Activated ERK2 interacts with and phosphorylates the docking protein GAB1

Grb2-associated binder 1 (GAB1) is a docking protein found to associate wit h 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 exp eriments with bacterially expressed MBD and fall-length GAB1 revealed the p resence of c-MET as web as phosphorylated ERK2 (pERK2). By using purified p ERK2 and non-pERK2, we found that GAB1 associates exclusively with the phos phorylated form of the enzyme and that this association does not require me diation by a third protein. When epitope-tagged GAB1 was cotransfected with constitutively active MEK1 into A293 cells, co-immunoprecipitation of GAB1 and pERK2 was observed, demonstrating that this interaction can occur in i ntact cells. In vitro, both the MBD and full-length GAB1 were found to be s ubstrates for activated ERK2. In intact cells, epitope-tagged GAB1 was foun d to be basally phosphorylated on serine with an increase following co-tran sfection with constitutively active MEK1 and the appearance of novel phosph orylation sites detected by phosphopeptide mapping. Thus, it appears that G AB1 can associate directly with phosphorylated ERK2 via the MET-binding dom ain and that GAB1 then acts as a substrate for the enzyme.