INSULIN-INDUCED TYROSINE DEPHOSPHORYLATION OF PAXILLIN AND FOCAL ADHESION KINASE REQUIRES ACTIVE PHOSPHOTYROSINE PHOSPHATASE 1D

Insulin stimulation of fibroblasts rapidly induces the tyrosine dephos phorylation of proteins of 68 kDa and 125 kDa, in addition to the tyro sine phosphorylation of the insulin receptor beta-chain, insulin recep tor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion k inase (pp125(FAK)) respectively. We have examined whether dephosphoryl ation of paxillin and pp125(FAK) requires interaction of the cells wit h the extracellular matrix. For this, cells were grown on poly(L-lysin e) plates, and the tyrosine phosphorylation of pp125(FAK) and paxillin was increased by addition of lysophosphatidic acid. Under these condi tions, insulin still induced the complete dephosphorylation of pp125(F AK) and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. W e also studied whether dephosphorylation of pp125(FAK) and paxillin re sults from the action of a phosphotyrosine phosphatase. It was found t hat phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, preve nted the insulin-induced dephosphorylation of pp125(FAK) and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine pho sphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125(FAK) requires active PTP 1D.