CYCLIC AMP-DEPENDENT PROTEIN KINASE STRUCTURE-FUNCTION

建议进行的研究旨在探讨 CAMP依赖的蛋白激酶催化亚单位 该酶的生物活性。此外，可能对 将检查该酶的生产情况。主要的方法是 包括分析催化亚单位突变以确定重要的 分子的功能结构域。突变株的分离将是 催化亚单位cDNAs序列的体外诱变。 改变的催化亚单位cDNA序列的功能将通过以下方法进行测试 导入哺乳动物的表达载体的构建 细胞。因此，拟议研究的第一个具体目标包括 含完整编码的催化亚单位cDNA克隆的分离 序列。在初步研究中，已分离出部分cdna，这 将其作为杂交探针，获得全长克隆。 全长克隆将被插入到表达载体中，该载体将 被导入至少两个cAMP反应细胞系用于分析 克隆的基因的功能。表示的后缀条件 C DNA克隆已确定，将随机突变的c DNA 通过亚硫酸氢盐处理和分离到的突变株导入 特色化的。对基因组催化亚单位序列的研究也将 以确定是否存在该基因的多个副本。 此外，对基因的内含子-外显子组织的分析可能 提供对酶的功能域的一些洞察。最后， 蛋白激酶基因表达的可能调节将通过以下方式进行研究 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 允许cAMP依赖的蛋白激酶参与的机制 生物过程的信号转导和调节。

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.