How protein kinase A (PKA) is reset to a resting, inactive state subsequent to 3'5'-cyclic adenosine monophosphate (cAMP)-mediated activation is unknown. The PKA regulatory subunits (RIα) mediate high-affinity interactions with cAMP (K d ∼1nM), thus precluding a spontaneous dissociation mechanism thereby functioning as a barrier to signal termination and reset. We have discovered the mechanism of cAMP-PKA type I termination leading to a reset of PKA by holoenzyme formation through the obligatory action of phosphodiesterases (PDEs). We report a catalytic subunit (Cα)-assisted mechanism for the reset of type I PKA and describe multiple structures of the reset PKA holoenzyme (RIα 2 :Cα 2 ) that capture an ensemble of multiple conformational end-states through integrative electron microscopy (EM) and structural mass spectrometry (MS) approaches. Together these complementary methods highlight the large conformational dynamics of RIα within the tetrameric reset PKA holoenzyme. Native MS revealed that PDEs are obligatory for formation of the ∼180 kDa heterotetrameric PKA holoenzyme complex. Pulse-labeling hydrogen/deuterium exchange MS(HDXMS) together with kinetics experiments revealed that Cα promoted dissociation of bound cAMP from RIα facilitating the subsequent cAMP hydrolysis by PDEs by a rapid process ( t = 4 min) thus highlighting the coordination between Cα and PDEs in resetting PKA. Cryo-EM of the end-state reset holoenzyme captured a 4.2Å resolution structure identifying a stable RIα CNB-A:Cα complex with the CNB-B (cAMP-binding domain B) adopting multiple conformations. Negative stain EM, crosslinking MS and HDXMS together revealed that each RIα 1 :Cα 1 chain was independently positioned attributable to N-terminal linker dynamics resulting in a dynamic holoenzyme with a broad spatial length spanning 160-320 Å. Overall, the cAMP-free reset PKA holoenzyme adopts multiple distinct conformations of RIα with contributions from the linker and CNB-B dynamics. Our findings highlight the interplay between RIα, Cα, and PDEs (PDE4 and PDE8) in cAMP-PKA signalosomes to offer a new paradigm for PDE-mediated regulation of cAMP-PKA signaling.
Venkatakrishnan et al. (Sun,) studied this question.