CAMP-DEPENDENT PROTEIN KINASE AND GENE EXPRESSION

8-Cl-cAMP的显著生长抑制作用与 其选择性结合和激活cAMP依赖性蛋白激酶 (PKA)同工酶：以高亲和力与RII调节亚基结合 对位点B具有低亲和力，使II型PKA保持在 全酶形式，同时以适度高的亲和力结合 位点A和位点B均与RI调节亚基连接， RI亚基的解离和I型PKA的下调。的 生长抑制和分化的细胞事件 由8-Cl-cAMP诱导的癌细胞包括快速核转位 RII-β的这种易位进入细胞核 与癌细胞中转录因子的增加相关 其特异性结合cAMP反应元件（CRE）。RI和RII 通过自身磷酸化和核转位来区分 特性. RII在蛋白水解位点具有自磷酸化位点， 敏感铰链区周围的R和C相互作用的网站，而RI有 假磷酸化位点。RII而不是RI含有核 定位信号，K K R K。RII-β的过表达通过一个 在几种癌细胞系中的表达载体导致了惊人的 PKA同工酶分布、生长停滞、分化和 逆变换生长抑制和逆转 与RII-β的核转位相关，因为突变的RII-β 不能转移到细胞核的β不能 诱导反向转化。cAMP通过PKA的激活， 参与真核细胞的转录调控。在这 作用机制，cAMP不改变CRE的结合亲和力- 结合蛋白质的CRE。相比之下，cAMP增强CRE结合， 原核细胞中的分解代谢物阻遏蛋白（CAP）， 调节几个操纵子的转录。结构性 CAP和PKA RII亚基cAMP结合位点的相似性 提示RII在真核基因中可能具有类似的作用， 调控在这里，我们报告说，RII-β亚基PKA是一个 能够在物理和功能上相互作用的转录因子 在CRE。与CREB/ATF相反，RII-β与CRE的结合 cAMP增强，此外，RII-β表现出 在一些实施方案中，本发明提供了作为Gal 4-RII-β融合蛋白的转录活性。这些 实验确定RII-β是替代途径的组成部分， 用于调节真核细胞中的CRE指导的转录。

The striking growth inhibitory effect of 8-Cl-cAMP has been related to its selective binding and activation of cAMP-dependent protein kinase (PKA) isozymes: It binds to RII regulatory subunit with a high affinity for Site B but with a low affinity for Site A, keeping type II PKA in the holoenzyme form, while binding with moderately high affinity for both Site A and Site B to RI regulatory subunit, facilitating dissociation of the RI subunit and down-regulation of type I PKA. The cellular events underlying growth inhibition and differentiation of cancer cells induced by 8-Cl-cAMP include a rapid nuclear translocation of RII-beta, and such translocation of RII-beta into the nucleus correlates with an increase in transcription factors in cancer cells that bind specifically to cAMP response element (CRE). The RI and RII are distinguished by their autophosphorylation and nuclear translocation properties. RII has an autophosphorylation site at a proteolytically sensitive hinge region around the R and C interaction site while RI has a pseudo-phosphorylation site. The RII but not the RI contains a nuclear location signal, K K R K. Overexpression of the RII-beta through an expression vector in several cancer cell lines results in a striking shift in PKA isozyme distribution, growth arrest, differentiation, and reverse transformation. The growth inhibition and reverse transformation correlates with nuclear translocation of RII-beta, as the mutant RII- beta which fails to translocate into the nucleus is incapable of inducing reverse transformation. cAMP through the activation of PKA, is involved in transcriptional regulation in eukaryotic cells. In this mechanism of action, cAMP does not alter the binding affinity of CRE- binding proteins to the CRE. In contrast, cAMP enhances the CRE-binding of the catabolite repressor protein (CAP) in prokaryotic cells and regulates the transcription of several operons. The structural similarity of the cAMP-binding sites in CAP and RII subunit of PKA suggests the possibility of a similar role for RII in eukaryotic gene regulation. Here we report that RII-beta subunit of PKA is a transcription factor capable of interacting physically and functionally with the CRE. In contrast to CREB/ATF, the binding of RII-beta to a CRE was enhanced by cAMP, and in addition, RII-beta exhibited transcriptional activity as a Gal4-RII-beta fusion protein. These experiments identify RII-beta as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.