Mitogenic roles of Gab1 and Grb10 as direct cellular partners in the regulation of MAP kinase signaling.

Functions of signaling mediators Grb10 or Gab1 have been described in mitogenesis but remained disconnected. Here, we report the peptide hormone-dependent direct association between Grb10 and Gab1 and their functional connection in mitogenic signaling via MAP kinase using cultured fibroblasts as a model. In response to PDGF-, IGF-I, or insulin increased levels of Grb10 potentiated cell proliferation or survival whereas dominant-negative, domain-specific Grb10 peptide mimetics attenuated cell proliferation. This response was sensitive to p44/42 MAPK inhibitor but not to p38 MAPK inhibitor. In response to IGF-I or insulin Raf-1, MEK 1/2, and p44/42 MAPK were regulated by Grb10 but not Ras or p38 MAPK. In response to PDGF MEK 1/2, p44/42 MAPK and p38 MAPK were regulated by Grb10 but not Ras or Raf-1. Peptide hormone-dependent co-immunoprecipitation of Grb10 and Gab1 was demonstrated and specifically blocked by a Grb10 SH2 domain peptide mimetic. This domain was sufficient for direct, peptide hormone-dependent association with Gab1 via the Crk binding region. In response to PDGF, IGF-I, or insulin, in a direct comparison, elevated levels of mouse Grb10 delta, or human Grb10 beta or zeta equally potentiated fibroblast proliferation. Proliferation was severely reduced by Gab1 gene disruption whereas an elevated Gab1 gene dose proportionally stimulated Grb10-potentiated cell proliferation. In conclusion, Gab1 and Grb10 function as direct binding partners in the regulation of the mitogenic MAP kinase signal. In cultured fibroblasts, elevated levels of human Grb10 beta, zeta or mouse Grb10 delta comparably potentiate mitogenesis in response to PDGF, IGF-I, or insulin.