Cardiac myosin binding protein-C phosphorylation as a function of multiple protein kinase and phosphatase activities

Thomas Kampourakis; Saraswathi Ponnam; Kenneth Campbell; Austin Wellette-Hunsucker; Daniel Koch

doi:10.1038/s41467-024-49408-5

Cardiac myosin binding protein-C phosphorylation as a function of multiple protein kinase and phosphatase activities

Thomas Kampourakis, Saraswathi Ponnam, Kenneth Campbell, Austin Wellette-Hunsucker, Daniel Koch

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C’s multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2 A (PP1 and PP2A), and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and various protein kinases known to phosphorylate cMyBP-C. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by potein kinase A. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results suggest non-redundant roles for PP1 and PP2A under both physiological and heart failure conditions, and emphasize the importance of phosphatases for cMyBP-C regulation.

Original language	English
Article number	5111
Journal	Nature Communications
Volume	15
Issue number	1
DOIs	https://doi.org/10.1038/s41467-024-49408-5
State	Published - Dec 2024

Bibliographical note

Publisher Copyright:
© The Author(s) 2024.

Funding

We would like to thank Aneta Koseska for helpful feedback on earlier versions of the manuscript, Frank Bergmann for help with the basico package, and Shodhan Rao for help with the derivation of the tQSSA rate law. LC-MS/MS analysis was performed by the University of York\u2019s Bioscience Technology Facility, Metabolomics and Proteomics Laboratory, Department of Biology, University of York, UK, using instrumentation within the York Center of Excellence in Mass Spectrometry (CoEMS). The York CoEMS was created thanks to a major capital investment through Science City York, supported by Yorkshire Forward with funds from the Northern Way Initiative, and subsequent support from EPSRC (EP/K039660/1; EP/M028127/1). We acknowledge the British Heart Foundation (grant nr: FS/16/3/31887, T.K.) and the European Molecular Biology Organization (grant nr: ALTF 310-2021, D.K.) for financial support.

Funders	Funder number
Northern Way Initiative
York University, New York, New York, USA bbbUniversity of Rochester, Rochester, New York
Engineering and Physical Sciences Research Council	EP/K039660/1, EP/M028127/1
Engineering and Physical Sciences Research Council
British Heart Foundation	FS/16/3/31887
British Heart Foundation
European Molecular Biology Organization	ALTF 310-2021
European Molecular Biology Organization

ASJC Scopus subject areas

General Chemistry
General Biochemistry, Genetics and Molecular Biology
General Physics and Astronomy

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abstract = "Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C{\textquoteright}s multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2 A (PP1 and PP2A), and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and various protein kinases known to phosphorylate cMyBP-C. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by potein kinase A. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results suggest non-redundant roles for PP1 and PP2A under both physiological and heart failure conditions, and emphasize the importance of phosphatases for cMyBP-C regulation.",

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AU - Ponnam, Saraswathi

AU - Campbell, Kenneth

AU - Wellette-Hunsucker, Austin

AU - Koch, Daniel

N1 - Publisher Copyright: © The Author(s) 2024.

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N2 - Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C’s multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2 A (PP1 and PP2A), and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and various protein kinases known to phosphorylate cMyBP-C. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by potein kinase A. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results suggest non-redundant roles for PP1 and PP2A under both physiological and heart failure conditions, and emphasize the importance of phosphatases for cMyBP-C regulation.

AB - Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C’s multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2 A (PP1 and PP2A), and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and various protein kinases known to phosphorylate cMyBP-C. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by potein kinase A. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results suggest non-redundant roles for PP1 and PP2A under both physiological and heart failure conditions, and emphasize the importance of phosphatases for cMyBP-C regulation.

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Cardiac myosin binding protein-C phosphorylation as a function of multiple protein kinase and phosphatase activities

Abstract

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