CELLULAR APPROACHES TO MYOSIN REGULATION

肌球蛋白在许多细胞过程中起着关键作用，包括 胞质分裂、细胞运动和发育调节的变化 细胞形态。网柄原基系统中的重链磷酸化 在控制肌球蛋白在收缩过程中的重新分布中起关键作用 事件，但关于细胞和分子的几乎一无所知 肌球蛋白重链激酶(MHCK)的特性 这些事件。最近开发的基因打靶技术，结合 分子遗传学、细胞生物学和生物化学方法 将允许我们在体外建立MHCK之间的关系 已观察到的活动及其在体内的功能 肌球蛋白定位调控中的蛋白质。最好的候选人之一 对于具有重要生理作用的网柄网柄藻来说，MHCK约为130 kDa 由Cote和Bukiejko提纯的激酶。这种激酶存在于 生长阶段的阿米巴虫，并在生长阶段增加 与活跃的细胞迁移相对应的发育。在体外， Dictyostelialmyosin被该激酶磷酸化后强烈抑制 在生理离子强度下形成细丝。体外靶点 该激酶的位点已被定位到三个苏氨酸残基 肌球蛋白重链的高位部分。在我的博士后工作期间，我使用了 定点突变以证明这三个残基是 关键参与调节肌球蛋白在体内的定位。同舟共济 这些结果表明，130 kDa的MHCK可能在 肌球蛋白在体内募集和组装的调控。具体的 这项建议的目的集中在分子遗传学和细胞生物学上 这个130 kDa的MHCK的特征。这些目标将包括：1) 编码MHCK和MHCK基因的Dictyostoma基因的分离 其核苷酸序列的测定，2)使用克隆的基因 通过基因打靶获得MHCK-细胞系并建立细胞 高表达活性激酶的细胞系，3)对这些细胞系的分析 研究运动、胞质分裂、趋化和发育行为 为了阐明MHCK在体内关于肌球蛋白的功能-- 中介角色和目标可能中介的角色 非肌球蛋白；4)MHCK基因的分子遗传操作 产生突变形式的激酶，目的是解剖 参与其特定方面的蛋白质的功能结构域 活体功能和调节其活动的机制。 这项研究将有助于确定细胞调节机制 肌球蛋白在活动期间的组织和组装成亚细胞结构 如细胞分裂、趋化和细胞分化。洞察 这些事件将对各种重要的 过程，从调节细胞生长和细胞分裂，到 肿瘤细胞的转移特性及其趋化行为 从白细胞到调节分化和发育的机制。

Myosin plays critical roles in many cellular processes, including cytokinesis, cell locomotion, and developmentally regulated changes in cell morphology. In the Dictyostelium system heavy chain phosphorylation plays a key role in controlling myosin redistribution during contractile events, but almost nothing is known regarding the cellular and molecular properties of the myosin heavy chain kinases (MHCKs) that participate in these events. Recently developed gene targeting technologies, combined with molecular genetic, cell biological, and biochemical methodologies will allow us to establish the relationship between in vitro MHCK activities that have been observed and the in vivo functions of these proteins in regulation of myosin localization. One of the best candidates for a Dictyostelium MHCK with important physiological roles is a 130 kDa kinase that was purified by Cote and Bukiejko. This kinase is present in growth phase amoebae, and increases in abundance during the phase of development corresponding to active cell migration. In vitro, phosphorylation of Dictyostelium myosin by this kinase strongly inhibits filament formation at physiological ionic strength. The in vitro target sites for this kinase have been mapped to three threonine residues in the tall portion of the myosin heavy chain. During my postdoctoral work I used site-directed mutagenesis to demonstrate that these three residues are critically involved in regulation of myosin localization in vivo. Together these results suggest that the 130 kDa MHCK may play a critical role in the regulation of myosin recruitment and assembly in vivo. The specific aims of this proposal focus on the molecular genetic and cell biological characterization of this 130 kDa MHCK. These aims will include: 1) isolation of the Dictyostelium gene that encodes this MHCK and determination of its nucleotide sequence, 2) use of the cloned gene to generate MHCK- cell lines via gene targeting, and establishment of cell lines that overexpress the active kinase, 3) analysis of these cells lines addressing motility, cytokinesis, chemotaxis, and developmental behavior to elucidate in vivo functions of the MHCK, with respect to myosin- mediated roles and with respect to possible roles mediated by targets other than myosin, and 4) molecular genetic manipulation of the MHCK gene to produce mutant forms of the kinase, with the goal of dissecting the functional domains of the protein involved in specific aspects of its in vivo function and mechanisms by which its activity is regulated. This research will help identify the cellular mechanisms that regulate myosin organization and assembly into subcellular structures during events such as cell division, chemotaxis, and cell differentiation. Insights into these events will have fundamental implications for a variety of important processes, ranging from regulation of cell growth and cell division, to metastatic properties of tumor cells and chemotactic behavior of leukocytes, to mechanisms regulating differentiation and development.