No 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.

We have overexpressed human cardiac troponin-I in Escherichia coli. Initially, protein expression was not detected in the bacterial cell extracts. Systematic deletion of the N-terminal region of the protein generated a series of truncated mutants which were expressed at varying levels in the bacteria. This allowed us to narrow the problem down to the first five codons in the gene sequence. In order to achieve expression at high levels, two base changes were required, in the second and the fourth codons of the cDNA sequence. The codon changes, (Ala2) GCG+GCC and (Gly4) GGG+ GGT, do not alter the coding potential of the DNA. We have also overexpressed the human cardiac isoform of troponin-C. Both proteins were purified using ion-exchange chromatography and have been proved to be biologically active. The recombinant troponin-I was able to bind to a troponin-C affinity column in the presence of 9 M urea in a calcium-dependent manner. The calcium-dependent troponin-I -troponin-C complex between both recombinant proteins was also demonstrated by alkaline-urea gel electrophoresis. In addition, troponin-I inhibited the acto-Sl MgATPase activity ; this inhibition was potentiated by the presence of tropomyosin and was reversed by the addition of troponin-C to the system. Biological activity was also demonstrated in vivo in that the recombinant proteins were able to restore the calcium-dependent force generation to calcium-insensitive skinned muscle fibres.Troponin and tropomyosin, located on the actin thin filament of vertebrate skeletal and cardiac muscles, are responsible for the calcium-dependent regulation of the actomyosin Mg-ATPase activity [l]. Troponin is a complex of three subunits ; troponin-C (Tn-C), the calcium-binding component, troponin-I (Tn-I), the inhibitory subunit, and troponin-T (Tn-T), which locates the troponin complex on the tropomyosin (TM) complex. Tn-C undergoes a conformational change upon the binding of calcium; this is transmitted through the troponin complex, weakening the Tn-I-actin interaction and causing TM to move away from the myosin-binding sites on actin [2]. The result is the formation of actomyosin crossbridges at these sites, the hydrolysis of Mg-ATP and force generation [3, 41. Cardiac and skeletal isoforms of Tn-I are highly similar in their sequences. The cardiac isoform differs from its skeletal counterpart in possessing a 30-33-amino-acid (speciesdependent) N-terminal extension [ 5 ] . This contains the cardiac-specific adjacent phosphorylation sites at Ser23-Ser24 in human and bovine sequences [6, 71. There is good evidence that this phosphorylation is involved in the modulaCorrespondence to I. P. Trayer,

A panel of seven monoclonal antibodies (mAbs) raised against cardiac troponin-I (CdTnI) isolated from canine and human hearts, which have been shown to be cardiac-specific but cross-species reactive [Cummins, B., Aukland, M. L. & Cummins, P. (1987) Amer. Heart J. 113, 1333Ϫ1344], were used in this study. These mAbs were tested against recombinant wild-type and mutant human CdTnI proteins to assess their value as probes for the phosphorylation status of CdTnI. Four mAbs were found to react positively with the recombinant wild-type protein and their epitopes were contained in residues 31Ϫ210 of the human cardiac protein. Two of these mAbs appeared to be directed against the same epitope site within this region. The remaining three mAbs only reacted against the recombinant wild-type protein when it was phosphorylated, showing that these three antibodies were directed against the phosphate group(s) on Ser23 and/or Ser24. In order to investigate this further, a series of single and double mutants of CdTnI were used in which either Ala (to direct the enzymatic phosphorylation) or Asp (to mimic the phosphate group) replaced the Ser23 and/or Ser24. It was found the all three mAbs were able to react with the mono-phosphorylated form of the [Ala23]CdTnI single mutant but not the mono-phosphorylated form of the [Ala24]CdTnI single mutant, showing that they specifically required phosphorylation at Ser24. Experiments with a synthetic peptide composed of residues 1Ϫ29 of human CdTnI confirmed these data. Two of the three phosphorylation-specific mAbs were able to react with mutants containing either two Asp residues replacing Ser23 and Ser24 or one Asp residue instead of Ser24, indicating that a negative charge at position Ser24 is sufficient to invoke a reaction. The other mAb was more specific in that it would only react with CdTnI species with a phosphate group on Ser24.Keywords : troponin-I ; phosphorylation; monoclonal antibody; phosphorylation-specific antibodies; cardiac muscle.Cardiac and skeletal muscle contraction involves the cyclic interaction of myosin crossbridges with actin involving the concomitant hydrolysis of MgATP. Troponin and tropomyosin, located on the actin thin filament, are responsible for the calciumdependent regulation of the contractile process by responding to Correspondence to I. P. Trayer,

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.