Cellular and Biochemical Studies of Myosin Assembly in Dictyostelium

描述(由申请人提供)：肌球蛋白II在细胞质分裂、细胞迁移和发育过程中的细胞形状变化中发挥重要作用。在所有这些情况下，肌球蛋白在细胞骨架中的动态局部组装对其细胞收缩作用是至关重要的。尽管空间和时间调控的组装很重要，但控制肌球蛋白II局部组装和分解的信号机制在任何系统中都不清楚。我们正在使用简单的盘状网囊阿米巴作为一个模型系统来识别调节肌球蛋白组装的信号通路。这种简单的阿米巴显示了多种形式的细胞运动、趋化和第二信使信号，类似于中性粒细胞或巨噬细胞等运动的哺乳动物细胞。肌球蛋白II在这个系统中的组装是由位于肌球蛋白尾端附近的一组定位的苏氨酸残基的磷酸化/去磷酸化来调节的。我们之前已经在这个系统中发现了一组新的肌球蛋白重链激酶(MHCKs)，它们通过肌球蛋白尾部定位的靶点的磷酸化参与肌球蛋白组装的体内控制。这些酶现在被认为是一个非常新颖的蛋白激酶家族的原型，存在于Dictyostelials和整个动物界，被称为“Alpha Kinase”。最近，1-哺乳动物α-激酶(TRPM7)参与了氧化应激诱导的细胞凋亡途径的激活，但其他哺乳动物-α-激酶的作用尚不清楚。我们的分子和基因组方法已经揭示了在这个简单的模式生物中总共有6个α-激酶。至少有2个DictyostelialalKinase似乎不是MHC激酶，但可能在细胞中发挥其他作用。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 这些研究包括：(1)进行生物化学/细胞分析以阐明自动磷酸化在MHCK激活中的作用；(2)进行研究以测试我们在早期工作中用生物化学方法确定的MHC磷酸酶的细胞作用，(3)进一步进行细胞/生化分析，以确定VwkA和AK1，以及(2)对其余两种尚不清楚细胞角色的网柄菌α激酶进行分析，以及(4)进行遗传筛选，以确定在从细胞分裂到渗透保护反应等各种环境中与肌球蛋白II相关的细胞功能有关的新基因。我们的研究将为了解肌球蛋白II在哺乳动物系统中的调控提供直接相关的重要基础。我们建议进行的研究将提供一个强大的平台，用于比较和深入了解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.