ENDOFIN IS A NOVEL COMPONENT IN EGF/EGFR ONCOGENIC SIGNALING TOY WEI YI B.SC (LIFE SCIENCES) (HONS) NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PATHOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I would like to express my gratitude and sincere thanks to my supervisor, Dr Lim Yoon Pin for giving me the opportunity to pursue my PhD studies in his laboratory and his kind patience and guidance throughout these years. My thanks also extend to my co-supervisor, Associate Prof. Richie Soong and my thesis advisory committee members, Associate Prof. Low Boon Chuan and Associate Prof. Shen Han-ming for their suggestions and advice. My heartfelt thanks to Dr Lim Shen Kiat and Dr Shirly Chong for their ready help and invaluable advice whenever needed and Ms Lee Huiyin for all the help she has given me throughout the years. I would also like to thank Dr Lim Shen Kiat and Emma May Stanford for taking their precious time to proof-read this thesis. I would like to thank all the past and present members of YPL lab for the wonderful working experience, especially Dr Bobby, Dr Yang Yixuan, Dr Man Xiaohui, Ms Choong Lee Yee, Mr Victor Tan, and Ms Emily Chen whom all have contributed to the success of this thesis in one way or another. It has been a real pleasure working with all of you throughout these years. My deepest gratitude to my dearest friends, Ms Chang Jaw-shin, Ms Lim Simin and Ms Peh Bee Keow for always being there to listen to my problems and give encouragement. Thanks for being such wonderful friends and I’ll always treasure our friendships. Lastly, I would like to express my most sincere thanks to my family, especially my parents for their constant moral support, my sister for her listening ears and my little brother for his IT expertise and help in drawing the diagrams. Without them, I would never been able to finish this. Thank You. Toy Weiyi January 2010 I TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS I II LIST OF FIGURES VI LIST OF TABLES VII ABBREVIATIONS VIII SUMMARY XII Chapter Introduction 1.1 Cellular communication and receptor tyrosine kinase 1.2 Receptor tyrosine kinase 1.3 Epidermal growth factor receptor 1.3.1 EGFR activation 1.3.2 EGFR signaling pathways 10 1.3.2.1 Ras/Raf/MEK/ERK pathway 10 1.3.2.2 PI3K/PDK1/Akt pathway 13 1.3.2.3 PLC-γ/DAG/IP3 pathway 16 1.3.2.4 JAK/STAT pathway 18 1.3.2.5 Src kinases 19 1.4 EGFR endocytosis 20 1.4.1 Trafficking of EGFR in the absence of ligands 21 1.4.2 Ligand-induced endocytosis 21 II 1.4.2.1 Clathrin-independent endocytosis 22 1.4.2.2 Clathrin-dependent endocytosis 23 Post-endocytic trafficking of EGFR 27 1.5.1 Trafficking of EGFR through the endosomes 27 1.5.2 EGFR sorting at MVBs 28 1.6 Receptor recycling 32 1.7 Endosomal signaling 32 1.7.1 Continuous EGFR signaling from endosomes 33 1.7.2 Endosomal-specific signaling 35 FYVE domain-containing proteins 36 1.8.1 The FYVE zinc-finger domain 36 1.8.2 FYVE domain proteins and membrane trafficking 37 1.8.3 FYVE domain proteins and signal transduction 40 1.8.4 FYVE domain proteins that have enzymatic activity 41 1.8.5 FYVE domain proteins and cytoskeleton regulation 42 1.5 1.8 1.9 Endofin 43 1.10 Research objectives 47 Chapter Materials and Methods 2.1 Chemicals and reagents 48 2.2 Antibodies 48 2.3 Plasmid constructs 49 2.4 Site-directed Mutagenesis 50 III 2.5 Cell culture 50 2.6 Transfection 51 2.7 Cell lysis 51 2.8 Subcellular fractionation 52 2.9 Immunoprecipitation 52 2.10 Immunoblotting 53 2.11 In vitro kinase assay 54 2.12 Immunofluorescence 54 2.13 Proliferation assay 56 Chapter Results 3.1 3.2 Characterization of Endofin phosphorylation 57 3.1.1 Tyrosine phosphorylation of Endofin occurs upon TGF-α stimulation and is dependent on EGFR activation 57 3.1.2 TGF-β does not induce tyrosine phosphorylation of Endofin 60 3.1.3 Endofin phosphorylation correlates closely to EGFR activity 61 3.1.4 Endofin is phosphorylated in the cytosol and clathrindependent endocytosis is essential for Endofin phosphorylation 64 3.1.5 EGF-induced PI3K activity and proper localization of Endofin are necessary for its tyrosine phosphorylation 69 3.1.6 EGF-dependent co-localization of Endofin with EGFR requires a functional FYVE domain 74 Determination of Endofin tyrosine phosphorylation site and function 78 3.2.1 78 Tyrosine 515 is a major phosphorylation site of Endofin IV 3.2.2 Phosphorylation at Y515 does not affect the localization of Endofin and its co-localization with EGFR 84 3.2.3 Endofin’s localization and phosphorylation increased the amplitude of EGF-induced MAPK pathway 87 3.2.4 Proliferation rates of Y515F and C753S expressing cells were elevated 94 Chapter Discussion 4.1 Endofin’s regulatory actions on EGFR signaling 96 4.2 Possible mechanisms utilized