Csk Enhances Insulin-Stimulated Dephosphorylation of Focal Adhesion Proteins

Abstract

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125 FAK ). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125 FAK . Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125 FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.Among the earliest cellular responses following insulin stimulation are the tyrosine phosphorylations of insulin receptor (IR) ␤ subunit as well as IR substrate 1 (IRS-1) (pp185) by the activated IR kinase activity (22, 23, 52, 64; for a review, see reference 62). IRS-1, a cytoplasmic protein found in most cells and tissues, contains more than 20 potential tyrosine phosphorylation sites; several of these sites have been shown to bind specifically to each of the Src homology 2 (SH2) domains of various signaling proteins such as phosphatidylinositol (PI) 3Ј-kinase, Ash/Grb2, Syp, and Nck (3,4,26,27,29,50,52,58). It is possible that the divergent structures and functions of the SH2 proteins which bind to IRS-1 mediate the pleiotropic responses of insulin; tyrosine phosphorylation of IRS-1 may act as a switch to control the interaction of IRS-1 with these signaling proteins (52). In addition to IRS-1, another substrate, pp190 (IRS-2), which was found in IRS-1-deficient mice and can substitute, at least in part, for the function of IRS-1, has been reported. IRS-2 also binds to PI 3Ј-kinase (2,53,55,59) and Ash/Grb2 (53, 59), suggesting that it too serves as an SH2-docking protein.

Opposite Effects of Insulin on Focal Adhesion Proteins in 3T3-L1 Adipocytes and in Cells Overexpressing the Insulin Receptor

“…Csk can inhibit Src activity by directly phosphorylating tyrosine residues on the C-terminal region of Src. Overexpression of Csk in CHO cells increases the amount of Csk associated with IRS-1, decreases the basal levels of tyrosine-phosphorylated FAK, and enhances the ability of insulin to stimulate the tyrosine dephosphorylation of FAK (Tobe et al, 1996). We speculate that a greater association between Csk and IRS-1 in CHO-IR cells occurs upon exposure to insulin, because of the supraphysiologically elevated number of insulin receptors.…”

“…tyrosine phosphorylation level of IRS-1 is markedly higher in the insulin receptor-overexpressing cell lines compared with the parental cell lines (Konstantopoulos and Clark, 1996; present study). Recently, it was shown that the SH2 domain of C-terminal Src kinase (Csk) associates with phosphorylated tyrosine residues on IRS-1 (Tobe et al, 1996). Csk can inhibit Src activity by directly phosphorylating tyrosine residues on the C-terminal region of Src.…”

Epidermal Growth Factor Modulates Tyrosine Phosphorylation of p130Cas

“…17); SHP2 is also a known component of the EGF receptor signaling pathway (38). Dephosphorylation may also take place through inactivation of tyrosine kinases; Tobe and co-workers (54) found that C-terminal Src kinase Csk is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of Src family kinases.…”

Tyrosine Phosphorylation of Paxillin and Focal Adhesion Kinase during Insulin-like Growth Factor-I-stimulated Lamellipodial Advance

“…However, the reported IGF-I-dependent dephosphorylation occurred in CHO cells transfected with the insulin receptor and does not occur via the IGF-IR. Also, insulin-stimulated FAK and paxillin dephosphorylation occurs in insulin receptor-transfected Swiss 3T3 fibroblasts (33) and CHO cells (34,35). This is surprising, given the many similarities between IGF-IR and insulin receptor structure and signal transduction (1).…”