Altun-Gultekin Z F, Chandriani S, Bougeret C, Ishizaki T, Narumiya S, de Graaf P, Van Bergen en Henegouwen P, Hanafusa H, Wagner J A, Birge R B
Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York, USA.
Mol Cell Biol. 1998 May;18(5):3044-58. doi: 10.1128/MCB.18.5.3044.
The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha3-integrin, and a higher-molecular-weight form of beta1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.
小GTP酶RhoA在由血清衍生因子（如溶血磷脂酸，LPA）激活的信号通路中发挥关键作用，包括在成纤维细胞中形成应力纤维以及在神经元细胞中引起神经突回缩和胞体变圆。此前，我们已经表明，在PC12细胞中异位表达v-Crk（一种含有SH2/SH3结构域的衔接蛋白），可增强神经生长因子（NGF）诱导的神经突生长，并在撤除NGF时促进细胞存活。在本研究中我们发现，当在含15%血清或含溶血磷脂酸的培养基中培养时，大多数表达v-Crk的PC12细胞（v-Crk PC12细胞）呈现扁平表型，具有宽阔的片状伪足，并且对NGF诱导的神经突生长具有抗性，除非撤除血清。用C3毒素处理细胞或Crk的SH2或SH3结构域发生突变，均可抑制v-Crk介导的细胞扁平化。用p160ROCK（Rho相关的含卷曲螺旋结构域的激酶）和v-Crk的表达质粒对293T细胞进行瞬时共转染，但不是v-Crk的SH2或SH3突变体，会导致p160ROCK的过度激活。此外，与表达突变型v-Crk的细胞或野生型细胞相比，v-Crk PC12细胞中磷脂酰肌醇-4,5-二磷酸的水平升高，这与PI(4)P5激酶是Rho的下游靶点一致。在PC12细胞中表达v-Crk不会导致Rac或Cdc42依赖性激酶PAK和S6激酶的激活，表明其对Rho具有特异性。与未观察到粘着斑和肌动蛋白应力纤维的天然PC12细胞不同，对v-Crk PC12细胞进行免疫组织化学分析发现，粘着斑复合物在细胞周边形成，并与肌动蛋白束相连。粘着斑的形成与粘着斑蛋白FAK、桩蛋白、α3整合素以及高分子量形式的β1整合素的表达上调相关。我们的结果表明，v-Crk激活Rho信号通路，并在PC12细胞粘着斑组装过程中作为一种支架蛋白发挥作用。
