In cAMP-Protein Kinase A (PKA) signaling, A-kinase anchoring protein scaffolds assemble PKA in close proximity to phosphodiesterases (PDE), kinase-substrates to form signaling islands or ‘signalosomes’. In its basal state, inactive PKA holoenzyme (R2:C2) is activated by binding of cAMP to regulatory (R)-subunits leading to dissociation of active catalytic (C)-subunits. PDEs hydrolyze cAMP-bound to the R-subunits to generate 5′-AMP for termination and resetting the cAMP signaling. Mechanistic basis for cAMP signaling has been derived primarily by focusing on the proteins in isolation. Here, we set out to simulate cAMP signaling activation-termination cycles in a signalosome-like environment with PDEs and PKA subunits in close proximity to each other. Using a combination of fluorescence polarization and amide hydrogen exchange mass spectrometry with regulatory (RIα), C-subunit (Cα) and PDE8 catalytic domain, we have tracked movement of cAMP through activation-termination cycles. cAMP signaling operates as a continuum of four phases: (1) Activation and dissociation of PKA into R- and C-subunits by cAMP and facilitated by substrate (2) PDE recruitment to R-subunits (3) Hydrolysis of cAMP to 5′-AMP (4) Reassociation of C-subunit to 5′-AMP-bound-RIα in the presence of excess ATP to reset cAMP signaling to form the inactive PKA holoenzyme. Our results demonstrate that 5′-AMP is not merely a passive hydrolysis end-product of PDE action. A ‘ligand-free’ state R subunit does not exist in signalosomes as previously assumed. Instead the R-subunit toggles between cAMP- or 5′-AMP bound forms. This highlights, for the first time, the importance of 5′-AMP in promoting adaptation and uncovers adenylate control in cAMP signaling.

中文翻译：

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cAMP信号传导中的腺苷酸控制：对信号小体适应性的影响。

在cAMP-蛋白激酶A（PKA）信号传导中，A激酶锚定蛋白支架将PKA组装在磷酸二酯酶（PDE），激酶底物的附近，从而形成信号岛或“信号体”。在其基础状态下，无活性的PKA全酶（R2：C2）通过将cAMP与调节性（R）-亚基结合而激活，从而导致活性催化（C）-亚基解离。PDE水解与R亚基结合的cAMP，以生成5'-AMP终止并重置cAMP信号。cAMP信号转导的机理基础主要是通过重点研究分离的蛋白质获得的。在这里，我们着手模拟在PDE和PKA亚基彼此接近的类信号体环境中的cAMP信号激活终止循环。使用荧光偏振和酰胺氢交换质谱与调节（RIα），C-亚基（Cα）和PDE8催化域的组合，我们跟踪了cAMP在激活终止循环中的运动。cAMP信号传导是四个阶段的连续体：（1）cAMP激活PKA并使其解离为R和C亚基，并受底物促进（2）PDE募集到R亚基（3）cAMP水解为5'- AMP（4）在过量的ATP存在下C亚基与5'-AMP结合的RIα的重新结合，以重置cAMP信号，形成无活性的PKA全酶。我们的结果表明5'-AMP不仅是PDE作用的被动水解终产物。如先前所假设的，在信号小体中不存在“无配体”状态的R亚基。相反，R-亚基在cAMP-或5'-AMP结合形式之间切换。