Identification and Characterization of a New Phosphorylation Site on Cardiac Myosin Binding Protein C

During recent years it has become increasingly evident that cardiac myosin binding protein C (cMyBP-C) exerts an important role in regulation of sarcomere function with consequences for in vivo cardiac performance. The functional role of cMyBP-C is tightly regulated by kinase-mediated phosphorylation, of which protein kinase A (PKA)-mediated phosphorylation is important during cardiac stress and exercise. To date, three serines have been identified on which cMyBP-C can be phosphorylated in vivo, all of which are located in the cardiac isoform specific M region, i.e. Ser273, Ser282 and Ser 302. However, previous experiments have indicated that at least one more phosphorylation site should exist in humans. The aim of the present study was to identify the unknown phosphorylation site on human cMyBP-C. cMyBP-C was semi-purified from human non-failing donor and end-stage failing cardiac tissue. Tandem mass spectrometry was used to identify phosphorylation sites. Besides the already known Ser282 site, a phosphorylation site was detected on Ser133 in donor tissue. For characterization of this site, a phospho-specific antibody was developed. Levels of Ser133 phosphorylation are lower in end-stage heart failure samples as well as in samples from hypertrophic and dilated cardiomyopathy patients compared to donor. Initial experiments suggest that unlike the three previously known phosphorylation sites, Ser133 is not phosphorylated by PKA.In conclusion, we identified a fourth phosphorylation site (Ser133) in human cMyBP-C, which is present in the Pro-Ala rich region that links the C0 and C1 domains. This site is less phosphorylated in various cardiac pathologies and does not seem to be a target of PKA.Supported by the 7th Framework Program of the European Union (“BIG-HEART,” grant agreement 241577).