THE FUNCTION OF TOM1-L1 IN BRIDGING EGFR SIGNALING AND ENDOCYTOSIS LIU NINGSHENG INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 THE FUNCTION OF TOM1-L1 IN BRIDGING EGFR SIGNALING AND ENDOCYTOSIS LIU NINGSHENG (M.Med. Southeast Univ.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements I would like to express my gratitude to all those who gave me the possibility to complete this thesis. My foremost thank goes to my supervisor: Prof. Hong Wanjin, for his patience and encouragement that carried me on through difficult times, and for his insights and suggestions that helped to shape my research skills. His valuable feedback contributed greatly to this dissertation. My committee members: Assoc. Prof. Cai Minjie and Assoc. Prof. Hunziker Walter, for their stimulating discussion and critique during my annual committee meeting. Their valuable feedback helped me to improve the dissertation in many ways. My past and present lab members: Dr. Seet Li Fong who introduced and helped me to start my graduate student life in Molecular and Cell Biology Science by teaching me molecular, cell biological and biochemical techniques without reservations. Her visionary thoughts and energetic working style have influenced me greatly as a biology scientist. Dr. Seet Li Fong, Dr. Loh Eva, Dr. Lim Kah Pang, Dr. Tham Jill, Dr. Lu Lei and Miss Ong Yan Shan for critical and careful reading of this thesis. Miss Ong Yan Shan for collaboration in High-Performance Liquid Chromatography (HPLC) in Figure 3.1. Dr. Tham Jill, Dr. Chan Siew Wee, Dr. Loh Eva, Miss Ong i Yan Shan, Miss Tran Thi Ton Hoai, Dr. Wang Tuanlao and Mr. Li Hongyu for sharing critical reagents for this study. I thank all the students and staffs in IMCB who gave me the possibility to complete this thesis. My appreciation also goes to the DNA sequencing and protein mass-spectrum unit of IMCB for their excellent services. Last but not least, I thank my grandparents, and my parents for always being there when I needed them most, and for supporting me through all these years. Liu Ningsheng 2007 ii Table of Contents Summary List of Tables List of Figures Abbreviations Chapter Introduction 1.1. Epidermal Growth Factor Receptor (EGFR) 1.1.1 EGFR Structure 1.1.2 Dimerization and Activation 1.1.3 Shc, Grb2 and the Ras/MAPK Pathway 1.1.3.1 Grb2 (Growth Factor Receptor-bound Protein 2) 1.1.3.2 The Src Family Kinase (SFK) 1.2. Endocytosis 1.2.1 The Classical Clathrin-dependent Endocytic Pathway 1.2.2 The Non-classical Clathrin-independent Endocytosis Pathway 1.2.3 EGFR and Lipid Raft 1.2.4 EGFR Sorting and Clathrin-dependent Endocytosis 1.2.5 EGFR Signaling during Trafficking 1.2.6 Ubiquitination and MVB Generation 1.3. TOM1 (Target of the Oncogene v-Myb 1) Family 1.4. Rational of this work Chapter Materials and Methods iii 2.1 cDNA Cloning and Sequencing 2.2 Plasmid Constructs 2.2.1 HA-TOM1-L1, HA-TOM1-L1 Y460F and GFP-TOM1-L1 2.2.2 HA-TOM1-L1 SH3, HA-TOM1-L1 Y392F and HA-TOM1-L1 Y460F & SH3 2.2.3 HA-TOM1-L1-PX and HA-TOM1-L1-PX FDPL450AAAA 2.2.4 GST-TOM1-L1 286-476 and other GST Deletion Constructs (316-476, 360-476, 384-476, 420-476,286-446,286-449,286-440) 2.2.5 GST-TOM1-L1 286-476 LPPL424AAAA, GST-TOM1-L1 286-476 HPAM431 AAAA, GST-TOM1-L1 286-476 DLQP438AAAA and GST-TOM1-L1 286-476 FDPL450AAAA 2.2.6 TOM1, m-TOM1-L1, m-TOM1-L1 Y392F, m-TOM1-L1 Y457F, m-TOM1-L1 DLQP437AAAA and m-TOM1-L1 FDLP449AAAA 2.3 Purification of GST-fusion Proteins 2.4 Immunization of Rabbits and Affinity Purification of Antibodies 2.5 Antibodies 2.6 Cell Culture 2.7 Transient and Stable Expression 2.8 Retroviral Infection 2.9 siRNA knockdown 2.10 EGF, PDGF-bb, or FGF2 Stimulation 2.11 EGF internalisation 2.12 Immunoprecipitation and Western Blot 2.13 Indirect Immunoflurescence Microscopy 2.14 Cytosol Extract iv 2.15 Gel Fractionating 2.16 Clathrin-Binding Assay 2.17 Cell Surface Biotinylation and Stripping 2.18 Biochemical Subcellular Fractionation 2.19 Ras Activation Assay 2.20 Soft Agar Assay for Colony Formation Chapter Characterization of Endogenous TOM1-L1 Complex and TOM1-L1 Antibodies 3.1 Endogenous TOM1-L1 is in a ~300 kDa Complex at A431 Cells. 