Cellular and Biochemical Studies of Myosin Assembly in Dictyostelium

描述（由申请人提供）：Myosin II在发育过程中细胞分裂、细胞迁移和细胞形状变化中起着重要作用。在所有这些情况下，肌凝蛋白进入细胞骨架的动态局部组装对其细胞收缩作用至关重要。尽管空间和时间调节组装的重要性，但控制肌球蛋白II的局部组装和拆卸的信号机制在任何系统中都不清楚。我们正在使用简单的变形虫盘基骨变形虫作为模型系统来识别调节肌球蛋白组装的信号通路。这种简单的变形虫表现出细胞运动性、趋化性和第二信使信号的形式，类似于中性粒细胞或巨噬细胞等运动哺乳动物细胞所表现出的形式。在这个系统中，肌凝蛋白II的组装是由位于肌凝蛋白尾部尖端附近的一组苏氨酸残基的磷酸化/去磷酸化调控的。我们之前已经在这个系统中发现了一组新的肌凝蛋白重链激酶（mhck），它们通过磷酸化肌凝蛋白尾部的标靶位点参与肌凝蛋白组装的体内控制。这些酶现在被认为是一个高度新颖的蛋白激酶家族的原型，该家族存在于盘基ostelium和整个动物王国中，被称为“α激酶”。最近，1哺乳动物α激酶（TRPM7）参与了氧化应激反应中凋亡通路的激活，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方法已经在盘基钢菌中发现了这个简单的模式生物共有6个α激酶。至少有两种盘基骨柱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。在本提案中，我们将：(1)使用

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.