Cellular and Biochemical Studies of Myosin Assembly in Dictyostelium

盘基网柄菌肌球蛋白组装的细胞和生化研究

• 批准号: 7931578
• 负责人: THOMAS EGELHOFF
• 金额: $ 8.41万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931578
• 关键词: Address; Affect; Affinity Chromatography; Amoeba genus; Animal Model; Animals; Apoptotic; Behavior; Binding; Biochemical; Biochemistry; Biological Models; Catalytic Domain; Cell Line; Cell Shape; Cell division; Cell physiology; Cells; Chemotaxis; Clone Cells; Complement; Complex; Cytokinesis; Cytoskeleton; Defect; Development; Dictyostelium; Dictyostelium discoideum; Enzymes; Eukaryota; Family; Filament; Fishes; Flow Cytometry; Fluorescence-Activated Cell Sorting; Foundations; Funding; GTPase-Activating Proteins; Genes; Genetic Screening; Genome; Genomics; Goals; Image; In Vitro; Insertional Mutagenesis; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Molecular; Myosin ATPase; Myosin Heavy Chains; Myosin Type II; Names; Null Lymphocytes; Organism; Osmotic Shocks; Oxidative Stress; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Property; Protein Dephosphorylation; Protein Kinase; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Regulation; Regulatory Pathway; Role; Second Messenger Systems; Series; Signal Pathway; Signal Transduction; Site; Suspension Culture; System; Tail; Tertiary Protein Structure; Testing; Threonine; Work; cell motility; in vivo; insight; light scattering; mRNA Differential Displays; macrophage; member; myosin heavy-chain kinase A; myosin-heavy-chain kinase; neutrophil; novel; prototype; response; restriction enzyme; second messenger

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.

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。该系统中的肌球蛋白II组件受到一组位于肌球蛋白尾巴尖端附近的映射苏氨酸残基的磷酸化/去磷酸化调节。 我们以前曾在该系统中鉴定出一组新型的肌球蛋白重链激酶（MHCK），它们通过肌球蛋白尾部映射的靶位点的磷酸化来参与肌球蛋白组装的体内控制。这些酶现在被公认为是在柱状层中以及被称为“α激酶”的动物界的高度新颖蛋白激酶家族的原型。最近，有1个哺乳动物α激酶（TRPM7）与氧化应激有关的凋亡途径激活与凋亡途径的活化有关，但是其他哺乳动物α激酶的作用在很大程度上是未知的。我们在Dictyostelium中的分子和基因组方法在这种简单的模型生物体中揭示了总共6种α激酶。至少有2个dictyosteliumα激酶似乎不是MHC激酶，但可能在细胞中起其他作用。我们提出了一系列补充方法，以了解所有这些酶的作用。在此提案中，我们将：（1）使用 生物化学/细胞分析以阐明自磷酸化在MHCK激活中的作用，（2）执行研究以测试我们在早期工作中在生化上确定生化的MHC磷酸酶的细胞作用，（3）对VWKA和AK1进行进一步的细胞/生物化学分析，对VWKA和AK1进行了2个依赖的良好孔和ALPHA KINASES（四个小细胞酶），不及时（不及时）（4个细胞）kilpha kilpha kilpha kinarel（不及时）（不及时（4）遗传筛选以鉴定在肌球蛋白II相关的细胞功能中涉及的新基因，从细胞分裂到渗透保护反应。我们的研究将为理解哺乳动物系统中的肌球蛋白II调节提供直接相关的重要基础。我们提出的研究解决了Dictyostelium Alpha激酶的完整补充，还将为比较α激酶在哺乳动物系统中的可能作用提供一个强大的平台。