Functional Antagonism of Protein Kinase C and A Phosphorylation of Cardiac Myosin Binding Protein-C

Heart muscle systolic and diastolic function is controlled on a beat-to-beat basis by the calcium-dependent activation of the contractile myofilaments but modulated by neurohumoral signaling pathways coupled to the activation of intracellular effector molecules such as protein kinases. Phosphorylation of myofilament regulatory proteins such as cardiac troponin I (cTnI) and cardiac myosin binding protein-C (cMyBP-C) has important regulatory function for the heart by controlling both cardiac inotropy and lusitropy. Sympathetic signaling activates both α- and β-adrenergic receptors on the surface of cardiomyocytes, which leads to an increase in cMyBP-C phosphorylation via protein kinase C (PKC)/D (PKD) and protein kinase A (PKA) signaling, respectively. However, the functional interactions between the PKC/PKD and PKA phosphorylation sites on cMyBP-C have remained uncharacterized. Here, using a combination of site-specific phosphorylation of recombinant N-terminal domains of cMyBP-C and in situ functional assays, we show that the PKC/PKD and PKA phosphorylation sites have antagonistic effects on myofilament activation. PKA phosphorylation on multiple sites in the N-terminal domains of cMyBP-C reduces both its activating and inhibiting effect on myofilament activation in the absence and presence of activator Ca2+, respectively. In contrast, PKC phosphorylation increases myofilament activation and blunts the inhibitory effect of PKA phosphorylation. Our results lead to a new model of phosphoregulation of cMyBP-C with important implications for both health and disease states of the heart.