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: 6795160
• 负责人: AUDREY G MINDEN
• 金额: $ 8.18万
• 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/6795160
• 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.

该提案中的实验旨在研究信号 当哺乳动物细胞为时会触发的转导级联 暴露于细胞外刺激。 了解这种信号如何 途径工作很重要，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 组成性激活。 该项目将重点放在分析 由哺乳动物丝氨酸/苏氨酸介导的信号通路 激酶与酵母信号传导酶Ste20相关。 特别 兴趣将是我们确定的一种新蛋白质Pak4，即 与蛋白激酶的PAK家族遥远有关。 角色的作用 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。 以下问题将被解决： 1）哪种映射激酶途径被PAK4激活和其他Ste20 激酶？ 这将涉及瞬态转染实验，其中 野生型或组成型活性突变体的表达向量 将Ste20相关的蛋白转染到哺乳动物细胞中。 他们会的 与表位的表达向量共转染，用于标记的JNK，ERK， 或p38 MAP激酶。 免疫复合激酶测定将进行 确定哪些MAP激酶途径被激活。实验 还将进行检查这些激活的机制 途径。 2）PAK4和其他Ste20（例如激酶）如何受到调节 细胞外刺激？ 免疫复合激酶测定将用于 检查响应于Ste20相关蛋白的激活 细胞外刺激，例如细胞因子和生长因子。 信号 还将研究其激活所需的酶。 3）是 PAK4由Rho家族的小GTP结合蛋白调节？ 序列分析表明，PAK4可能是GTP结合的新目标 蛋白质。 实验将旨在确定是否 与Rho的GTP结合蛋白相互作用并被激活 家庭。 4）PAK4和其他的直接下游目标是什么 Ste20喜欢激酶？ 实验旨在确定Ste20是否 相关的蛋白质直接激活已知的信号酶，例如MEKK。 实验还旨在识别新型底物。 这会 参与使用酵母两个混合系统来识别交互 蛋白质和筛选表达式文库以识别直接目标。