NOVEL SAPK ACTIVATING KINASE IN RENAL EPITHELIAL STRESS

肾小管上皮损伤导致显著的细胞表型 变化包括形态、细胞骨架组织的变化， 基因表达的诱导被认为是调控的关键 细胞对损伤的早期反应。已经取得了重大进展 定义组件并了解的功能 细胞内信号转导通路导致调节 其他系统中的类似过程。应激激活蛋白激酶 途径，特别是SAPKs(应激激活蛋白激酶/c-jun N-末端激酶)被多个细胞应激快速激活 包括缺血/再灌流在内的刺激物可引起肾脏损伤。 此外，SAPKs被GTP结合的rac1和cdc42Hs激活，Rho- 就像GTP酶一样，最初被描述为 细胞骨架组织。因此，有人提出， SAPKs激活的信号转导途径调控 可能会引发肾脏对损伤的部分早期反应。我们有 从胚胎肾脏中鉴定、克隆并初步鉴定 一种新的丝氨酸/苏氨酸蛋白激酶，称为DLK。DLK是 一个结构独特的蛋白激酶亚家族，名为混合血统 激活剂。 如本文所示，DLK可有效激活p46SAPK和p38MAPK，但不能 ERK2在细胞培养中过表达。DLK似乎以 SAPK途径的近端激活物，可能是近端效应器 对于rac1和cdc42hs。在正常成人肾脏中，DLK表达于 近端肾小管上皮，仅存在于根尖下 亚细胞室。鉴于它在肾脏中的定位，它的 激活SAPK和p38MAPK的能力，以及它作为 的效应，我们假设DLK代表一个 一个或多个信号转导通路的近端组件 参与调节肾小管上皮细胞对 细胞压力或损伤。这项提议主要是为了调查 DLK的基本生化和调控，并将开始 将这些发现应用于近端肾小管上皮细胞。具体来说， 该项目将：具体目标1：确定具体的下游 DLK的底物(S)，并定义DLK的下游途径 激活p46SAPK和p38 MAPK。具体目标2：调查 DLK活性的调节既受潜在上游刺激又受 蛋白磷酸酶2B(钙调神经磷酸酶)。具体目标3：调查 DLK亮氨酸拉链结构域的性质及其鉴定 结构域相互作用蛋白。

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.