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 prot ein 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 ph osphate/mol and by PKC to 2.0 mol phosphate/mol. Tryptic phosphopeptid es from MyBP-C were purified by successive iron iminodiacetate column chromatography and reversed-phase high-performance liquid chromatograp hy, Three phosphopeptides purified from PKA-phosphorylated MyBP-C cont ained phosphoserine [T1, (RTS[P]LAGGGR) and T2, (KRDS[P]FLR)] or phosp hothreonine (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 i dentified in the N-terminus of the cDNA deduced amino acid sequence (S -265, S-300, and T-274, respectively). The PKC-specific site in peptid e T2a was at position S-1169. cDNA clones encoding rat cardiac MyBP-C were isolated, and the segment corresponding to PKA and major PKC phos phorylation sites was sequenced. Chicken cardiac MyBP-C has a threonin e at position 274 (CT3), whereas rat cardiac MyBP-C has a serine at th e corresponding position. Only chicken cardiac MyBP-C had a phosphoryl atable residue at the position corresponding to S-1169, All of the car diac MyBP-C phosphorylation sites are absent in known sequences of ske letal muscle MyBP-C isoforms. (C) 1998 Academic Press.