-catalyzed phosphorylation of PTPa) were observed. The observation that fyn and sre exhibit feedback on P T P a by effecting its phosphorylation, and that has been reported to be important for P T P a action in various signaling pathways (Yang et al., 1998; Zheng and Shalloway, 2001; 155 Chen et a l , 2006) make it difficult to address the regulation of N R 2 A / B phosphorylation by P T P a i n the presence o f S F K s . However, it is possible that N R 2 phosphorylation is regulated by kinase (fyn/src)-phosphatase (PTPa) interactions of a catalytic and/or physical nature. Although no differences in vitro phosphatase and kinase activities were detected, it is possible that P T P a and S F K s exhibit inter-regulation in a cellular environment, for example as mediated through other proteins or even by P T P / S F K cellular localization. For instance, P T P a may inhibit src activity while having no effect on or enhancing fyn activity towards N R 2 , and/or src may enhance while fyn inhibits or does not affect P T P a phosphatase activity towards N R 2 . Although these studies in cell culture revealed an unexpected effect of P T P a as a negative regulator of src-mediated N R 2 phosphorylation, the parallel studies in mice reveal that P T P a is clearly a physiological upstream positive regulator of N M D A R tyrosine phosphorylation. In the physiological system, P T P a may modulate N M D A R phosphorylation and action via its action on more than one S F K . P T P a action via fyn has been confirmed, since fyn-mediated tyrosine phosphorylation of the N R 2 B subunit is reduced in PTPa" 7" hippocampus (Petrone et al., 2003). Furthermore, fyn"7" mice exhibit a partially similar phenotype to PTPa" 7" mice, including aberrant hippocampal development, impaired L T P , and impaired spatial learning (Grant et al., 1992). To date, no studies have reported any neuroanatomical or behavior abnormalities in mice deficient in other neuronal SFKs . The phenotype of combined src/fyn/yes knockout mice (Stein et al., 1994) indicates that these S F K s have partially overlapping and redundant functions. Further studies are required to investigate the role of P T P a in regulation of N M D A R function via individual SFKs . 156 Besides SFKs , I have identified another potential intermediate molecule in PTPa-mediated regulation of N M D A R tyrosine phosphorylation and function. Autophosphorylation of Pyk2 at its key activation site, Tyr402, was reduced in PTPa" 7" mice. Co-expression of P T P a in H E K 2 9 3 cells enhanced the association of fyn and Pyk2, suggesting that the ablation of P T P a in mice may also affect Pyk2 and S F K association. Similar to PTPa" 7" mice, Pyk2 phosphorylation in hippocampi o f fyn"7" mice is also decreased (Corvol et al., 2005), indicating that P T P a and fyn could potentially regulate Pyk2 phosphorylation by similar mechanisms. How the absence of fyn causes a reduction in Pyk2 phosphorylation is still unclear since the S F K inhibitor PP2 inhibits integrin-stimulated N M D A R tyrosine phosphorylation, but not Pyk2 autophosphorylation (Bernard-Trifilo et al., 2005). Furthermore, Huang et al. (2001) have reported that in N M D A R signaling, Pyk2 can function upstream of sre to up-regulate N M D A R function. Pyk2 is closely related to F A K and regulation of F A K by P T P a has been demonstrated, albeit v ia the SFKs that function upstream of F A K . In integrin signaling, PTPa-activated sre and fyn promote F A K autophosphorylation and F A K association with fyn and sre (Zeng et al., 2003). However, although Pyk2 and F A K are similar in regions of sequence identity, overall structure, and functions, they are distinct in their regulation and signaling (Girault et al., 1999). Thus further studies are required to investigate the nature of the potential PTPa-catalyzed activation of Pyk2. Altogether, this study suggests that P T P a positively affects Pyk2 activation by modulating Pyk2 autophosphorylation and its association with SFKs and that the altered Pyk2 activity may contribute to reduced N M D A R phosphorylation in PTPa" 7" mice. 157 The findings that P T P a tyrosine phosphorylation can up-regulate N R 2 phosphorylation in co-transfected cells and that P T P a tyrosine phosphorylation in mouse synaptosomes is developmentally down-regulated during postnatal development indicate a potential mechanism to limit or prevent over-stimulation of SFK-mediated N R 2 phosphorylation, and thus N M D A R activity. Altogether, the studies in Chapter 4 and 5 have revealed a role of P T P a in the regulation of N M D A R tyrosine phosphorylation, and implicated fyn, src, yes, lck, and/or Pyk2 in the action of P T P a on the N M D A R . Furthermore, the tyrosine phosphorylation of P T P a may play a role in modulating P T P a actions on SFK-mediated N M D A R tyrosine phosphorylation. 