Multiplicity of Regulatory Subunit Conformations Defines Structural Ensemble of Reset Protein Kinase A Holoenzyme

How protein kinase A (PKA) is reset to a basal state
following
3′5′-cyclic adenosine monophosphate (cAMP)-mediated
activation is unknown. Here we describe the mechanism of cAMP-PKA
type I signal 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 for the first time multiple structures
of the reset PKA holoenzyme (RIα2:Cα2) that capture an ensemble of multiple conformational end-states
through integrative electron microscopy and structural mass spectrometry
approaches. Together these complementary methods highlight the large
conformational dynamics of the regulatory subunit (RIα) within
the tetrameric reset PKA holoenzyme. The cAMP-free reset PKA holoenzyme
adopts multiple distinct conformations of RIα with contributions
from the N-terminal linker and CNB-B dynamics. Our findings highlight
the interplay between RIα, Cα, and PDEs (PDE8) in cAMP-PKA
signalosomes to offer a new paradigm for PDE-mediated regulation of
cAMP-PKA signaling.