THE UNIQUE AMINO-TERMINAL PEPTIDE OF CARDIAC TROPONIN-I REGULATES MYOFIBRILLAR ACTIVITY ONLY WHEN IT IS PHOSPHORYLATED

Protein kinase A (PKA) dependent phosphorylation of C-protein and card iac troponin I (cTnI) is known to be associated with a reduced sensiti vity to Ca2+. We have investigated the relative importance of each of these sites of phosphorylation in this effect by use of extraction/rec onstitution experiments and mutagenesis of recombinant cTnI. Condition s developed for extraction of troponin (Tn) complex also resulted in e xtraction of C-protein. A truncated cTnI (cTnI/NH2) lacking the 32 ami no acids in the unique amino terminal extension of cTnI was engineered and expressed. In contrast to native cTnI, cTnI/NH2, which lacks Ser( 23) and Ser(24), was not phosphorylated by PKA either in pure form or after incorporation into the myofilament lattice. The relation between pCa(-log molar free Ca2+ concentration) and MgATPase activity of non- phosphorylated native myofibrils or non-phosphorylated myofibrils reco nstituted with cTnI, but lacking C-protein, was the same and could not be distinguished from that of control or PKA-treated myofibrils into which we exchanged cTnI with cTnI/NH2. However, PKA-dependent phosphor ylation of either native myofibrils or reconstituted myofibrils contai ning cTnI but lacking C-protein resulted in an identical and significa nt rightward shift of pCa(50) (half-maximally activating pCa) in the p Ca-activity relationship. Our results strongly indicate that phosphory lation of cTnI at Ser residues in the unique amino terminal extension of the molecule is both necessary and sufficient for the decrease in m yofilament Ca2+-sensitivity associated with PKA-dependent phosphorylat ion. (C) 1995 Academic Press Limited