Cellular and Biochemical Studies on Novel MHC Kinases

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通过一组位于肌球蛋白尾端附近的定位苏氨酸残基的磷酸化/去磷酸化来调节的。在之前的研究中，我们重点研究了肌球蛋白重链激酶A(MHCK A)酶的生物化学和细胞生物学，它通过肌球蛋白尾部定位的靶点的磷酸化参与体内肌球蛋白组装的控制。MHCK A现在被认为是一个高度新颖的蛋白激酶家族的原型，该家族存在于网柄苔藓和整个动物界。我们现在已经确定了该激酶家族的另外几个成员，初步数据表明，其中至少有两个成员也是MHC激酶。我们有证据表明，在趋化过程中，这两种酶的动态定位控制，而且我们有证据表明，脂质信号通路和酸性磷脂可能调节这些酶的活性。下一个资助阶段的研究将集中在这些酶的细胞作用上，使用基因打靶来了解每一种激酶的相对细胞作用，并使用结构域解剖方法来了解这些酶动态招募到细胞皮质的机制，并解决这些酶被酸性磷脂激活的机制。遗传学方法也将被用来识别参与肌球蛋白II组装和定位控制的新基因。

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.Under previous funding, we focused on the biochemistry and cell biology of the enzyme myosin heavy chain kinase A (MHCK A), which participates in the in vivo control of myosin assembly via phosphorylation of the mapped target sites in the myosin tail. MHCK A is now recognized as the prototype for a highly novel family of protein kinases present in Dictyostelium and throughout the animal kingdom. We have now identified several additional Dictyostelium members of this kinase family; our preliminary data indicate that at least two of these are also MHC kinases. We have evidence for dynamic localization control of two of these kinases during chemotaxis, and we have evidence that lipid signaling pathways and acidic phospholipids may regulate the activity of these enzymes. Studies proposed for the next funding period will focus on the cellular roles of these enzymes, using gene targeting to understand the relative cellular roles of each kinase, and using domain dissection approaches to understand the mechanisms involved in the dynamic recruitment of these enzymes to the cell cortex and to address the mechanism of activation of these enzymes by acidic phospholipids. Genetic approaches will also be used to identify new genes that participate in the control of myosin II assembly and localization.