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
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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 : short communication. / Kuster, Diederik W D; Sequeira, Vasco; Najafi, Aref; Boontje, Nicky M; Wijnker, Paul J M; Witjas-Paalberends, E Rosalie; Marston, Steven B; Dos Remedios, Cristobal G; Carrier, Lucie; Demmers, Jeroen A A; Redwood, Charles; Sadayappan, Sakthivel; van der Velden, Jolanda.
In: CIRC RES, Vol. 112, No. 4, 15.02.2013, p. 633-9.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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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 -