Abl tyrosine kinase phosphorylates nonmuscle Myosin light chain kinase to regulate endothelial barrier function.
Abl tyrosine kinase phosphorylates nonmuscle Myosin light chain kinase to regulate endothelial barrier function.
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DOI：
10.1091/mbc.e09-10-0876
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发表时间：
2010-11-15
影响因子：
3.3
通讯作者：
Garcia JG
中科院分区：
文献类型：
--
作者：
Dudek SM;Chiang ET;Camp SM;Guo Y;Zhao J;Brown ME;Singleton PA;Wang L;Desai A;Arce FT;Lal R;Van Eyk JE;Imam SZ;Garcia JG
本研究通过质谱鉴定了多个新型 c-Abl 介导的 nmMLCK 磷酸化位点，并检查了它们对 nmMLCK 功能和人肺内皮屏障调节的影响。数据表明 Abl 激酶通过 nmMLCK 和肌动蛋白结合蛋白 cortactin 的磷酸化在血管屏障调节中发挥重要作用。非肌肉肌球蛋白轻链激酶 (nmMLCK) 是一种对血管稳态至关重要的多功能细胞骨架蛋白，受到酪氨酸磷酸化的高度调节。我们通过质谱分析鉴定了多个新型 c-Abl 介导的 nmMLCK 磷酸化位点（包括 Y231、Y464、Y556、Y846），并检查了它们对 nmMLCK 功能和人肺内皮细胞 (EC) 屏障调节的影响。 nmMLCK 的酪氨酸磷酸化增加了激酶活性，逆转了 nmMLCK 介导的 Arp2/3 介导的肌动蛋白聚合抑制，并增强了与关键肌动蛋白结合磷酸酪氨酸蛋白 Cortactin 的结合。使用 1-磷酸鞘氨醇 (S1P)（一种有效的屏障增强激动剂）进行 EC 攻击，导致 c-Abl 和磷酸化 nmMLCK 募集到富含小窝蛋白的微结构域中，Abl 激酶活性快速增加，以及 c-Abl 空间靶向促进屏障的皮质肌动蛋白结构。相反，减少 EC (siRNA) 中的 c-Abl 表达显着减弱 S1P 介导的皮质肌动蛋白形成，降低 EC 弹性模量（通过原子力显微镜评估），减少 nmMLCK 和皮质蛋白酪氨酸磷酸化，并减弱 S1P 介导的屏障增强。这些研究表明 Abl 激酶通过 nmMLCK 的翻译后修饰在血管屏障调节中发挥重要作用，并强烈支持 c-Abl-cortactin-nmMLCK 相互作用作为基于皮质肌动蛋白的细胞骨架重排的新决定因素，这对 S1P 介导的 EC 屏障增强至关重要。
This study identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy and examined their influence on nmMLCK function and human lung endothelial barrier regulation. The data indicate an essential role for Abl kinase in vascular barrier regulation via phosphorylation of nmMLCK and the actin-binding protein cortactin. Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.