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.

Human cardiac myosin–binding protein C restricts actin structural dynamics in a cooperative and phosphorylation-sensitive manner

“…The first three serines were found to be phosphorylated, but no phosphorylation was detected on Ser-311. This specific serine in mouse and rat has been observed to be phosphorylated by others (39, 40) and by results from our lab using three distinct lines of mice (not shown). Human cMyBP-C has not been reported to be phosphorylated in vivo on this site and our in vitro results suggest that it is not a target of PKA.…”

“…In control experiments, we performed LC-MS/MS on PKA-treated C0–C2 to confirm phosphorylation of the four putative target sites (Ser-275, Ser-284, Ser-304, and Ser-311). All of these serines have been demonstrated to be phosphorylated in mouse and rat cMyBP-C (39, 40). The first three serines were found to be phosphorylated, but no phosphorylation was detected on Ser-311.…”

Angiotensin II type 1 receptor and the activation of Myosin Light-Chain Kinase and Protein Kinase C-βII: Mini Review

“…Protein phosphorylation [15] does though appear to be strongly involved as it is generally agreed that it is central to strength of contraction. In the case of MyBP-C and its functional insistence on Mg 2+ -Ca 2+ exchange [6] one might expect the observed change in contraction on its PKA induced phosphorylation [21] but not in FSLH. Kinase action on the troponins does also modulate contraction strength [15], but again coupling with length induced changes has not been directly demonstrated and common PKA or PKC are not involved [1][2][3][4].…”

“…Generally in striated muscle MLCK is Ca 2+ and calmodulin dependent but is also constitutively active, i.e. it has been found to have a variable fractions of its maximal activity in the absence of Ca 2+ and calmodulin [21,24,25]. The variability probably arises with the level of self-phosphorylation reported.…”

MyBP-C: one protein to govern them all

“…The differential binding of cMyBP-C N-terminal regions to both myosin and to actin suggests a very important regulatory role for this protein, shuttling between thick and thin filaments, modulated by phosphorylation/load. In a very recent study, this was investigated at multiple levels to demonstrate that hierarchical phosphorylation imbues specific properties on cMyBP-C. At low phosphorylation levels the myosin heads are released and cMyBP-C can participate in thin filament activation, whereas at higher phosphorylation levels the activation of actin is blunted, facilitating diastole (Ponnam et al 2019). This has added a more nuanced appreciation of earlier data showing high levels of phosphorylation decreased the interaction between cMyBP-C and the thin filament (Previs et al 2016;Weith et al 2012a, b).…”