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: 6513320
• 负责人: AUDREY G MINDEN
• 金额: $ 25.82万
• 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 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6513320
• 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.

本方案中的实验旨在研究信号 当哺乳动物细胞被激活时， 暴露于细胞外刺激。 了解这种信号是如何 通路的工作是重要的，因为在临床疾病如癌症中， 信号转导途径可能受到不适当的调节， 组成性激活。 本项目将重点分析 哺乳动物丝氨酸/苏氨酸介导的信号通路 与酵母信号传导酶STE 20相关的激酶。 特别 我们感兴趣的是PAK 4，这是我们已经发现的一种新蛋白质，也就是说 与蛋白激酶PAK家族有远亲关系。 角色为 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。 将讨论以下问题： 1)哪些MAP激酶通路被PAK 4和其他STE 20激活， 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 将STE 20相关蛋白转染到哺乳动物细胞中。 他们将 与表位标记的JNK，ERK， 或p38 MAP激酶。 将进行免疫复合物激酶试验 以确定哪些MAP激酶通路被激活。实验 也将进行研究的机制，激活这些 途径。2)PAK 4和其他STE 20样激酶是如何被 细胞外刺激？ 免疫复合物激酶测定将用于 检查STE 20相关蛋白的激活， 细胞外刺激物如细胞因子和生长因子。 信令 还将研究其活化所需的酶。 3)是 PAK 4受Rho家族小GTP结合蛋白的调节？ 序列分析表明PAK 4可能是GTP结合的新靶点 proteins. 将设计实验来确定它是否 与Rho的GTP结合蛋白相互作用并被其激活， 家人 4)PAK 4和其他药物的直接下游靶点是什么？ STE 20类似激酶？ 设计实验以确定STE 20是否 相关蛋白质直接激活已知的信号传导酶如MEKK。 实验也被设计来识别新的基板。 这将 涉及使用酵母双杂交系统来鉴定相互作用 蛋白质，并筛选表达文库以鉴定直接靶标。