REGULATION OF CGMP DEPENDENT PROTEIN KINASE

和cAMP一样，cGMP也是一个重要的第二信使，它调节广泛的 多种生理过程，而cGMP依赖的蛋白激酶是 CGMP行动的主要调解人。这样做的长期目标是 研究是为了破译生理调节机制。 CGMP依赖的蛋白激酶。CGMP作用的特异性将被研究 通过检测cGMP/cAMP的选择性来激活酶，并通过 两个可能的细胞因子的磷酸化及其作用的研究 底物。 CGMP依赖的蛋白激酶被认为是cGMP- 心房利钠等激动剂引起的平滑肌松弛 利钠肽和一氧化氮。模拟这些作用的药物 包括硝基血管扩张剂(例如，硝酸甘油)、甲基黄嘌呤(例如， 咖啡因)，可能还有通过cGMP“交叉激活”的β-激动剂-- CAMP依赖的蛋白激酶。这些代理通常用于 缓解胸痛、哮喘、男性阳萎和高血压。这个 激酶也可能参与神经功能，如记忆。 人型Ibeta cGMP依赖的蛋白激酶将过度表达 在SF9细胞中利用杆状病毒感染来做结构/功能 学习。该酶的cGMP/cAMP选择性将用Site- 基于cGMP结合模型化结构的定向突变 网站。 第一个要研究的底物将是激酶本身。 (自动磷酸化)。将尝试分离磷酸盐和磷酸盐 酶的脱磷形式的层析用于这些 学习。自身磷酸化在催化自身抑制中的作用 将会被检查。自动磷酸化的功能效应也将是 使用磷酸蛋白磷酸酶进行研究。32P并入 将在以下情况下测定完整的平滑肌细胞中的酶 用升高cGMP或cAMP的药物治疗。这些元素包括 自抑制结构域将通过部分蛋白分解和 诱变。 第二个要研究的底物将是cGMP结合 磷酸二酯酶。基于磷酸化位点周围的序列 在这种酶中，合成肽将被用来识别 有助于其对蛋白质磷酸化的效力和特异性 激活剂。这种磷酸二酯酶的磷酸化是否激活了 将通过测量cGMP的变化来研究完整细胞中的酶 对激活cGMP依赖的蛋白激酶的cGMP类似物的反应。

Like cAMP, cGMP is an important second messenger that modulates a wide variety of physiological processes, and cGMP-dependent protein kinase is a major mediator of cGMP action. The long-term objective of this investigation is to decipher the mechanisms of physiological regulation of cGMP-dependent protein kinase. Specificity of cGMP action will be studied by examining the cGMP/cAMP selectivity for activation of the enzyme and by studying the phosphorylation and role of two of its putative cellular substrates. cGMP-dependent protein kinase is believed to be the mediator of cGMP- induced relaxation of smooth muscle caused by agonists such as atrial natriuretic peptide and nitric oxide. Drugs that mimic these effects include nitrovasodilators (e.g., nitroglycerin), methylxanthines (e.g., caffeine), and perhaps beta-agonists through "cross-activation" of cGMP- dependent protein kinase by cAMP. These agents are commonly used for relief of chest pain, asthma, male impotence, and high blood pressure. The kinase may also be involved in neural functions such as memory. The human type Ibeta cGMP-dependent protein kinase will be overexpressed in SF9 cells using baculovirus infection in order to do structure/function studies. cGMP/cAMP selectivity of the enzyme will be studied using site- directed mutagenesis based on modeled structures of the cGMP-binding sites. The first substrate to be studied will be the kinase itself (autophosphorylation). Attempts will be made to separate the phospho- and dephospho-forms of the enzyme chromatographically to use for these studies. The role of autophosphorylation in autoinhibition of catalysis will be examined. Functional effects of autophosphorylation will also be studied using phosphoprotein phosphatases. 32p incorporation into the enzyme will be determined in intact smooth muscle cells following treatment with agents that elevate cGMP or cAMP. The elements of the autoinhibitory domain will be defined by partial proteolysis and mutagenesis. The second substrate to be studied will be a cGMP binding phosphodiesterase. Based on sequences surrounding the phosphorylation site of this enzyme, synthetic peptides will be used to identify elements that contribute to its potency and specificity for phosphorylation by protein kinases. Whether phosphorylation of this phosphodiesterase activates the enzyme in intact cells will be studied by measuring changes in cGMP in response to cGMP analogs that activate cGMp-dependent protein kinase.