Gene Structure and Transciptional Regulation

描述(申请人提供)：趋化性，或引导细胞向小分子配体运动，在许多细胞和生理反应中发挥关键作用，包括癌细胞的转移，免疫中的中性粒细胞和巨噬细胞的移动，胚胎细胞在发育过程中的迁移，以及发育过程中网柄基底壳的聚集。细胞必须能够对较浅的细胞外趋化物质梯度做出反应，并将其转化为细胞内陡峭的信号成分梯度，这一梯度控制着F-肌动蛋白在细胞前沿(以及较小程度的细胞后部)的空间受限聚合，以及肌球蛋白II在细胞后部的组装和收缩。这需要整合几条信号转导途径，其中许多信号转导途径在网柄基菌和人类之间是保守的。最近的研究发现，磷脂酰肌醇3-激酶(PI3K)和MAP激酶作为细胞对定向信号反应的关键调节因子而级联。 这项建议侧重于进一步分析Dictyostelialmek1/ERK1 MAP激酶级联及其在控制趋化作用中的作用。该提案利用可用的生化、遗传和细胞生物学方法来剖析这个实验系统中的调控途径。我们已经证明了MEK1及其下游的MAP激酶ERK1是正常趋化所必需的，并且这两个组分都定位于趋化细胞的前沿。我们发现，控制这些成分的亚细胞定位需要MEK1的可逆SUMO化，而这一途径的适应是正常发育所必需的，涉及ERK1和PI3K途径的反馈调节。我们的目标是通过确定MEK1/ERK1缺失细胞的趋化功能缺陷来阐明MEK1和ERK1是如何调节趋化作用的。我们将通过双杂交筛选来鉴定和检测ERK1底物的功能，然后通过生化方法和突变筛选来检测它们的功能。此外，我们还将阐明控制MEK1去SUMO化和途径适应的机制。最后，我们将确定SMEK1，一个进化保守的MEK1的第二位点抑制因子，它是我的实验室发现的，在调节MEK1/ERK1途径和趋化作用中的作用。本申请中提出的工作将为控制这一高度进化保守的细胞生物学过程的机制提供新的和重要的见解，从而为阐明各种人类疾病的细胞基础提供必要的背景，包括那些影响癌细胞天然免疫和转移的疾病。

DESCRIPTION (provided by applicant): Chemotaxis, or directed cell movement toward a small molecule ligand, plays a key role in many cellular and physiological responses, including metastasis of cancer cells, movement of neutrophils and macrophage in immunity, migration of embryonic cells during development, and aggregation of Dictyostelium during development. Cells must be able to respond to a shallow extracellular chemoattractant gradient and convert this into a steep intracellular gradient of signaling components, which controls the spatially restricted polymerization of F-actin at the leading edge (and to a lesser degree the cell's posterior) and assembly and contraction of myosin II at the cell's posterior. This requires the integration of several signal transduction pathways, many of which are conserved between Dictyostelium and man. Recent findings have established that phosphatidylinositol 3-kinase (PI3K) and MAP kinases cascades as key regulators of a cell's responses to directional signals. This proposal focuses on the further analysis of the Dictyostelium MEK1/ERK1 MAP kinase cascade and its role in controlling chemotaxis. The proposal takes advantage of the biochemical, genetic, and cell biological approaches available to dissect regulatory pathways in this experimental system. We have demonstrated that MEK1 and its downstream MAP kinase ERK1 are required for proper chemotaxis and that both components localize to the leading edge in chemotaxing cells. We have discovered that the control of the subcellular localization of these components requires the reversible SUMOylation of MEK1 and that adaptation of this pathway, which is essential for proper development, involves feedback regulation by the ERK1 and PI3K pathways. Our goal is to elucidate how MEK1 and ERK1 regulate chemotaxis by determining which chemotaxis functions are defective in mek1/erk1 null cells. We will identify and examine the function of ERK1 substrates through two-hybrid screens and then examine their function through biochemical approaches, and mutant screens. In addition, we will elucidate the mechanisms that control MEK1 deSUMOylation and pathway adaptation. Lastly, we will determine the role of SMEK1, an evolutionarily-conserved, second-site suppressor of MEK1 that was identified in my lab in regulating the MEK 1/ERK1 pathway and chemotaxis. The work proposed in this application should provide new and important insights into mechanisms that control this highly evolutionarily conserved cell biological process, and thus provide the needed background to elucidate the cellular basis underlying a variety of human diseases, including those affecting innate immunity and metastasis of cancer cells.