Patwardhan Parag, Shen Yongquan, Goldberg Gary S, Miller W Todd
Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook, New York 11794.
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084.
J Biol Chem. 2006 Jul 28;281(30):20689-20697. doi: 10.1074/jbc.M602311200. Epub 2006 May 17.
Cas is a multidomain signaling protein that resides in focal adhesions. Cas possesses a large central substrate domain containing 15 repeats of the sequence YXXP, which are phosphorylated by Src. The phosphorylation sites are essential for the roles of Cas in cell migration and in regulation of the actin cytoskeleton. We showed previously that Src catalyzes the multisite phosphorylation of Cas via a processive mechanism. In this study, we created mutant forms of Cas to identify the determinants for processive phosphorylation. Mutants containing single or multiple YXXP mutations were phosphorylated processively by Src, suggesting that individual sites are dispensable. The results also suggest that there is no defined order to the Cas phosphorylation events. We also studied the effects of these mutations by reintroducing Cas into Cas-deficient fibroblasts. Mutants lacking some or all YXXP sites augment the ability of Src to promote anchorage-independent growth. On the other hand, deletion of YXXP sites compromises the ability of Cas to promote tumor cell migration.
Cas是一种存在于粘着斑中的多结构域信号蛋白。Cas拥有一个大的中央底物结构域，其中包含15个YXXP序列重复，这些序列被Src磷酸化。磷酸化位点对于Cas在细胞迁移和肌动蛋白细胞骨架调节中的作用至关重要。我们之前表明，Src通过一种持续性机制催化Cas的多位点磷酸化。在本研究中，我们创建了Cas的突变形式以确定持续性磷酸化的决定因素。含有单个或多个YXXP突变的突变体被Src持续性磷酸化，这表明单个位点是可有可无的。结果还表明，Cas磷酸化事件没有明确的顺序。我们还通过将Cas重新引入缺乏Cas的成纤维细胞中来研究这些突变的影响。缺少一些或所有YXXP位点的突变体增强了Src促进不依赖贴壁生长的能力。另一方面，YXXP位点的缺失损害了Cas促进肿瘤细胞迁移的能力。
