PINI and MEK2 regulate BPGAPI induced ERK activation and cell migration

PIN1 AND MEK2 REGULATE BPGAP1-INDUCED ERK ACTIVATION AND CELL MIGRATION PAN QIURONG, CATHERINE [B.Sc. (Hons), NUS] A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGMENTS I would like to express my deepest gratitude and appreciation to my supervisor, A/P Low Boon Chuan, for giving me the opportunity to work on this challenging project. Without your great patience, kind guidance, insightful suggestions and motivating encouragement, this work would not have been possible. I truly enjoy our project discussions where many fascinating ideas budded from those discussions. Your cheerful optimism and passion in teaching have touched and inspired me greatly. I certainty have learned tons of invaluable knowledge from you during the training. I am also very grateful to Dr Liou Yih-Cherng for his invaluable insights and knowledge. Also, without your generous gifts of plasmids and reagents, this work would not have made such a great progress. I also like to sincerely thank Xia Yun and Yang Qiaoyun from Dr Liou‟s laboratory for their kind guidance and patience in teaching me the technical applications. I would like to thank to my colleagues for their kind help and friendship, Dr Zhou Yiting, Dr Jan Paul Buschdorf, Dr Liu Lihui, Dr Soh Jim Kin Unice, Dr Chew Lili, Dr Zhu Shizhen, Dr Zhong Dandan, Dr Liu Xinjun, Aarthi Ravichandran, Sharmy Jennifer James, Chew Ti Weng, Chin Fei Li Jasmine, Leow Shu Ting, Sun Wei, Toh Pei Chern Pearl, Guo Kunyao Alvin and Tan Yong Wah. Special thanks to my benchmate, Lim Gim Keat Kenny, for being my listening ear and a great companion. I would also like to thank our lab manager, Tan Jee Hian Allan, for taking care of our group delicately. During my training, our laboratory has moved from Block S2 to Brenner Centre under the wing of Research Centre of Excellence (RCE) in Mechanobiology. Though I really missed those great times when we were in Block S2 wing, there are also many things in RCE-Mechanobiology to look forward. Special thanks to Dr Tan Yuen Peng, who is in-charge of the Core facilities of RCEMechanobiology, for taking care of us like a big sister over in Brenner Centre. I truly valued the great friendship, discussion and also jokes that we shared. Without your perpetual encouragement and kind help, this work would also not have been possible. I would like to acknowledge and thank the Ministry of Education, Singapore, for awarding me the research scholarship. This work was supported by grants from Academic Research Fund (Ministry of Education, Singapore and the National University of Singapore) and also the Biomedical Research Council of Singapore. Last but not least, I want to thank beloved family members for their constant support and endurance during my training. Deepest thanks to my parents for their unconditional love and care, especially my daddy for fetching me home during late nights and my mummy for preparing my daily packed lunch to school. This work has specially dedicated to you all! i TABLE OF CONTENTS Page ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii SUMMARY vii LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS 1. xiv INTRODUCTION 1.1 Signal transduction 1.1.1 1.1.2 1.1.3 Overview of signal transduction Components of signaling networks Overview of the Ras/MAPK signaling cascade 1.1.3.1 Mechanisms of intracellular signal transduction 1.1.3.1.1 Roles of protein interaction domains in signal transduction 1.1.3.1.1.1 Multidomain scaffold proteins 1.1.3.2 Regulation of intracellular signal transduction 1 4 11 1.2 Rho GTPases 15 1.2.1 1.2.2 1.2.3 1.2.4 1.2.5 1.2.6 15 16 18 21 24 27 Rho GTPase family The Rho GTPase cycle Regulation of Rho GTPases Crosstalk: Family reunion of Ras and Rho GTPases Cellular functions of Rho GTPases Pathological roles of Rho GTPases 1.3 The Rho GTPase-activating proteins (RhoGAPs) 1.3.1 1.3.2 1.3.3 1.3.4 1.3.5 1.3.6 RhoGAP family Mechanism of Rho GTPase-activating reaction Abundance RhoGAP members: The more the merrier? Crosstalks: Promiscuous Rho GTPases and RhoGAPs Regulation of RhoGAPs Cellular functions of RhoGAPs 1.3.6.1 RhoGAPs in cell growth and differentiation 1.3.6.2 RhoGAPs in cell migration 