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

TY - JOUR

T1 - 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

T2 - short communication

AU - Kuster, Diederik W D

AU - Sequeira, Vasco

AU - Najafi, Aref

AU - Boontje, Nicky M

AU - Wijnker, Paul J M

AU - Witjas-Paalberends, E Rosalie

AU - Marston, Steven B

AU - Dos Remedios, Cristobal G

AU - Carrier, Lucie

AU - Demmers, Jeroen A A

AU - Redwood, Charles

AU - Sadayappan, Sakthivel

AU - van der Velden, Jolanda

N1 - Wijnker für: Vrije Univ Amsterdam, Med Ctr, Inst Cardiovasc Res, Dept Physiol, Amsterdam, Netherlands

PY - 2013/2/15

Y1 - 2013/2/15

N2 - 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 3β (GSK3β) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3β showed phosphorylation on Ser133. In addition, GSK3β phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A-induced phosphorylation at Ser304. GSK3β treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment.CONCLUSIONS: GSK3β 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 GSK3β at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

AB - 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 3β (GSK3β) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3β showed phosphorylation on Ser133. In addition, GSK3β phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A-induced phosphorylation at Ser304. GSK3β treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment.CONCLUSIONS: GSK3β 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 GSK3β at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

KW - Amino Acid Sequence

KW - Cardiomyopathy, Dilated

KW - Carrier Proteins

KW - Glycogen Synthase Kinase 3

KW - Heart Ventricles

KW - Humans

KW - Molecular Sequence Data

KW - Myocardial Contraction

KW - Myocardial Ischemia

KW - Peptide Fragments

KW - Phosphorylation

KW - Phosphoserine

KW - Protein Processing, Post-Translational

KW - Protein Structure, Tertiary

KW - Recombinant Proteins

KW - Sarcomeres

KW - Tandem Mass Spectrometry

U2 - 10.1161/CIRCRESAHA.112.275602

DO - 10.1161/CIRCRESAHA.112.275602

M3 - SCORING: Journal article

C2 - 23277198

VL - 112

SP - 633

EP - 639

JO - CIRC RES

JF - CIRC RES

SN - 0009-7330

IS - 4

ER -