Role of cGMP signaling in Dictyostelium chemotaxis

cGMP 信号在盘基网柄菌趋化性中的作用

• 批准号: 6720815
• 负责人: JANET L SMITH
• 金额: $ 34.43万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6720815
• 关键词: Dictyostelium; binding proteins; biological signal transduction; chemotaxis; cyclic GMP; immunoprecipitation; mitogen activated protein kinase; myosins; polymerase chain reaction; protein kinase; protein localization; protein structure function; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): The goal of the project is to understand the signaling pathways that control cytoskeletal changes during processes such as smooth muscle contraction and cell migration. In eukaryotes, cell migration in response to extracellular signals controls processes such as wound healing, embryogenesis, axonal guidance, metastasis of cancer cells, and aggregation of Dictyostelium cells to form a multicellular organism. In Dictyostelium, cGMP signaling plays a vital role in coordinating cytoskeletal rearrangements during chemotaxis, and is implicated in regulating myosin II and other unidentified targets. A bioinformatics approach was used to identify genes for cGMP-binding proteins, with the expectation that one or more of them would have a severe chemotaxis defect. One of these proteins - GbpC - is largely responsible for transmitting the downstream effects of cGMP during chemotaxis. GbpC is the Dictyostelium homolog of a novel family of protein kinases that is evolutionarily conserved throughout the metazoan lineage. These proteins contain leucine-rich repeats, a Ras-like domain, and a protein kinase domain in the MEKK family. GbpC additionally contains, at its C terminus, a RasGEF domain and two cGMP-binding binding sites, and therefore has properties of the cyclic nucleotide regulated GEFs found in higher eukaryotes. GbpC is required for chemoattractant-elicited recruitment of myosin to the cytoskeleton, a process regulated by threonine phosphorylation of the myosin heavy chain (MHC). The subcellular localization of myosin in GbpC-null cells will be examined, and localization of GbpC itself will be determined. Changes in MHC threonine phosphorylation and the regulation of MHC kinases and phosphatase during chemotaxis in wild-type and gbpC cells will be examined. These studies will lead to basic insight into how myosin dynamics in the cell are controlled during chemotaxis, and into GbpC's regulatory role during this process. The juxtaposition of cGMP, RasGEF, Ras and protein kinase domains on the GbpC protein suggests that GbpC may be activated by a novel intramolecular signaling mechanism. This model will be tested using in vivo complementation assays and in vitro protein kinase and GTPase assays. Since GbpC's protein kinase domain is in the MEKK family, it may play a role in the activation of MAP kinase pathways during chemotaxis, and activation of these pathways by GbpC will be tested using a gbpC- strain. These studies will reveal upstream signaling pathways for myosin II and ERK regulation, and will elucidate the role of the key regulatory protein GbpC in the complex network of signaling needed to achieve directional movement toward a chemoattractant.

描述（由申请人提供）：该项目的目的是了解控制过程中控制细胞骨架变化的信号通路，例如平滑肌收缩和细胞迁移。在真核生物中，对细胞外信号响应的细胞迁移控制着伤口愈合，胚胎发生，轴突引导，癌细胞转移以及dictyostelium细胞的聚集以形成多细胞生物体。在Dictyostelium中，CGMP信号在趋化过程中在协调细胞骨架重排方面起着至关重要的作用，并且与调节肌球蛋白II和其他未识别靶标有关。使用生物信息学方法来鉴定CGMP结合蛋白的基因，并期望其中一种或多种会有严重的趋化性缺陷。这些蛋白质之一-GBPC-主要负责在趋化过程中传播CGMP的下游效应。 GBPC是一种新型蛋白激酶家族的dictyostelium同源物，该家族在整个后生谱系中是进化保守的。这些蛋白质包含富含亮氨酸的重复序列，一个RAS样结构域和MEKK家族中的蛋白激酶结构域。 GBPC还包含在其C末端，一个RASGEF结构域和两个CGMP结合位点，因此具有在较高真核生物中发现的环状核苷酸调节的GEF的特性。 GBPC是将肌球蛋白介绍为细胞骨架的趋化因素募集所必需的，这是由肌球蛋白重链（MHC）的苏氨酸磷酸化调节的过程。将检查肌球蛋白在GBPC无效细胞中的亚细胞定位，并将确定GBPC本身的定位。将检查MHC苏氨酸磷酸化的变化以及在野生型和GBPC细胞中趋化过程中MHC激酶和磷酸酶的调节。这些研究将导致对细胞中肌球蛋白动力学在趋化性期间的控制以及在此过程中GBPC的调节作用中如何控制的基本见解。 GBPC蛋白上CGMP，RASGEF，RAS和蛋白激酶结构域的并置表明GBPC可以通过一种新型分子内信号传导机制激活GBPC。该模型将使用体内互补测定以及体外蛋白激酶和GTPase分析进行测试。由于GBPC的蛋白激酶结构域位于MEKK家族中，因此它可能在趋化过程中的MAP激酶途径的激活中起作用，并且将使用GBPC对这些途径的激活进行测试。这些研究将揭示肌球蛋白II和ERK调节的上游信号传导途径，并将阐明关键调节蛋白GBPC在实现趋化剂方向运动所需的复杂信号网络中的作用。