30 30 30 31 32 34 36 37 38 ii 1.3.6.3 RhoGAPs in exocytosis, endocytosis and intracellular molecule trafficking 1.3.6.4 RhoGAPs in neuronal morphogenesis 1.3.6.5 RhoGAPs in tumour suppression 1.3.7 Pathological roles of RhoGAPs 38 1.3 BPGAP1 (BNIP-2 and Cdc42GAP Homology (BCH) domaincontaining, Proline –rich and Cdc42GAP-like protein subtype 1) 43 1.4.1 1.4.2 Overview of BPGAP Multidomain BPGAP1 1.4.2.1 BCH domain 1.4.2.2 Proline-rich region 1.4.2.3 RhoGAP domain 1.4.3 Molecular mechanism of BPGAP1 function 1.4.4 Pin1: A potential regulator of BPGAP1 1.4.4.1 Structural analysis of Pin1 1.4.4.2 Substrate recognition 1.4.4.3 Regulation of Pin1 1.5 Hypothesis/Objectives 39 40 41 43 44 44 47 49 50 52 53 54 55 57 2. MATERIALS AND METHODS 2.1. Plasmid construction 2.1.1. Bacteria strains 2.1.2. Expression vectors 2.1.2.1.pXJ40 expression plasmids containing Flag and HA epitopes 2.1.2.2.pET-42 2.1.3. Plasmid constructs 2.1.3.1.BPGAP1 constructs 2.1.3.1.1. Generation of BPGAP1 truncations mutants 2.1.3.1.2. Generation of BPGAP1 deletion mutants 2.1.3.1.3. Generation of BPGAP1 point mutants 2.1.3.2.Pin1 constructs 2.1.3.3.Mek constructs 2.1.3.4.c-Raf construct 2.1.4. DNA sequencing 2.2. Cell culture and transfection 2.2.1. Transfection 2.2.2. EGF stimulation of cells 59 59 59 59 60 60 60 60 61 62 62 63 63 64 64 65 65 iii 2.3. Generation of stable Pin1 knockdown lines 65 2.4. Pull-down and co-immunoprecipitation 66 2.4.1. Mammalian cell lysate preparation 2.4.2. Pull-down assays 2.4.2.1.Pull down by mammalian-expressed proteins 2.4.2.2.Pull down by recombinant proteins 2.4.3. Co-immunoprecipitation 66 66 66 67 68 2.5. Phosphatase treatment 68 2.6. RhoA activation assay (RBD assay) 68 2.7. Western transfer and immunodetection 69 2.8. Immunofluorescence 70 2.9. Cell migration 71 3. RESULTS 3.1. RhoGAP domain of BPGAP1 harbors a cryptic Pin1-binding site 73 3.1.1. Potential Pin1-bining sites in BPGAP1 3.1.2. Pin1 interacts with BPGAP1 specifically at the GAP domain of BPGAP1 3.1.3. Distinct binding profiles of WW and PPI domain of Pin1 on BPGAP1 3.1.4. Pin1 targets an unorthodox site on the RhoGAP domain 73 74 3.2. Pin1 independently suppresses BPGAP1-induced acute Erk activation and increases its RhoGAP activity 88 3.2.1. Pin1 suppresses BPGAP1-induced acute Erk activation 3.2.2. Pin1 increases the RhoGAP activity of BPGAP1 3.2.3. Pin1 suppresses BPGAP1-induced acute Erk activation independent of its RhoGAP activity 88 91 93 3.3. Activate Mek2 forms ternary complex with Pin1 and BPGAP1 3.3.1. Activated Mek2 enhances BPGAP1 and Pin1 interaction 3.3.2. BPGAP1 exhibits differential binding profile to Mek2 3.3.3. Pin1 exhibits differential binding profile to Mek1/2 3.3.4. The ternary complex of Pin1-active Mek2-BPGAP1 forms downstream of active Raf and independent of active Erk 77 79 94 94 96 97 99 iv 3.4. Active Mek2 as a dynamic regulatory scaffold for BPGAP1 and Pin1 interaction 101 3.4.1. Active Mek2-induced binding of BPGAP1 and Pin1 is independent of phosphorylation 3.4.2. Optimal binding between BPGAP1 and Pin1 requires appropriate concentration of active Mek2 3.4.3. The BPGAP1-active Mek2 complex leads to Pin1 re-distribution to cytosol 101 3.5. Active Mek2 releases the autoinhibited proline-rich region to promote concert-binding of WW and PPI domains to BPGAP1 112 3.5.1. Different domains of Pin1 exhibit differential binding profiles to BPGAP1 under the influence of Mek2-SD 3.5.2. Active Mek2-induced binding of BPGAP1 and WW domain of Pin1 is independent of phosphorylation 3.5.3. WW domain of Pin1 binds to the non-phosphorylated PPLP motif in BPGAP1 upon active Mek2 stimulation 3.5.4. Proline-rich region mediates autoinhibition within BPGAP1 3.5.5. WW and PPI domains of Pin1 act in concert to target BPGAP1 upon active Mek2 stimulation 112 3.6. Physiological impacts by Pin1/Mek2 regulation on functions of BPGAP1 126 3.6.1. WW and PPI domains of Pin1 act in concert to suppress BPGAP-induced acute Erk activation 3.6.2. Pin1 suppresses enhanced cell motility induced by BPGAP1 and active Mek2 126 4. 