Multiplicity of regulatory subunit conformations define structural ensemble of reset Protein Kinase A holoenzyme

ABSTRACT: 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 signal termination leading to 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 holoenzyme that capture an ensemble of conformational end-states through the integration of electron microscopy with structural mass spectrometry.Together, integrative cryo-EM with mass spectrometry showcases the high domain-specific dynamics of the regulatory subunit (RIα) and their interactions with Cα. Our integrated structure and dynamics approaches reveal that the tetrameric cAMP-free reset holoenzyme adopts multiple, distinct conformations of the RIα with contributions from the N-terminal linker and CNB-B domains. Our findings highlight the interplay between RIα, Cα and PDEs (PDE8) in signalosomes and the impact of substrate and nucleotides and offer a new paradigm for PDE-mediated regulation of cAMP-PKA signaling.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: Varun Venkatakrishnan  

LAB HEAD: Ganesh Srinivasan

PROVIDER: PXD061315 | Pride | 2025-07-14

REPOSITORIES: Pride