Regulation and function of LIM-kinases and cofilin in platelets and endothelial cells

血小板和内皮细胞在动脉粥样硬化和动脉粥样硬化血栓形成的发病机制中起着重要作用。我的研究小组的一个重点是了解调节这些细胞中肌动蛋白动力学的信号机制。它们是血小板形态改变、伸展和聚集、内皮细胞收缩、迁移、滤过泡形成和凋亡的基础。基于我们以前的研究表明Rho/Rho激酶信号通路的激活在血小板形状改变和内皮细胞收缩中起中心作用，我们最近集中研究Rho激酶介导的LIM激酶的激活，其通过肌动蛋白解聚蛋白cofilin的磷酸化调节肌动蛋白动力学。我们发现Rho激酶/LIMK/cofilin通路在凝血酶刺激的血小板和内皮细胞中受到不同的调节。此外，我们观察到在内皮细胞中，凝血酶在LIMK 2转染的细胞中诱导了水泡样结构，而在LIMK 1转染的细胞中没有。此外，我们发现LIMK 2穿梭于内皮细胞的细胞质和细胞核之间。我们已经在LIMK 2中鉴定了特异的核和核仁定位序列，以及一个PKC依赖的磷酸化位点（Ser-283），它调节LIMK 2的核输入。由于肌动蛋白、cofilin和其他细胞骨架蛋白也存在于细胞核中，我们认为LIMK/cofilin通路不仅在细胞质中起作用，而且在细胞核中也起作用。我们的主要目标是：（a）阐明LIMK 1/LIMK 2和cofilin磷酸酶弹弓在调节受刺激的血小板和内皮细胞中cofilin磷酸化和肌动蛋白中的作用;（B）明确LIMK 2在内皮细胞中对核质穿梭的调节;（c）了解LIMK 2和cofilin的核功能;（d）阐明LIMK 2依赖性bleB形成的机制和生理作用。

Platelets and endothelial cells play a central role in the pathogenesis of atherosclerosis andatherothrombosis. One focus of my research group is to understand the signalling mechanismswhich regulate the actin dynamics in these cells. They underlie shape change, spreading andaggregation of platelets, and contraction, migration, bleb formation and apoptosis of endothelialcells. Based on own previous studies which showed that activation of the Rho/Rho-Kinasesignalling pathway plays a central role for platelet shape change and endothelial cell contraction, weconcentrated our studies recently on Rho-kinase mediated activation of LIM-kinases, whichregulate actin dynamics through phosphorylation of the actin-depolymerising protein cofilin. Wefound that the Rho-kinase/LIMK/cofilin pathway is differentially regulated in thrombin-stimulatedplatelets and endothelial cells. Additionally we observed in endothelial cells, that thrombin inducedbleb like structures in LIMK2 but not LIMK1 transfected cells. Furthermore, we found that LIMkinase2 (LIMK2) shuttles between the cytoplasm and nucleus of endothelial cells. We haveidentified in LIMK2 specific nuclear and nucleolar localization sequences, and a PKC-dependentphosphorylation site (Ser-283), which regulate the nuclear import of LIMK2. Since actin, cofilin, andother cytoskeleton proteins are also known to be present in the nucleus, we propose that theLIMK/cofilin pathway functions not only in the cytoplasm, but also in the nucleus. Our main goalsare: (a) to clarify the role of LIMK1/LIMK2 and the cofilin phosphatase slingshot for the regulation ofcofilin phosphorylation and actin in stimulated platelets and endothelial cells; (b) to define theregulation of nucleocytoplasmic shuttling of LIMK2 in endothelial cells, and (c) to understand thenuclear function of LIMK2 and cofilin; (d) to clarify the mechanism and physiological role of LIMK2-dependent bleb formation.