Functional interactions of protein tyrosine phosphatase alpha (PTPa) and src in mouse development and integrin singaling investigation of double PTPa src deficient mice and cells

Functional Interactions of Protein Tyrosine Phosphatase Alpha (PTPα) and Src in Mouse Development and Integrin Signaling: Investigation of Double PTPα/Src-Deficient Mice and Cells CHEN MIN NATIONAL UNIVERSITY OF SINGAPORE 2007 Functional Interactions of Protein Tyrosine Phosphatase Alpha (PTPα) and Src in Mouse Development and Integrin Signaling: Investigation of Double PTPα/Src-Deficient Mice and Cells CHEN MIN (M.Sc., Shanghai Medical University) (B.Med., Shanghai Medical University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 i Acknowledgements I would like to take this opportunity to express my sincere gratitude to my supervisor, Professor Catherine J PALLEN, although word is not always enough Thanks to her great scientific guidance, encouragement, and endless patience, I can go through my graduate study, and complete my thesis with her kind help and careful proofreading I am thankful for my supervisory committee members, Dr Pauline JOHNSON and Dr Michael COX, in Canada; Dr Xinmin Cao, Dr Kongpeng LAM, and Dr Borluen Tang in Singapore Their thoughtful ideas and broad knowledge helped me a lot for the progress of my project I was so luck to have a chance to work with such nice people either in Singapore or in Canada I am thankful for their generosity for sharing the reagents and information, and their wisdom and broad knowledge made my life in the lab more interesting I don’t think I have guts to pursue the degree without the persistent and strong support from my husband, Yan XU I want to let him know that his understanding is always precious, and I really appreciate that I am forever indebted to my parents I want to say thanks to them for their understanding and encouragement I am so grateful for my parents-in-law to take care of my adorable son without any complaint ii Table of Contents Acknowledgements…………………………………………………………………… i Table of contents……………………………………………………………………….ii List of tables………………………………………………………………………….viii List of figures……………………………………………………………………… ix Abbreviations………………………………………………………………………… xi Summary…………………………………………………………………………… xiii CHAPTER1 Introduction ……………………………………………………………1 1.1 Protein phosphorylation……………………………………………… 1.2 Protein tyrosine phosphatase (PTP) superfamily 1.3 Catalytic mechanism of protein tyrosine phosphatases………… 1.4 Receptor-like PTPs and their features ……………………………… 1.5 Receptor-like tyrosine phosphatase alpha (PTPα) ……………………8 1.5.1 Overview of PTPα …………………………………………… 1.5.2 Biological properties of PTPα ……………………………… 10 1.5.2.1 Substrates of PTPα.………………………………10 1.5.2.2 Biological functions of PTPα …………………… 10 1.5.3 Combinatorial regulation of PTPα catalytic activity and specificity …………………………………………………….13 1.5.3.1 1.5.3.2 1.5.3.3 1.5.3.4 1.5.3.5 1.6 Dimerization………………………………… 13 Phosphorylation…………………………………….14 Protein-protein interactions…… ………………… 16 Proteolysis …………….………………………… 18 Oxidation……………………………………………18 PTPs involved in regulating Src family kinases (SFKs)………… .18 1.6.1 Structure and regulation of SFKs …………………………….19 1.6.2 Regulation of SFK activity by PTPs ……………………… 23 iii 1.6.2.1 PTPα… …………………………………………….23 1.6.2.2 CD45……………………………………………… 24 1.6.2.3 PTP1B………………………………………………26 1.6.2.4 SHP1 and SHP2…………………………………….27 1.7 Integrin signaling…………………………………………….…… 28 1.7.1 Kinases in integrin-mediated signaling……………………… 31 1.7.1.1 Focal adhesion kinase (FAK)…………………….…31 1.7.1.2 SFKs in integrin signaling……………………….….34 1.7.2 PTPs in integrin signaling………………………… ……… 36 1.7.2.1 PTPα …………………………………………….