THE ROLE OF PAXILLIN SUPERFAMILY MEMBERS- HIC-5 AND LEUPAXIN IN B CELL ANTIGEN RECEPTOR SIGNALING CHEW SUK PENG NATIONAL UNIVERSITY OF SINGAPORE 2007 THE ROLE OF PAXILLIN SUPERFAMILY MEMBERS- HIC-5 AND LEUPAXIN IN B CELL ANTIGEN RCEPTOR SIGNALING CHEW SUK PENG BSc PHARMACY (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE ACKNOWLEDGEMENTS I would like to express my heartfelt appreciation to my supervisor Associate Prof Lam Kong Peng for his guidance and critical comments throughout the entire project I’m grateful to my fellow colleagues especially, Ng Chee Hoe, Andy Tan Hee Meng and Dr Joy Tan En Lin for their technical assistance I’m also thankful to other members of the lab including Dr Wong Siew Cheng, Lee Koon Guan, Dr Yap An Teck, Dr Hou Jian Xin and Dr Xu Sheng Li for their constant insightful comments and suggestions to my project Special thanks to attachement students Lin You Bin, Xianne Leong, Lionel Low and Sharon Goh for their friendship and encouragement Appreciation is also extended to lab biologists Chew Weng Keong, Tan Kar Wai, Chan Siow Teng and Elaine Tan for their contribution in managing the lab and allowing smooth progress of the project To my family members my mom, my aunt, my uncle and cousins thanks for their encouragement, moral supports, love and concerns especially for taking good care of me and tolerating my busy schedule and occasional bad temper and mood swing My fellow PhD mates from A*Star Graduate Scholarship, especially Pauline Tay, Liu Mei Hui, Tam Wai Leong, Dave Aw, Cecilia Lee, Lee Terk Shuen, Harmeet Singh, Adrian Mathew Mak, Emril Mohamad Ali, Fong Siew Wan and Sebastian Ku, I truly cherish their constant support and occasional social meetings to complain and listen to each other about difficulties and stress in research My personal friends, Franck M, Harry Chua, Angel Choong, Kristie Ong, Eryn Chew, Angela Koo, Jessey Ding, Lynda Lee, Chin Woey, Jacqueline Chong, Jerry Tan, Lim Thian Yew, Dave Chia and Simon Heng, thanks for their constant support and having the faith in me to complete my PhD Finally, special thanks to a special friend, Jackson Chiam, for his love and support I thank God for without His grace and blessing I would not have come this far Also thanks to my church friends especially Grace, Cecilia, Sabrina, Victor and Carmen for their constant prayers TABLE OF CONTENTS SUMMARY i ABBREVIATIONS iii LIST OF SCHEMATIC DIAGRAMS AND TABLES v LIST OF FIGURES vi LIST OF PUBLICATIONS ix CHAPTER 1: INTRODUCTION 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 The immune system Innate and adaptive immunity B cell antigen receptor signaling pathways 1.3.1 Signaling via PI3-K pathway 1.3.2 Signaling via PLCγ2 pathway 1.3.3 Signaling via Ras/Raf/Erk pathway 1.3.4 Signaling via Vav/Rac pathway Src family kinases Adaptor proteins in lymphocyte signaling 1.5.1 Bam32 1.5.2 TAPP & Negative regulatory pathways of B cell antigen receptor signaling 1.6.1 Protein tyrosine phosphatases (PTPs) 1.6.1.1 SHP-1 1.6.1.2 PEP 1.6.2 Lipid Phophatases 1.6.2.1 SHIP-1 1.6.2.2 PTEN 1.6.3 Protein tyrosine kinases 1.6.3.1 Csk 1.6.3.2 Lyn 1.6.4 Cbl Family of Ubiquitin Ligases 1.6.4.1 C-Cbl 1.6.4.2 Cbl-b Paxillin superfamily members 1.7.1 Paxillin 1.7.2 Hic-5 1.7.3 Leupaxin Rationale and aims of this project 1 10 12 13 17 20 23 26 26 26 28 29 29 31 32 32 33 35 35 36 36 39 40 42 43 CHAPTER 2: MATERIAL AND METHODS 2.1 2.2 2.3 2.4 2.5 2.6 List of antibodies for Immuno-fluorescence, BCR stimulation, Immunoprecipitation and Immuno-blotting List of primers Molecular cloning methodology 2.3.1 Buffers and solutions 2.3.2 Plasmids DNA constructs 2.3.3 Extraction of RNA 2.3.4 First strand cDNA synthesis 2.3.5 Polymerase chain reaction 2.3.6 DNA sequencing 2.3.7 Restriction digestion of DNA 2.3.8 Agarose gel electroporesis 2.3.9 Elution of DNA from agarose gel 2.3.10 Dephosphorylation of plasmid DNA 2.3.11 Ligation of DNA 2.3.12 Preparation of DH5α competent cells 2.3.13 Transformation of DH5α by heat shock method 2.3.14 Bacterial DNA mini-prep by alkaline lysis 2.3.15 Bacterial maxi-prep using Qiagen Maxi-prep columns Mammalian cell culture methodology 2.4.1 Cell culture media 2.4.2 Purification of splenic B cells 2.4.3 Transfection of HEK 293T cells 2.4.4 Transfection of A20 B cells 2.4.5 Stimulation of A20/ BJAB cells Molecular and cellular immunology methodology 2.5.1 Flow cytometry 2.5.2 BCR-induced IL-2 production 2.5.3 BCR-induced activation of IL-2 promoter 2.5.4 Confocal microscopy Protein methodology 2.6.1 Buffers and solutions 2.6.2 Immunoprecipitation 2.6.3 Western blotting 2.6.4 Isolation of membrane fraction 45 46 47 47 50 50 51 52 53 54 55 55 56 56 57 58 58 59 59 59 60 61 62 62 62 62 63 63 64 65 65 66 67 68 CHAPTER 3: THE ROLE OF HIC-5 IN B CELL RECEPTOR SIGNALING 3.1 Introduction 3.2 Results 3.2.1 Yeast-two-Hybrid using B cells adaptor protein, Bam32 as a bait 3.2.2 Interaction of Bam32 with Lyn 3.2.3 Interaction of Bam32 with Hic-5 and its homologue, paxillin 69 72 72 72 74 3.3 3.4 3.5 3.2.4 Interaction of Bam32 homologues: TAPP1 and TAPP2, with Hic-5 and paxillin 75 3.2.5 PH domain of Bam32 mediates binding to Hic-5 and paxillin 78 3.2.6 Interaction of Hic-5 and paxillin with Lyn is independent of Bam32 81 3.2.7 Bam32 competes with Hic-5 and paxillin to interact with Lyn 82 3.2.8 Tyrosine phosphorylation of Hic-5 and paxillin by Lyn in HEK293T cells 85 3.2.9 BCR-induced tyrosine phosphorylation of Hic-5 87 3.2.10 BCR-induced interaction of Hic-5 with Lyn 90 3.2.11 Hic-5 was recruited to the plasma membrane upon BCR ligation 91 3.2.12 Inhibition of JNK and p38 activation by Hic-5 in A20 B cells 94 Discussion 97 Future directions 102 Conclusion 104 CHAPTER 4: THE ROLE OF LEUPAXIN IN B CELL RECEPTOR SIGNALING 4.1 4.2 4.3 4.4 4.5 Introduction 105 Results 107 4.2.1 Sequence consensus between human and mouse leupaxin 107 4.2.2 Leupaxin is tyrosine phosphorylated upon BCR ligation in human BJAB B cells 109 4.2.3 Leupaxin is recruited to the plasma membrane upon BCR ligation in human BJAB B cells 112 4.2.4 Leupaxin interacts with Lyn 113 4.2.5 Leupaxin interacts with Lyn through its LD3 domain 117 4.2.6 Lyn phosphorylates leupaxin at tyrosine 72 119 4.2.7 Selective inhibition of JNK, p38 and Akt pathways by leupaxin in A20 B cells 123 4.2.8 Leupaxin inhibits IL-2 production in A20 B cells 128 4.2.9 Tyrosine 72 of leupaxin is important for its inhibitory function 132 Discussion 137 Future directions 141 Conclusion 144 CONCLUSION 145 LIST OF REFERENCES 146 PUBLICATIONS SUMMARY Adaptor proteins play an important role in B cell antigen receptor (BCR) signaling by mediating intermolecular interactions in a spatial and temporal manner One of these adaptor proteins, Bam32, has been shown to regulate BCR signaling On the other hand, the role of paxillin superfamily of adaptor proteins in BCR signaling has not been studied previously Paxillin superfamily members consist of paxillin, Hic-5 and leupaxin based on their homology in multiple