Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
J Mol Cell Cardiol. 2012 Dec;53(6):809-19. doi: 10.1016/j.yjmcc.2012.08.022. Epub 2012 Sep 4.
The ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ-containing scaffolding protein that regulates a variety of physiological functions. In the vasculature, EBP50 promotes neointima formation following arterial injury. In this study the role of EBP50 on vascular smooth muscle cell (VSMC) migration was characterized. The spreading and motility of primary VSMC isolated from EBP50 knockout (KO) mice were significantly reduced compared to wild-type (WT) cells. EBP50-null VSMC had fewer and larger focal adhesions than wild-type cells. Assembly and disassembly of focal adhesion-assessed by live-cell total internal reflection fluorescence imaging-in response to epidermal growth factor (EGF) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with EGF receptor via the PDZ2 domain and with focal adhesion kinase (FAK) via the C-terminal ERM domain. EBP50 promoted the formation of a complex containing both EGF receptor and FAK. Phosphorylation of Tyr-925 of FAK in response to EGF was significantly reduced in KO cell compared to WT cells. The residence time of FAK in focal adhesions-determined by fluorescence recovery after photobleaching-was increased in WT cells. Collectively, these studies indicate that EBP50, by scaffolding EGF receptor and FAK, facilitates activation of FAK, focal adhesion turnover, and migration of VSMC.
埃兹蛋白-根蛋白-膜突蛋白结合磷蛋白 50（EBP50）是一种含有 PDZ 结构域的支架蛋白，调节多种生理功能。在血管中，EBP50 促进动脉损伤后的新生内膜形成。在这项研究中，研究了 EBP50 对血管平滑肌细胞（VSMC）迁移的作用。与野生型（WT）细胞相比，从 EBP50 敲除（KO）小鼠分离的原代 VSMC 的铺展和迁移能力显著降低。EBP50 缺失的 VSMC 比 WT 细胞具有更少且更大的焦点黏附。通过活细胞全内反射荧光成像评估的焦点黏附的组装和拆卸，对表皮生长因子（EGF）的反应在 KO 细胞中显著减少。免疫沉淀实验表明，EBP50 通过 PDZ2 结构域与 EGF 受体相互作用，通过 C 末端 ERM 结构域与粘着斑激酶（FAK）相互作用。EBP50 促进包含 EGF 受体和 FAK 的复合物的形成。与 WT 细胞相比，KO 细胞中 EGF 诱导的 FAK 酪氨酸残基 925 的磷酸化显著减少。通过荧光恢复后光漂白测定的 FAK 在焦点黏附中的停留时间在 WT 细胞中增加。总之，这些研究表明，EBP50 通过支架 EGF 受体和 FAK，促进 FAK 的激活、焦点黏附的转化以及 VSMC 的迁移。