….36 1.7.2.2 SHP2……………………………………………… 38 1.7.2.3 PTP1B…………………………………………… 39 1.7.2.4 PTP-PEST……………………………………… 41 1.7.2.5 PTEN……………………………………………….42 1.7.3 Summary of integrin-induced signaling events… ………….43 1.8 Research rationale and objectives…………………………………… 44 CHAPTER 2: Materials and Methods…………………………………………… 47 2.1 Mouse genotyping……………………… 47 2.1.1 DNA extraction from mouse tail tips or embryonic yolk sacs………………………………………………… 47 2.1.2 Genotyping for PTPα and Src ……………… 47 2.2 Generation of PTPα/Src double mutant mice………………….… .48 2.3 Embryonic dissection………………………………………….…… 50 2.4 Mouse growth observation………………………………………… 50 2.5 Histological staining……………………………………….………….50 2.6 Experiments with mouse embryonic fibroblasts…………………… 51 2.6.1 Derivation of mouse embryonic fibroblasts………………… 51 2.6.2 Other mouse embryonic fibroblasts………………………… 52 2.6.3 Cell proliferation assay……………………………………… 52 iv 2.6.4 Cells stimulation with extracellular matrix (ECM) components………………………………………………………… 53 2.6.5 Cell adhesion, spreading and migration assays……………….53 2.6.5.1 Cell adhesion assay ……………………………… 53 2.6.5.2 Cell spreading assay …………………………… ….54 2.6.5.3 Cell migration assay……………………………….54 2.7 Immunofluorescent staining……………………………….………….55 2.8 Protein analysis……………………………………………………….56 2.8.1 2.8.2 Determination of protein concentration …… …….……… 56 2.8.3 Immunoblotting……………………………………… … 57 2.8.4 Immunoprecipitation …………………………… …… .57 2.8.5 2.9 Cell lysis……………………………….………… …………56 Quantification of proteins…………………………… … .58 Transient transfection…………………………………….………… 58 2.9.1 2.9.2 2.10 Plasmids amplification and purification ……… …….………58 Cell culture and transient transfection………………………59 Treatment of cells with inhibitors……………………………… .60 2.10.1 2.10.2 2.11 PP2 and PP3 treatment………………………… ……… 60 Cytochalasin D treatment…………………… … ……… 60 PTPα adenovirus expression system…………………… .61 2.11.1 Generation of pKS-PTPαY789F (∆ Pac) ………………….61 2.11.2 pAdEasy transfer vector (pShuttleCMV) subcloning …… 62 2.11.3 Generation of pAdEasy recombinant plasmids in bacterial cells …… .63 2.11.4 Transfection of recombinant pAdEasy plasmid into Qbi-293A cells……… …………………………………………… 63 2.11.5 Confirmation of adenoviral-mediated PTPα expression… 64 2.11.6 Amplification of viral particles …………… ………… 64 v 2.11.7 Cesium chloride (CsCl) purification of recombinant adenovirus ……………………….……………… …… 65 2.11.8 Viral particles titration.……….………… ……………….66 2.11.9 Re-introduction of wild type and mutant PTPα into PTPα-/cells by recombinant adenovirus infection …… ……… 67 CHAPTER 3: Characterization of PTPα-/-Src-/- Double Knockout Mice …… 68 3.1 Overview …………………………………………………………… 68 3.2 Results ……………………………………………………………… 68 3.2.1 3.2.2 Post-natal survival and growth of double mutant PTPα-/-Src-/mice ……………………………………… ………… .72 3.2.3 3.3 Combined ablation of PTPα and Src does not result in embryonic lethality … ………………………………… 69 The combined ablation of PTPα and Src does not affect organogenesis ………………………………………… .76 Discussion…………………………………………………………….81 CHAPTER 4: Phenotypes of PTPα/Src Double Mutant Mouse Embryonic fibroblasts (α/s DKO)……… 86 4.1 Overview ……………………………………………………….… 86 4.2 Results …………………………………………………………… .87 4.2.1 4.2.2 α/s DKO cells are defective in fibronectin-induced cell adhesion and spreading ………………………………… 91 4.2.3 Integrin-induced cytoskeletal organization is altered in α/s DKO cells …………………………………… ………… 95 4.2.4 Integrin-induced FAK tyr397 phosphorylation is not affected in α/s DKO cells …… … ……………….101 4.2.5 4.3 Mouse embryonic fibroblasts deficient in both PTPα and Src display a distinctive morphology after spontaneous immortalization … .87 Constitutive activation of Erk is a consequence of the combined absence of PTPα and Src……………………….104 Discussion………………………………… 105 vi CHAPTER 5: Integrin-induced PTPα Tyrosine Phosphorylation is Required for cytoskeletal Reorganization and Cell Migration ……………… 112 5.1 Overview ……………………………………………………………112 5.2 Results ………………………………………………………………113 5.2.1 5.2.2 SFKs are essential for FN-induced PTPα tyrosine phosphorylation ………………………………….