amino (N)-terminal leucine (L)- and aspartate (D)-rich sequences (LD domains) and carboxyl (C)-terminal lin-11, isl-1, mec-3 (LIM) domains Both LD and LIM domains allow protein-protein interactions The role of paxillin superfamily adaptor proteins, in particular paxillin and Hic-5, is well established in growth factor and integrin mediated signaling pathways In this thesis, the potential role of paxillin superfamily members - Hic-5 and leupaxin in BCR signaling were explored The project was initiated by a yeast-two-hybrid screen using Bam32 as a bait, which identified Hic-5 and Lyn as potential binding partners Later we found that Hic-5 can also interact with Lyn, which is a critical Src-family kinase in BCR signaling Our current discoveries lead us to a model where Hic-5 is recruited to the plasma membrane and binds Lyn upon BCR signaling Following that Hic-5 is tyrosine phosphorylated and hence activated by Lyn By overexpression in mouse A20 lymphoma B cells, we showed that Hic-5 is a negative regulator in BCR signaling specifically in the phosphorylation of JNK and p38 MAPK Bam32 by competing with Hic-5 to bind Lyn regulates the inhibitory function of Hic-5 i specifically in BCR-induced phosphorylation of p38 MAPK Despite the current findings, the detailed mechanism of the function of Hic-5 in BCR signaling remains to be elucidated The role of another member of paxillin superfamily proteins- leupaxin was explored in our current project as well First we showed that leupaxin (LPXN) is tyrosine-phosphorylated and recruited to the plasma membrane of human BJAB lymphoma cells upon BCR stimulation, and interacts with Lyn in a BCR-inducible manner LPXN contains four leucine-rich sequences termed LD motifs and serial truncation and specific domain deletion of LPXN indicated that its LD3 was involved in the interaction with Lyn Of a total of 11 tyrosine (Y) sites on LPXN, we mutated Y22, Y72, Y198 and Y257 to phenylalanine (F) and demonstrated that LPXN was phosphorylated by Lyn only at Y72 and this tyrosine site was proximal to the LD3 domain of LPXN, which is the domain responsible for its interaction with Lyn The overexpression of LPXN in A20 B cells led to the suppression of BCR-induced activation of JNK, p38 MAPK and to a lesser extent, Akt but not Erk and NFkB, suggesting that LPXN could selectively repress BCR signaling We further showed that LPXN suppressed the secretion of IL-2 by BCR-activated A20 B cells and this inhibition was abrogated in the Y72F LPXN mutant, indicating that the phosphorylation of Y72 is critical for the biological function of LPXN in B cells In conclusion, we discovered a previously unknown inhibitory function of paxillin superfamily adaptor proteins in BCR signaling ii ABBREVIATIONS ARF BCR BLNK Btk Csk DAG DNA Dok ERK FACS FAK FITC GDP Grb2 GTP HA HPK1 I(1,3,4,5)P4 Ig IL IP3 IRS ITAM ITIM JNK LD LIM LPXN Lyn MAPK MHC NFAT NF-κB PEP 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62 62 63 63 64 65 65 66 67 68 CHAPTER 3: THE ROLE OF HIC- 5 IN B CELL RECEPTOR SIGNALING 3 .1 Introduction 3.2... Ligases 1. 6.4 .1 C-Cbl 1. 6.4.2 Cbl -b Paxillin superfamily members 1. 7 .1 Paxillin 1. 7.2 Hic- 5 1. 7.3 Leupaxin Rationale and aims of this project 1 10 12 13 17 20 23 26 26 26 28 29 29 31 32 32 33 35 35. .. 3 .5 3.2.4 Interaction of Bam32 homologues: TAPP1 and TAPP2, with Hic- 5 and paxillin 75 3.2 .5 PH domain of Bam32 mediates binding to Hic- 5 and paxillin 78 3.2.6 Interaction of Hic- 5 and paxillin