CSK ENHANCES INSULIN-STIMULATED DEPHOSPHORYLATION OF FOCAL ADHESION PROTEINS

Insulin has pleiotropic effects on the regulation of cell physiology t hrough binding to its receptor. The wide variety of tyrosine phosphory lation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the m ultiple functions of insulin. Recent studies have shown that activatio n of the insulin receptor leads to the regulation of focal adhesion pr oteins, such as a dephosphorylation of focal adhesion kinase (pp125(FA K)). We show here that C-terminal Src kinase (Csk), which phosphorylat es C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-s timulated dephosphorylation of focal adhesion proteins. We demonstrate d that the overexpression of Csk enhanced and prolonged the insulin-in duced dephosphorylation of pp125(FAK). Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a k inase-negative mutant of Csk was able to inhibit the insulin-induced d ephosphorylation of pp125(FAK) and paxillin. Although we have shown th at the Csk Src homology 2 domain can bind to several tyrosine-phosphor ylated proteins, including pp125(FAK) and paxillin, a majority of prot ein 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 adhesi on proteins. We therefore propose that the kinase activity of Csk, thr ough the insulin-induced complex formation of Csk with IRS-1, is invol ved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activi ty of c-Src family kinases.