Krishnamurthy Srinath, Moorthy Balakrishnan Shenbaga, Xin Xiang Lim, Xin Shan Lim, Bharatham Kavitha, Tulsian Nikhil Kumar, Mihalek Ivana, Anand Ganesh S
Department of Biological Sciences, National University of Singapore, Singapore; Mechanobiology Institute, National University of Singapore, Singapore.
Department of Biological Sciences, National University of Singapore, Singapore.
Biophys J. 2014 Sep 16;107(6):1426-40. doi: 10.1016/j.bpj.2014.07.050.
Cyclic 3'5' adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.
环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）信号传导是介导细胞对激素刺激反应的基本调节途径。该途径通过cAMP与PKA调节亚基的高亲和力结合而被激活，当cAMP从PKA解离时信号终止。尽管激活阶段的步骤已被充分理解，但对于信号终止/重置如何发生却知之甚少。由于cAMP与PKA的亲和力很高（KD约为低纳摩尔），结合的cAMP不易从PKA上解离，因此就产生了一个问题，即紧密结合的cAMP如何从PKA上释放出来以重置其信号状态，从而对后续刺激做出反应。最近的研究表明，磷酸二酯酶（PDEs）可以催化结合的cAMP解离，从而在cAMP信号脱敏/终止中发挥积极作用。这是通过与PKA调节亚基的直接相互作用实现的，从而促进cAMP的解离和水解。在本研究中，我们通过酰胺氢/氘交换质谱、肽阵列和计算对接相结合的方法，绘制了哺乳动物cAMP信号传导中特定环核苷酸磷酸二酯酶（PDE8A）与PKA调节亚基（RIα亚型）之间的直接相互作用。通过肽阵列和氢/氘交换质谱探测的PDE8A:RIα复合物的相互作用界面，汇集了跨越磷酸二酯酶活性位点和RIα的cAMP结合位点的区域。计算对接与酰胺氢/氘交换质谱相结合，提供了一个模型，用于从RIα的两个串联cAMP结合结构域平行解离结合的cAMP。活性位点偶联表明底物通道在依赖PDE的cAMP与PKA结合的解离和水解中起作用。据我们所知，这是PDEs在哺乳动物系统中直接与cAMP受体蛋白相互作用的第一个实例，并突出了RIα的一类全新的结合伙伴。这项研究还强调了结构质谱与计算对接相结合在绘制瞬时信号蛋白复合物动力学方面的应用。总之，这些结果揭示了磷酸二酯酶在调节微结构域中cAMP的局部浓度和信号重置方面的新的关键作用。
