SEQUENTIAL PHOSPHORYLATION OF ADJACENT SERINE RESIDUES ON THE N-TERMINAL REGION OF CARDIAC TROPONIN-I - STRUCTURE-ACTIVITY IMPLICATIONS OF ORDERED PHOSPHORYLATION

We have used NMR spectroscopy to monitor the phosphorylation of a pept ide corresponding to the N-terminal region of human cardiac troponin-I (residues 17-30), encompassing the two adjacent serine residues of th e dual phosphorylation site. An ordered incorporation of phosphate cat alysed by PKA was observed, with phosphorylation of Ser-24 preceding t hat of Ser-23. Diphosphorylation induced a conformational transition i n this region, involving the specific association of the Arg-22 and Se r-24P side-chains, and maximally stabilised when both phosphoserines w ere in the di-anionic form. The results suggest that the second phosph orylation at Ser-23 of cardiac troponin-I is of particular significanc e in the mechanism by which adrenaline regulates the calcium sensitivi ty of the myofibrillar actomyosin Mg-ATPase.