H+ IS INVOLVED IN THE ACTIVATION OF CALCINEURIN BY CALMODULIN

We have studied recently the surface hydrophobic properties of several calmodulin (CaM) target enzymes and found that a certain amphipathic domain(s) of the enzyme was exposed in response to a H+ increase withi n the neutral pH range. The exposed domain appeared to be related to t heir CaM-binding domains and associated with their activation by CaM ( Huang, S., Carlson, G. M., and Cheung, W,Y. (1994) J. Biol. Chem. 269, 7631-7638). In this paper, the involvement of H+ in the activation of calcineurin (CaN), one of the CaM target enzymes, was further studied . Using dansylated CaM to monitor its conformational change, we found that the binding of Ca2+ to CaM occurred at a lower range of Ca2+ conc entrations (pCa from 7 to 6) than the formation of CaM CaN complex (pC a from 6 to 5). However, addition of H+ within the neutral pH range sh ifted the formation of CaM CaN complex to the lower range of Ca2+ conc en trations. Similarly, the addition of H+ shifted the Ca2+ requiremen t for enzyme activation to a lower Ca2+ range. These results show that the interaction of CaM with CaN could be initiated either by Ca2+ or by H+ in the presence of suboptimal Ca2+, suggesting a positive cooper ative effect between Ca2+ and H+. The CaM-stimulated activity of CaN w as accompanied by a time-dependent autoinactivation. Kinetic analysis showed that the autoinactivation initiated by Ca2+ or by H+ was biphas ic and that the decay rate constants were comparable, In heavy water ( D2O), the Ca2+-induced formation of CaM CaN complex as well as the act ivity of CaN were decreased, indicating a solvent deuterium isotope ef fect. The same solvent isotope effect was observed in the H+-induced f ormation of CaM CaN complex and in the H+-induced enzyme activity. Whe n the enzyme was rendered CaM-independent by limited proteolysis, the isotope effect was abolished, suggesting that H+ was involved in the C aM-dependent and not the CAM-independent activity. Collectively, these data suggest that the interaction of CaM with CaN depends on the two cations: Ca2+ exposes an amphipathic domain on CaM, and H+ exposes the CaM-binding domain on the target enzyme.