p130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading
p130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading
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DOI：
10.1091/mbc.e03-09-0689
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发表时间：
2004-02-01
影响因子：
3.3
通讯作者：
Defilippi, P
中科院分区：
文献类型：
--
作者：
Di Stefano, P;Cabodi, S;Defilippi, P
整合素介导的细胞粘附刺激控制细胞增殖、迁移和存活的信号传导途径的级联，主要通过信号传导分子的酪氨酸磷酸化。p130 Cas最初被鉴定为v-Src的主要底物，是与几种蛋白质相互作用的支架分子，并且在细胞粘附和有丝分裂原处理后介导多种细胞事件。在这里，我们描述了一种新的p130 Cas-associated蛋白命名为p140 Cap Cas-associated蛋白）作为一种新的酪氨酸磷酸化分子参与整合素和表皮生长因子（EGO依赖的信号。通过亲和层析的人ECV 304细胞提取物上的MBP-p130 Cas柱，然后质谱基质辅助激光解吸电离/飞行时间分析，我们确定p140帽作为蛋白迁移在140 kDa。我们检测了它在人、小鼠和大鼠细胞以及不同小鼠组织中的表达。内源性和转染的p140 Cap蛋白在ECV 304和人胚肾293细胞中与p130 Cas共免疫沉淀，并通过其羧基末端区域与p130 Cas结合。通过免疫荧光分析，我们证明，在ECV 304细胞铺在纤连蛋白，内源性p140帽共定位与p130 Cas在核周区域以及在板状伪足。此外，p140 Cap与皮质肌动蛋白和肌动蛋白应力纤维共分布，但不与局灶性粘连共分布。我们还表明，p140 Cap是酪氨酸磷酸化的细胞粘附整合素配体在15分钟内。p140 Cap酪氨酸磷酸化也通过EGF受体依赖性机制响应于EGF而被诱导。有趣的是，p140 Cap在NIH 3 T3和ECV 304细胞中的表达延迟了细胞粘附到纤连蛋白的早期阶段中细胞铺展的开始。因此，p140 Cap是一种与p130 Cas和肌动蛋白细胞骨架结构相关的新蛋白。它的酪氨酸磷酸化的整合素介导的粘附和EGF刺激，并参与细胞铺展基质蛋白表明，p140帽起着控制肌动蛋白细胞骨架组织响应粘附和生长因子信号的作用。
Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGO-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coirnmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.