………115 5.2.3 Catalytically inactive mutant PTPαDM or Tyr789 mutant PTPαY789F is not phosphorylated upon integrin stimulation……………………………………… …… 116 5.2.4 Integrin-induced PTPα phosphorylation is dependent on an intact actin cytoskeleton and FAK …… ……………… 119 5.2.5 A PTPα adenoviral expression system efficiently reintroduces wild type and mutant forms of PTPα into PTPα-/fibroblasts………………………………………… … 120 5.2.6 PTPα phosphorylation at Tyr789 is not required for integrininduced Src/Fyn activation and FAK or paxillin phosphorylation ……………………… …………… 121 5.2.7 PTPα Tyr789 phosphorylation is required for integrin induced cell spreading and cytoskeletal organization…………… 126 5.2.8 PTPα Tyr789 phosphorylation is required for integrinstimulated cell migration …… ………………………… 129 5.2.9 The cell detachment-induced dephosphorylation of PTPα is not due to auto-dephosphorylation ……………………… 130 5.2.10 5.3 Integrin-induced tyrosine phosphorylation of PTPα … …113 SHP2 is not the phosphatase responsible for detachmentinduced dephosphorylation of PTPα …………………131 Discussion ……………………………………………………… 133 CHAPTER 6: GENREAL DISSCUSSION and CONCLUSIONS……………136 6.1 Roles of PTPα and Src in embryonic development ……………… 136 6.1.1 PTPα is not essential for embryonic development, but is required for normal hippocampal development and proper function ………………………………………………… 137 vii 6.1.2 6.1.3 6.2 SFKs play essential but redundant roles in embryonic development ………………….………………………… 139 A combined deficiency in PTPα and Src does not affect mouse embryonic development, but does increase postnatal mortality 141 The roles of PTPα and Src in integrin signaling ……………… …143 6.2.1 6.2.2 Role of PTPα as an activator of SFKs in integrin signaling………………………………………………… 145 6.2.3 6.3 SFKs are required for integrin signaling ………………….143 Additional roles of PTPα-mediated SFK activation in integrin signaling ……………… ………………………….…….146 Regulation of PTPα by integrin stimulation………………….…… 151 6.3.1 PTPα is tyrosine phosphorylated upon integrin stimulation, but this is not required for SFK activation … ….……… 151 6.3.2 PTPα Y789 phosphorylation is required for integrinstimulated cell spreading and migration.………………… 155 6.3.3 Two roles of PTPα in integrin signaling …………….… 156 6.3.4 The reciprocal link between integrin-induced PTPα phosphorylation and cytoskeletal organization may underlie the defects observed in α/s DKO cells ……………….157 6.4 Overall summary ………………………………………………… 159 6.5 Future directions ………………………………………….…………162 Reference ………………………………………………………………………… 164 Publications ……………………………………………………………………… 199 viii LIST of TABLES 2.1 Primer sequences used in PCR reactions for mouse PTPα and Src genotyping…………………………………………………………………….49 2.2 Sequences of primers used to generate the PTPαY789F mutant ……….……62 3.1 Embryos obtained from heterozygous PTPα/Src intercrosses (PTPα+/-Src+/-) x (PTPα+/-Src+/-) ………………………………………………………….…… 70 3.2 Embryos obtained from homozygous PTPα and heterozygous Src intercrosses (PTPα-/-Src+/-) x (PTPα-/-Src+/-) …………………………………… …… 71 3.3 Pups obtained from homozygous PTPα and heterozygous Src intercrosses (PTPα-/-Src+/-) x (PTPα-/-Src+/-)……………………………………………….73 3.4 Weights of organs…………………………………………………………… 79 3.5 Organ weight as a 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integrin-stimulated neuroblastoma cell motility Oncogene 2007; (in press) .. .Functional Interactions of Protein Tyrosine Phosphatase Alpha (PTPα) and Src in Mouse Development and Integrin Signaling: Investigation of Double PTPα /Src- Deficient Mice and Cells CHEN MIN... integrin signaling, and plays a negative feedback role in orchestrating integrin signaling To determine how PTPα is regulated upon integrin stimulation and how a signal emanating from integrin. .. Phosphorylation of PTPα at Ser180 and Ser204 reduces the affinity of Grb2 SH2 binding to phospho-Tyr789 of PTPα without reducing the affinity of Src SH2 binding, resulting in less Grb2 and more Src binding