title: Novel binding partners and differentially regulated phosphorylation sites clarify Eps8 as a multi-functional adaptor.
abstract: Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.
authors.affiliation: Cancer Research UK Growth Factor Signalling Group, School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Debbie L
authors.initials: DL
authors.lastname: Cunningham
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Creese
authors.forename: Giulio
authors.initials: G
authors.lastname: Auciello
authors.forename: Steve M M
authors.initials: SM
authors.lastname: Sweet
authors.forename: Tulin
authors.initials: T
authors.lastname: Tatar
authors.forename: Joshua Z
authors.initials: JZ
authors.lastname: Rappoport
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Grant
authors.forename: John K
authors.initials: JK
authors.lastname: Heath
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003598:Cytoskeletal Proteins; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009842:Oligopeptides / Q000032:analysis; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D040901:Proteomics; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; C489989:EPS8 protein, human; D009842:Oligopeptides; D010750:Phosphoproteins; D017468:Receptors, Fibroblast Growth Factor; D019000:Phosphotyrosine; D019061:src-Family Kinases