Effects of protein kinase A phosphorylation on signaling between cardiac troponin I and the N-terminal domain of cardiac troponin C.

During beta-adrenergic stimulation of the heart, there is a decrease in myofilament Ca2+ sensitivity mediated by the protein kinase A-(PKA-) induced phosphorylation of troponin I (cTnI). Phosphorylation, which occurs at Ser 23 and Ser 24 in an amino-terminal extension unique to cTnI, decreases the Ca2+ affinity of the amino-terminal regulatory site of cardiac troponin C (cTnC). In view of the antiparallel organization of the cTnI-cTnC complex [Krudy, G. A., Kleerekoper, Q., Guo, X., Howarth, J. W., Solaro, R. J., and Rosevear, P. R. (1994) J. Biol. Chem. 269, 23731-23735], it is not clear how the phosphorylation signal at one end of the complex affects the Ca2+ binding site at the other end. To address this question, we probed the interaction between cTnI and cTnC fragments, cTnC1-89 and cTnC90-162 (recombinant peptides corresponding to the N- and C-domains of cTnC). cTnI-Cys 5 mutant (S5C/C81I/C98S) and cTnC1-89 were fluorescently labeled with IAANS. When cTnI was phosphorylated, the affinity of Ca2+ for the cTnI-cTnC1-89 complex decreased significantly as indicated by a shift in the pCa50 value from 6.65 to 5.25. Upon phosphorylation, the affinity of cTnI for cTnC1-89 decreased by 3.8-fold in the absence of Ca2+ and 1.7-fold in the presence of Ca2+. In contrast to the case with full-length cTnC, neither cTnC1-89 nor cTnC90-162 induced significant structural changes in cTnI-Cys 5 as determined from intersite distance measurements between Cys 5 and Trp 192. Moreover, neither fragment of cTnC could significantly restore Ca2+ regulation of force generation, when exchanged into fiber bundles from which cTnC had been extracted. Our findings indicate that the transduction of PKA-induced phosphorylation signal from cTnI to the regulatory site of cTnC involves a global change in cTnI structure.