103 109 113 115 117 121 128 DISCUSSION 4.1. Novel regulatory mechanism for BPGAP1 130 4.2. Molecular mechanism of interaction between BPGAP1 and Pin1, in the presence of active Mek2 131 4.2.1. Roles of BPGAP1 4.2.2. Roles of Pin1 4.2.3. Roles of active Mek2: A dynamic regulatory scaffold 4.3. Impact of BPGAP1, Pin1 and active Mek2 interaction on cell signaling and cellular dynamics 4.3.1. BPGAP1 and Pin1 as novel modulators for Mek/Erk signaling 4.3.2. Cell motility 133 134 136 139 139 140 v 4.4. Future Perspectives: Searching the missing links… 143 4.5. Conclusion 146 REFERENCES 147 vi SUMMARY BPGAP1 is a multidomain Rho GTPase-activating Protein (RhoGAP) that inactivates Rho small GTPases i.e. a major class of molecular switches that control cell dynamics. We previously showed that BPGAP1 induces cell protrusions and cell migration via the interplay of its BCH domain, Proline-Rich Region (PRR; 176PPPTKTPPPRPPLP-189) and the GAP domain. Through the PRR, BPGAP1 mediates translocation of cortactin from the cytosol to the membrane periphery for cell migration and it also engages EEN/endophilin II to increase EGF receptor internalization and Erk activation. Intriguingly, its GAP domain could also independently lead to Erk activation by yet unknown mechanism(s). Since the PRR of BPGAP1 contains multiple potential binding sites for SH3, WW or EVH1 domains, it could become a target for further regulation by proteins containing these domains. By candidate approach, the peptidyl-prolyl cis/trans isomerase Pin1 was identified as one putative partner for BPGAP1. Pin1 is a ubiquitous regulator of protein conformation that utilizes its WW domain to target phosphorylated Ser/ThrPro motif and help isomerise the phosphorylated Ser/Thr-Pro peptide bond through its peptidyl-prolyl cis/trans isomerase (PPI) domain. It regulates diverse cellular functions including cell proliferation, cell stress, neuronal function and survival. However, no direct functional link between Pin1 and Rho/RhoGAP signaling has been established. Here, I have demonstrated that the RhoGAP domain of BPGAP1 interacted with Pin1, leading to enhanced RhoGAP activity of BPGAP1 towards Rho and also suppression of BPGAP1-induced acute (but not chronic) Erk activation. Interestingly, BPGAP1 also interacted with either wildtype or constitutive-active vii Mek2 i.e. the upstream activator of Erk, but not with kinase-dead Mek2. However, only active Mek2 could interact with Pin1 thus bridging Pin1 and BPGAP1 in a scaffold manner through a process that is independent of protein phosphorylation but involving at least release of an autoinhibited proline-rich motif, 186-PPLP-189 in BPGAP1. In addition, active Mek2 and BPGAP1 together promoted Pin1 redistribution from the nucleus. Further mutagenesis studies supported a concerted binding by WW and PPI domain of Pin1 to two non-canonical sites in BPGAP1 i.e. the 186-PPLP-189 motif and 256-DDYGD-260 motif which reside in the PRR and RhoGAP domain, respectively. Consequently, single mutations of WW (S16E, S16A or W34A) or deletion of “DDYGD” motif on BPGAP1 alone, were sufficient to abolish binding between Pin1 and BPGAP1. Importantly, Pin1-knockdown augmented BPGAP1 ability to enhance acute Erk activation when cells were challenged with EGF whereas reintroducing wildtype Pin1, but not its catalytic mutant, Pin1-H157A, reversed the effect and also inhibited BPGAP1/Mek2-induced cell migration. These findings provide the first evidence that BPGAP1 potentiates acute Erk activation in response to EGF, and this process is kept in check, possibly by a feedback mechanism involving Mek2 acting as a scaffold to recruit Pin1 to the BPGAP1/Mek2 complex. Pin1 binding to its two unorthodox motifs on the PRR and RhoGAP domain is essential to suppress the acute enhancement of Erk signaling but enhancing the RhoGAP activity and consequently reducing cell motility. 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Anticancer Res. 29, 119-123. 165 [...]... 