Fincham V J, Chudleigh A, Frame M C
The Beatson Institute for Cancer Research, CRC Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK.
J Cell Sci. 1999 Mar;112 ( Pt 6):947-56. doi: 10.1242/jcs.112.6.947.
The v-Src oncoprotein perturbs the dynamic regulation of the cellular cytoskeletal and adhesion network by a mechanism that is poorly understood. Here, we have examined in detail the effects of a temperature-dependent v-Src protein on the regulation of p190 RhoGAP, a GTPase activating protein (GAP) that has been implicated in disruption of the organised actin cytoskeleton, and addressed the dependence of v-Src-induced stress fibre loss on inhibition of Rho activity. We found that activation of v-Src induced association of tyrosine phosphorylated p190 with p120(RasGAP) and stimulation of p120(RasGAP)-associated RhoGAP activity, although p120(RasGAP) itself was not a target for phosphorylation by v-Src in chicken embryo cells. These events required the catalytic activity of v-Src and were linked to loss of actin stress fibres during morphological transformation and not mitogenic signalling. Furthermore, these effects were rapidly reversible since switching off v-Src led to dissociation of the p190/p120(RasGAP) complex, inactivation of p120(RasGAP)-associated RhoGAP activity and re-induction of actin stress fibres. In addition, transient transfection of Val14-RhoA, a constitutively active Rho protein that is insensitive to RhoGAPs, suppressed v-Src-induced stress fibre loss and cell transformation. Thus, we show here for the first time that an activated Src kinase requires the inactivation of Rho-mediated actin stress fibre assembly to induce its effects on actin disorganisation. Moreover, our work supports p190 as a strong candidate effector of v-Src-induced cytoskeletal disruption, most likely mediated by antagonism of the cellular function of Rho.
v-Src癌蛋白通过一种尚不清楚的机制扰乱细胞细胞骨架和粘附网络的动态调节。在这里，我们详细研究了温度依赖性v-Src蛋白对p190 RhoGAP调节的影响，p190 RhoGAP是一种GTP酶激活蛋白（GAP），与有组织的肌动蛋白细胞骨架的破坏有关，并探讨了v-Src诱导的应力纤维丧失对Rho活性抑制的依赖性。我们发现v-Src的激活诱导酪氨酸磷酸化的p190与p120（RasGAP）结合，并刺激与p120（RasGAP）相关的RhoGAP活性，尽管在鸡胚细胞中p120（RasGAP）本身不是v-Src磷酸化的靶点。这些事件需要v-Src的催化活性，并与形态转化过程中肌动蛋白应力纤维的丧失有关，而与促有丝分裂信号无关。此外，这些效应是快速可逆的，因为关闭v-Src会导致p190/p120（RasGAP）复合物解离、与p120（RasGAP）相关的RhoGAP活性失活以及肌动蛋白应力纤维的重新诱导。此外，组成型活性Rho蛋白Val14-RhoA（对RhoGAP不敏感）的瞬时转染抑制了v-Src诱导的应力纤维丧失和细胞转化。因此，我们首次表明，活化的Src激酶需要使Rho介导的肌动蛋白应力纤维组装失活，才能诱导其对肌动蛋白解聚的作用。此外，我们的工作支持p190作为v-Src诱导的细胞骨架破坏的有力候选效应物，最有可能是通过拮抗Rho的细胞功能介导的。
