Dataset Information

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

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 45

Publications

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

Venkatakrishnan Varun V Laremore Tatiana N TN Buckley Theresa S C TSC Armache Jean-Paul JP Anand Ganesh S GS

Journal of the American Chemical Society 20250416 17

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 (RIα<sub>2</sub>:Cα<s ...[more]

PMID: 40241376

Similar Datasets

Project description:Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3’5’cyclic AMP (cAMP)-dependent Protein Kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in Acrodysostosis. How mutations on PDEs and RIα interfere with regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit. PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα:PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: Translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis Step 2) Processivity: Binding of free cAMP from the cytosol at both CNBs of RIα and Step 3) Product (5’AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that trigger subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, (PDE8 T690P) and RIα (T207A) that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and Fluorescence Polarization (FP) reveal that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5’AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5’AMP release from the PDE, T207A greatly slowed release of substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα:PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.

Project description:Besozzi2012 - Oscillatory regimes in the Ras/cAMP/PKA pathway in S.cerevisiae Mechanistic model of the Ras/cAMP/PKA in yeast S.cerevisiae. The Ras/cAMP/PKA pathway plays a major role in the regulation of metabolism, stress resistance and cell cycle progress and is tightly regulated by multiple feedback loops, exerted by the protein kinase A (PKA). This model investigates the dynamics of the second messenger cAMP on Ras/cAMP/PKA pathway, to determine the effects of the feedback mechanisms on establising stable oscillatory regimes. The model has been defined according to the stochastic formulation of chemical kinetics [Gillespie DT, 1977] , which requires to specify the set of molecular species occurring in the pathway and their respective interactions, formally described as a set of biochemical reactions. The volume considered for this system is 30fL; this value can be used to convert the model into the deterministic formulation. This model is described in the article: The role of feedback control mechanisms on the establishment of oscillatory regimes in the Ras/cAMP/PKA pathway in S. cerevisiae. Besozzi D, Cazzaniga P, Pescini D, Mauri G, Colombo S, Martegani E. EURASIP J Bioinform Syst Biol. 2012 Jul 20;2012(1):10. Abstract: In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway is involved in the regulation of cell growth and proliferation in response to nutritional sensing and stress conditions. The pathway is tightly regulated by multiple feedback loops, exerted by the protein kinase A (PKA) on a few pivotal components of the pathway. In this article, we investigate the dynamics of the second messenger cAMP by performing stochastic simulations and parameter sweep analysis of a mechanistic model of the Ras/cAMP/PKA pathway, to determine the effects that the modulation of these feedback mechanisms has on the establishment of stable oscillatory regimes. In particular, we start by studying the role of phosphodiesterases, the enzymes that catalyze the degradation of cAMP, which represent the major negative feedback in this pathway. Then, we show the results on cAMP oscillations when perturbing the amount of protein Cdc25 coupled with the alteration of the intracellular ratio of the guanine nucleotides (GTP/GDP), which are known to regulate the switch of the GTPase Ras protein. This multi-level regulation of the amplitude and frequency of oscillations in the Ras/cAMP/PKA pathway might act as a fine tuning mechanism for the downstream targets of PKA, as also recently evidenced by some experimental investigations on the nucleocytoplasmic shuttling of the transcription factor Msn2 in yeast cells. This model is hosted on BioModels Database and identified by: BIOMD0000000478 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

			Action	DRS
	Ca_state.csv	Csv
	HDX_raw.zip	Other
	PLGS.zip	Other
	R1-alpha-1_tryp_BS3-XL_3of10uL-08.msf	Msf
	R1-alpha-1_tryp_BS3-XL_3of10uL-08.msfView	Msf