Ouwens D M, Mikkers H M, van der Zon G C, Stein-Gerlach M, Ullrich A, Maassen J A
Department of Medical Biochemistry, Sylvius Laboratory, University of Leiden, The Netherlands.
Biochem J. 1996 Sep 1;318 ( Pt 2)(Pt 2):609-14. doi: 10.1042/bj3180609.
Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor beta-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(L-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 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 pp125FAK requires active PTP 1D.
胰岛素刺激成纤维细胞除了能使胰岛素受体β链、胰岛素受体底物1和2以及Shc发生酪氨酸磷酸化外，还能迅速诱导68 kDa和125 kDa蛋白质的酪氨酸去磷酸化。使用特异性抗体，分别将68 kDa和125 kDa蛋白质鉴定为桩蛋白和粘着斑激酶（pp125FAK）。我们研究了桩蛋白和pp125FAK的去磷酸化是否需要细胞与细胞外基质相互作用。为此，将细胞培养在聚（L-赖氨酸）平板上，通过添加溶血磷脂酸增加pp125FAK和桩蛋白的酪氨酸磷酸化。在这些条件下，胰岛素仍能诱导pp125FAK和桩蛋白完全去磷酸化，表明该过程可独立于整合素与细胞外基质蛋白的相互作用而发生。我们还研究了pp125FAK和桩蛋白的去磷酸化是否由磷酸酪氨酸磷酸酶的作用引起。发现磷酸酪氨酸磷酸酶抑制剂苯砷氧化物可阻止胰岛素诱导的pp125FAK和桩蛋白去磷酸化。此外，在表达磷酸酪氨酸磷酸酶1D（PTP 1D）显性负突变体的细胞中，这种胰岛素诱导的去磷酸化也受到损害。因此，我们已确定桩蛋白是胰岛素去磷酸化的靶点。此外，我们已获得证据表明胰岛素介导的桩蛋白和pp125FAK去磷酸化需要活性PTP 1D。
