Lin T H, Aplin A E, Shen Y, Chen Q, Schaller M, Romer L, Aukhil I, Juliano R L
Department of Pharmacology, University of North Carolina, Chapel Hill 27599, USA.
J Cell Biol. 1997 Mar 24;136(6):1385-95. doi: 10.1083/jcb.136.6.1385.
Integrin-mediated cell adhesion causes activation of MAP kinases and increased tyrosine phosphorylation of focal adhesion kinase (FAK). Autophosphorylation of FAK leads to the binding of SH2-domain proteins including Src-family kinases and the Grb2-Sos complex. Since Grb2-Sos is a key regulator of the Ras signal transduction pathway, one plausible hypothesis has been that integrin-mediated tyrosine phosphorylation of FAK leads to activation of the Ras cascade and ultimately to mitogen activated protein (MAP) kinase activation. Thus, in this scenario FAK would serve as an upstream regulator of MAP kinase activity. However, in this report we present several lines of evidence showing that integrin-mediated MAP kinase activity in fibroblasts is independent of FAK. First, a beta1 integrin subunit deletion mutant affecting the putative FAK binding site supports activation of MAP kinase in adhering fibroblasts but not tyrosine phosphorylation of FAK. Second, fibroblast adhesion to bacterially expressed fragments of fibronectin demonstrates that robust activation of MAP kinase can precede tyrosine phosphorylation of FAK. Finally, we have used FRNK, the noncatalytic COOH-terminal domain of FAK, as a dominant negative inhibitor of FAK autophosphorylation and of tyrosine phosphorylation of focal contacts. Using retroviral infection, we demonstrate that levels of FRNK expression sufficient to completely block FAK tyrosine phosphorylation were without effect on integrin-mediated activation of MAP kinase. These results strongly suggest that integrin-mediated activation of MAP kinase is independent of FAK and indicate the probable existence of at least two distinct integrin signaling pathways in fibroblasts.
整合素介导的细胞黏附导致丝裂原活化蛋白激酶（MAP激酶）的激活以及粘着斑激酶（FAK）酪氨酸磷酸化增加。FAK的自身磷酸化导致包括Src家族激酶和Grb2 - Sos复合物在内的SH2结构域蛋白的结合。由于Grb2 - Sos是Ras信号转导途径的关键调节因子，一个合理的假说是，整合素介导的FAK酪氨酸磷酸化导致Ras级联反应的激活，并最终导致丝裂原活化蛋白（MAP）激酶的激活。因此，在这种情况下，FAK将作为MAP激酶活性的上游调节因子。然而，在本报告中，我们提供了几条证据表明，成纤维细胞中整合素介导的MAP激酶活性独立于FAK。首先，影响假定的FAK结合位点的β1整合素亚基缺失突变体支持黏附的成纤维细胞中MAP激酶的激活，但不支持FAK的酪氨酸磷酸化。其次，成纤维细胞与细菌表达的纤连蛋白片段的黏附表明，MAP激酶的强烈激活可以先于FAK的酪氨酸磷酸化。最后，我们使用了FAK的非催化性COOH末端结构域FRNK，作为FAK自身磷酸化和粘着斑酪氨酸磷酸化的显性负性抑制剂。通过逆转录病毒感染，我们证明足以完全阻断FAK酪氨酸磷酸化的FRNK表达水平对整合素介导的MAP激酶激活没有影响。这些结果强烈表明，整合素介导的MAP激酶激活独立于FAK，并表明成纤维细胞中可能存在至少两条不同的整合素信号通路。
