Cellular and Biochemical Studies on Novel MHC Kinases

新型 MHC 激酶的细胞和生化研究

• 批准号: 6781911
• 负责人: THOMAS EGELHOFF
• 金额: $ 30.6万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6781911
• 关键词: Dictyostelium; catalyst; cell biology; cell motility; enzyme mechanism; enzyme structure; enzyme substrate; gene expression; gene mutation; gene targeting; green fluorescent proteins; immunoelectron microscopy; microorganism genetics; molecular cloning; myosins; phosphorylation; protein biosynthesis; protein kinase; protein localization; protein sequence; protein structure function; tissue /cell culture

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.

描述（由申请人提供）：肌球蛋白II在发育过程中的细胞因子，细胞迁移和细胞形状变化中扮演基本角色。在所有这些设置中，都可以很好地确定，肌球蛋白在细胞骨架中的动态局部组装对于其细胞收缩作用至关重要。尽管在空间和时间上受到时间调节的组装的重要性，但在任何系统中都不了解控制肌球蛋白II的局部组装和拆卸的信号传导机制。我们正在使用简单的Amoeba Dictyostelium Discoideum作为模型系统，用于识别调节肌球蛋白组装的信号通路。这种简单的变形虫显示了类似于哺乳动物细胞（如中性粒细胞或巨噬细胞）的细胞运动，趋化性和第二信使信号的形式。 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肌球蛋白尾部的位置。现在，MHCK A被公认为是在Dictyostelium和整个动物界中存在的高度新颖蛋白激酶家族的原型。现在，我们已经确定了该激酶家族的其他几个dictyostelium成员。我们的初步数据表明，其中至少两个也是MHC激酶。我们有证据表明在趋化性过程中对这些激酶中的两种激酶进行动态定位控制，并且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。在下一个资金期间提出的研究将重点关注这些酶的细胞作用，使用基因靶向来了解每种激酶的相对细胞作用，并使用域解剖方法来了解这些酶在细胞皮质中的动态募集的机制，并通过酸性磷酸化的糖脂激活机制来解决细胞皮质的机制。遗传方法还将用于确定参与肌球蛋白II组装和定位的新基因。