Cardiac Myosin-Binding Protein C (MyBP-C): Identification of Protein Kinase A and Protein Kinase C Phosphorylation Sites

• Published in: Archives of biochemistry and biophysics 1998-10, Vol.358 (2), p.313-319
• Main Authors: Mohamed, Ali S., Dignam, John David, Schlender, Keith K.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites - drug effects; cardiac muscle; Carrier Proteins - metabolism; Chickens; Cyclic AMP-Dependent Protein Kinases - metabolism; Enzyme Activation; Humans; Hydrolysis; Mice; Molecular Sequence Data; Myocardium - enzymology; Myocardium - metabolism; myofibrils; myosin binding protein-C; Myosins - metabolism; Peptide Mapping; Phosphopeptides - metabolism; Phosphorylation; Protein Kinase C - metabolism; protein phosphorylation; Rats; Sequence Analysis; Trypsin - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1998.0857

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 substratein vivoandin vitrofor 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.