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Abstract:
Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a crucial determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C’s multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2A (PP1 and PP2A) and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and protein kinases A (PKA), C and RSK2. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by PKA. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results emphasize the importance of phosphatases for cMyBP-C regulation and prompt us to propose reciprocal relationships between cMyBP-C m-motif conformation, phosphorylation state and myofilament function.
