Cellular Regulation of a Myosin II Heavy Chain Kinase

描述（由申请人提供）：本提案中描述的研究的主要目的是深入了解在非肌肉细胞背景下调节肌球蛋白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的信号通路知之甚少。在简单的阿米巴Dictyosteelium中的研究已经确定，肌球蛋白II双极丝组装和拆卸的动态过程影响肌球蛋白II在细胞中的时间和空间定位。更具体地，肌球蛋白重链（MHC）的“尾”段中的位点的磷酸化导致细胞骨架相关肌球蛋白丝分解成可溶性的单体细胞质池。MHC激酶A（MHCK A）是网骨藻中通过MHC磷酸化驱动肌球蛋白II丝解聚的关键酶，并且被组织成三个功能结构域：a）N-末端卷曲螺旋区，B）与任何常规蛋白激酶无关的新激酶结构域，和c）在其C-末端的WD-重复结构域。在化学引诱物刺激后，MHCK A迅速募集到细胞皮层，并且优先募集到伪足形成的位点。这种招聘，这是肌动蛋白依赖的，是由MHCK A的卷曲螺旋结构域冥想，并建议发挥重要作用，在调节局部分解肌球蛋白II丝在皮质网站。这里描述的实验将探索MHCK A募集到细胞的肌动蛋白丰富的皮质的机制和功能后果。为此，将解决以下问题：1）MHCK A的卷曲螺旋结构域的哪一部分介导细胞皮质的募集？MHCK A卷曲螺旋结构域截短物将在网骨藻细胞中表达为GFP融合蛋白，然后测试趋化因子刺激的向富含肌动蛋白的皮质的易位。2)卷曲螺旋结构域单独能够直接与肌动蛋白丝相互作用吗？将在肌动蛋白结合的共沉降试验中检测含有整个卷曲螺旋结构域的MHCK A的截短物。3)MHCK A的活性是否受其与肌动蛋白丝相互作用的调节？将进行激酶测定以探索肌动蛋白丝对纯化的MHCK A体外活性的影响。补充实验将检查MHCK A易位到肌动蛋白丰富的结构在调节激酶激活（自磷酸化）中的作用。这些研究的结果将有助于我们了解的机制控制本地化肌球蛋白II组装在非肌肉细胞的背景下。这些信息对于理解调节细胞收缩过程（如胞质分裂和细胞迁移）的分子因子至关重要，并最终有助于我们理解这些过程在表现出不受控制的细胞分裂和转移的癌细胞中如何出错。在另一个方面，这些研究对于理解在免疫系统细胞如中性粒细胞的趋化过程中肌动蛋白-肌球蛋白重组是如何调节的是重要的。

DESCRIPTION (provided by applicant): The broad aim of the studies described in this proposal is to gain 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 ll 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 consequences 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? Kinase assays will be performed to explore the effect of actin filaments on the activity of purified MHCK A in vitro. Complimentary experiments will examine the role of MHCK A translocation to actin-rich structures in regulating the activation (autophosphorylation) 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 i chemotaxis in immune system cells such as neutrophils.