Analyses of a Novel Signaling Molecule, Cas

新型信号分子 Cas 的分析

基本信息

批准号：
09044271
负责人：
HIRAI Hisamaru
金额：
$ 5.44万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044271/
关键词：
Cas SH3 subcellular localization focal adhesion knockout mouse myofibril シグナル伝達チロシンリン酸化

项目摘要

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

p13OCas 是一种接头蛋白，在底物结构域中具有一个 SH3 结构域，后跟多个 SH2 结合基序。它还包含 C 末端附近的酪氨酸残基和富含脯氨酸的序列，它们是 p13OCas 的 SH2 和 SH3 结构域的结合位点。 Cas 分别被证明在整合素刺激下被诱导酪氨酸磷酸化，我们检查了 Gas 的亚细胞定位并确定了其定位到焦点所需的区域。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 527F-c-Src 转化细胞中，Gas 主要定位于足小体。在表达细胞中观察到 Cas 定位于粘着斑。激酶阴性 527F/295M-c-Src Cas 的 SH3 结构域对于其定位于粘着斑是必需的。未转化的细胞中，SH3 结构域和 Cas 的 C 端 Src 结合结构域在 527F-c-Src-transform 细胞和纤连蛋白刺激的细胞中都是必需的。此外，Gas 在粘着斑中的定位被废除。 Src 阴性细胞。这些结果表明 Cas 的 SH3 结构域以及 Cas 与 Src 激酶的关联在 Gas 定位到粘着斑中发挥着关键作用。在体内，我们产生了缺乏气体的小鼠，它们在子宫内死亡，表现出明显的全身充血和生长迟缓，心脏发育不良，心脏的电子显微镜分析显示肌原纤维紊乱和破坏。此外，Cas 缺陷的原代成纤维细胞中肌动蛋白应力纤维的形成严重受损，而且 Cas 缺陷的原代成纤维细胞中活化的 Src 的表达也没有受到影响。诱导完全转化的表型，可能是由于足小体中肌动蛋白细胞骨架的积累不足。这些发现明确了心血管发育、肌动蛋白丝组装和 Src 诱导的转化中的气体功能。