Phosphorylation of Both Serine Residues in Cardiac Troponin I Is Required to Decrease the Ca Affinity of Cardiac Troponin C

• Published in: The Journal of biological chemistry Vol. 270; no. 51; p. 30773
• Main Authors: Ren Zhang, JiaJu Zhao, James D. Potter
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 22.12.1995
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.51.30773

The phosphorylation of cardiac muscle troponin I (CTnI) at two adjacent N-terminal serine residues by cAMP-dependent protein kinase (PKA) has been implicated in the inotropic response of the heart to β-agonists. Phosphorylation of these residues has been shown to reduce the Ca affinity of the single Ca -specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca dissociation from this site (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J.(1982) J. Biol. Chem. 257, 260-263). Recent studies (Zhang, R., Zhao, J., and Potter, J. D.(1995) Circ. Res. 76, 1028-1035) have correlated this increase in Ca dissociation with a reduced Ca sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation. To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine. The wild type and the mutated CTnIs were expressed in Escherichia coli and purified. Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca sensitivity of force development seen in a skinned cardiac muscle preparation. The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca sensitivity. The results also suggest that one of the serines ( 23 ) may be constitutively phosphorylated and that serine 22 may be functionally more important.