MIXED LINEAGE KINASE, SPRK IN STRESS SIGNALING

依据：SPRK是混合谱系激酶（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， 它们的内部含有丝氨酸/苏氨酸和酪氨酸激酶 催化域申请人及其同事， 证明SPRK是SAPK/JNK通路的上游激活剂。 本申请的目标：确定SPRK作为一种 SAPK/JNK活性的调节剂，并阐明信号通路 参与SPRK激活SAPK/JNK。 具体目的：具体目的1：鉴定激活SPRK的激动剂 在原地。申请方将确定诱导激活的激动剂 的SPRK。将检查已知刺激SAPK/JNK通路的激动剂 包括生长因子（例如EGF和IGF-1）、炎性细胞因子 (e.g. TNF-α和IL-1 β）和细胞毒性刺激（例如UV辐射， ATP耗竭和渗透压休克）。一旦激活SPRK的激动剂 这些激动剂参与调节SPRK的机制 将被审查。具体目标2：阐明蛋白质的相互作用 与SPRK通过其SH 3域：申请人建议a）识别 与SPRK的SH 3结构域结合的蛋白; B）定义功能性的 5个氨基酸插入物在SPRK的SH 3结构域中的作用，和c） 阐明在具体目标1中鉴定的SPRK激动剂的作用， 通过与SPRK的SH 3结构域结合来进行信号传导。具体目标3： 识别SPRK和小GTP结合蛋白之间的相互作用： 申请人建议a）检查SPRK和G之间的相互作用 蛋白质Rho、Ras、Rac和Cdc 42和B）决定了 SPRK和GTP结合蛋白之间的相互作用。申请人 预期GTP结合蛋白可以上调或抑制 激动剂诱导的SPRK活化。 技术方法：为了实现这些具体目标，申请人将使用 广泛的技术方法，他有广泛的 体验.这些包括激酶测定，免疫印迹， 免疫沉淀，用重组DNA转染细胞， 酵母双杂交系统 提案的长期目标：确定激活 SAPK/JNK通过SPRK和鉴定结合SPRK的蛋白质， 规范其活动。申请人承认，他的做法可能会导致 新信号蛋白的鉴定。如果新的蛋白质 鉴定的这些将由申请人克隆和测序。

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.