by Endofin in the regulation of MAPK signaling 99 4.3 Future areas of research 101 References 103 Publications 122 V LIST OF FIGURES 1.3 Schematic representation of EGFR structural domains 1.3.1 Schematic representation of phosphorylated tyrosine residues on EGFR and its binding substrates 1.3.2.1 Ras/Raf/MEK/ERK pathway 12 1.3.2.2 PI3K/PDK1/Akt 15 1.3.2.3 PLC-γ/DAG/IP3 pathway 17 1.5 Schematic diagram of receptor endocytosis 31 1.9 Schematic representation of the various domains and interacting partners of Endofin 46 3.1.1 Tyrosine phosphorylation of Endofin occurs upon TGF-α stimulation and is dependent on EGFR activation 59 3.1.2 Endofin is not phosphorylated upon TGF-β stimulation 60 3.1.3 Endofin phosphorylation is dependent on EGFR activity 63 3.1.4A Phosphorylated Endofin was detected in non-nuclear intracellular compartment 66 3.1.4B-C Endofin phosphorylation is dependent on EGFR endocytosis 67 3.1.4D-E Endofin phosphorylation requires EGFR clathrin-dependent endocytosis 68 3.1.5A 71 Endofin phosphorylation is dependent on PI3K activity 3.1.5B-C C753S Endofin mutant is unable to localize at the early endosomes 72 3.1.5D Proper localization of Endofin is essential for its phosphorylation 73 3.1.6A EGFR and Endofin co-localize at EEA1-positive endosomes 76 3.1.6B Co-localization of Endofin with EGFR requires its FYVE domain 77 3.2.1A-B Y515 of Endofin is a major phosphorylation site for EGFR 82 VI 3.2.1C-D Endofin is a direct substrate of EGFR 83 3.2.2A-B Endosomal localization of Endofin is not dependent on its Y515 phosphorylation 85 3.2.2C Co-localization of Endofin with EGFR does not require Y515 phosphorylation 86 3.2.3A Endofin expression and transfection efficiency in various cell lines 88 3.2.3B Endofin overexpression has no effect on EGFR modulation or signaling 88 3.2.3C-D Mislocalization of Endofin amplified EGF-induced MAPK signaling 90 3.2.3E-G Y515F mutant increased MAPK2 activity in Endofin knockdown cells 93 3.2.4 Y515F and C753S expressing cells have increased proliferation rates 95 4.1 Schematic diagram of Endofin’s role in EGFR signaling 98 LIST OF TABLES 3.2.1 Predicted tyrosine phosphorylation sites on Endofin 79 VII ABBREVIATIONS ARF1 ATP bFGF BMP BSA C degree Celsius 3' untranslated region arachidonic acid AKR mouse T-cell lymphoma-derived oncogenic product adaptor protein complex adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper-containing ½ ADP-ribosylation factor adenosine triphosphate basic fibroblast growth factor bone morphogenetic protein bovine serum albumin cysteine Ca2+ c-Cbl CCP CDE CIE CO2 Cox II CR C-terminal DAG DH DN DNA DTT E. coli ECL EDTA EEA1 EGF EGFR EHD ERC ERK ESCRT F FAB FBS FYVE GDP calcium c-Casitas B-lineage lymphoma clathrin-coated pits clathrin-dependent endocytosis clathrin-independent endocytosis carbon dioxide cytochrome-c oxidase subunit II cysteine-rich domain carboxyl (COOH)-terminal 1,2-diacylglycerol Dbl homology dominant-negative deoxyribonucleic acid dithiothreitol Escherichia coli enhanced chemiluminescence ethylene-diamine tetra-acetic acid early endosomal antigen epidermal growth factor epidermal growth factor receptor epsin-homology domain endocytic recycling compartment extracellular signal-regulated kinase endosomal sorting complex required for transport complexes phenylalanine F-actin filament-binding domain fetal bovine serum Fab1p, YOTB, Vac1p EEA1 guanosine diphosphate °C 3'UTR AA Akt AP2 APPL VIII GEF GFP GM-CSF GPCR Grb2 GTP GTPase HA HGF HRP Hrs IGF-1 ILV IP IP3 JAK JM JNK kDa LB LBPA L MAPK MEK MEM mg μg MgCl2 mL μl mM μM MTM MTS MVB Na3VO4 NaCL NaF ng NID N-terminal PA PBD guanine-nucleotide exchange factor green fluorescent protein granulocyte-macrophage colony-stimulating factor G-protein coupled receptors growth factor receptor-bound protein guanosine triphosphate guanosine triphosphatase haemagglutinin hepatocyte growth factor horseradish peroxidase hepatocyte growth factor-regulated tyrosine kinase substrate insulin growth factor intraluminal vesicles immunoprecipitation inositol 1,3,5-trisphosphate Janus kinase juxtamembrane region c-Jun N-terminal kinase kilo Dalton Luria Bertani lysobisphosphatic acid leucine mitogen-activated protein kinase mitogen activated extracellular signal regulated kinase modified eagles medium milligram microgram magnesium chloride millilitre microlitre millimolar micromolar myotubularin 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt multivesicular bodies sodium orthovanadate sodium chloride sodium fluoride nanogram non-ionic denaturing amino (NH2)-terminal phosphatidic acid protein phosphatase catalytic subunit (PP1c) binding domain IX Futter CE, Collinson LM, Backer JM, Hopkins CR (2001). 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Proteomics 7: 2384-97. 