Modulation of Prostaglandins by Arsenic

描述（由申请人提供） 砷是一种普遍存在的无机环境污染物。 长期接触砷通常会导致外周血管疾病，以及皮肤癌、肺癌、膀胱癌和肾癌。 最近的证据表明，亚砷酸盐刺激环氧合酶（COX-II）的表达，表明亚砷酸盐影响前列腺素的合成。 研究人员发表的砷研究表明丝氨酸/苏氨酸激酶MEKK 4参与砷信号转导。 为了进一步了解砷引起其有害作用的分子机制以及MEKK 4的活性如何导致砷毒性，研究人员的努力集中在表征MEKK 4在血管平滑肌细胞中的活性，因为它似乎在COX-II的上游起作用。 该假说认为前列腺素生物合成途径受MEKK 4调节，砷通过MEKK 4调节前列腺素稳态而影响血管系统。 具体目标是： 1. 鉴定和表征MEKK 4的酪氨酸磷酸化。 我们的数据表明Pyk 2磷酸化MEKK 4。 该位点未知，将通过LC-MS鉴定。然后，突变该位点，表征MEKK 4催化活性，以评估磷酸化位点的意义。 2. 研究SHP-2与MEKK 4相互作用的机制以及亚砷酸盐如何抑制SHP-2活性。 SHP-2以刺激依赖性方式与MEKK 4结合。 细胞内钙促进MEKK 4的去磷酸化，而亚砷酸盐抑制MEKK 4的酪氨酸去磷酸化。 3. 鉴定和表征MEKK 4底物。 候选蛋白质包括MAP激酶家族的成员或将使用“栓系”MEKK 4方法和LC-MS的修改来鉴定的新蛋白质。MEKK 4可能磷酸化并调节胞质磷脂酶A2（cPLA 2），其是参与前列腺素生物合成的关键酶，其由磷酸化调节。 4. 表征MEKK 4调节前列腺素合成的机制。 MEKK 4的激酶失活突变体作为显性负性蛋白发挥作用，研究人员已经表明，这种蛋白的表达抑制Cox-II启动子/荧光素酶嵌合质粒的转录。 该结果表明内源性MEKK 4在调节前列腺素合成的途径的上游起作用。 这项研究将进一步了解砷诱导的信号传导过程的重要性，以及它如何对外周血管系统产生不利影响并促进组织损伤。

DESCRIPTION (provided by applicant) 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.