Cellular and Biochemical Studies on Novel MHC Kinases

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。在这个系统中的肌球蛋白II组装是由磷酸化/去磷酸化的一组映射的苏氨酸残基，位于附近的尖的肌球蛋白tail.Under以前的资金，我们专注于生物化学和细胞生物学的酶肌球蛋白重链激酶A（MHCK A），参与在体内控制肌球蛋白组装通过磷酸化的映射的靶位点在肌球蛋白尾巴。MHCK A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。我们现在已经确定了这个激酶家族的几个额外的Dictyosteoprotein成员;我们的初步数据表明，其中至少有两个也是MHC激酶。我们有证据表明，在趋化过程中，这些激酶的动态本地化控制，我们有证据表明，脂质信号通路和酸性磷脂可以调节这些酶的活性。下一个资助期的研究将侧重于这些酶的细胞作用，使用基因靶向来了解每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向细胞皮质的动态招募机制，并解决酸性磷脂激活这些酶的机制。遗传学方法也将被用来确定新的基因，参与控制肌球蛋白II组装和本地化。

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.