Analyses of a Novel Signaling Molecule, Cas

p13 OCas是一种衔接蛋白，其具有SH 3结构域，随后在底物结构域中具有多个SH 2结合基序。它还含有一个酪氨酸残基和一个富含脯氨酸的序列的C端附近，这是结合位点的SH 2和SH 3结构域的Src激酶，分别。Cas在整合素刺激后被诱导酪氨酸磷酸化。我们研究了气体的亚细胞定位，并确定其本地化所需的区域，以局灶性粘连。在非转化细胞中，Cas主要定位于细胞质，部分定位于粘着斑。然而，在527 F-c-Src转化的细胞中，Gas主要定位于podosomes。在表达激酶阴性527 F/295 M-c-Src的细胞中观察到Cas定位于粘着斑。Cas的SH 3结构域对于其在非转化细胞中定位于粘着斑是必需的，而Cas的SH 3结构域和C末端Src结合结构域两者在527 F-c-Src-Cas中都是必需的。 ...更多信息 RMED细胞和纤连蛋白刺激的细胞。此外，在Src阴性细胞中，Gas局部粘连被消除。这些结果表明Cas的SH 3结构域和Cas与Src激酶的结合在Gas定位于粘着斑中起关键作用。为了确定其在体内的作用，我们产生了缺乏Gas的小鼠。缺乏气体的胚胎在子宫内死亡，表现出明显的全身充血和生长迟缓。组织学上，心脏发育不良，血管明显扩张。心脏的电子显微镜分析显示肌原纤维的解体和Z盘的破坏。此外，肌动蛋白应力纤维的形成严重受损的Cas缺陷的原代成纤维细胞。此外，表达激活的Src在Cas缺陷的原代成纤维细胞没有诱导一个完全转化的表型，可能是由于不足的积累肌动蛋白细胞骨架的podosomes。这些发现已经定义了Gas在心血管发育、肌动蛋白丝组装和Src诱导的转化中的功能。少

p13OCas is an adapter protein that has an SH3 domain followed by multiple SH2 binding motifs in the substrate domain. It also contains a tyrosine residue and a proline-rich sequence near the C terminus, which are the binding sites for the SH2 and SH3 domains of Src kinase, respectively. Cas was shown to be inducibly tyrosine phosphorylated upon integrin stimulation. We examined the subcellular localization of Gas and determined the regions required for its localization to focal adhesions. In nontransformed cells, Cas was localized predominantly to the cytoplasm and partially to focal adhesions. However, in 527F-c-Src-transformed cells, Gas was localized mainly to podosomes. The localization of Cas to focal adhesions was observed in cells expressing the kinase-negative 527F/295M-c-Src. The SH3 domain of Cas is necessary for its localization to focal adhesions in nontransformed cells while both the SH3 domain and the C-terminal Src binding domain of Cas are required in 527F-c-Src-transfo … More rmed cells and fibronectin-stimulated cells. In addition, the localization of Gas to focal adhesions was abolished in Src-negative cells. These results demonstrate that the SH3 domain of Cas and the association of Cas with Src kinase play a pivotal role in the localization of Gas to focal adhesions. To determine its role in vivo, we generated mice lacking Gas. Gas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Gas function in cardiovascular development, actin filament assembly and Src-induced transformation. Less