Endofin is a novel component in EGR EGFR oncogenic signaling

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). Human VPS34 is required for internal vesicle formation within multivesicular endosomes. J Cell Biol 155: 125164. Futter CE, Felder S, Schlessinger J, Ullrich A, Hopkins CR (1993). Annexin I is phosphorylated in the multivesicular body during the processing of the epidermal growth factor receptor. J Cell Biol 120: 77-83. Galperin E, Sorkin A (2008). Endosomal targeting of MEK2 requires RAF, MEK kinase activity and clathrin-dependent endocytosis. Traffic 9: 1776-90. Gaullier JM, Simonsen A, D'Arrigo A, Bremnes B, Stenmark H (1999). FYVE finger proteins as effectors of phosphatidylinositol 3-phosphate. Chem Phys Lipids 98: 8794. Gaullier JM, Simonsen A, D'Arrigo A, Bremnes B, Stenmark H, Aasland R (1998). FYVE fingers bind PtdIns(3)P. Nature 394: 432-3. Grassot J, Mouchiroud G, Perriere G (2003). RTKdb: database of Receptor Tyrosine Kinase. Nucleic Acids Res 31: 353-8. Grassot, J.; Gouy, M.; Perrière, G.; and Mouchiroud, G. (2006). Origin and Molecular Evolution of Receptor Tryosine Kinases with Immunoglobulin-Like Domains. Molecular Biology and Evolution. 23(6): 1232-1241. Graus-Porta D, Beerli RR, Daly JM, Hynes NE (1997). ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. Embo J 16: 1647-55. Gruenberg J, Stenmark H (2004). The biogenesis of multivesicular endosomes. Nat Rev Mol Cell Biol 5: 317-23. Haigler HT, McKanna JA, Cohen S (1979). Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J Cell Biol 81: 382-95. Hallberg B, Rayter SI, Downward J (1994). Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation. J Biol Chem 269: 3913-6. Hanahan D. (2000). The Hallmarks of Cancer. Cell 100(1): 57-70. Haslekas C, Breen K, Pedersen KW, Johannessen LE, Stang E, Madshus IH (2005). The inhibitory effect of ErbB2 on epidermal growth factor-induced formation of clathrin-coated pits correlates with retention of epidermal growth factor receptorErbB2 oligomeric complexes at the plasma membrane. Mol Biol Cell 16: 5832-42. Hasseine LK, Murdaca J, Suavet F, Longnus S, Giorgetti-Peraldi S, Van Obberghen E (2007). Hrs is a positive regulator of VEGF and insulin signaling. Exp Cell Res 313: 1927-42. 107 Haugh JM, Huang AC, Wiley HS, Wells A, Lauffenburger DA (1999). Internalized epidermal growth factor receptors participate in the activation of p21(ras) in fibroblasts. J Biol Chem 274: 34350-60. Hayakawa A, Hayes S, Leonard D, Lambright D, Corvera S (2007). Evolutionarily conserved structural and functional roles of the FYVE domain. Biochem Soc Symp: 95-105. Hayakawa A, Kitamura N (2000). Early endosomal localization of hrs requires a sequence within the proline- and glutamine-rich region but not the FYVE finger. J Biol Chem 275: 29636-42. He J, Vora M, Haney RM, Filonov GS, Musselman CA, Burd CG et al (2009). Membrane insertion of the FYVE domain is modulated by pH. Proteins 76: 852-60. Henis YI, Hancock JF, Prior IA (2009). Ras acylation, compartmentalization and signaling nanoclusters (Review). Mol Membr Biol 26: 80-92. Hernandez-Sotomayor SM, Carpenter G (1993). Non-catalytic activation of phospholipase C-gamma in vitro by epidermal growth factor receptor. Biochem J 293 ( Pt 2): 507-11. Ho J, Kong JW, Choong LY, Loh MC, Toy W, Chong PK et al (2009). Novel breast cancer metastasis-associated proteins. J Proteome Res 8: 583-94. Hofer F, Berdeaux R, Martin GS (1998). Ras-independent activation of Ral by a Ca(2+)-dependent pathway. Curr Biol 8: 839-42. Honegger AM, Dull TJ, Felder S, Van Obberghen E, Bellot F, Szapary D et al (1987). Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell 51: 199-209. Huang F, Goh LK, Sorkin A (2007). EGF receptor ubiquitination is not necessary for its internalization. Proc Natl Acad Sci U S A 104: 16904-9. Huang F, Jiang X, Sorkin A (2003). Tyrosine phosphorylation of the beta2 subunit of clathrin adaptor complex AP-2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking. J Biol Chem 278: 43411-7. Huang F, Khvorova A, Marshall W, Sorkin A (2004). Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference. J Biol Chem 279: 16657-61. Huang F, Kirkpatrick D, Jiang X, Gygi S, Sorkin A (2006). Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol Cell 21: 737-48. Huang F, Sorkin A (2005). Growth factor receptor binding protein 2-mediated recruitment of the RING domain of Cbl to the epidermal growth factor receptor is essential and sufficient to support receptor endocytosis. Mol Biol Cell 16: 1268-81. 108 Hubbard SR, Till JH (2000). Protein tyrosine kinase structure and function. Annu Rev Biochem 69: 373-98. Huber C, Martensson A, Bokoch GM, Nemazee D, Gavin AL (2008). FGD2, a CDC42-specific exchange factor expressed by antigen-presenting cells, localizes to early endosomes and active membrane ruffles. J Biol Chem 283: 34002-12. Hurley JH, Emr SD (2006). The ESCRT complexes: structure and mechanism of a membrane-trafficking network. Annu Rev Biophys Biomol Struct 35: 277-98. Ikeda W, Nakanishi H, Tanaka Y, Tachibana K, Takai Y (2001). Cooperation of Cdc42 small G protein-activating and actin filament-binding activities of frabin in microspike formation. Oncogene 20: 3457-63. Ikonomov OC, Sbrissa D, Shisheva A (2001). Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5kinase PIKfyve. J Biol Chem 276: 26141-7. Jiang X, Huang F, Marusyk A, Sorkin A (2003). Grb2 regulates internalization of EGF receptors through clathrin-coated pits. Mol Biol Cell 14: 858-70. Jiang X, Sorkin A (2003). Epidermal growth factor receptor internalization through clathrin-coated pits requires Cbl RING finger and proline-rich domains but not receptor polyubiquitylation. Traffic 4: 529-43. Johannessen LE, Pedersen NM, Pedersen KW, Madshus IH, Stang E (2006). Activation of the epidermal growth factor (EGF) receptor induces formation of EGF receptor- and Grb2-containing clathrin-coated pits. Mol Cell Biol 26: 389-401. Johannessen LE, Ringerike T, Molnes J, Madshus IH (2000). Epidermal growth factor receptor efficiently activates mitogen-activated protein kinase in HeLa cells and Hep2 cells conditionally defective in clathrin-dependent endocytosis. Exp Cell Res 260: 136-45. Jones JT, Akita RW, Sliwkowski MX (1999). Binding specificities and affinities of egf domains for ErbB receptors. FEBS Lett 447: 227-31. Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW (2003). Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 284: 31-53. Kane LP, Shapiro VS, Stokoe D, Weiss A (1999). Induction of NF-kappaB by the Akt/PKB kinase. Curr Biol 9: 601-4. Kholodenko, B.N. (2006). Cell-Signalling dynamics in time and space. Nature Reviews Molecular Cell Biology. 7: 165-176. 109 Kil SJ, Carlin C (2000). EGF receptor residues leu(679), leu(680) mediate selective sorting of ligand-receptor complexes in early endosomal compartments. J Cell Physiol 185: 47-60. Kim Y, Ikeda W, Nakanishi H, Tanaka Y, Takekuni K, Itoh S et al (2002). Association of frabin with specific actin and membrane structures. Genes Cells 7: 413-20. King, M.W. (2010). Mechanisms of Signal Transduction. The Medical Biochemistry Page. Indiana University, School of Medicine http://themedicalbiochemistrypage.org/signal-transduction.html Kloth MT, Laughlin KK, Biscardi JS, Boerner JL, Parsons SJ, Silva CM (2003). STAT5b, a Mediator of Synergism between c-Src and the Epidermal Growth Factor Receptor. J Biol Chem 278: 1671-9. Komada M, Kitamura N (1995). Growth factor-induced tyrosine phosphorylation of Hrs, a novel 115-kilodalton protein with a structurally conserved putative zinc finger domain. Mol Cell Biol 15: 6213-21. Kornblum HI, Hussain R, Wiesen J, Miettinen P, Zurcher SD, Chow K et al (1998). Abnormal astrocyte development and neuronal death in mice lacking the epidermal growth factor receptor. J Neurosci Res 53: 697-717. Krishna M, Narang H (2008). The complexity of mitogen-activated protein kinases (MAPKs) made simple. Cell Mol Life Sci 65: 3525-44. Kumari S, Mayor S (2008). ARF1 is directly involved in dynamin-independent endocytosis. Nat Cell Biol 10: 30-41. Kutateladze T, Overduin M (2001). Structural mechanism