CYCLIC AMP REGULATION OF PROTEIN SECRETION

CAMP依赖的蛋白激酶(PKA)的激活代表了 细胞表面受体与腺苷环化酶偶联的机制 能够调节细胞功能。细胞的磷酸化 底物蛋白质，如转录因子、离子通道和 细胞骨架蛋白由PKA催化(C)亚单位触发 细胞基因转录、兴奋性和形态的变化。 在之前的赠款期间，我们将PKA法规描述为 AtT-20细胞的蛋白质分泌和抑制物的分离cDNA PKA蛋白。蛋白激酶抑制物(PKI)是一种小分子蛋白质 能够与催化(C)亚单位有效和特异地相互作用 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 细胞对cAMP升高的反应尚不清楚。近期工作 在该地区已经支持了PKI蛋白的生理作用。这个 为新的资助期提出的研究将结合生化， 分子生物学、免疫学和细胞生物学技术 阐明PKI在调节细胞对PKA反应中的作用 激活。 具体地说，将分离已知PKI的cdna和基因组克隆。 亚型以及新的PKI亚型。这些克隆的DNA将被 用于定量检测不同组织和细胞中PKI基因的表达水平 经Northern印迹分析和原位杂交鉴定。DNA将会 也可用于产生针对特定PKI亚型的抗体 激素作用后发育过程中PKI蛋白的定量研究 刺激，以及在细胞周期中。编码基因的DNA的转染 将使用PKI异构体和C亚基来鉴定C-PKI复合体 在体内形成并鉴定PKI和C亚基的特异性突变体 从而改变C-PKI的相互作用。这些突变体以及正常的PKI 蛋白质将被用来确定PKI亚单位表达对C 亚基的稳定性和活性。最后，C-PKI的作用 对特定细胞反应的相互作用，如基因转录 蛋白质的分泌将会被确定。 这些调查的结果将是对 PKI蛋白在调节细胞免疫应答中的作用 激活PKA信号转导通路。远景目标 这项工作的关键是理解PKA信号的所有分量 关键细胞的转导系统及其调控机制 蛋白质分泌等过程。

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.