Department of Biological Sciences, National University of Singapore, Singapore.
Mol Cell Proteomics. 2011 Feb;10(2):M110.002295. doi: 10.1074/mcp.M110.002295. Epub 2010 Oct 5.
Although extensive structural and biochemical studies have provided molecular insights into the mechanism of cAMP-dependent activation of protein kinase A (PKA), little is known about signal termination and the role of phosphodiesterases (PDEs) in regulatory feedback. In this study we describe a novel mode of protein kinase A-anchoring protein (AKAP)-independent feedback regulation between a specific PDE, RegA and the PKA regulatory (RIα) subunit, where RIα functions as an activator of PDE catalysis. Our results indicate that RegA, in addition to its well-known role as a PDE for bulk cAMP in solution, is also capable of hydrolyzing cAMP-bound to RIα. Furthermore our results indicate that binding of RIα activates PDE catalysis several fold demonstrating a dual function of RIα, both as an inhibitor of the PKA catalytic (C) subunit and as an activator for PDEs. Deletion mutagenesis has localized the sites of interaction to one of the cAMP-binding domains of RIα and the catalytic PDE domain of RegA whereas amide hydrogen/deuterium exchange mass spectrometry has revealed that the cAMP-binding site (phosphate binding cassette) along with proximal regions important for relaying allosteric changes mediated by cAMP, are important for interactions with the PDE catalytic domain of RegA. These sites of interactions together with measurements of cAMP dissociation rates demonstrate that binding of RegA facilitates dissociation of cAMP followed by hydrolysis of the released cAMP to 5'AMP. cAMP-free RIα generated as an end product remains bound to RegA. The PKA C-subunit then displaces RegA and reassociates with cAMP-free RIα to regenerate the inactive PKA holoenzyme thereby completing the termination step of cAMP signaling. These results reveal a novel mode of regulatory feedback between PDEs and RIα that has important consequences for PKA regulation and cAMP signal termination.
尽管广泛的结构和生化研究为 cAMP 依赖性蛋白激酶 A（PKA）激活的机制提供了分子见解，但对于信号终止和磷酸二酯酶（PDE）在调节反馈中的作用知之甚少。在这项研究中，我们描述了一种新的蛋白激酶 A 锚定蛋白（AKAP）独立的调节反馈模式，即特定的 PDE RegA 和 PKA 调节（RIα）亚基之间的调节反馈，其中 RIα 作为 PDE 催化的激活剂发挥作用。我们的结果表明，RegA 除了作为溶液中 bulk cAMP 的众所周知的 PDE 之外，还能够水解与 RIα 结合的 cAMP。此外，我们的结果表明，RIα 的结合激活了 PDE 催化作用几倍，表明 RIα 具有双重功能，既是 PKA 催化（C）亚基的抑制剂，又是 PDE 的激活剂。缺失突变已将相互作用的位点定位到 RIα 的一个 cAMP 结合结构域和 RegA 的催化 PDE 结构域上，而酰胺氢/氘交换质谱已显示 cAMP 结合位点（磷酸结合盒）以及对于中继 cAMP 介导的变构变化很重要的近端区域，对于与 RegA 的 PDE 催化结构域的相互作用很重要。这些相互作用的位点以及 cAMP 解离速率的测量表明，RegA 的结合促进了 cAMP 的解离，随后释放的 cAMP 被水解为 5'AMP。作为终产物生成的无 cAMP 的 RIα 仍然与 RegA 结合。然后，PKA C 亚基取代 RegA 并与无 cAMP 的 RIα 重新结合，以再生无活性的 PKA 全酶，从而完成 cAMP 信号终止步骤。这些结果揭示了 PDE 和 RIα 之间的一种新的调节反馈模式，这对 PKA 调节和 cAMP 信号终止具有重要意义。
