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

Abstract

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.

Grb10 is a dual regulator of receptor tyrosine kinase signaling

“…Furthermore, in response to PDGF, IGF-1 and insulin, Grb10 potentiates cell proliferation [15]. This biological effect is mediated through association of Grb10 with Gab1.…”

Tissue-specific regulation and function of Grb10 during growth and neuronal commitment

“…Accordingly, overexpression of Grb10 inhibits the PI3K/AKT and MAPK pathways whereas Grb10 deficiency increases the insulin-dependent phosphorylation of proteins within these pathways, including AKT and MAPK1 (26)(27)(28)(29). Conflicting studies, however, have reported that Grb10 has a positive effect on insulin signaling (9,30). It has been proposed that these results are due to the overexpression of different Grb10 isoforms and/or the use of cell types that are not insulin-sensitive (25).…”

Regulation of insulin and type 1 insulin‐like growth factor signaling and action by the Grb10/14 and SH2B1/B2 adaptor proteins

“…Paradoxically, stimulatory effects of Grb10 over‐expression on mitogenic effects of insulin and IGF–1 in NIH3T3 fibroblasts and on metabolic effects of insulin in L6 myoblasts and 3T3L1 adipocytes have also been reported. Whether, as previously proposed , these opposite effects reflect the use of different Grb10 isoforms and domains, cell lines that are not representative of insulin target cells, and/or non‐physiological levels of Grb10 expression remains unclear.…”