Identification of sites phosphorylated in bovine cardiac troponin I and troponin T by protein kinase C and comparative substrate activity of synthetic peptides containing the phosphorylation sites

• Published in: The Journal of biological chemistry 1989-12, Vol.264 (34), p.20778-20785
• Main Authors: Noland, T.A., Raynor, R.L., Kuo, J.F.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cattle; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; heart; Kinetics; Molecular Sequence Data; Myocardium - metabolism; Oligopeptides - chemical synthesis; Oligopeptides - metabolism; Peptide Fragments - isolation & purification; Phosphopeptides - isolation & purification; Phosphorylation; protein kinase C; Protein Kinase C - metabolism; Substrate Specificity; Transferases; Troponin - metabolism; Troponin I; Troponin T
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)47130-5

As an extension of our previous reports that cardiac and skeletal muscle troponin I (Tn-I) and troponin T (Tn-T) are excellent substrates for protein kinase C (PKC) (Katoh, N., Wise, B. C., and Kuo, J. F. (1983) Biochem. J. 209, 189-195; Mazzei, G. J., and Kuo, J. F. (1984) Biochem. J. 218, 361-369), we have now determined that PKC phosphorylated serine 43 (and/or serine 45), serine 78, and threonine 144 in the free Tn-I subunit and threonine 190, threonine 199, and threonine 280 in the free Tn-T subunit of bovine cardiac troponin. PKC appeared to phosphorylate the same sites of the subunits present in the form of the troponin complex, as indicated by the similarity in the two-dimensional phosphopeptide maps. Although some of the phosphorylation sites were shared by other classes of protein kinases, PKC exhibited a distinct substrate specificity. It was also noted that phosphorylated serine and threonine residues in Tn-I and Tn-T had neighboring basic amino acid residues separated by 1 or 2 other residues both at the amino and carboxyl termini, in agreement with the conclusion of House et al. (House, C., Wettenhall, R. E. H., and Kemp, B. E. (1987) J. Biol. Chem. 262, 772-777) based upon their studies on other substrate proteins. Several peptides having sequences around the phosphorylating sites have been synthesized. The phosphorylation experiments indicated that these peptides were substrates for PKC, and their relative substrate activity (determined by the ratios of Vmax/Km) compared with other proteins, in descending order, was Tn-I = Tn-I(134-154) > Tn-T ≫ histone H1 > Tn-I(33-35) ≈ Tn-T(268-284) > Tn-T(179-198) ≈ Tn-T(191-209). It is suggested that PKC phosphorylation of Tn-I and Tn-T could be biologically significant in terms of possible modifications in interactions among the individual contractile protein components as well as the Ca2+ sensitivity and activity of actomyosin ATPase.