of endosome docking by the FYVE domain. Science 291: 1793-6. Kutateladze TG (2006). Phosphatidylinositol 3-phosphate recognition and membrane docking by the FYVE domain. Biochim Biophys Acta 1761: 868-77. Langelier C, von Schwedler UK, Fisher RD, De Domenico I, White PL, Hill CP et al (2006). Human ESCRT-II complex and its role in human immunodeficiency virus type release. J Virol 80: 9465-80. Langlois WJ, Sasaoka T, Saltiel AR, Olefsky JM (1995). Negative feedback regulation and desensitization of insulin- and epidermal growth factor-stimulated p21ras activation. J Biol Chem 270: 25320-3. Lawe DC, Chawla A, Merithew E, Dumas J, Carrington W, Fogarty K et al (2002). Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1. J Biol Chem 277: 8611-7. 110 Leaman DW, Pisharody S, Flickinger TW, Commane MA, Schlessinger J, Kerr IM et al (1996). Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor. Mol Cell Biol 16: 369-75. Lee JW, Soung YH, Kim SY, Nam HK, Park WS, Nam SW et al (2005). Somatic mutations of EGFR gene in squamous cell carcinoma of the head and neck. Clin Cancer Res 11: 2879-82. Lee MK, Pardoux C, Hall MC, Lee PS, Warburton D, Qing J et al (2007). TGF-beta activates Erk MAP kinase signalling through direct phosphorylation of ShcA. Embo J 26: 3957-67. Lemmon, M.A. and Schlessinger, J. (2010). Cell Signaling by Receptor Tyrosine Kinases. Cell. 141:1117-1134. Li H, Villalobo A (2002). Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor. Biochem J 362: 499-505. Lindmo K, Stenmark H (2006). Regulation of membrane traffic by phosphoinositide 3-kinases. J Cell Sci 119: 605-14. Llado A, Timpson P, Vila de Muga S, Moreto J, Pol A, Grewal T et al (2008). Protein kinase Cdelta and calmodulin regulate epidermal growth factor receptor recycling from early endosomes through Arp2/3 complex and cortactin. Mol Biol Cell 19: 1729. Lombardo CR, Consler TG, Kassel DB (1995). In vitro phosphorylation of the epidermal growth factor receptor autophosphorylation domain by c-src: identification of phosphorylation sites and c-src SH2 domain binding sites. Biochemistry 34: 1645666. Lorenzo O, Urbe S, Clague MJ (2005). Analysis of phosphoinositide binding domain properties within the myotubularin-related protein MTMR3. J Cell Sci 118: 2005-12. Lorenzo O, Urbe S, Clague MJ (2006). Systematic analysis of myotubularins: heteromeric interactions, subcellular localisation and endosome related functions. J Cell Sci 119: 2953-9. Luetteke NC, Qiu TH, Fenton SE, Troyer KL, Riedel RF, Chang A et al (1999). Targeted inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development. Development 126: 2739-50. Luetteke NC, Qiu TH, Peiffer RL, Oliver P, Smithies O, Lee DC (1993). TGF alpha deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice. Cell 73: 263-78. Mann GB, Fowler KJ, Gabriel A, Nice EC, Williams RL, Dunn AR (1993). Mice with a null mutation of the TGF alpha gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation. Cell 73: 249-61. 111 Mao W, Irby R, Coppola D, Fu L, Wloch M, Turner J et al (1997). Activation of cSrc by receptor tyrosine kinases in human colon cancer cells with high metastatic potential. Oncogene 15: 3083-90. Mao Y, Nickitenko A, Duan X, Lloyd TE, Wu MN, Bellen H et al (2000). Crystal structure of the VHS and FYVE tandem domains of Hrs, a protein involved in membrane trafficking and signal transduction. Cell 100: 447-56. Marais R, Light Y, Mason C, Paterson H, Olson MF, Marshall CJ (1998). Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C. Science 280: 109-12. Martin-Nieto J, Villalobo A (1998). The human epidermal growth factor receptor contains a juxtamembrane calmodulin-binding site. Biochemistry 37: 227-36. Martin NP, Mohney RP, Dunn S, Das M, Scappini E, O'Bryan JP (2006). Intersectin regulates epidermal growth factor receptor endocytosis, ubiquitylation, and signaling. Mol Pharmacol 70: 1643-53. Masui H, Castro L, Mendelsohn J (1993). Consumption of EGF by A431 cells: evidence for receptor recycling. J Cell Biol 120: 85-93. Mayor S, Pagano RE (2007). Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 8: 603-12. McBride HM, Rybin V, Murphy C, Giner A, Teasdale R, Zerial M (1999). Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13. Cell 98: 377-86. McClellan M, Kievit P, Auersperg N, Rodland K (1999). Regulation of proliferation and apoptosis by epidermal growth factor and protein kinase C in human ovarian surface epithelial cells. Exp Cell Res 246: 471-9. Mettlen M, Pucadyil T, Ramachandran R, Schmid SL (2009). Dissecting dynamin's role in clathrin-mediated endocytosis. Biochem Soc Trans 37: 1022-6. Miaczynska M, Christoforidis S, Giner A, Shevchenko A, Uttenweiler-Joseph S, Habermann B et al (2004). APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment. Cell 116: 445-56. Miettinen PJ, Berger JE, Meneses J, Phung Y, Pedersen RA, Werb Z et al (1995). Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 376: 337-41. Misra S, Hurley JH (1999). Crystal structure of a phosphatidylinositol 3-phosphatespecific membrane-targeting motif, the FYVE domain of Vps27p. Cell 97: 657-66. 112 Miura S, Takeshita T, Asao H, Kimura Y, Murata K, Sasaki Y et al (2000). Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA. Mol Cell Biol 20: 9346-55. Mizuno E, Kawahata K, Kato M, Kitamura N, Komada M (2003). STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitininteracting motif. Mol Biol Cell 14: 3675-89. Mizuno E, Kawahata K, Okamoto A, Kitamura N, Komada M (2004). Association with Hrs is required for the early endosomal localization, stability, and function of STAM. J Biochem 135: 385-96. Morino C, Kato M, Yamamoto A, Mizuno E, Hayakawa A, Komada M et al (2004). A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor. Exp Cell Res 297: 380-91. Mosesson Y, Chetrit D, Schley L, Berghoff J, Ziv T, Carvalho S et al (2009). Monoubiquitinylation regulates endosomal localization of Lst2, a negative regulator of EGF receptor signaling. Dev Cell 16: 687-98. Motley A, Bright NA, Seaman MN, Robinson MS (2003). Clathrin-mediated endocytosis in AP-2-depleted cells. J Cell Biol 162: 909-18. Murk JL, Humbel BM, Ziese U, Griffith JM, Posthuma G, Slot JW et al (2003). Endosomal compartmentalization in three dimensions: implications for membrane fusion. Proc Natl Acad Sci U S A 100: 13332-7. Nada S, Hondo A, Kasai A, Koike M, Saito K, Uchiyama Y et al (2009). The novel lipid raft adaptor p18 controls endosome dynamics by anchoring the MEK-ERK pathway to late endosomes. Embo J 28: 477-89. Naslavsky N, Boehm M, Backlund PS, Jr., Caplan S (2004). Rabenosyn-5 and EHD1 interact and sequentially regulate protein recycling to the plasma membrane. Mol Biol Cell 15: 2410-22. Naslavsky N, McKenzie J, Altan-Bonnet N, Sheff D, Caplan S (2009). EHD3 regulates early-endosome-to-Golgi transport and preserves Golgi morphology. J Cell Sci 122: 389-400. Nesterov A, Carter RE, Sorkina T, Gill GN, Sorkin A (1999). Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis. Embo J 18: 2489-99. Nielsen E, Christoforidis S, Uttenweiler-Joseph S, Miaczynska M, Dewitte F, Wilm M et al (2000). Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain. J Cell Biol 151: 601-12. Nishibe S, Wahl MI, Hernandez-Sotomayor SM, Tonks NK, Rhee SG, Carpenter G (1990). Increase of the catalytic activity of phospholipase C-gamma by tyrosine phosphorylation. Science 250: 1253-6. 113 Normanno N, Bianco C, Strizzi L, Mancino M, Maiello MR, De Luca A et al (2005). The ErbB receptors and their ligands in cancer: an overview. Curr Drug Targets 6: 243-57. Normanno N, De Luca A, Bianco C, Strizzi L, Mancino M, Maiello MR et al (2006). Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366: 2-16. Olayioye MA, Graus-Porta D, Beerli RR, Rohrer J, Gay B, Hynes NE (1998). ErbB-1 and ErbB-2 acquire distinct signaling properties dependent upon their dimerization partner. Mol Cell Biol 18: 5042-51. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P et al (2006). Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127: 635-48. Olson MF, Pasteris NG, Gorski JL, Hall A (1996). Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development, are upstream regulators of Rho GTPases. Curr Biol 6: 1628-33. Omerovic J, Prior IA (2009). Compartmentalized signalling: Ras proteins and signalling nanoclusters. Febs J 276: 1817-25. Orth JD, Krueger EW, Weller SG, McNiven MA (2006). A novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization. Cancer Res 66: 3603-10. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB (1999). NFkappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401: 82-5. Pasteris NG, Cadle A, Logie LJ, Porteous ME, Schwartz CE, Stevenson RE et al (1994). Isolation and characterization of the faciogenital dysplasia (Aarskog-Scott syndrome) gene: a putative Rho/Rac guanine nucleotide exchange factor. Cell 79: 669-78. Patki V, Lawe DC, Corvera S, Virbasius JV, Chawla A (1998). A functional PtdIns(3)P-binding motif. Nature 394: 433-4. Pawson T (1995). Protein modules and signalling networks. Nature 373: 573-80. Pawson T, Gish GD, Nash P (2001). SH2 domains, interaction modules and cellular wiring. Trends Cell Biol 11: 504-11. Pawson T and Scott JD (1997). Signaling Through Scaffold, Anchoring, and Adaptor Proteins. Science 278: 2075-2080. Prenzel N, Zwick E, Leserer M, Ullrich A (2000). Tyrosine kinase signalling in breast cancer. Epidermal growth factor receptor: convergence point for signal integration and diversification. Breast Cancer Res 2: 184-90. 114 Pullan L, Mullapudi S, Huang Z, Baldwin PR, Chin C, Sun W et al (2006). The endosome-associated protein Hrs is hexameric and controls cargo sorting as a "master molecule". Structure 14: 661-71. Raiborg C, Bache KG, Gillooly DJ, Madshus IH, Stang E, Stenmark H (2002). Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes. Nat Cell Biol 4: 394-8. Raiborg C, Bache KG, Mehlum A, Stang E, Stenmark H (2001a). Hrs recruits clathrin to early endosomes. Embo J 20: 5008-21. Raiborg C, Bremnes B, Mehlum A, Gillooly DJ, D'Arrigo A, Stang E et al (2001b). FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes. J Cell Sci 114: 2255-63. Raiborg C, Malerod L, Pedersen NM, Stenmark H (2008). Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking. Exp Cell Res 314: 80113. Rane SG, Reddy EP (2002). JAKs, STATs and Src kinases in hematopoiesis. Oncogene 21: 3334-58. Rayala SK, Hollander P, Balasenthil S, Molli PR, Bean AJ, Vadlamudi RK et al (2006). Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) interacts with PELP1 and activates MAPK. J Biol Chem 281: 4395-403. Razi M, Futter CE (2006). Distinct roles for Tsg101 and Hrs in multivesicular body formation and inward vesiculation. Mol Biol Cell 17: 3469-83. Ren Y, Cheng L, Rong Z, Li Z, Li Y, Zhang X et al (2008). hSef potentiates EGFmediated MAPK signaling through affecting EGFR trafficking and degradation. Cell Signal 20: 518-33. Robinson DR, Wu YM, Lin SF (2000). The protein tyrosine kinase family of the human genome. Oncogene 19: 5548-57. Roxrud I, Raiborg C, Pedersen NM, Stang E, Stenmark H (2008). An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor. J Cell Biol 180: 1205-18. Runyan CE, Schnaper HW, Poncelet AC (2005). The role of internalization in transforming growth factor beta1-induced Smad2 association with Smad anchor for receptor activation (SARA) and Smad2-dependent signaling in human mesangial cells. J Biol Chem 280: 8300-8. Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ et al (2005). Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23: 94-101. 115 Salomon DS, Brandt R, Ciardiello F, Normanno N (1995). Epidermal growth factorrelated peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 19: 183-232. Sankaran VG, Klein DE, Sachdeva MM, Lemmon MA (2001). High-affinity binding of a FYVE domain to phosphatidylinositol 3-phosphate requires intact phospholipid but not FYVE domain oligomerization. Biochemistry 40: 8581-7. Sasaki Y, Sugamura K (2001). Involvement of Hgs/Hrs in signaling for cytokinemediated c-fos induction through interaction with TAK1 and Pak1. J Biol Chem 276: 29943-52. Sasaoka T, Langlois WJ, Leitner JW, Draznin B, Olefsky JM (1994). The signaling pathway coupling epidermal growth