Activated ERK2 interacts with and phosphorylates the docking protein GAB1.

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.