C SUBUNITS OF PROTEIN KINASE A

蛋白激酶 A 的 C 亚基

• 批准号: 6386854
• 负责人: Stephen Jack Beebe
• 金额: $ 17.89万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386854
• 关键词: chimeric proteins; cyclic AMP; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; gene expression; genetic transcription; isozymes; molecular cloning; phosphorylation; protein kinase A; protein structure function; tissue /cell culture; transfection

DESCRIPTION: Protein kinases phosphorylate intracellular substrates to regulate cell functions such as gene transcription. Kinase crystal structures indicate that they share similar structures and catalytic mechanisms. The kinase C-subunit of cAMP-dependent protein kinase (PKA) is used as a kinase model. This project proposes to utilize two natural catalytic (C) subunit variants, C-alpha and C-gamma and chimeric mutants of them, to identify structures that specify the regulation of gene transcription. C-alpha and C-gamma are highly homologous (83 percent identity), yet functionally distinct isozymes, thereby providing targets for structure-function differences. Different cAMP-mediated phenotypes are observed in C-gamma- versus C-alpha-expressing stable Kin8 clones. In transiently co-transfected cells, the isozymes exhibit remarkable differences in the regulation of CRE-reporter gene activity. In vitro, the isozymes exhibit differences in kinetics, substrate, and pseudosubstrate specificities. It is hypothesized that the isozyme-specific phenotypes are due to differences between C-alpha and C-gamma in the recognition of substrates and/or pseudosubstrates. The specific aims are: (1) to utilize native and mutant C-gamma and C-alpha isozymes and differences in CRE-reporter gene activity to define kinase structures that determine the regulation of CRE-gene transcription in intact cells; (2) to utilize recombinant C-gamma and C-alpha subunits in vitro to define differences in the isozymes for the recognition of substrates and pseudosubstrate inhibitors; and (3) to determine the specificities of mutants that differ for CRE-gene expression in intact cells.

描述：蛋白激酶磷酸化细胞内底物至 调节细胞功能，例如基因转录。 激酶晶体 结构表明它们具有相似的结构和催化 机制。 CAMP依赖性蛋白激酶（PKA）的激酶C-亚基是 用作激酶模型。 该项目建议利用两个自然 催化（C）亚基变体，C-Alpha和C-Gamma和嵌合突变体的突变体 它们，确定指定基因调节的结构 转录。 C-Alpha和C-Gamma高度同源（83％ 身份），但功能上不同的同工酶，从而为 结构功能差异。 不同的营地介导的表型是 在C-gamma-与C-Alpha表达稳定的KIN8克隆中观察到。 在 瞬时共转染的细胞，同工酶表现出显着的 Cre-Reporter基因活性调节的差异。 体外， 同工酶在动力学，底物和伪基底表现出差异 特殊性。 假设同工酶特异性表型是 由于C-Alpha和C-Gamma之间的差异， 底物和/或伪造物。 具体目的是：（1）使用天然和突变的C-Gamma和C-Alpha 同工酶和Cre-Reporter基因活性的差异以定义激酶 确定完整中Cre-Gene转录调控的结构 细胞； （2）在体外利用重组C-Gamma和C-Alpha亚基 定义识别底物的同工酶的差异和 假基底抑制剂； （3）确定 在完整细胞中Cre-Gene表达不同的突变体。