title: 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.
abstract: BACKGROUND
  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.
  
  
  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.
authors.affiliation: Cell and Molecular Physiology, Health Science Division, Loyola University of Chicago, 2160 First Ave, Bldg 102, Room 4637, Maywood, IL 60153, USA. dkuster@lumc.edu
authors.forename: Diederik W D
authors.initials: DW
authors.lastname: Kuster
authors.forename: Vasco
authors.initials: V
authors.lastname: Sequeira
authors.forename: Aref
authors.initials: A
authors.lastname: Najafi
authors.forename: Nicky M
authors.initials: NM
authors.lastname: Boontje
authors.forename: Paul J M
authors.initials: PJ
authors.lastname: Wijnker
authors.forename: E Rosalie
authors.initials: ER
authors.lastname: Witjas-Paalberends
authors.forename: Steven B
authors.initials: SB
authors.lastname: Marston
authors.forename: Cristobal G
authors.initials: CG
authors.lastname: Dos Remedios
authors.forename: Lucie
authors.initials: L
authors.lastname: Carrier
authors.forename: Jeroen A A
authors.initials: JA
authors.lastname: Demmers
authors.forename: Charles
authors.initials: C
authors.lastname: Redwood
authors.forename: Sakthivel
authors.initials: S
authors.lastname: Sadayappan
authors.forename: Jolanda
authors.initials: J
authors.lastname: van der Velden
mesh_terms: D000595:Amino Acid Sequence; D002311:Cardiomyopathy, Dilated / Q000378:metabolism / Q000473:pathology; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006352:Heart Ventricles / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009200:Myocardial Contraction / Q000502:physiology*; D017202:Myocardial Ischemia / Q000378:metabolism / Q000473:pathology; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012518:Sarcomeres / Q000502:physiology; D053719:Tandem Mass Spectrometry; D000099163:Myosin Binding Protein C
chemical_list: D002352:Carrier Proteins; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D010446:Peptide Fragments; D010768:Phosphoserine; D011994:Recombinant Proteins; D000099163:Myosin Binding Protein C; C000605506:GSK3B protein, human