CYCLIC AMP-DEPENDENT PROTEIN KINASE STRUCTURE-FUNCTION

建议的研究旨在探讨 cAMP依赖性蛋白激酶的催化亚基， 酶的生物活性。 此外，可能的监管 将检测酶的产生。 主要方法将 包括分析催化亚基突变体以确定重要的 分子的功能结构域。 隔离变种人 通过催化亚基cDNA序列的体外诱变来促进。 改变的催化亚基cDNA序列的功能将通过 将被引入哺乳动物细胞的表达载体的构建 细胞 因此，拟议研究的第一个具体目标涉及 分离含有完整编码的催化亚基cDNA克隆 顺序 在初步研究中，已经分离了部分cDNA， cDNA将用作杂交探针以获得全长克隆。 将全长克隆插入表达载体中，所述表达载体将 被引入至少两种cAMP-应答细胞系中用于分析 克隆基因的功能。 表达式的条件之后， cDNA克隆已经确定，cDNA中的随机突变将被 通过亚硫酸氢盐处理引入，分离出突变体， 表征了 基因组催化亚基序列的研究也将在 以确定是否存在多个基因拷贝。 此外，对该基因的内含子-外显子组织的分析可以 提供了一些酶的功能域的见解。 最后 蛋白激酶基因表达的可能调节将通过以下方法进行检测： cAMP处理的细胞和组织中mRNA水平的杂交分析。 这些研究的总体目标是增加对 使cAMP依赖性蛋白激酶参与 信号转导和生物过程的调节。

The proposed studies seek to examine the structural features of the catalytic subunit of cAMP-dependent protein kinase which are necessary for biological activity of the enzyme. In addition, the possible regulation of the production of the enzyme will be examined. The principal approach will involve analysis of catalytic subunit mutants to determine important functional domains of the molecule. The isolation of mutants will be facilitated by in vitro mutagenesis of catalytic subunit cDNA sequences. The function of altered catalytic subunit cDNA sequences will be tested by construction of expression vectors which will be introduced into mammalian cells. Thus, the first specific aim of the proposed studies involves isolation of catalytic subunit cDNA clones containing the complete coding sequence. In preliminary studies a partial cDNA has been isolated and this cDNA will be used as a hybridization probe to obtain a full-length clone. The full-length clone will be inserted in an expression vector which will be introduced into at least two cAMP-responsive cell lines for analysis of the function of the cloned gene. After conditions for expression of the cDNA clone have been determined, random mutations in the cDNA will be introduced by treatment with bisulfite and mutants isolated and characterized. Studies of genomic catalytic subunit sequences will also be performed to determine if there are multiple copies of the gene. Furthermore, analysis of the intron-exon organization of the gene may provide some insight into functional domains of the enzyme. Finally, the possible regulation of protein kinase gene expression will be examined by hybridization analysis of mRNA levels in cAMP treated cells and tissues. The overall goal of these studies is to increase the understanding of the mechanisms which allow cAMP-dependent protein kinase to participate in signal transduction and regulation of biological processes.