MIXED LINEAGE KINASE, SPRK IN STRESS SIGNALING

基本原理：SPRK是混合血统蛋白激酶(MLK)家族的成员， 新出现并迅速扩大的激酶家族，其独特之处在于 它们的体内同时含有丝氨酸/苏氨酸和酪氨酸激酶 催化域。申请人，连同他的同事，已经 证明SPRK是SAPK/JNK通路的上游激活物。 应用的目标：确定SPRK作为一种 SAPK/JNK活性调节因子及其信号转导途径 参与SAPK/JNK的SPRK激活。 具体目标：具体目标1：确定激活SPRK的激动剂 在原地。申请者将确定诱导激活的激动剂(S) SPRK的。已知的刺激SAPK/JNK途径的激动剂将被检测 包括生长因子(如EGF和IGF-1)、炎性细胞因子 (例如，肿瘤坏死因子-α和IL-1β)和细胞毒性刺激(例如，紫外线辐射， ATP耗竭和渗透性休克)。一旦激活SPRK的激动剂 确定了这些激动剂调节SPRK的机制 将会被检查。特定目标2：阐明蛋白质之间的相互作用 通过其SH3域使用SPRK：申请人提议a)识别 与SPRK的SH3结构域结合的蛋白质；b)定义了 5个氨基酸插入片段在SPRK和c)SH3结构域中的作用 阐明SPRK激动剂的作用，在特定的目标1，在 通过与SPRK的SH3结构域结合来传递信号。具体目标3： 确定SPRK和小GTP结合蛋白之间的相互作用： 申请人建议a)审查SPRK和G之间的互动 Rho、Ras、Rac和Cdc42和b)蛋白决定了 SPRK与GTP结合蛋白的相互作用。申请人 预测GTP结合蛋白可能上调或抑制 激动剂诱导SPRK激活。 技术方法：为了实现这些特定目标，申请者将使用 广泛的技术方法，他拥有广泛的 经验。这些方法包括激酶分析、免疫印迹、 免疫沉淀法、重组DNA转染法和 酵母双杂交系统。 该提案的长期目标：确定激活的激动剂 SAPK/JNK通过SPRK并鉴定与SPRK和JNK结合的蛋白 规范其活动。申请人认识到他的方法可能会导致 用于鉴定新的信号蛋白。如果新的蛋白质 申请人将对已鉴定的这些基因进行克隆和测序。

RATIONALE: SPRK is a member of Mixed Lineage Kinase (MLK) family, an emerging and rapidly enlarging family of kinases, which are unique in that they contain both serine/threonine and tyrosine kinases within their catalytic domains. The applicant, together with his co-workers, has demonstrated that SPRK is an upstream activator of the SAPK/JNK pathway. GOAL OF THE APPLICATION: To determine the physiological role of SPRK as a regulator of SAPK/JNK activity and to elucidate the signaling pathways involved in SPRK activation of SAPK/JNK. SPECIFIC AIMS: Specific aim 1: To identify the agonists that activate SPRK in situ. The applicant will identify the agonist(s) that induce activation of SPRK. Agonists known to stimulate the SAPK/JNK pathway will be examined including growth factors (e.g. EGF and IGF-1), inflammatory cytokines (e.g. TNF-alpha, and IL-1beta) and cytotoxic stimuli (e.g. UV radiation, ATP depletion and osmotic shock). Once the agonists that activate SPRK are identified, the mechanisms involved in regulating SPRK by these agonists will be examined. Specific aim 2: To elucidate interactions of proteins with SPRK via its SH3 domain: The applicant proposes to a) identify the proteins that bind to the SH3 domain of SPRK; b) define the functional role of the five amino acid inserts in the SH3 domain of SPRK and c) elucidate the role of agonists of SPRK, identified in specific aim 1, in signaling via binding to the SH3 domain of SPRK. Specific aim 3: To identify interactions between SPRK and the small GTP-binding proteins: The applicant proposes to a) examine interactions between SPRK and the G proteins Rho, Ras, Rac and Cdc42 and b) determine the functional effect of interactions between SPRK and the GTP-binding proteins. The applicant anticipate that GTP-binding proteins may either up-regulate or inhibit agonist-induced SPRK activation. TECHNICAL APPROACH: To achieve these specific aims, the applicant will use a broad range of technical approaches with which he has extensive experience. These include kinase assays, immunoblotting, immunoprecipitation, transfection of cells with recombinant DNA and the yeast two-hybrid system. LONG TERM GOALS OF THE PROPOSAL: to identify the agonists that activate SAPK/JNK via SPRK and to identify the proteins that bind to SPRK and regulate its activity. The applicant recognizes that his approach may lead to the identification of novel signaling proteins. If novel proteins are identified these will be cloned and sequenced by the applicant.