Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling
Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling
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DOI：
10.1111/j.1742-4658.2006.05112.x
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发表时间：
2006-02-01
期刊：
影响因子：
5.4
通讯作者：
Suh, PG
中科院分区：
文献类型：
--
作者：
Kim, E;Lee, S;Suh, PG
穹窿是高度保守的，普遍存在的核糖核蛋白（RNP）粒子，具有未知的功能。对于三种蛋白质种类（TEP 1、VPARP和MVP）和构成穹窿的小RNA，独特的100-kDa主要穹窿蛋白（MVP）的表达足以形成基本穹窿结构。为了鉴定和表征与Src的Src同源性2（SH 2）结构域相互作用并可能调节Src活性的蛋白质，我们使用了使用GST-Src-SH 2融合蛋白的下拉测定。我们在人胃组织中发现MVP作为Src-SH 2结合蛋白。在253 J胃癌细胞中也观察到Src和MVP的相互作用。使用免疫荧光显微镜的亚细胞定位研究表明，表皮生长因子（EGF）的刺激触发MVP易位从细胞核的胞质溶胶和核周区域，它与Src共定位。我们发现，Src和MVP之间的相互作用是严重依赖于Src活性和蛋白质（MVP）酪氨酰磷酸化，这是由EGF刺激诱导。我们的研究结果还表明MVP是Src的一种新底物，并且以EGF依赖的方式磷酸化。有趣的是，纯化的MVP抑制Src在体外酪氨酸激酶活性的浓度依赖性的方式。MVP过表达下调Src过表达细胞中EGF依赖的ERK激活据我们所知，这是MVP与参与不同细胞信号通路的蛋白酪氨酸激酶相互作用的第一份报告。看来MVP是Src介导的信号级联的新调节剂。
Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST-Src-SH2 fusion proteins. We found MVP as a Src-SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner. MVP overexpression downregulates EGF-dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src-mediated signalling cascades.