CYCLIC AMP REGULATION OF PROTEIN SECRETION

cAMP依赖性蛋白激酶（PKA）的激活是 细胞表面受体与腺苷酸环化酶偶联的机制 能够调节细胞功能。 磷酸化的细胞 底物蛋白质，如转录因子、离子通道和 细胞骨架蛋白的催化（C）PKA触发亚基 细胞基因转录、兴奋性和形态学的变化。 在上一个赠款期间，我们的特点PKA调节 AtT-20细胞中的蛋白质分泌和抑制剂的分离cDNA PKA蛋白。 蛋白激酶抑制剂（PKIs）是一种小分子蛋白质 能够与催化（C）亚基有效且特异性地相互作用 PKA抑制细胞蛋白的磷酸化。 这些PKI 蛋白质在进化上是保守的，但它们在调控中的确切作用 对cAMP升高的细胞反应尚不清楚。 最近的工作 支持了PKI蛋白的生理作用。 的 在新的资助期内提出的研究将联合收割机与生物化学， 分子生物学、免疫学和细胞生物学技术， 阐明PKIs在调节细胞对PKA反应中的作用 activation. 具体地，将分离已知PKI的cDNA和基因组克隆。 异构体以及新的PKI异构体。 这些克隆的DNA 用于定量各种组织和细胞中PKI mRNA的水平 通过北方印迹分析和原位杂交技术对10个株系进行鉴定。 DNA会 也可用于产生特异性PKI同种型的抗体， 定量的PKI蛋白在发展过程中，激素后， 刺激，以及在细胞周期中。 编码以下的DNA的转染 PKI异构体和C亚基将用于鉴定C-PKI复合物 并鉴定PKI和C亚基的特异性突变体 这改变了C-PKI交互。 这些突变体沿着着正常的PKI 蛋白质将被用于定义PKI亚基表达对C 亚基稳定性和活性。 最后，C-PKI的作用 对特定细胞反应的相互作用，如基因转录 并测定蛋白质分泌。 这些调查的结果将是详细了解 PKI蛋白在调节细胞对 PKA信号转导通路的激活。 远景目标 这项工作的重点是了解PKA信号的所有组成部分 关键细胞信号转导系统及其调控机制 如蛋白质分泌过程。

Activation of cAMP-dependent protein kinase (PKA) represents the major mechanism by which cell surface receptors coupled to adenylate cyclase are able to regulate cellular functions. The phosphorylation of cellular substrate proteins such as transcription factors, ion channels, and cytoskeletal proteins by the catalytic (C) subunit of PKA triggers changes in cellular gene transcription, excitability, and morphology,. During the previous grant period we characterized PKA regulation of protein secretion in AtT-20 cells and isolated cDNAs for an inhibitor protein of PKA. The protein kinase inhibitors (PKIs) are small proteins able to potently and specifically interact with the catalytic (C) subunit of PKA to inhibit the phosphorylation of cellular proteins. These PKI proteins are evolutionarily conserved but their exact role in regulated cellular responses to elevation of cAMP is not understood. Recent work in the area has supported a physiological role for the PKI proteins. The research proposed for the new grant period will combine biochemical, molecular biological, immunological and cell biological techniques to elucidate the role of PKIs in regulating cellular responses to PKA activation. Specifically, cDNA and genomic clones will be isolated for the known PKI isoforms as well as for novel PKI isoforms. These cloned DNAs will be used to quantitate the level of PKI mRNA in various tissues and cell lines by Northern blot analysis and in situ hybridization. The DNAs will also be used to generate antibodies to the specific PKI isoforms for quantitation of the PKI proteins during development, after hormonal stimulation, and during the cell cycle. Transfection of DNAs coding for the PKI isoforms and C subunit will be used to identify C-PKI complexes formed in vivo and to identify specific mutants of both PKI and C subunit which alter C-PKI interactions. These mutants along with the normal PKI proteins will be used to define effect of PKI subunit expression on C subunit stability and activity. Finally, the effects of C-PKI interactions on specific cellular responses such as gene transcription and protein secretion will be determined. The result of these investigations will be a detailed understanding of the role of PKI proteins in modulating the cellular response to activation of the PKA signal transduction pathway. The long-range goal of this work is to understand all of the components of the PKA signal transduction system and their mechanisms of control of key cellular processes such as protein secretion.