Analyses of a Novel Signaling Molecule, Cas

P13OCas是一种具有SH3结构域和底物结构域中多个SH2结合基序的适配蛋白。它还在C末端附近含有一个酪氨酸残基和一个富含Pro的序列，这两个序列分别是Src激酶SH2和SH3结构域的结合部位。在整合素刺激下，CAS被诱导酪氨酸磷酸化。我们研究了GAS的亚细胞定位，并确定了其定位到焦点粘连所需的区域。在未转化的细胞中，Cas主要定位于细胞质，部分定位于灶性粘连。然而，在527F-c-Src转化的细胞中，Gas主要定位于足体。在表达激酶阴性的527F/295M-c-Src的细胞中，观察到Cas定位于局灶性粘连。在未转化的细胞中，CaS的SH3结构域是其与焦点粘连定位所必需的，而在527F-c-Src-Transfo…中，CaS的SH3结构域和C末端的Src结合域都是必需的更多的rmed细胞和纤维连接蛋白刺激的细胞。此外，在Src阴性细胞中，Gas对局灶性粘连的定位被取消。这些结果表明，CaS的SH3结构域和CaS与Src激酶的结合在GAS对局灶性粘连的定位中起着关键作用。为了确定它在体内的作用，我们培育了缺乏气体的小鼠。缺气胚胎在宫内死亡，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，在CaS缺乏的原代成纤维细胞中，肌动蛋白应激纤维的形成严重受损。此外，在CaS缺乏的原代成纤维细胞中表达激活的Src并没有诱导完全转化的表型，这可能是由于足体中肌动蛋白细胞骨架的积累不足所致。这些发现明确了Gas在心血管发育、肌动蛋白细丝组装和Src诱导的转化中的作用。较少

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