3.23 WW and PPI domains of Pin1 act in concert to target BPGAP1 upon active Mek2 stimulation 124 Figure 3.24 WW and PPI domains of Pin1 act in concert to suppress BPGAP1 -induced acute Erk activation 127 Figure 3.25 Pin1 suppresses enhanced cell motility induced by BPGAP1 and Mek2- SD 129 Figure 4.1 A schematic model of Mek2 acting as a scaffold to promote the binding between Pin1 and BPGAP1 and suppresses... Active Erk then phosphorylates serine or threonine residues at Ser/Thr-Pro motif in many different proteins including nuclear transcription factors to mediate diverse cellular responses: cell growth and differentiation, gene expression, mitosis, cell motility, metabolism, cell survival and apoptosis, and embryogenesis (Roberts and Der, 2007; Lu and Xu, 2006) For example, Erk has been shown to enhance cell. .. Mek2 -BPGAP1 forms downstreanm of active Raf and independent of active Erk 100 Figure 3.15 Active Mek2- induced binding between BPGAP1 and Pin1 is independent of phosphorylation 102 Figure 3.16 Optimal binding between BPGAP1 and Pin1 requires appropriate stoichiometric concentrations with active Mek2 104 Figure 3.17 Active Mek2 acts as a novel scaffold protein to stimulate interaction between Pin1 and. .. INTRODUCTION structure and biochemical properties This includes G-protein-coupled receptors (GPCR), receptor tyrosine kinase (RTK), integrins, Toll-like receptors and ligandgated ion channels, Wnt receptors, cytokines receptors and death receptors One of the classical membrane receptors is RTK which consists of intracellular kinase domain and an extracellular domain that binds ligand Upon ligand stimulation,... 3.8 Pin1 suppresses BPGAP1 -induced acute Erk activation 90 Figure 3.9 Pin1 increases the RhoGAP activity of BPGAP1 92 Figure 3.10 Pin1 suppresses BPGAP1 -induced acute Erk activation independent of its RhoGAP activity 93 Figure 3.11 Active Mek2 enhances BPGAP1 and Pin1 interaction 95 Figure 3.12 Binding profile of BPGAP1 with wildtype and various mutants of Mek2 97 Figure 3.13 Differential Mek1/2 binding... from extracellular stimuli that bind to their specific receptors and trigger the signaling cascade Generally, there are two types of extracellular stimuli, namely, the hydrophobic ligand e.g steroid hormones which can diffuse across the plasma membrane and binds to the cytosolic nuclear receptor, and more commonly, the hydrophilic ligand e.g hormone, growth factors, cytokines, chemokines and neurotransmitters... between signaling proteins and also the efficiency by increasing the proximity and concentration of signaling proteins In addition, scaffold proteins can insulate the signaling proteins from inactivation and/ or degradation For example, upon cell activation, KSR (Kinase suppressor of Ras) translocates to the plasma membrane and assembles the three-tier MAPKs in close proximity for the activation MAPK pathway... Growth Factor) stimulation induces the sustained Erk activation and results in differentiation while EGF stimulation induced transient Erk activation which results in proliferation, in the neuronal differentiation model- PC12 cell (pheochromocytoma of the rat adrenal medulla) (Gotoh et al., 1990; Nguyen et al., 1993; Marshall, 1995) Therefore, the observed cellular effect i.e differentiation elicited by... refers to the process by which the cell senses and relays external signal to intracellular to elicit specific response required for the adaption to the changing environment Generally, signal transduction involves the transmission of extracellular stimuli (or ligands) to receptors which then triggers intracellular signaling cascades to amplify the initial signal within the cell interior, leading to immediate... Raf-1, Mek and Erk to the adhesion complexes in response to HGF stimulation (Hepatocyte growth factor) The activated Erk then phosphorylates paxillin at Ser83, which induces the binding of FAK (Focal adhesion kinase) to the scaffold-paxillin and leads to the activation of PI3-kinase (Phosphatidylinositol 3-kinase) and small GTPase, Rac (Ishibe et al., 2003; Ishibe et al., 2004) On the other hand, the . PIN1 AND MEK2 REGULATE BPGAP1 -INDUCED ERK ACTIVATION AND CELL MIGRATION PAN QIURONG, CATHERINE [B.Sc. (Hons),. diverse cellular responses: cell growth and differentiation, gene expression, mitosis, cell motility, metabolism, cell survival and apoptosis, and embryogenesis (Roberts and Der, 2007; Lu and Xu,. reversed the effect and also inhibited BPGAP1 /Mek2- induced cell migration. These findings provide the first evidence that BPGAP1 potentiates acute Erk activation in response to EGF, and this process