NOVEL SAPK ACTIVATING KINASE IN RENAL EPITHELIAL STRESS

新型 SAPK 激活肾上皮应激激酶

• 批准号: 2612865
• 负责人: LAWRENCE B. HOLZMAN
• 金额: $ 20.92万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 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 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2612865
• 关键词: active sites; calcineurin; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; epithelium; guanosinetriphosphatases; laboratory rabbit; mitogen activated protein kinase; protein kinase; protein localization; protein purification; renal tubule; stress; tissue /cell culture; transfection

Renal tubular epithelial injury results in dramatic cellular phenotypic changes including alternations in morphology, cytoskeletal organization, and induction of gene expression thought to be critical for regulating the cell's early response to injury. Significant progress has been made in defining the components and understanding the function of intracellular signal transduction pathways that lead to regulation of similar processes in other systems. The stress activated protein kinase pathway and in particular SAPKs (stress activated protein kinases/c-jun N-terminal kinases) are rapidly activated by multiple cell stressing stimuli including ischemia/reperfusion induced injury of the kidney. Moreover, the SAPKs are activated by GTP-bound Rac1 and Cdc42Hs, Rho- like GTPases that were originally described as regulators of cytoskeletal organization. Therefore, it has been proposed that regulation of signal transduction pathways leading to SAPKs activation may initiate part of the kidney's early response to injury. We have identified, cloned from embryonic kidney, and initially characterized a novel serine/threonine protein kinase called DLK. DLK is a member of a structurally unique subfamily of protein kinases named mixed lineage kinases. As presented herein, DLK potently activates p46SAPK and p38 mapk but not ERK2 when overexpressed in cell culture. DLK appears to participate as a proximal activator of the SAPK pathway and may be a proximal effector for Rac1 and Cdc42Hs. In normal adult kidney, DLK is expressed in the proximal tubular epithelium where it is present only within a subapical subcellular compartment. Given its localization within the kidney, its ability to activate SAPK and p38mapk, and its potential role as an effector of Rac1 and Cdc42Hs, we hypothesize that DLK represents a proximal component of a signal transduction pathway or pathways that participates in modulating the response of the tubular epithelium to cellular stress or injury. This proposal seeks primarily to investigate the fundamental biochemistry and regulation of DLK and will begin to apply these findings to the proximal tubular epithelium. Specifically, this project will: Specific Aim 1: Identify the specific downstream substrate(s) of DLK and define the downstream pathway through which DLK activates p46SAPK and p38 mapk. Specific Aim 2: Investigate the regulation of DLK activity both by potential upstream stimuli and by protein phosphatase 2B (calcineurin). Specific Aim 3: Investigate the properties of DLK's leucine zipper domain and identify leucine zipper domain interacting proteins.

肾小管上皮细胞损伤导致细胞表型改变 变化包括形态学、细胞骨架组织、 以及诱导基因表达， 细胞对损伤的早期反应 取得了重大进展 在定义组件和理解的功能， 细胞内信号转导途径，导致调节 其他系统中的类似过程。 应激激活蛋白激酶 途径，特别是SAPKs（应激活化蛋白激酶/c-jun N-末端激酶）被多细胞应激快速激活 包括缺血/再灌注的刺激诱导的肾损伤。 此外，SAPKs被GTP结合的Rac1和Cdc42Hs、Rho- 比如GTP酶，最初被描述为 细胞骨架组织因此，有人提出， 调节导致SAPKs活化的信号转导途径 可能引发肾脏对损伤的部分早期反应。 我们有 从胚胎肾中鉴定、克隆并初步鉴定 一种新的丝氨酸/苏氨酸蛋白激酶DLK。DLK是 一个结构独特的蛋白激酶亚家族，名为混合谱系 激酶。 如本文所述，DLK有效地激活p46SAPK和p38 mapk，但不激活p46SAPK和p38 mapk。 当在细胞培养物中过表达时，ERK 2。 DLK似乎参与了 SAPK通路的近端激活剂，可能是近端效应物 对于Rac1和Cdc42H。 在正常成人肾脏中，DLK表达于 近端小管上皮，仅存在于根尖下 亚细胞区室鉴于其在肾脏内的定位， 激活SAPK和p38mapk的能力，以及其作为一种免疫调节剂的潜在作用。 Rac 1和Cdc 42 HS的效应子，我们假设DLK代表一种 信号转导通路的近端组分， 参与调节肾小管上皮细胞对 细胞应激或损伤。 这项建议主要是为了调查 DLK的基本生物化学和调节，并将开始 将这些发现应用于近端肾小管上皮细胞。 具体地说， 本项目将：具体目标1：确定具体的下游 DLK的底物，并定义DLK通过其 激活p46 SAPK和p38 mapk。 具体目标2：调查 通过潜在的上游刺激和通过 蛋白磷酸酶2B（钙调磷酸酶）。 具体目标3：调查 DLK的亮氨酸拉链结构域的性质和鉴定亮氨酸拉链 结构域相互作用蛋白。