SIGNALING BY A NEW MAMMALIAN STE20 RELATED KINASE

一种新的哺乳动物 STE20 相关激酶的信号传导

• 批准号: 6173345
• 负责人: AUDREY G MINDEN
• 金额: $ 24.34万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6173345
• 关键词: 3T3 cells; SDS polyacrylamide gel electrophoresis; autoradiography; biological signal transduction; cell line; cytokine; enzyme activity; enzyme induction /repression; gel electrophoresis; growth factor receptors; guanine nucleotide binding protein; image processing; immunoprecipitation; mitogen activated protein kinase; phosphorylation; protein kinase; transfection; tumor necrosis factor alpha; western blottings; yeast two hybrid system

The experiments in this proposal are designed to investigate the signal transduction cascades that are triggered when mammalian cells are exposed to extracellular stimuli. Understanding how such signaling pathways work is important because in clinical disorders such as cancer, signal transduction pathways may be improperly regulated or constitutively activated. This project will focus on the analysis of signaling pathways that are mediated by mammalian serine/threonine kinases related to the yeast signaling enzyme STE20. Of particular interest will be PAK4, a new protein that we have identified, that is distantly related to the PAK family of protein kinases. The roles for these proteins will be investigated in cultured mammalian fibroblasts and macrophage cell lines. The following questions will be addressed: 1) Which MAP Kinase pathways are activated by PAK4 and other STE20 like kinases? This will involve transient transfection experiments in which expression vectors for wild-type or constitutively active mutants of STE20 related proteins are transfected into mammalian cells. They will be co-transfected with expression vectors for epitope tagged JNK, ERK, or p38 MAP Kinases. Immune complex kinase assays will be carried out to determine which MAP Kinase pathways become activated. Experiments will also be carried out to examine the mechanism of activation of these pathways. 2) How are PAK4 and other STE20 like kinases regulated by extracellular stimuli? Immune complex kinase assays will be used to examine the activation of STE20 related proteins in response to extracellular stimuli such as cytokines and growth factors. Signaling enzymes required for their activation will also be investigated. 3) Is PAK4 regulated by small GTP binding proteins of the Rho family? Sequence analysis suggests that PAK4 may be a new target for GTP binding proteins. Experiments will be designed to determine whether it interacts with, and is activated by, GTP binding proteins of the Rho family. 4) What are the direct downstream targets of PAK4 and other STE20 like kinases? Experiments are designed to determine whether STE20 related proteins directly activate known signaling enzymes such as MEKK. Experiments are also designed to identify novel substrates. This will involve using the yeast two hybrid system to identify interacting proteins, and screening expression libraries to identify direct targets.

本提案中的实验旨在研究信号 当哺乳动物细胞被激活时触发的转导级联 暴露于细胞外刺激。 了解此类信号如何发出 通路的作用很重要，因为在癌症等临床疾病中， 信号转导途径可能受到不当调节或 本构激活。 该项目将重点分析 由哺乳动物丝氨酸/苏氨酸介导的信号通路 与酵母信号酶 STE20 相关的激酶。 特别是 我们感兴趣的是 PAK4，这是我们已经鉴定出的一种新蛋白质，即 与蛋白激酶 PAK 家族关系较远。 的角色 这些蛋白质将在培养的哺乳动物成纤维细胞中进行研究 和巨噬细胞系。 将解决以下问题： 1) PAK4 和其他 STE20 等激活哪些 MAP 激酶途径 激酶？ 这将涉及瞬时转染实验，其中 野生型或组成型活性突变体的表达载体 STE20相关蛋白被转染到哺乳动物细胞中。 他们会 与表位标记为 JNK、ERK 的表达载体共转染， 或 p38 MAP 激酶。 将进行免疫复合物激酶测定 以确定哪些 MAP 激酶途径被激活。实验 还将进行这些激活机制的研究 途径。 2) PAK4 和其他 STE20 样激酶是如何被调节的 细胞外刺激？ 免疫复合物激酶测定将用于 检查 STE20 相关蛋白的激活反应 细胞外刺激，例如细胞因子和生长因子。 信令 还将研究其激活所需的酶。 3) 是 PAK4 受 Rho 家族小 GTP 结合蛋白调节？ 序列分析表明PAK4可能是GTP结合的新靶点 蛋白质。 将设计实验来确定是否 与 Rho 的 GTP 结合蛋白相互作用并被其激活 家庭。 4) PAK4等的直接下游靶标是什么 STE20 类似激酶吗？ 实验旨在确定 STE20 是否 相关蛋白直接激活已知的信号酶，例如 MEKK。 实验还旨在识别新的底物。 这将 涉及使用酵母两种杂交系统来识别相互作用 蛋白质，并筛选表达文库以确定直接目标。