6 . 3 Significance of the regulation of NMDAR tyrosine phosphorylation by PTPa in NMDAR-related processes Previous studies have reported that mice lacking P T P a exhibited defects in learning and memory, hippocampal neuron migration, and L T P (Petrone et al., 2003; Skelton et al., 2003), processes that are dependent on N M D A R function. Moreover, studies from hippocampal neurons have revealed a positive role for P T P a in regulation of N M D A R -mediated whole-cell currents and induction of synaptic L T P in hippocampal neurons (Lei et al., 2002). These lines of evidence indicate an involvement of P T P a in NMDAR-re la ted processes. The results of studies described in Chapter 4 suggest that the defects reported in PTPa-nul l mice may be linked to impaired N M D A R tyrosine phosphorylation. Furthermore, this investigation also reveals that fyn, src, yes, lck, and Pyk2 are candidate kinase 158 intermediates for PTPa-dependent regulation o f N M D A R tyrosine phosphorylation. It has been shown that sre and fyn phosphorylate N M D A R , resulting in enhanced N M D A R activity ( Y u et a l , 1997; Okumura-Noji, 1995) and altered synaptic localization and surface expression of the receptor (Grosshans et al., 2002; Prybylowski et al., 2005). This suggests that alteration of N M D A R tyrosine phosphorylation in mice lacking P T P a may affect N M D A R activity and/or its localization or expression at synapses, and therefore impact on NMDAR-media ted processes. P T P a action on NMDAR-unrela ted signaling mechanisms may further contribute to the lack o f synaptic plasticity and the impairment of memory in PTPa" 7" mice. For instance, suppression of specific potassium channels has been reported to impair L T P in C A 1 hippocampal neurons (Meir i et al., 1998). P T P a is capable of dephosphorylating and activating potassium channels (Tsai et al., 1999; hnbrici et al., 2000). This suggests that P T P a may also regulate L T P in C A 1 through its action on potassium channels. Furthermore, integrin signaling has been implicated in the multiple stages of the development o f forebrain lamination (Magdaleno and Curran, 2001). Moreover, it has been reported that synaptic integrins activate local tyrosine kinases including F A K , Pyk2, and S F K s , resulting in enhanced N M D A R tyrosine phosphorylation and, consequently, NMDAR-media ted function in mature hippocampal synapses (Bernard-Trifilo et al., 2005). A role for P T P a in integrin signaling in the non-neuronal fibroblast system has been revealed (Su et al., 1999; Zeng et al., 2003). This suggests that P T P a may also be involved in these NMDAR-re la ted processes via its action on a neuronal integrin signaling pathway. N M D A R function is tightly regulated in normal organisms. In humans, hypofunction o f N M D A R s has been implicated in multiple neuronal abnormalities including schizophrenia 159 (Laruelle et al., 2003). In contrast, the excessive and prolonged activation of N M D A R s results in neuronal cell death, a cause of various neuropathies, ranging from acute hypoxic-ischemia brain injury to chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease (Lynch and Guttmann, 2002). A number of N M D A R antagonists have been developed and tested in pre-clinical and clinical studies for their potential neuroprotective activity in neurodegenerative disorders. However, most of these drugs have been unsuccessful because of their strong impact on excitatory synaptic transmission, which causes unacceptable adverse effects. Memantine is the only N M D A R antagonist reported to be effective in inhibiting activated channels with low side effects as observed in human clinical trials for treatment of moderate to severe Alzheimer's disease (Chen and L ip ton, 2006). Thus, the search for drugs that limit excito toxicity without harmful consequences is on-going. P T P a is highly expressed in the brain and its expression and phosphorylation are tightly controlled during development. The lack o f P T P a cannot be compensated for by other cellular molecules in the regulation of N M D A R activity, suggesting a unique role of P T P a in N M D A R tyrosine phosphorylation and function. Thus, abnormalities in regulation of P T P a expression and activity may underlie some N M D A R -associated pathologies. This indicates the potential utility of P T P a as a potential target for PTP-targeted therapeutics for the treatment of neurodegenerative disorders. 