Cardiac myosin-binding protein C (MyBP-C): identification of protein kinase A and protein kinase C phosphorylation sites.
Myosin binding protein C (MyBP-C) is a major myofibril-associated protein in cardiac muscle which is subject to reversible phosphorylation. Cardiac MyBP-C is a substrate in vivo and in vitro for cAMP-dependent protein kinase (PKA) and calcium/phospholipid-dependent protein kinase (PKC). Chicken cardiac MyBP-C was phosphorylated by PKA to 3.0 mol phosphate/mol and by PKC to 2.0 mol phosphate/mol. Tryptic phosphopeptides from MyBP-C were purified by successive iron iminodiacetate column chromatography and reversed-phase high-performance liquid chromatography. Three phosphopeptides purified from PKA-phosphorylated MyBP-C contained phosphoserine [T1, (RTS[P]LAGGGR) and T2, (KRDS[P]FLR)] or phosphothreonine (CT3, MT[P]SAFL). PKC phosphorylated two of the same sites (T1 and T2) as PKA and an additional site [T2a (TGTTYKPPS[P]YK)]. PKA phosphorylation sites corresponding to peptides T1, T2, and T3 were identified in the N-terminus of the cDNA deduced amino acid sequence (S265, S300, and T274, respectively). The PKC-specific site in peptide T2a was at position S1169. cDNA clones encoding rat cardiac MyBP-C were isolated, and the segment corresponding to PKA and major PKC phosphorylation sites was sequenced. Chicken cardiac MyBP-C has a threonine at position 274 (CT3), whereas rat cardiac MyBP-C has a serine at the corresponding position. Only chicken cardiac MyBP-C had a phosphorylatable residue at the position corresponding to S1169. All of the cardiac MyBP-C phosphorylation sites are absent in known sequences of skeletal muscle MyBP-C isoforms.
Department of Pharmacology, Department of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, Ohio, 43614-5804, USA.
A S
AS
Mohamed
J D
JD
Dignam
K K
KK
Schlender
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002645:Chickens; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology* / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism; D000099163:Myosin Binding Protein C
D002352:Carrier Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D009218:Myosins; D010748:Phosphopeptides; D011493:Protein Kinase C; D014357:Trypsin; D000099163:Myosin Binding Protein C