ERK regulates the hepatocyte growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase

Based on our previous observations that active ERK associates with and phos phorylates Gab1 in response to HGF, and the prediction that the ERK phospho rylation site is adjacent to one of the phosphatidylinositol 3-kinase (PI3K ) SH2 binding motifs, we examined the possibility that ERK phosphorylation can regulate the Gab1/PI3K association. The HGF-mediated association of Gab 1 with either full-length GST-p85 or its isolated N- or C-terminal SH2 doma ins was inhibited by similar to 50% in the setting of ERK inhibition, a res ult confirmed by coimmunoprecipitation of the native proteins. A 14-amino a cid peptide encoding (YVPMTP477)-Y-472 (one of the major p85 binding sites in Gab1 and the predicted ERK phosphorylation site) was synthesized with ei ther phosphotyrosine alone (pY), or phosphotyrosine + phosphothreonine (pYT ). In both pull-down assays and competition assays, pYT demonstrated a high er affinity for p85 than did pY alone. Finally, examination of the phosphor ylation state of Akt after HGF stimulation revealed that ERK inhibition res ulted in a decrease in Akt activation at both 5 and 10 min. These results s uggest that activated ERK can phosphorylate Gab1 in response to HGF stimula tion and thereby potentiate the Gab1/P13K association and subsequent P13K a ctivation.