REDUCED CELL ATTACHMENT AND PHOSPHORYLATION OF FOCAL ADHESION KINASE ASSOCIATED WITH EXPRESSION OF A MUTANT INSULIN-RECEPTOR

Insulin signaling results in rapid changes to the cell cytoskeleton, a nd it has recently been shown that insulin stimulates the dephosphoryl ation of the cytoskeletal-associated tyrosine kinase, focal adhesion k inase (pp125(FAK)). We report here that mutation of two tryptic cleava ge sites (Lys(164) and Lys(582) --> Asn; 2N) in the insulin receptor a lpha-subunit results in a cell-line (CHO.2N-10) with altered morpholog y associated with an increase in cell size, a decrease in cell adhesiv eness, and a decrease in pp125(FAK) tyrosine phosphorylation in the ab sence of insulin (45.2 +/- 9.7% compared to nontransfected Chinese ham ster ovary (CHO) cells). In contrast to pp125(FAK) paxillin phosphoryl ation was similar in all cell Lines despite lower levels (61.0 +/- 10. 4% compared to CHO cells) of paxillin protein in CHO.2N-10 cells. We o bserved comparable protein levels of pp125(FAK) and the structural foc al adhesion protein, vinculin, in all cell Lines. Despite underphospho rylation of pp125(FAK) in the basal state, insulin stimulation of CHO. 2N-10 cells still resulted in dephosphorylation of pp125(FAK). CHO.2N- 10 and CHO.T (overexpress wild-type insulin receptor) cells have simil ar insulin binding characteristics, insulin-stimulated autokinase and peptide phosphorylation, and insulin-stimulated pp185/IRS-1 phosphoryl ation. Our results suggest that the insulin receptor may play an impor tant role in cell-matrix interactions, such as modulating cell adhesio n and inducing cell architecture changes.