Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca2+ affinity of cardiac troponin C

Ren Zhang, JiaJu Zhao, James D. Potter

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Abstract

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 Ca2+ affinity of the single Ca2+-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca2+ 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 Ca2+ dissociation with a reduced Ca2+ 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 Ca2+ 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 Ca2+ sensitivity. The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.

Original languageEnglish
Pages (from-to)30773-30780
Number of pages8
JournalJournal of Biological Chemistry
Volume270
Issue number51
StatePublished - Dec 22 1995
Externally publishedYes

Fingerprint

Troponin C
Phosphorylation
Troponin I
Serine
Muscle
Myocardium
Protein Kinases
Muscle Relaxation
Cyclic AMP-Dependent Protein Kinases
Alanine
Escherichia coli

ASJC Scopus subject areas

  • Biochemistry

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Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca2+ affinity of cardiac troponin C. / Zhang, Ren; Zhao, JiaJu; Potter, James D.

In: Journal of Biological Chemistry, Vol. 270, No. 51, 22.12.1995, p. 30773-30780.

Research output: Contribution to journalArticle

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abstract = "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 Ca2+ affinity of the single Ca2+-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca2+ 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 Ca2+ dissociation with a reduced Ca2+ 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 Ca2+ 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 Ca2+ sensitivity. The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.",
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