Cellular Regulation of a Myosin II Heavy Chain Kinase

描述(由申请人提供)： 这项建议中描述的研究的广泛目的是深入了解在非肌肉细胞环境中调节肌球蛋白II细丝功能定位的鲜为人知的过程。关键的细胞事件，如细胞分裂和细胞运动，依赖于肌球蛋白II在细胞内的正确组织和定位；然而，在这些细胞事件中调节肌球蛋白II的信号通路却知之甚少。对简单阿米巴的研究已经证实，肌球蛋白II双极细丝的组装和分解的动态过程会影响肌球蛋白II在细胞中的时空定位。更具体地说，肌球蛋白重链(MHC)“尾部”片段的磷酸化导致细胞骨架相关的肌球蛋白细丝分解成一个可溶的、细胞质的单体池。MHC蛋白激酶A(MHC Kinase A，MHCK A)是一种通过MHC的磷酸化作用来驱动肌球蛋白II细丝分解的关键酶，它由三个功能区组成：a)N端卷曲区域，b)一个与任何传统蛋白激酶无关的新的激动域，c)其C端的WD-重复结构域。MHCK A在化学诱导剂刺激下迅速招募到细胞皮质，并优先招募到伪足形成部位。这种依赖肌动蛋白的募集是由MHCK A的卷曲线圈结构域控制的，并被认为在调节皮质部位肌球蛋白II细丝的局部分解中发挥重要作用。这里描述的实验将探索MHCK A重新聚集到细胞富含肌动蛋白的皮质的机制和功能后果。为此，将解决以下问题：1)MHCK A的卷曲线圈结构域的哪一部分介导向细胞皮质的募集？MHCK A螺旋卷曲结构域截断将以GFP融合蛋白的形式在Dictyostelials细胞中表达，然后测试化学诱导剂刺激的易位到富含肌动蛋白的皮质。2)卷曲线圈结构域是否能够与肌动蛋白细丝直接相互作用？包含整个卷曲卷曲结构域的MHCK A的截断将在共沉淀分析中进行肌动蛋白结合的测试。3)MHCK A的活性是否受其与肌动蛋白细丝相互作用的调节？MHCKA活性的几种分析将被用来确定MHCKA移位到肌动蛋白细丝是否影响该激酶的活性。这些研究的结果将有助于我们理解在非肌肉细胞环境中控制肌球蛋白II局部组装的机制。这些信息对于理解调控细胞收缩过程的分子因素至关重要，如胞质分裂和细胞迁移，并最终有助于我们理解这些过程是如何在表现出不受控制的细胞分裂和转移的癌细胞中出错的。另一方面，这些研究对于理解在中性粒细胞等免疫系统细胞的趋化过程中如何调控肌球蛋白重组很重要。

DESCRIPTION (provided by applicant): The broad aim of the studies described in this proposal is to gain an insight on the poorly understood processes regulating the functional localization of myosin II filaments in a nonmuscle cell context. Critical cellular events, such as cell division and cell locomotion, rely on the correct organization and localization of myosin II within the cell; however, the signaling pathways regulating myosin II during these cellular events are poorly understood. Studies in the simple amoeba Dictyostelium have established that the dynamic process of myosin II bipolar filament assembly and disassembly influences the temporal and spatial localization of myosin II in a cell. More specifically, the phosphorylation of sites in the "tail" segment of myosin heavy chain (MHC) results in the disassembly of cytoskeleton-associated myosin filaments into a soluble, cytoplasmic pool of monomers. MHC Kinase A (MHCK A) is a key enzyme driving myosin II filament disassembly through MHC phosphorylation in Dictyostelium and is organized into three functional domains: a) an N-terminal coiled-coil region, b) a novel kinase domain unrelated to any conventional protein kinases, and c) a WD-repeat domain at its C-terminus. MHCK A is rapidly recruited to the cell cortex upon chemoattractant stimulation, and is preferentially recruited to sites of pseudopod formation. This recruitment, which is actin-dependent, is meditated by the coiled-coil domain of MHCK A and is proposed to play an important role in regulating localized disassembly of myosin II filaments at cortical sites. The experiments described here will explore the mechanism and functional consequence of MHCK A recruitment to the actin-rich cortex of the cell. To this end, the following questions will be addressed: 1) What part of the coiled -coil domain of MHCK A mediates recruitment to the cell cortex? MHCK A coiled-coil domain truncations will be expressed as GFP fusion proteins in Dictyostelium cells and then tested for chemoattractant-stimulated translocation to the actin-rich cortex. 2) Is the coiled-coil domain alone able to interact directly with actin filaments? Truncations of MHCK A containing the entire coiled-coil domain will be tested in co-sedimentation assays for actin-binding. 3) Is the activity of MHCK A regulated by its interaction with actin filaments? Several assays of MHCK A activity will be used to determine if MHCKA translocation to actin filaments affects the activity of the kinase. Results from these studies will contribute to our understanding of the mechanisms controlling localized myosin II assembly in a nonmuscle cell context. This information is critical for understanding the molecular factors regulating cellular contractile processes such as cytokinesis and cell migration and ultimately contributes to our understanding of how these processes go awry in cancer cells exhibiting uncontrolled cell division and metastasis. In another context, these studies are important for understanding how acto-myosin reorganization is regulated during the process of chemotaxis in immune system cells such as neutrophils.