Cellular and Biochemical Studies of Myosin Assembly in Dictyostelium

描述（由申请方提供）：肌球蛋白II在发育过程中的胞质分裂、细胞迁移和细胞形状变化中发挥重要作用。在所有这些设置中，它是公认的，肌球蛋白到细胞骨架的动态局部组装是至关重要的细胞收缩作用。尽管空间和时间调节组装的重要性，控制肌球蛋白II的局部组装和拆卸的信号转导机制在任何系统中都不清楚。我们正在使用简单的阿米巴Dictyosteelium discoideum作为一个模型系统，以确定调节肌球蛋白组装的信号通路。这种简单的变形虫表现出与活动的哺乳动物细胞（如中性粒细胞或巨噬细胞）相似的细胞运动性、趋化性和第二信使信号。肌球蛋白II在这个系统中的组装是由一组映射的苏氨酸残基，位于肌球蛋白尾部的尖端附近的磷酸化/去磷酸化调节。 我们以前已经确定了一组新的肌球蛋白重链激酶（MHCKs）在这个系统中，参与在体内控制肌球蛋白组装通过磷酸化的映射靶位点的肌球蛋白尾巴。这些酶现在被认为是存在于网骨藻和整个动物王国中的高度新颖的蛋白激酶家族的原型，称为“α激酶”。最近，1哺乳动物α激酶（TRPM 7）已被牵连在细胞凋亡途径激活响应氧化应激，但其他哺乳动物α激酶的作用在很大程度上是未知的。我们的分子和基因组的方法在网骨藻揭示了6 α激酶在这个简单的模式生物。至少有2种Dictyosteoprotein α激酶似乎不是MHC激酶，但可能在细胞中起其他作用。我们提出了一系列互补的方法来深入了解所有这些酶的作用。在本建议中，我们将：（1）使用 生物化学/细胞分析以阐明自磷酸化在MHCK活化中的作用，（2）进行研究以测试我们在早期工作中以生物化学方式鉴定的MHC磷酸酶的细胞作用，（3）对VwkA和AK 1进行进一步的细胞/生物化学分析，这两种剩余的网骨藻α激酶的细胞作用还不清楚，和（4）进行遗传筛选以鉴定在从细胞分裂到渗透保护反应的设置中涉及肌球蛋白II相关细胞功能的新基因。我们的研究将为了解哺乳动物系统中肌球蛋白II的调节提供直接相关的重要基础。我们提出的研究，解决完整的网骨藻α激酶也将提供一个强大的平台，比较和洞察α激酶在哺乳动物系统中的可能作用。

DESCRIPTION (provided by applicant): Myosin II plays fundamental roles in cytokinesis, cell migration, and cell shape changes during development. In all these settings, it is well established that dynamic localized assembly of myosin into the cytoskeleton is critical for its cellular contractile roles. Despite the importance of spatially and temporally regulated assembly, the signaling mechanisms that control localized assembly and disassembly of myosin II are not understood in any system. We are using the simple amoeba Dictyostelium discoideum as a model system for identifying signaling pathways that regulate myosin assembly. This simple amoeba displays forms of cellular motility, chemotaxis, and second messenger signaling similar to those displayed by motile mammalian cells such as neutrophils or macrophages. Myosin II assembly in this system is regulated by phosphorylation/dephosphorylation of a set of mapped threonine residues that lie near the tip of the myosin tail. We have previously identified a set of novel myosin heavy chain kinases (MHCKs) in this system, that participate in the in vivo control of myosin assembly via phosphorylation of the mapped target sites in the myosin tail. These enzymes are now recognized as the prototype for a highly novel family of protein kinases present in Dictyostelium and throughout the animal kingdom known as "Alpha kinases". Recently, 1 mammalian alpha kinase (TRPM7) has been implicated in apoptotic pathway activation in response to oxidative stress, but roles of other mammalian alpha kinases are largely unknown. Our molecular and genomic approaches in Dictyostelium have revealed a total of 6 alpha kinases in this simple model organism. At least 2 of the Dictyostelium alpha kinases appear NOT to be MHC kinases, but likely serve other roles in the cell. We propose a series of complementary approaches to gain insights into the roles of all of these enzymes. In this proposal we will: (1) use biochemistry/cellular analysis to elucidate the role of autophosphorylation in MHCK activation, (2) perform studies to test the cellular role of an MHC phosphatase that we identified biochemically in earlier work, (3) perform further cellular/biochemical analysis of VwkA and AK1, 2 remaining Dictyostelium alpha kinases for which cellular roles are not well understood, and (4) perform genetic screens to identify new genes involved in myosin II-related cellular functions in settings ranging from cell division to osmotic protective responses. Our studies will provide an important foundation of direct relevance for understanding myosin II regulation in mammalian systems. The studies we propose addressing the full complement of Dictyostelium alpha kinases will also provide a powerful platform for comparisons and insights into possible roles of alpha kinases in mammalian systems.