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Updated: May 11, 2025

Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Multiplicity of Regulatory Subunit Conformations Defines Structural Ensemble of Reset Protein Kinase A Holoenzyme
Varun Venkatakrishnan1, Tatiana N Laremore2, Theresa S C Buckley1
1Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
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 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.
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