122 [...]... (amino acids 622-644), followed by an intracellular domain that contains a juxtamembrane region, a tyrosine kinase and a C-terminal region which consists of several phosphorylation sites (Normanno et al., 2005)(Fig 1.3) The extracellular N-terminal sequence of EGFR, also known as ectodomain, contains 4 domains, L1, CR1, L2 and CR2 The ligand-binding pocket is located between L1 and L2 domains and a. .. enhance the amplitude of the MAPK cascade and increase cell proliferation, suggesting a possible role of Endofin in the modulation of MAPK pathway Collectively, this study has identified a novel signaling cascade involving EGFR, PI3K, Endofin and MAPK in the EGFR signaling network XII Chapter 1 Introduction 1.1 Cellular communication Cellular communication is a core aspect of a functional biological... endocytosis, a CIE pathway that involves CDC42, ADP-ribosylation factor 1 (ARF1), actin and another mode of CIE which is dynamin-independent and is associated with ARF6 GTPase (Kumari and Mayor, 2008; Mayor and Pagano, 2007) CIE of EGFR was first observed in studies performed in A4 31 cells Plasma membrane ruffling and formation of labeled EGF-containing micro- and macropinocytic vesicles with no clathrin coat... phosphotyrosine binding sites on the intracellular portion of EGFR This suggests that differential binding patterns of the substrates can result in diverse signaling outputs from the receptor 9 1.3.2 EGFR signaling pathways Based on the diversity of protein complexes formed at EGFR, multiple signaling pathways can be elicited from the activated EGFR The major signaling pathways activated by EGFR are the Ras/Raf/MEK/ERK,... of signaling networks, with each signaling pathway described as a “signal transduction” (King, 2010) A signal transduction begins with the extracellular signals, usually in the form of endocrine, paracrine hormones, or signaling molecules, binding to specific proteins on the cell membrane The binding of these specific membrane proteins activates signaling cascades that transverse across the cytosol and... PI3K/PDK1/Akt, PLCγ/DAG/IP3 and JAK/STAT pathways All of these signaling pathways contribute to the regulation of cellular processes such as cell proliferation, survival, adhesion and migration (Yarden and Sliwkowski, 2001) 1.3.2.1 Ras/Raf/MEK/ERK pathway The Ras/Raf/MEK/ERK pathway is one of the best characterized signaling pathways emanating from EGFR (Fig 1.3.2.1) This pathway begins with the adaptor... leads to the engagement of other signaling pathways such as JNK and MAPK pathways (Marais et al., 1998; McClellan et al., 1999) In addition to its function as a PKC activator, DAG is also suggested to be a negative modulator of Rac signaling (Wang et al., 2006) 16 Fig 1.3.2.3 PLC-γ/DAG/IP3 pathway EGFR- activated PLC-γ catalyses the hydrolysis of PIP2 to generate second messengers IP3 and DAG IP3 binds... transcription (STAT) proteins, in particular STAT1, STAT3 and STAT5, have been implicated in EGFR signaling as well In general, growth factor or cytokine signaling results in the binding of STAT proteins to the growth factor receptor or cytokine receptor/Janus kinase (JAK) complex via its SH2 domains and subsequent phosphorylation of crucial tyrosine residues on STAT proteins by the kinases The activated STAT proteins... plasma membrane (Mettlen et al., 2009) Dynamin is phosphorylated by Src kinase at Tyr597 and this phosphorylation was shown to be necessary for dynamin’s self-assembly and GTPase activity Expression of an Y597F dynamin mutant inhibited EGFR internalization, therefore indicating the dependency of EGFR endocytosis on Src kinase activity (Ahn et al., 2002) In EGFR degradation, Cbl, an E3 ubiquitin ligase,... of Endofin s functions is very limited, especially with respect to EGFR signaling In this study, an attempt was made to map the signaling events associated with Endofin following activation of EGFR with EGF Tyrosine phosphorylation of Endofin was shown to be dependent on clathrindependent endocytosis of EGFR and EGFR activity Phosphatidylinositol 3-kinase activity and FYVE domain-mediated localization . amino acids), a transmembrane segment (amino acids 622-644), followed by an intracellular domain that contains a juxtamembrane region, a tyrosine kinase and a C-terminal region which consists. 1998). In addition, mice with an EGFR kinase domain mutation or expressing a dominant-negative EGFR exhibit impaired ductal growth, indicating that EGFR is essential for promoting ductal growth in. proliferation, suggesting a possible role of Endofin in the modulation of MAPK pathway. Collectively, this study has identified a novel signaling cascade involving EGFR, PI3K, Endofin and MAPK in