3.2 Specific of anti-TOM1-L1 and anti-p-TOM1-L1. Chapter TOM1-L1 is Tyrosine Phosphorylated by EGF, PDGF, and FGF via a Src/Fyn-dependent Pathway 4.1 Tyrosine Phosphorylation of TOM1-L1 by Fyn Mediates its Association with Grb2 and PI3K-p85 4.2 Tyrosine Phosphorylation of TOM1-L1 by Src at the Putative SH2 Binding Site. 4.3 TOM1-L1 is Tyrosine Phosphorylated by EGF via a Src-Dependent Pathway and Important for Interaction with Grb2. 4.4 TOM1-L1 is also Tyrosine Phosphorylated by PDGF and FGF via a Src-Dependent Pathway. Chapter TOM1-L1 Mediates the Endocytosis of EGFR 5.1 EGF-Stimulated Tyr-Phosphorylation of TOM1-L1 is Transient and Correlates with its Transient Interaction with EGFR. 5.2 Endogenous Localisation of TOM1-L1. 5.3 Temporal Correlation between the Association of TOM1-L1 with Cellular Membranes and EGF Stimulation of A431 Cells. 5.4 TOM1-L1 is Recruited to EGF Receptor-containing Early Endosomes in response to v EGF. 5.5 Mutant Forms of TOM1-L1 Defective in Tyr-Phosphorylation or Interaction with Grb2 Inhibit Endocytosis of EGFR. 5.6 siRNA-Mediated Knockdown of TOM1-L1 Inhibits Endocytosis of EGFR. Chapter The C-Terminal Tail of Tom1L1 Harbors a Novel Clathrin- Interacting Motif Important for Mediating EGFR Endocytosis 6.1 The C-terminal Tail of TOM1-L1 Harbors a Novel Clathrin-interacting Motif. 6.2 TOM1-L1’s Clathrin Binding Motif is Important for its Role in Mediating EGFR Endocytosis. 6.3 Effect of Depletion of AP2, Clathrin, Cbl, Grb2 or TOM1-L1 on EGFR and TfnR Endocytosis. Chapter TOM1-L1 Interacts with Ubiquitin, Hrs and STAM, and Mediates Degradation of EGFR 7.1 TOM1 Family Proteins Interact with ubiquitin. 7.2 TOM1-L1 Interacts with Hrs, STAM. 7.3 Hrs Recruits TOM1-L1 to Endosomes. 7.4 Knockdown of both TOM1-L1 and Hrs further Delays EGFR degradation. Chapter TOM1-L1 is a Negative Regulator in Src Kinase Signaling 8.1 TOM1-L1 Inhibits the Activation of Ras upon EGF Stimulation. 8.2 TOM1-L1 Inhibits the Colony Formation in A431 Cells. Chapter Discussion Chapter 10 Conclusion and future perspectives References vi SUMMARY The molecular mechanism governing ligand-stimulated endocytosis of receptor tyrosine kinases remains elusive. I show here that EGF stimulates transient tyrosine-phosphorylation of TOM1-L1(TOM-Like 1) by the Src family kinases, resulting in its transient interaction with the activated EGF (Epidermal Gowth Factor) receptor (EGFR) bridged by the receptor-bound Grb2 (Growth Factor Receptor-Bound protein 2). Cytosolic TOM1-L1 is recruited onto the plasma membrane and subsequently redistributes with EGFR into the early endosome. Mutant forms of TOM1-L1 defective in tyrosine-phosphorylation or interaction with Grb2 is incapable of interaction with EGFR and inhibits endocytosis of EGFR. In addition, siRNA (small interference RNA)-mediated knockdown of TOM1-L1 inhibits endocytosis of EGFR. The C-terminal tail of TOM1-L1 contains a novel clathrin-interacting motif, which is important for exogenous TOM1-L1 to rescue endocytosis of EGFR in TOM1-L1 knocked-down cells. These results suggest that EGF triggers a transient association of EGFR with TOM1-L1 to engage the endocytic machinery for endocytosis of the ligand-receptor complex. 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Science.292:1716-8. 148 [...]... lysine residues and one histidine and the head group of the lipid ARH, Dab2 PTB domain Direct contact between basic residues in the PTB domain and Numb lipid-binding pocket and PtdIns(4,5)P2 β-arrestin Arginine/lysin High-affinity basic PtdIns(4,5)P2 binding site at the e residues C-terminal domain of β-arrestin Epsin ENTH The ENTH domain binds both the head group and domain glycerol backbone of PtdIns(4,5)P2... TOM1- L1 Delayed EGF-Induced Degradation and Endocytosis of EGFR Figure 6.1 TOM1- L1 Contains a Novel Clathrin-Binding Motif Figure 6.2 TOM1- L1 s Clathrin Binding Motif is Important for its Role in Mediating