MIXED LINEAGE KINASE, SPRK IN STRESS SIGNALING

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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.