title: Activated ERK2 interacts with and phosphorylates the docking protein GAB1.
abstract: Grb2-associated binder 1 (GAB1) is a docking protein found to associate with the activated c-MET receptor via the MET-binding domain (MBD) and appears to be critical for the tubulogenic actions of this receptor. Pull-down experiments with bacterially expressed MBD and full-length GAB1 revealed the presence of c-MET as well as phosphorylated ERK2 (pERK2). By using purified pERK2 and non-pERK2, we found that GAB1 associates exclusively with the phosphorylated form of the enzyme and that this association does not require mediation by a third protein. When epitope-tagged GAB1 was co-transfected with constitutively active MEK1 into A293 cells, co-immunoprecipitation of GAB1 and pERK2 was observed, demonstrating that this interaction can occur in intact cells. In vitro, both the MBD and full-length GAB1 were found to be substrates for activated ERK2. In intact cells, epitope-tagged GAB1 was found to be basally phosphorylated on serine with an increase following co-transfection with constitutively active MEK1 and the appearance of novel phosphorylation sites detected by phosphopeptide mapping. Thus, it appears that GAB1 can associate directly with phosphorylated ERK2 via the MET-binding domain and that GAB1 then acts as a substrate for the enzyme.
authors.affiliation: Division of Nephrology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA. broshan@caregroup.harvard.edu
authors.forename: B
authors.initials: B
authors.lastname: Roshan
authors.forename: C
authors.initials: C
authors.lastname: Kjelsberg
authors.forename: K
authors.initials: K
authors.lastname: Spokes
authors.forename: A
authors.initials: A
authors.lastname: Eldred
authors.forename: C S
authors.initials: CS
authors.lastname: Crovello
authors.forename: L G
authors.initials: LG
authors.lastname: Cantley
mesh_terms: D000818:Animals; D002460:Cell Line; D004285:Dogs; D004789:Enzyme Activation; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D010750:Phosphoproteins; D019950:Mitogen-Activated Protein Kinase 1