CAMP DEPENDENT PROTEIN KINASES AS MEDIATORS OF RECEPTOR ACTION

不同的神经递质的不同生化作用， 刺激cAMP产生的激素已经被提出， 通过特定PKA亚型的位点特异性定位。 II型PKA的定位是通过蛋白质-蛋白质 调节亚基（RII）和特异性A激酶之间的相互作用 锚定蛋白（AKAP）。 在过去的资助期间，我们 表明PKA锚定通过蛋白质的相互作用进行。 RII二聚体与AKAP上的两亲性螺旋，阐明了作用 选择AKAP靶向PKA到特定亚细胞 隔室，并证明破坏PKA锚定， 解偶联细胞内的某些cAMP反应事件。 计划 下一个供资期的重点是扩大这些调查范围。 要求1 将通过测量RII上的AKAP结合位点， 通过等离子体共振测定AKAP结合亲和力的变化， 在选定的RII突变体中进行平衡透析， RII片段与AKAP肽复合时的结构 多维核磁共振 目标2将定义过氧化物酶体靶向 AKAP 220上的序列，一种与 过氧化物酶体 目的3将确定PKA定位的操作 通过确定是否正确的PKA改变cAMP反应事件 锚定促进两种生理靶的磷酸化： 转录因子CREB参与调节 基因表达和MAP激酶级联的酶， 调节细胞生长。

The diverse biochemical effects of different neurotransmitters or hormones that stimulate cAMP production have been proposed to occur through the site-specific localization of particular PKA subtypes. Localization of the type II PKA is maintained through protein-protein interactions between the regulatory subunit (RII) and specific A-kinase anchoring proteins (AKAPs). During the past funding period we have demonstrated that PKA anchoring proceeds through interaction of the RII dimer with an amphipathic helix on the AKAP, elucidated the roles of selected AKAPs in targeting PKA to specific subcellular compartments, and demonstrated that disruption of PKA anchoring uncouples certain cAMP-responsive events inside cells. Plans for the next funding period focus on extending these lines of inquiry. Aim 1 will structurally elucidate the AKAP-binding site on RII by measuring changes in AKAP-binding affinity by plasmon resonance and equilibrium dialysis in selected RII mutants and by completing the structure of the RII fragment when complexed to an AKAP peptide by multidimensional NMR. Aim 2 will define the peroxisomal targeting sequence on AKAP220, a novel AKAP which associates with peroxisomes. Aim 3 will establish if manipulation of PKA localization alters cAMP-responsive events by determining whether correct PKA anchoring facilitates the phosphorylation of two physiological targets: the transcription factor CREB which is involved in the regulation of gene expression, and enzymes of the MAP kinase cascade which modulate cell growth.