6.4 Future directions Accumulating evidence of the association of P T P a with various cell surface receptors, including F3/contactin, integrins, N C A M , and the N M D A R itself (Zeng et al., 160 1999; Le i et al., 2002; von Wichert et al., 2003; Bodrikov et al., 2005), suggests that P T P a action as an activator of S F K s is regulated by its interactions with ligand-stimulated receptors. Further studies are required to determine which of the multiple neuronal receptors that regulate SFK-catalyzed N M D A R tyrosine phosphorylation and function/localization (Salter and Kal ia , 2004) may do so via a P T P a - m e d i a t e d signaling mechanism. Proper N M D A R function is important for neuronal development, synaptic formation and plasticity (McBa in and Mayer, 1994). Deregulation of N M D A R function has been implicated in numerous neurodegenerative diseases such as hypoxic-ischemia brain injury, Alzheimer's disease, Parkinson's disease, and Huntington's disease (Lynch and Guttmann, 2002). The function of P T P a in normal physiology has been demonstrated. Further studies should focus on the dysfunction of P T P a underlying NMDAR-associa ted signaling defects manifested as disease symptoms/states. Lastly, P T P a is found to differentially regulate N M D A R phosphorylation in a fyn-and src-dependent manner in a H E K 2 9 3 cell culture system. To provide genetic evidence for a function o f P T P a upstream of sre and fyn, disruption of both P T P a and fyn, or both P T P a and sre, in mice would be potential systems to understand the physiological regulation of N M D A R tyrosine phosphorylation and function by the specific phosphatase/kinase signaling modules of P T P a / f y n and P T P a / s r c . 6.5 O v e r a l l s u m m a r y This study indicates a role for P T P a in N M D A R signaling via its action on S F K s but eliminates its function in insulin signaling, in particular, its action as an IR phosphatase. This 161 further supports the emerging concept that P T P a is the best-characterized PTP regulator o f S F K s and that P T P a participates in multiple signaling systems mainly via its upstream action on S F K s . However, within the complex setting of the whole organism, or even within a cell, P T P a is unlikely to be the sole regulator of SFKs since several other PTPs such as PTP1B, SHP1/2 and PTPe are also capable of activating SFKs (Roskoski et al., 2005). It is possible that in some cases, these PTPs may uniquely control the actions of a specific S F K in individual signaling pathways, indicating the independent and distinct roles of each PTP . However, in other cases, individual PTPs may act through a common S F K substrate to regulate a single pathway, indicating an overlapping function of these PTPs. Although some PTPs target the same substrate, it does not necessarily imply that these PTPs are fully redundant as their expression patterns, physical access to the substrate, and the regulation o f their catalytic activities may vary. If the lack of P T P a is partially compensated for by other PTPs, mutant animals with ablated P T P a expression may display a relatively weak phenotype. Thus, gene knockouts in animals may result in compensatory effects, thereby limiting the ability o f this method to evaluate the exact role of P T P a in cell signaling. Several other approaches have been employed to overcome the limitations of such single gene knockout studies. The combined ablation of PTPs whose roles overlap in part is an alternative method to evaluate the precise physiological function of a specific PTP. In addition, a chemical approach using selective PTP inhibitors is a method with many advantages such as simplicity, speed, tenability, and reversibility; however, it is a challenge to design potent and selective P T P inhibitors since the active sites of P T P superfamily members are highly 162 conserved. Thus, several approaches must be utilized to investigate and determine the specific functions of PTPa in normal physiology. 163 7 REFERENCES Adams J. P. and Sweatt J. D . (2002) Molecular psychology: roles for the E R K M A P kinase cascade in memory. 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