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核心作用。我的研究小组的一个重点是了解调控这些细胞中肌动蛋白动态的信号机制。它们导致了血小板的形状变化、扩散和聚集，以及内皮细胞的收缩、迁移、气泡形成和凋亡。在以往研究表明Rho/Rho-Kinase信号通路的激活在血小板形状改变和内皮细胞收缩中起核心作用的基础上，我们最近集中研究了Rho-Kinase介导的Lim-Kinase的激活，它通过肌动蛋白解聚蛋白cofilin的磷酸化来调节肌动蛋白的动力学。我们发现，Rho-Kinase/LIMK/Cofilin通路在凝血酶刺激的血小板和内皮细胞中有不同的调节作用。此外，我们在内皮细胞中观察到，凝血酶诱导了LIMK2细胞中的泡状结构，但不能诱导LIMK1转基因细胞。此外，我们还发现LIMK2(LIMK2)在内皮细胞的胞浆和胞核之间穿梭。我们已经在LIMK2中确定了特定的核和核仁定位序列，以及调控LIMK2核输入的PKC依赖的磷酸化位点(Ser-283)。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途径不仅在细胞质中发挥作用，而且在细胞核中也发挥作用。我们的主要目标是：(A)阐明LIMK1/LIMK2和cofilin磷酸酶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)确定LIMK2在内皮细胞中核质穿梭的调控；以及(C)了解LIMK2和cofilin的核功能；(D)阐明依赖LIMK2的BLOB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.