GSK3β Phosphorylates Newly Identified Site in the Proline-Alanine-Rich Region of Cardiac Myosin-Binding Protein C and Alters Cross-Bridge Cycling Kinetics in Human

Rationale: Cardiac myosin-binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C. Objective: To identify and characterize additional phosphorylation sites in human cMyBP-C. Methods and Results: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine-rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3 beta (GSK3 beta) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3 beta showed phosphorylation on Ser133. In addition, GSK3 beta phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A-induced phosphorylation at Ser304. GSK3 beta treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. Conclusions: GSK3 beta phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine-rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3 beta at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics. (Circ Res. 2013; 112:633-639.)