FUNCTIONAL ANALYSIS OF CY-SUBUNIT OF CAMP PROTEIN KINASE

CAMP蛋白激酶CY亚基的功能分析

• 批准号: 3304048
• 负责人: Stephen Jack Beebe
• 金额: $ 14.3万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1995-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304048
• 关键词: animal tissue; clone cells; cyclic AMP; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; gene expression; isozymes; molecular cloning; northern blottings; phosphorylation; protein kinase A; protein purification; protein sequence; protein structure function; recombinant DNA; tissue /cell culture; transfection; transfection /expression vector; western blottings

The cAMP-dependent protein kinase holoenzyme (PKA) is composed of a two monomeric catalytic (C) subunits, which are released and activated when cAMP binds to the dimeric regulatory (R) subunit. The PKA C-subunits are prototypes for the kinase enzyme class, which covalently modify several types of protein substrates via a phosphorylation mechanism. One general type of phosphoprotein is rate-limiting enzymes of metabolism, which are modified by phosphorylation. Another general type of phosphoprotein substrate regulates more sustained effects through the regulation of gene transcription. These proteins are either induced by and/or phosphorylated by a PKC C-subunit mediated mechanism. At least three distinct C-subunit isozymes, designated C-alpha, C-beta, and C-gamma, carry out these cAMP- regulated responses. Based on sequence homology, C-gamma is clearly a PKA C-subunit. Recently, a rabbit anti-bovine heart C-subunit antibody was used to recognize 40 KDa C-gamma and C-alpha in vitro translation products. The specific aims of this proposal are designed to express C-gamma, C-beta, and C-alpha in E. Coli and mammalian cells and to characterize and compare them in vitro and in intact cells. The full-length human (h) C-gamma cDNA, as well as C-beta and C-alpha, will be expressed in E. Coli using the pT7-7 vector and the T7 promotor. The cDNAs will also be expressed in PKA deficient (Kin8) Y1 adrenal cells using the vectors pCMV-neo, which contains the cytomegalovirus promotor, and pZEM3-neo, which contains the metallothionine promotor. C-gamma will be analyzed for binding to the regulatory subunit, activation of the holoenzyme and phosphotransferase activity. The C-subunit isozymes will be compared regarding their peptide substrate inhibitor peptide specificity. In addition, C-gamma will be tested for the regulation of intact cell responses by reversion of a steroid synthesis deficiency in a PKA-deficient (Kin8) Y1 adrenal cell line. To test the role of C-gamma in regulating transcription, expression vectors containing the C-gamma coding sequence will be co-transfected with a fusion gene consisting of the enkephalin promotor coupled to a chloramphenicol acetyltransferase reporter gene. These studies should help elucidate potential differences among the C-subunit isozymes and determine if they have specialized roles in regulating specific cell functions.

cAMP依赖性蛋白激酶全酶（PKA）由两个 单体催化（C）亚基，当释放和激活时 cAMP与二聚体调节（R）亚基结合。 PKA C-Subunits是 激酶类的原型，该酶类别共价修改了几个 蛋白质底物的类型通过磷酸化机制。 一个将军 磷蛋白的类型是代谢的限速酶， 通过磷酸化修饰。 另一种一般类型的磷蛋白 底物通过调节基因来调节更持续的效果 转录。 这些蛋白质由和/或磷酸化诱导 通过PKC C-Subunit介导的机制。 至少三个不同的c-subunit 指定为C-Alpha，C-Beta和C-Gamma的同工酶进行这些营地 受管制的响应。 基于序列同源性，C-Gamma显然是PKA c-subunit。 最近，一种兔抗牛心脏C-亚基抗体是 用于在体外翻译产品中识别40 kDa c-gamma和c-alpha。 该提案的具体目的旨在表达C-Gamma，C-Beta， 大肠杆菌和哺乳动物细胞中的c-alpha，并表征和比较 它们在体外和完整的细胞中。 全长人（H）C-Gamma cDNA， 以及C-Beta和C-Alpha，将使用PT7-7在大肠杆菌中表达 向量和T7启动子。 cDNA也将在PKA中表达 缺乏（KIN8）Y1肾上腺细胞使用载体PCMV-NEO，该细胞 包含巨细胞病毒启动子和PZEM3-neo，其中包含 金属氨酸启动子。 C-Gamma将分析与 调节亚基，全酶和磷酸转移酶的激活 活动。 将在其肽上比较C-亚基同工酶 底物抑制剂肽特异性。 此外，C-Gamma将会 测试了通过恢复完整的细胞反应的调节 PKA缺陷型（KIN8）Y1肾上腺细胞中的类固醇合成缺乏症 线。 为了测试C-Gamma在调节转录中的作用，表达 包含C-GAMMA编码顺序的向量将与 由邻链蛋白启动子组成的融合基因 氯霉素乙酰转移酶报告基因。 这些研究应该有助于 阐明C-亚基同工酶之间的潜在差异并确定 如果它们在调节特定细胞功能方面具有专门的作用。