Arsenic (As) is an inorganic environmental contaminant of major concern due to its ubiquitous presence. Chronic exposure to arsenic frequently results in peripheral vascular disease, as well as skin, lung, bladder, and kidney cancer. Recent evidence demonstrates that arsenite stimulates cyclooxygenase (COX-II) expression suggesting that arsenite affects prostaglandin synthesis. The investigator's published studies with arsenic have demonstrated that the serine/threonine kinase, MEKK4, is involved in arsenic signal transduction. To further understand the molecular mechanism by which arsenic causes its deleterious effects and how the activity of MEKK4 contributes to arsenic toxicity, efforts by the investigators have focused on characterizing the activity of MEKK4 in vascular smooth muscle cells since it appears to function upstream of COX-II. The hvpothesis of this proposal is that the prostaglandin biosynthetic pathway is regulated by MEKK4 and that arsenic affects the vascular system by modulating prostaglandin homeostasis through MEKK4.
The Specific Aims are to: 1. To identify and characterize tyrosine phosphorylation of MEKK4. Our data indicate that Pyk2 phosphorylates MEKK4. This site is not known and will be identified by LC-MS. Then, the site will be mutated and MEKK4 catalytic activity will be characterized to assess the significance of the phosphorylation site. 2. To characterize the mechanism by which SHP-2 interacts with MEKK4 and how arsenite inhibits SHP-2 activity. SHP-2 associates with MEKK4 in a stimulus-dependent manner. Intracellular calcium promotes dephosphorylation of MEKK4, while arsenite inhibits the tyrosine dephosphorylation of MEKK4. 3. To identify and characterize the MEKK4 substrate. Candidate proteins include members of the MAP kinase family or novel proteins that will be identified using a modification of the """"""""tethered"""""""" MEKK4 approach and LC-MS. It is possible that MEKK4 phosphorylates and regulates cytosolic phospholipase A2 (cPLA2), a key enzyme involved in prostaglandin biosynthesis that is regulated by phosphorylation. 4. To characterize the mechanism by which MEKK4 regulates prostaglandin synthesis. The kinase-inactive mutant of MEKK4 functions as a dominant-negative protein, and the investigators have shown that expression of this protein inhibits transcription of a Cox-II promoter/luciferase chimeric plasmid. This result suggests that endogenous MEKK4 functions upstream of pathways that regulate prostaglandin synthesis. The proposed research will provide a further understanding of the importance of the arsenic-induced signaling processes and how it adversely affects the peripheral vascular system and promotes tissue injury.
