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.
Highlights
Contraction of cardiac muscle is initiated by activation of the actin-containing thin filaments, but is modulated by structural changes in the myosin-containing thick filaments
Multiple conserved phosphorylation sites have been identified in close proximity to each other within cardiac myosin binding protein-C (cMyBP-C)’s regulatory mmotif [13]
Each phosphorylation site is a substrate for a different set of protein kinases, suggesting that cMyBP-C might act as a central signaling hub within the sarcomere, integrating different signaling pathways to control contractile function
Summary
Contraction of cardiac muscle is initiated by activation of the actin-containing thin filaments, but is modulated by structural changes in the myosin-containing thick filaments. Myosin interactions of cMyBP-C’s N terminus are generally associated with an inhibitory effect on contractility, and both structural and functional studies suggest that cMyBP-C stabilizes the thick filament OFF state by tethering myosin head domains to the surface of the thick filament backbone [9, 10]. The results lead to a model of regulation by cMyBP-C phosphorylation through altered affinity of cMyBP-C’s N terminus for thin and thick filaments, as well as their structures and associated regulatory states Impairment of these mechanisms is likely to underlie the functional effects of mutations in filament proteins associated with cardiomyopathy. R.L.M. is a guest editor invited by the Editorial Board
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