The Effects of Phosphorylation of Cardiac Troponin-I on Its Interactions with Actin and Cardiac Troponin-C

Abstract

It is known that the phosphorylation of two serine residues on the NH2-terminal extension specific to cardiac troponin-I (Tn-I) modulates the calcium-dependent activation of the myofilaments. The process by which this occurs remains an unsolved puzzle. We have applied a dissective approach to study the effect of this phosphorylation on the interactions between Tn-I and its partner proteins, actin and troponin-C (Tn-C). Using N-[14C]ethylmaleimide-labelled Tn-I in sedimentation assays with F-actin, we found that the dephosphorylated Tn-I binds to F-actin with pronounced positive cooperativity, both in the absence and the presence of tropomyosin. Phosphorylation of the protein slightly weakens the interaction in the absence of tropomyosin, but the cooperativity remained. In the presence of tropomyosin, phosphorylation of the Tn-I also appeared to slightly weaken the interaction as well but, more significantly, the cooperativity was eliminated. These data can only be explained simply by a cooperative interaction between the monomer units in the actin filament. The interactions between cardiac Tn-I and Tn-C were studied by labelling the Tn-C with the fluorescent probe dansyl aziridine. As expected, the binding of the dephosphorylated Tn-I to Tn-C was strengthened by over 20-fold upon the addition of calcium to the assay. Phosphorylation of the protein, however, had a dramatic effect on the interaction in that it appeared to desensitise the complex to the effect of calcium: the Ka values obtained for both interactions (+/- Ca2+) were almost identical. These results clearly indicate that the phosphorylation of Tn-I in the cardiac system has dramatic effects on the isolated inter-protein interactions. We also discuss the possible significance of such an effect on the interactions of the isolated proteins in their roles within the intact cardiac regulatory complex.

Bisphosphorylation of cardiac troponin I modulates the Ca2+‐dependent binding of myosin subfragment S1 to reconstituted thin filaments

“…31p_ NMR spectra showed that only phosphate groups present in the bisphosphorylared species interact with another cTn subunit [6]. Recent studies have shown that bisphosphorylation decreases the affinity of cTnI for cTnC and for actin [28]. However, there exist three further cTnI states, namely the two monophosphorylated ones and the dephosphorylated one as well as multiple cTnT states whose functions are still not known to date.…”

Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin‐I mutants and troponin‐C

“…In the heart, phosphorylation of TnI by PKA occurs following fladrenergic stimulation [17] and may be considered as an adaptive mechanism to prevent overstimulation. The phosphorylation of cardiac TnI has been shown to reduce the affinity of TnC for Ca 2+ [18], thus, promoting more rapid relaxation [9]. Different TnI isoforms from various tissues have been overexpressed in bacterial systems [19,20] and a number of mutants have been produced in order to investigate the structur~func-tion relationship in TnI [7,10,[21][22][23].…”

Power Output Is Increased After Phosphorylation of Myofibrillar Proteins in Rat Skinned Cardiac Myocytes

“…Phosphorylation of these residues causes folding of TnI at its N-terminal extension. 30 This conformational change weakens the cooperative binding of TnI to actin-tropomyosin 31 and reduces the affinity of TnI for TnC, especially in the presence of Ca 2ϩ . 32,33 Because the interaction between TnI and TnC increases Ca 2ϩ binding, reduced TnI/TnC binding lowers the affinity of TnC for Ca 2ϩ .…”