Site-specific phosphorylation of myosin binding protein-C coordinates thin and thick filament activation in cardiac muscle

Saraswathi Ponnam; Ivanka Radkova Sevrieva; Yin-Biao Sun; Malcolm Irving; Thomas Kampourakis

doi:https://doi.org/10.1073/pnas.1903033116

Site-specific phosphorylation of myosin binding protein-C coordinates thin and thick filament activation in cardiac muscle

Saraswathi Ponnam, Ivanka Radkova Sevrieva, Yin-Biao Sun, Malcolm Irving, Thomas Kampourakis

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Abstract

The heart's response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Cε (PKCε), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C's multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C's N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKCε, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C's regulatory N-terminal domains in defined phosphorylation states for protein-protein interaction studies with isolated cardiac native thin filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing thick filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the thick filaments and on troponin C in the thin filaments to monitor structural changes in the myofilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function.

Original language	English
Pages (from-to)	15485-15494
Number of pages	10
Journal	Proceedings of National Academy of Sciences USA
Volume	116
Issue number	31
DOIs	https://doi.org/10.1073/pnas.1903033116
Publication status	Published - 30 Jul 2019

Keywords

cardiac muscle regulation
myosin binding protein-C
phosphorylation

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title = "Site-specific phosphorylation of myosin binding protein-C coordinates thin and thick filament activation in cardiac muscle",

abstract = "The heart's response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Cε (PKCε), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C's multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C's N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKCε, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C's regulatory N-terminal domains in defined phosphorylation states for protein-protein interaction studies with isolated cardiac native thin filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing thick filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the thick filaments and on troponin C in the thin filaments to monitor structural changes in the myofilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function.",

keywords = "cardiac muscle regulation, myosin binding protein-C, phosphorylation",

author = "Saraswathi Ponnam and Sevrieva, {Ivanka Radkova} and Yin-Biao Sun and Malcolm Irving and Thomas Kampourakis",

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AU - Ponnam, Saraswathi

AU - Sevrieva, Ivanka Radkova

AU - Sun, Yin-Biao

AU - Irving, Malcolm

AU - Kampourakis, Thomas

PY - 2019/7/30

Y1 - 2019/7/30

N2 - The heart's response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Cε (PKCε), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C's multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C's N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKCε, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C's regulatory N-terminal domains in defined phosphorylation states for protein-protein interaction studies with isolated cardiac native thin filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing thick filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the thick filaments and on troponin C in the thin filaments to monitor structural changes in the myofilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function.

AB - The heart's response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Cε (PKCε), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C's multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C's N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKCε, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C's regulatory N-terminal domains in defined phosphorylation states for protein-protein interaction studies with isolated cardiac native thin filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing thick filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the thick filaments and on troponin C in the thin filaments to monitor structural changes in the myofilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function.

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JO - Proceedings of National Academy of Sciences USA

JF - Proceedings of National Academy of Sciences USA

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ER -

Site-specific phosphorylation of myosin binding protein-C coordinates thin and thick filament activation in cardiac muscle

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