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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM055835-01A1
Application #
2761329
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Anderson, James J
Project Start
1999-03-01
Project End
2004-02-29
Budget Start
1999-03-01
Budget End
2000-02-29
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Jin, Huajun; Kanthasamy, Arthi; Harischandra, Dilshan S et al. (2014) Histone hyperacetylation up-regulates protein kinase C? in dopaminergic neurons to induce cell death: relevance to epigenetic mechanisms of neurodegeneration in Parkinson disease. J Biol Chem 289:34743-67
Rana, Ajay; Rana, Basabi; Mishra, Rajakishore et al. (2013) Mixed Lineage Kinase-c-Jun N-Terminal Kinase Axis: A Potential Therapeutic Target in Cancer. Genes Cancer 4:334-41
Ghosh, Anamitra; Saminathan, Hariharan; Kanthasamy, Arthi et al. (2013) The peptidyl-prolyl isomerase Pin1 up-regulation and proapoptotic function in dopaminergic neurons: relevance to the pathogenesis of Parkinson disease. J Biol Chem 288:21955-71
Leicht, Deborah T; Balan, Vitaly; Zhu, Jun et al. (2013) MEK-1 activates C-Raf through a Ras-independent mechanism. Biochim Biophys Acta 1833:976-86
Viswakarma, Navin; Jia, Yuzhi; Bai, Liang et al. (2013) The Med1 subunit of the mediator complex induces liver cell proliferation and is phosphorylated by AMP kinase. J Biol Chem 288:27898-911
Kanthasamy, Anumantha; Jin, Huajun; Anantharam, Vellareddy et al. (2012) Emerging neurotoxic mechanisms in environmental factors-induced neurodegeneration. Neurotoxicology 33:833-7
Rangasamy, Velusamy; Mishra, Rajakishore; Sondarva, Gautam et al. (2012) Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function. Proc Natl Acad Sci U S A 109:8149-54
Thylur, Ramesh P; Senthivinayagam, Subramanian; Campbell, Edward M et al. (2011) Mixed lineage kinase 3 modulates ?-catenin signaling in cancer cells. J Biol Chem 286:37470-82
Jin, Huajun; Kanthasamy, Arthi; Anantharam, Vellareddy et al. (2011) Transcriptional regulation of pro-apoptotic protein kinase Cdelta: implications for oxidative stress-induced neuronal cell death. J Biol Chem 286:19840-59
Rangasamy, Velusamy; Mishra, Rajakishore; Mehrotra, Suneet et al. (2010) Estrogen suppresses MLK3-mediated apoptosis sensitivity in ER+ breast cancer cells. Cancer Res 70:1731-40

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