factor receptors to activation of p21ras. J Biol Chem 269: 32621-5. Sato K, Sato A, Aoto M, Fukami Y (1995). c-Src phosphorylates epidermal growth factor receptor on tyrosine 845. Biochem Biophys Res Commun 215: 1078-87. Sbrissa D, Ikonomov OC, Shisheva A (2002). Phosphatidylinositol 3-phosphateinteracting domains in PIKfyve. Binding specificity and role in PIKfyve. Endomenbrane localization. J Biol Chem 277: 6073-9. Schlessinger, J. and Ullrich, A. (1992). Growth factor signalling by receptor tyrosine kinases. Neuron 9(3): 383-391. Schulze WX, Deng L, Mann M (2005). Phosphotyrosine interactome of the ErbBreceptor kinase family. Mol Syst Biol 1: 2005 0008. Sebastian S, Settleman J, Reshkin SJ, Azzariti A, Bellizzi A, Paradiso A (2006). The complexity of targeting EGFR signalling in cancer: from expression to turnover. Biochim Biophys Acta 1766: 120-39. Seet LF, Hong W (2001). Endofin, an endosomal FYVE domain protein. J Biol Chem 276: 42445-54. Seet LF, Hong W (2005). Endofin recruits clathrin to early endosomes via TOM1. J Cell Sci 118: 575-87. Seet LF, Liu N, Hanson BJ, Hong W (2004). Endofin recruits TOM1 to endosomes. J Biol Chem 279: 4670-9. Shi W, Chang C, Nie S, Xie S, Wan M, Cao X (2007). Endofin acts as a Smad anchor for receptor activation in BMP signaling. J Cell Sci 120: 1216-24. Sibilia M, Steinbach JP, Stingl L, Aguzzi A, Wagner EF (1998). A strain-independent postnatal neurodegeneration in mice lacking the EGF receptor. Embo J 17: 719-31. Sibilia M, Wagner EF (1995). Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 269: 234-8. 116 Sigismund S, Argenzio E, Tosoni D, Cavallaro E, Polo S, Di Fiore PP (2008). Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev Cell 15: 209-19. Sigismund S, Woelk T, Puri C, Maspero E, Tacchetti C, Transidico P et al (2005). Clathrin-independent endocytosis of ubiquitinated cargos. Proc Natl Acad Sci U S A 102: 2760-5. Simonsen A, Gaullier JM, D'Arrigo A, Stenmark H (1999). The Rab5 effector EEA1 interacts directly with syntaxin-6. J Biol Chem 274: 28857-60. Slieker LJ, Martensen TM, Lane MD (1986). Synthesis of epidermal growth factor receptor in human A431 cells. Glycosylation-dependent acquisition of ligand binding activity occurs post-translationally in the endoplasmic reticulum. J Biol Chem 261: 15233-41. Sorkin A, Goh LK (2008). Endocytosis and intracellular trafficking of ErbBs. Exp Cell Res 314: 3093-106. Sorkin A, Krolenko S, Kudrjavtceva N, Lazebnik J, Teslenko L, Soderquist AM et al (1991a). Recycling of epidermal growth factor-receptor complexes in A431 cells: identification of dual pathways. J Cell Biol 112: 55-63. Sorkin A, Mazzotti M, Sorkina T, Scotto L, Beguinot L (1996). Epidermal growth factor receptor interaction with clathrin adaptors is mediated by the Tyr974containing internalization motif. J Biol Chem 271: 13377-84. Sorkin A, McClure M, Huang F, Carter R (2000). Interaction of EGF receptor and grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy. Curr Biol 10: 1395-8. Sorkin A, von Zastrow M (2009). Endocytosis and signalling: intertwining molecular networks. Nat Rev Mol Cell Biol 10: 609-22. Sorkin A, Waters C, Overholser KA, Carpenter G (1991b). Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis. J Biol Chem 266: 8355-62. Sorkin AD, Teslenko LV, Nikolsky NN (1988). The endocytosis of epidermal growth factor in A431 cells: a pH of microenvironment and the dynamics of receptor complex dissociation. Exp Cell Res 175: 192-205. Sorkina T, Huang F, Beguinot L, Sorkin A (2002). Effect of tyrosine kinase inhibitors on clathrin-coated pit recruitment and internalization of epidermal growth factor receptor. J Biol Chem 277: 27433-41. Soubeyran P, Kowanetz K, Szymkiewicz I, Langdon WY, Dikic I (2002). Cbl-CIN85endophilin complex mediates ligand-induced downregulation of EGF receptors. Nature 416: 183-7. 117 Stang E, Blystad FD, Kazazic M, Bertelsen V, Brodahl T, Raiborg C et al (2004). Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits. Mol Biol Cell 15: 3591-604. Stern KA, Visser Smit GD, Place TL, Winistorfer S, Piper RC, Lill NL (2007). Epidermal growth factor receptor fate is controlled by Hrs tyrosine phosphorylation sites that regulate Hrs degradation. Mol Cell Biol 27: 888-98. Stoscheck CM, Carpenter G (1984a). Characterization of the metabolic turnover of epidermal growth factor receptor protein in A-431 cells. J Cell Physiol 120: 296-302. Stoscheck CM, Carpenter G (1984b). Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts. J Cell Biol 98: 1048-53. Stover DR, Becker M, Liebetanz J, Lydon NB (1995). Src phosphorylation of the epidermal growth factor receptor at novel sites mediates receptor interaction with Src and P85 alpha. J Biol Chem 270: 15591-7. Sun L, Carpenter G (1998). Epidermal growth factor activation of NF-kappaB is mediated through IkappaBalpha degradation and intracellular free calcium. Oncogene 16: 2095-102. Tanos BE, Pendergast AM (2007). Abi-1 forms an epidermal growth factor-inducible complex with Cbl: role in receptor endocytosis. Cell Signal 19: 1602-9. Taylor GS, Maehama T, Dixon JE (2000). Inaugural article: myotubularin, a protein tyrosine phosphatase mutated in myotubular myopathy, dephosphorylates the lipid second messenger, phosphatidylinositol 3-phosphate. Proc Natl Acad Sci U S A 97: 8910-5. Teis D, Taub N, Kurzbauer R, Hilber D, de Araujo ME, Erlacher M et al (2006). p14MP1-MEK1 signaling regulates endosomal traffic and cellular proliferation during tissue homeostasis. J Cell Biol 175: 861-8. Teis D, Wunderlich W, Huber LA (2002). Localization of the MP1-MAPK scaffold complex to endosomes is mediated by p14 and required for signal transduction. Dev Cell 3: 803-14. Thien CB, Walker F, Langdon WY (2001). RING finger mutations that abolish c-Cbldirected polyubiquitination and downregulation of the EGF receptor are insufficient for cell transformation. Mol Cell 7: 355-65. Threadgill DW, Dlugosz AA, Hansen LA, Tennenbaum T, Lichti U, Yee D et al (1995). Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 269: 230-4. 118 Tice DA, Biscardi JS, Nickles AL, Parsons SJ (1999). Mechanism of biological synergy between cellular Src and epidermal growth factor receptor. Proc Natl Acad Sci U S A 96: 1415-20. Tong J, Taylor P, Peterman SM, Prakash A, Moran MF (2009). Epidermal growth factor receptor phosphorylation sites Ser991 and Tyr998 are implicated in the regulation of receptor endocytosis and phosphorylations at Ser1039 and Thr1041. Mol Cell Proteomics 8: 2131-44. Torii S, Kusakabe M, Yamamoto T, Maekawa M, Nishida E (2004). Sef is a spatial regulator for Ras/MAP kinase signaling. Dev Cell 7: 33-44. Tosoni D, Cestra G (2009). CAP (Cbl associated protein) regulates receptor-mediated endocytosis. FEBS Lett 583: 293-300. Tran DD, Russell HR, Sutor SL, van Deursen J, Bram RJ (2003). CAML is required for efficient EGF receptor recycling. Dev Cell 5: 245-56. Troyer KL, Luetteke NC, Saxon ML, Qiu TH, Xian CJ, Lee DC (2001). Growth retardation, duodenal lesions, and aberrant ileum architecture in triple null mice lacking EGF, amphiregulin, and TGF-alpha. Gastroenterology 121: 68-78. Tsukazaki T, Chiang TA, Davison AF, Attisano L, Wrana JL (1998). SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor. Cell 95: 779-91. Urbe S, Mills IG, Stenmark H, Kitamura N, Clague MJ (2000). Endosomal localization and receptor dynamics determine tyrosine phosphorylation of hepatocyte growth factor-regulated tyrosine kinase substrate. Mol Cell Biol 20: 7685-92. Vieira AV, Lamaze C, Schmid SL (1996). Control of EGF receptor signaling by clathrin-mediated endocytosis. Science 274: 2086-9. Wang H, Yang C, Leskow FC, Sun J, Canagarajah B, Hurley JH et al (2006). Phospholipase Cgamma/diacylglycerol-dependent activation of beta2-chimaerin restricts EGF-induced Rac signaling. Embo J 25: 2062-74. Wang Y, Pennock S, Chen X, Wang Z (2002). Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival. Mol Cell Biol 22: 7279-90. Wang Z, Moran MF (1996). Requirement for the adapter protein GRB2 in EGF receptor endocytosis. Science 272: 1935-9. Wang Z, Zhang L, Yeung TK, Chen X (1999). Endocytosis deficiency of epidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGF stimulation. Mol Biol Cell 10: 1621-36. Ware MF, Tice DA, Parsons SJ, Lauffenburger DA (1997). Overexpression of cellular Src in fibroblasts enhances endocytic internalization of epidermal growth factor receptor. J Biol Chem 272: 30185-90. 119 White IJ, Bailey LM, Aghakhani MR, Moss SE, Futter CE (2006). EGF stimulates annexin 1-dependent inward vesiculation in a multivesicular endosome subpopulation. Embo J 25: 1-12. Wilde A, Beattie EC, Lem L, Riethof DA, Liu SH, Mobley WC et al (1999). EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake. Cell 96: 677-87. Wiley HS (2003). Trafficking of the ErbB receptors and its influence on signaling. Exp Cell Res 284: 78-88. Wiley HS, Herbst JJ, Walsh BJ, Lauffenburger DA, Rosenfeld MG, Gill GN (1991). The role of tyrosine kinase activity in endocytosis, compartmentation, and downregulation of the epidermal growth factor receptor. J Biol Chem 266: 11083-94. Williams RL, Urbe S (2007). The emerging shape of the ESCRT machinery. Nat Rev Mol Cell Biol 8: 355-68. Xie W, Paterson AJ, Chin E, Nabell LM, Kudlow JE (1997). Targeted expression of a dominant negative epidermal growth factor receptor in the mammary gland of transgenic mice inhibits pubertal mammary duct development. Mol Endocrinol 11: 1766-81. Yamakami M, Yoshimori T, Yokosawa H (2003). Tom1, a VHS domain-containing protein, interacts with tollip, ubiquitin, and clathrin. J Biol Chem 278: 52865-72. Yamauchi T, Ueki K, Tobe K, Tamemoto H, Sekine N, Wada M et al (1997). Tyrosine phosphorylation of the EGF receptor by the kinase Jak2 is induced by growth hormone. Nature 390: 91-6. Yamazaki T, Zaal K, Hailey D, Presley J, Lippincott-Schwartz J, Samelson LE (2002). Role of Grb2 in EGF-stimulated EGFR internalization. J Cell Sci 115: 1791802. Yang J, Kim O, Wu J, Qiu Y (2002). Interaction between tyrosine kinase Etk and a RUN domain- and FYVE domain-containing protein RUFY1. A possible role of ETK in regulation of vesicle trafficking. J Biol Chem 277: 30219-26. Yarden Y (2001). The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur J Cancer 37 Suppl 4: S3-8. Yarden Y, Sliwkowski MX (2001). Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2: 127-37. Yarden Y, Ullrich A (1988). Growth factor receptor tyrosine kinases. Annu Rev Biochem 57: 443-78. 120 Yokouchi M, Kondo T, Sanjay A, Houghton A, Yoshimura A, Komiya S et al (2001). Src-catalyzed phosphorylation of c-Cbl leads to the interdependent ubiquitination of both proteins. J Biol Chem 276: 35185-93. Yoon S, Seger R (2006). The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24: 21-44. Zandi R, Larsen AB, Andersen P, Stockhausen MT, Poulsen HS (2007). Mechanisms for oncogenic activation of the epidermal growth factor receptor. Cell Signal 19: 2013-23. Zerial M, McBride H (2001). Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2: 107-17. Zhang F, Qiu T, Wu X, Wan C, Shi W, Wang Y et al (2009). Sustained BMP signaling in osteoblasts stimulates bone formation by promoting angiogenesis and osteoblast differentiation. J Bone Miner Res 24: 1224-33. Zoncu R, Perera RM, Balkin DM, Pirruccello M, Toomre D, De Camilli P (2009). A phosphoinositide switch controls the maturation and signaling properties of APPL endosomes. Cell 136: 1110-21. 121 PUBLICATIONS Toy W, Lim SK, Loh MC, Lim YP EGF-induced tyrosine phosphorylation of Endofin is dependent on PI3K activity and proper localization to endosomes. (2010) Cell Signal 22: 437-46. (Online publication) Ho J, Kong JW, Choong LY, Loh MC, Toy W, Chong PK et al (2009). Novel breast cancer metastasis-associated proteins. J Proteome Res 8: 583-94. Choong LY, Lim S, Loh MC, Man X, Chen Y, Toy W et al (2007). Progressive loss of epidermal growth factor receptor in a subpopulation of breast cancers: implications in target-directed therapeutics. Mol Cancer Ther 6: 2828-42. Chen Y, Low TY, Choong LY, Ray RS, Tan YL, Toy W et al (2007a). Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells. 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