EGFR Endocytosis Figure 6.3 Effects of Protein Depletion on EGFR and TfnR Endocytosis Figure 7.1 TOM1 Family Interacts with Ubiquitin Figure 7.2 TOM1- L1 Interacts with Hrs, STAM Figure 7.3 Effect of. .. basic sidechains (mainly two clusters of lysine residues at each end of the binding domain) on the N terminus of the α subunit and a cluster of conserved lysine residues at the surface of μ2 C-terminal domain AP180/ ANTH Two α helices (α helices 1 and 2) out of the nine that CALM domain comprise the ANTH contact the membrane The interaction takes place between the solvent exposed sidechains of three consecutive... as the Golgi by the clathrin-dependent pathway (Gruenberg and Stenmark, 2004, Schlessinger et al., 2002, Bishop, 2003) Table 2 Phosphatidylinositol (4, 5)-bisphosphate (PtdIns (4,5)P2) Binding Domains found in Clathrin Adaptor Proteins Clathrin adaptor AP2 PtdIns(4,5)P2 binding domain α core μ2 C-terminal domain Binding Properties Binding takes place between the phosphate groups of PtdIns(4,5)P2 and. .. PtdIns(4,5)P2 in a basic pocket formed by the first four of the eight α helices in this domain Upon binding of the ENTH domain to the membrane, a new α helix forms called helix 0 (residues 3-15), which inserts its outer hydrophobic surface into the hydrophobic phase of the membrane and its inner basic surface augments the PtdIns(4,5)P2 head group binding pocket, anchoring the epsin protein to the membrane... (Hunter and Cooper, 1985, Parsons and Weber, 1989, Smart et al., 1981) A conserved tyrosine residue (Y527 in Src) that lies at the end of the C-terminal is essential for auto-regulation in SFKs (Brown and Cooper, 1996) For Src, the SH3 and SH2 domains are at the back of the kinase domain The SH2 domain binds to pY527 while the SH3 domain interacts with a short polyproline type II helix between the SH2 and. .. or Cbl-induced ubiquitinations of receptors (via direct association of Cbl with RTKs) (Yoon et al., 1995, De Sepulveda et al., 1999) Grb2 activates Ras/MAPK signaling pathway via Sos On the other hand it also promotes the ubiquitination and endocytosis of receptors through recruiting the Cbl-CIN85 (Cbl-interacting protein of 85 kDa)-endophilin (End) complex, and binding to Sprouty, SHIPs, SOCS and Ack,... Overexpression of HA-tagged Hrs on Endosomes and TOM1- L1 Licalization Analyized by Immunoflurescence microscpy Figure 7.4 Simultaneous Knockdown of Both TOM1- L1 and Hrs Causes a Delay in EGFR Greater than Knockdown of either TOM1- L1 or Hrs Alone Figure 8.1 TOM1- L1 Inhibits the Activation of Ras upon EGF Stimulation Figure 8.2 TOM1- L1 inhibits the Colony Formation in A431 Cells Figure 9.1 A Proposed Model for TOM1- L1. .. membrane location and ligand-binding They have a single transmembrane domain and a large cytoplasmic region that contains a tyrosine kinase and multiple phosphorylation sites (Slieker et al., 1986) The structure of the mature EGFR (ErbB1) receptor is represented as below Figure 1.1 Domain Organization of EGF Receptor Abbreviations: I and III: ligand binding domains; II and IV: cysteine-rich domains; TM: transmembrane... involved in endocytosis and subsequent sorting of internalized receptor to MVB (Citri and Yarden, 2006; Rubin et al., 2005) Src collaborates with the ubiquitination and proteosomal degradation of Cbl, thereby attenuating the level of Cbl and postponing EGFR downregulation, hence forth promoting the recycling of receptors back to the plasma membrane and extends EGFR signaling (Bao et al., 2000, Muthuswamy et . THE FUNCTION OF TOM1- L1 IN BRIDGING EGFR SIGNALING AND ENDOCYTOSIS LIU NINGSHENG (M.Med. Southeast Univ.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE. with EGFR and inhibits endocytosis of EGFR. In addition, siRNA (small interference RNA)-mediated knockdown of TOM1- L1 inhibits endocytosis of EGFR. The C-terminal tail of TOM1- L1 contains a novel. THE FUNCTION OF TOM1- L1 IN BRIDGING EGFR SIGNALING AND ENDOCYTOSIS LIU NINGSHENG INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE