Role of stat3 in cell migration

A CYTOPLASMIC ROLE OF STAT3 IN DIRECTIONAL CELL MIGRATION PRESTON TERK SHIN TENG B.Sc. (Hons), UNIVERSITY OF MELBOURNE A THESIS SUBMITTED FOR THE DEGREE OF DOCTORAL OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgement First and foremost, I wish to express my appreciation to all the people that I have met and interacted during the candidature of my PhD, for the completion of this thesis would not have been possible without you all. In particular, I would like to thank A*STAR (Agency for Science, Technology and Research) for providing me the opportunity to work with my supervisor, Associate Professor Xinmin Cao, whom I am indebted to for her endless patience, support and guidance throughout this course. In addition, my gratitude goes to members of my thesis advisory committee, Associate Professor Bor Luen Tang, Associate Professor Thomas Leung and Dr Jinrong Peng, as well as Associate Professor Edward Manser and Dr Jackson Zhao for their advices given. Next, I would to thank the past and present members of the laboratory for their help and discussion. Special thanks go to Dr Dominic Ng and Miss Baohong Lin for the stimulating feedbacks and assistances. In addition, I am grateful to Dr Cheh Peng Lim for the reading of this thesis. I would also like to thank Professor Valeria Poli for providing the Stat3-deficient cells used in this research. Lastly, I would like to thank my family and friends for their support and encouragement given. Once again, heaps of thanks to everyone for accompanying me through this enriching journey. i Table of Contents Acknowledgement i Table of Contents ii List of Figures vii List of tables x Abbreviations xi Summary xiv Chapter 1: Introduction 1.1 Signal transduction 1.2 Signal transducer and Activator of Transcription (STAT) 1.3 Discovery of STAT proteins 1.4 Functional domains of STAT proteins 1.4.1 N-terminal domain 1.4.2 Coiled-coil domain 1.4.3 DNA-binding domain 1.4.4 The linker domain 1.4.5 The SH2 domain 1.4.6 The transactivation domain 1.5 Phosphorylation of STATs is a key regulatory mechanism 10 1.6 Regulation of STAT proteins 11 1.6.1 STAT activation by cytokine signalling 12 ii 1.6.2 STAT activation by growth factor signalling and other kinases 1.7 Negative regulation of STAT signalling 14 14 1.7.1 Suppressor of cytokine signalling (SOCS) 15 1.7.2 Protein inhibitors of activated STAT (PIAS) 15 1.7.3 Ubiquitination and degradation of STAT proteins 16 1.7.4 Dephosphorylation of STAT proteins and JAKs 16 1.8 Functions of STAT proteins 17 1.8.1 Stat1 18 1.8.2 Stat2 19 1.8.3 Stat3 19 1.8.4 Stat4 and Stat6 20 1.8.5 Stat5a and Stat5b 21 1.9 STAT proteins and tumourigenesis 1.9.1 Stat3 in oncogenesis 1.10 STAT3 and motility 22 22 25 1.10.1 Role of STAT3 in cell migration under physiological conditions 25 1.10.2 Role of Stat3 in cell invasion and metastasis 27 1.10.3 Cytoplasmic role of Stat3 in regulating microtubules 28 1.11 Cell migration 29 1.11.1 Process of cell migration 29 1.11.2 Coupling membrane protrusion and adhesion in cell migration 32 1.11.3 Rho family of small GTPases 32 1.11.4 Regulation of Rho GTPases 34 iii 1.11.5 Rho GTPases and the actin cytoskeleton 36 1.11.6 Rho GTPases and cell polarity 39 1.11.7 Regulation of Rho GTPases activation 44 1.11.7.1 Rho GTPases and integrin signalling 45 1.11.8 Rho GTPases and focal adhesion disassembly 50 1.11.9 Dysregulation of the Rho GTPases and cell migration 53 1.12 Objectives of study 54 Chapter 2: Materials and Methods 56 2.1 Chemical and reagents 57 2.2 Antibodies 57 2.3 Cell Cultures 58 2.4 Molecular cloning 58 2.4.1 Construction of expression plasmids 58 2.4.2 Experiments involved in construction of expression plasmids 59 2.4.2.1 Polymerase chain reaction (PCR) 59 2.4.2.2 Restriction endonuclease digestion of DNA 59 2.4.2.3 DNA agarose gel electrophoresis 60 2.4.2.4 Gel extraction of DNA and ligation 60 2.4.3 Bacterial transformation 61 2.4.4 Plasmid DNA preparation and purification 62 2.5 DNA plasmids and siRNA transfections of mammalian cells 63 2.6 Retroviral transductions and generation of stable cell lines 63 iv 2.7 Cell lysis and immunoprecipitation 64 2.8 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis 65 2.9 Cell migration and spreading analysis 66 2.9.1 Wound healing assay 66 2.9.2 Random migration assay 67 2.9.3 Cell spreading assay 68 2.10 Immunofluorescence 68 2.11 Generation and purification of bacterial recombinant GST proteins 69 2.12 Rho GTPases activity assays 70 2.12.1 Conventional pulldown assay 70 2.12.2 Rho G-LISA™ activation assay 71 2.13 Luciferase assay 72 2.14 Statistical analysis 72 Chapter 3: Results 73 3.1 Stat3-deficient murine embryonic fibroblasts are impaired in directional migration 74 3.2 Loss of Stat3 expression promotes random migration 76 3.3 ΔSt3 MEFs are impaired in polarisation of the MTOC 77 3.4 ΔSt3 MEFs are compromised in polarisation of the actin cytoskeleton 80 3.5 Stat3 differentially regulates the microtubule and actin cytoskeleton 82 v 3.6 ΔSt3 MEFs displayed elevation of Rac1 activity 84 3.7 Stat3 expression is implicated in the regulation of Rac1 activity in MCF-7 cells 87 3.8 Integrin and FAK signalling are not affected in ΔSt3 MEFs 88 3.9 Restoration of Stat3 expression in ΔSt3 MEFs 91 3.10 Stat3 regulates Rac1 activation to modulate persistence in migration 93 3.11 Rac1 activity is the determinant of persistence in directional migration 97 3.12 Stat3 affects organisation of the actin cytoskeleton through regulation of Rac1 activation 100 3.13 Stat3 interacts with β-Pix, a Rac1/Cdc42 GEF 104 3.14 Functional analysis of Stat3/β-Pix interaction 110 Chapter 4: Discussion 114 References 128 vi List of Figures Figure 1.1. Comparison of the structures of STAT dimer-DNA complex. Figure 1.2. A schematic representation of the functional domains of Stat3. Figure 1.3. Canonical JAK/STAT pathway in cytokine signalling 13 Figure 1.4. Schematic diagram illustrating the four basic steps of cell migration 31 Figure 1.5. Classical actin-based cytoskeletal structures mediated by Rho GTPases 33 Figure 1.6. The Rho GTPase cycle 35 Figure 1.7. Tread milling model of actin polymerisation at the lamellipodia 37 Figure 1.8. A schematic diagram illustrating wound healing assay Figure 1.9. Reorientation of the microtubule organisation centre and Golgi apparatus during wound-induced migration 41 (MTOC) 42 Figure 1.10. Coupling of cell adhesion to membrane protrusion mediated by integrin signalling. 46 Figure 1.11. Regulation of focal adhesion disassembly 51 vii Figure 1.12. Rho GTPases regulate adhesion and reorganisation of the microtubule and actin cytoskeleton during cell migration 53 Figure 3.1. ΔSt3 MEF exhibited altered morphology during migration. 75 Figure 3.2. Effect of Stat3 on directional persistence of migration 78 Figure 3.3. ΔSt3 MEFs are defective in MTOC polarisation 79 Figure 3.4. Effect of Stat3 on organisation of the actin cytoskeleton 82 Figure 3.5. Stat3 differentially regulates the microtubule and actin cytoskeleton 83 Figure 3.6 ΔSt3 MEFs exhibit elevated Rac1 activation during integrinmediated adhesion and migration. 86 Figure 3.7. Knockdown of Stat3 expression in MCF-7 cells increases Rac1 activation. 89 Figure 3.8. Effect of Stat3 on FAK signalling during integrin mediated adhesion 90 Figure 3.9. Restoration of Stat3 expression in ΔSt3 MEFs 92 Figure 3.10. Expression of GFP-Y705F Stat3 in ΔSt3 MEFs regulates Rac1 activation and restores persisted migration 96 Figure 3.11. Increase of Rac1 activity promotes random migration 99 viii Figure 3.12 Stat3 mediate stress fibre formation 104 Figure 3.13. Stat3 interacts with β-Pix 109 Figure 3.14. 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Science 264, 95-98. 154 [...]... also participates in cell invasion and motility Although a majority of these studies has implicated and emphasised the importance of Stat3 transcriptional activity in cell migration, little is known about how Stat3 regulates cell migration In this study, we used Stat3- deficient and wild type Stat3- expressing murine embryonic fibroblasts as a model to investigate the role of Stat3 in cell migration We have... properties of the various domains in STAT proteins are illustrated in Figure 1.2 Figure 1.1 Comparison of the structures of STAT dimer-DNA complex (A) Ribbon diagram illustrating the STAT3 dimer bound to DNA The coiledcoil domain is in purple, the DNA-binding domain in red, the linker domain in green and the SH2 domain in orange DNA is represented by the purple hollow core between the DNA-binding domains... the activation of STAT proteins Typically, the SH2 domain plays an important role in mediating protein-protein interaction by binding to specific phospho-tyrosine containing motifs Upon specific ligandreceptor interaction, the receptors are activated by phosphorylation and this in turn mediates the recruitment of STAT proteins via binding of the SH2 domain Consequentially, this results in STAT phosphorylation... essential role in integrating signals from extracellular stimuli such as growth factors, cytokines, chemokines, hormones and extracellular matrix to mediate a variety of cellular responses including development, differentiation, cell growth, proliferation, immune responses and cell migration The dogma of signal transduction involves the binding of an extracellular ligand to its receptor and this interaction... domain is a conserved arginine residue located in the phospho-tyrosine binding pocket, which coordinates the formation of a hydrogen bond formed between the phosphate group of the phospho-tyrosine residue and the SH2 domain to result in high affinity binding The importance of this arginine is highlighted by mutational studies whereby mutation of this conserved arginine residue in Stat1 and 3 prevent... et al., 2004) Instead of acting as a dominant negative protein, Stat3 is capable of rescuing the embryonic lethal phenotype of Stat3- deficient mice as well as inducing the expression of specific Stat3 target genes Nevertheless, Stat3 still exhibits non-redundant function as compared to Stat3 STAT homologues have also been identified in invertebrates as well as other vertebrates including chicken,... domains Reprinted from Becker et al., 1998 with permission from Nature Publishing Group (B) Ribbon diagram illustrating the STAT1 dimer bound to DNA, which shows similarity to the STAT3 -DNA complex The coiled-coil domain is in green, the DNA-binding domain in pink, the linker domain in orange and the SH2 domain in cyan DNA is represented by the grey hollow core between the DNA-binding domains Reprinted... STAT activation by cytokine signalling Cytokine signalling plays an essential role in mediating a variety of cell responses such as cell growth, development and immune responses In general, cytokines can be categorised into two different groups based on their interaction with cytokine receptors (Schindler and Strehlow, 2000) For example, type I cytokines, which include interleukins (IL-2 to 7, IL-9,... and these proteins are also known as JAK-binding protein (JAB)/STAT-induced STAT inhibitor (SSI)/cytokine-inducible SH2 domaincontaining proteins (CIS) (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997; Yoshimura et al., 1995) In general, the SOCS proteins consist of a variable Nterminal domain, a SH2 domain and a conserved C-terminal SOCS box (Hilton et al., 1998) The SOCS proteins negatively... 15 Chapter 1: Introduction al., 1998a), whereas PIAS3 binds to activated Stat3 to inhibit its DNA-binding activity, thus resulting in the suppression of Stat3 transcriptional activity (Chung et al., 1997) 1.7.3 Ubiquitination and degradation of STAT proteins As compared to the negative regulation of STAT proteins by SOCS and PIAS, the role of the ubiquitin-proteasome pathway in regulating STATs via . proteins and tumourigenesis 22 1.9.1 Stat3 in oncogenesis 22 1.10 STAT3 and motility 25 1.10.1 Role of STAT3 in cell migration under physiological conditions 25 1.10.2 Role of Stat3 in cell invasion. how Stat3 regulates cell migration. In this study, we used Stat3- deficient and wild type Stat3- expressing murine embryonic fibroblasts as a model to investigate the role of Stat3 in cell migration. . 1.10.3 Cytoplasmic role of Stat3 in regulating microtubules 28 1.11 Cell migration 29 1.11.1 Process of cell migration 29 1.11.2 Coupling membrane protrusion and adhesion in cell migration 32 1.11.3