Baron V, Calléja V, Ferrari P, Alengrin F, Van Obberghen E
INSERM, U145, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cédex 2, France.
J Biol Chem. 1998 Mar 20;273(12):7162-8. doi: 10.1074/jbc.273.12.7162.
The focal adhesion kinase p125(Fak) is a widely expressed cytosolic tyrosine kinase, which is involved in integrin signaling and in signal transduction of a number of growth factors. In contrast to tyrosine kinase receptors such as the platelet-derived growth factor and the hepatocyte growth factor receptors, which induce p125(Fak) phosphorylation, insulin has been shown to promote its dephosphorylation. In this study, we compared p125(Fak) phosphorylation in insulin-stimulated cells maintained in suspension or in an adhesion state. We found that, in nonattached cells, insulin promotes p125(Fak) phosphorylation, whereas dephosphorylation occurred in attached cells. This was observed in Rat-1 fibroblasts overexpressing the insulin receptor, as well as in Hep G2 hepatocytes and in 3T3-L1 adipocytes expressing more natural levels of insulin receptors. Insulin-induced p125(Fak) phosphorylation correlated with an increase in paxillin phosphorylation, indicating that p125(Fak) kinase activity may be stimulated by insulin. Mixing of purified insulin or insulin-like growth factor-I (IGF-I) receptors with p125(Fak) resulted in an increase in p125(Fak) phosphorylation. Using a kinase-deficient p125(Fak) mutant, we found that this protein is a direct substrate of the insulin and IGF-I receptor tyrosine kinases. This view is supported by two additional findings. (i) A peptide corresponding to p125(Fak) sequence comprising amino acids 568-582, which contains tyrosines 576 and 577 of the kinase domain regulatory loop, is phosphorylated by the insulin receptor; and (ii) p125(Fak) phosphorylation by the insulin receptor is prevented by addition of this peptide. Finally, we observed that p125(Fak) phosphorylation by the receptor results in its activation. Our results show that the nature of the cross-talk between the insulin/IGF-I receptors and p125(Fak) is dependent on the cell architecture, and hence the interaction of the insulin/IGF-I signaling system with the integrin system will vary accordingly.
粘着斑激酶p125（Fak）是一种广泛表达的胞质酪氨酸激酶，参与整合素信号传导以及多种生长因子的信号转导。与诸如血小板衍生生长因子和肝细胞生长因子受体等能诱导p125（Fak）磷酸化的酪氨酸激酶受体不同，胰岛素已被证明可促进其去磷酸化。在本研究中，我们比较了悬浮状态或粘附状态下胰岛素刺激细胞中p125（Fak）的磷酸化情况。我们发现，在未附着细胞中，胰岛素促进p125（Fak）磷酸化，而在附着细胞中则发生去磷酸化。在过表达胰岛素受体的大鼠-1成纤维细胞以及表达更接近天然水平胰岛素受体的Hep G2肝细胞和3T3-L1脂肪细胞中均观察到这种现象。胰岛素诱导的p125（Fak）磷酸化与桩蛋白磷酸化增加相关，表明p125（Fak）激酶活性可能受到胰岛素的刺激。纯化的胰岛素或胰岛素样生长因子-I（IGF-I）受体与p125（Fak）混合导致p125（Fak）磷酸化增加。使用激酶缺陷型p125（Fak）突变体，我们发现该蛋白是胰岛素和IGF-I受体酪氨酸激酶的直接底物。另外两个发现支持了这一观点。（i）包含激酶结构域调节环酪氨酸576和577的对应于p125（Fak）序列568-582位氨基酸的肽段被胰岛素受体磷酸化；（ii）添加该肽段可阻止胰岛素受体对p125（Fak）的磷酸化。最后，我们观察到受体对p125（Fak）的磷酸化导致其激活。我们的结果表明，胰岛素/IGF-I受体与p125（Fak）之间相互作用的性质取决于细胞结构，因此胰岛素/IGF-I信号系统与整合素系统的相互作用也将相应地有所不同。
