The goal of Dr. Klee and her colleagues is to elucidate the mechanism of stimulus-response coupling mediated by Ca2+ and calmodulin. The regulation of the calmodulin-stimulated protein phosphatase, calcineurin, recently identified as the target of immunosuppressive drugs, is used as a model system. The role of the regulatory subunit, calcineurin B, in the folding and activation of calcineurin, and the mechanism of the Ca2+/ calmodulin inactivation of calcineurin by the superoxide anion have been the focus of attention during the past year. Reconstitution of recombinant calcineurin A beta, solubilized in 6M guanidine-HCl, into a fully active and soluble enzyme was shown by Ren Hao to require calcineurin B and calmodulin. Calcineurin B mutants in either one of the four Ca2+ sites, expressed in E. coli by Zhong-Hua and characterized by Xutong Wang, were tested for their ability to replace wild type calcineurin B in this reconstitution assay by Aram Lee. Failure to reconstitute active enzyme with a calcineurin B mutant deficient in Ca2+ binding to the second Ca2+ site indicates that Ca2+ binding to this low affinity site is either required for reconstitution or activation of calcineurin. In collaboration with Dr. J. Zweier and colleagues, Manik Ghosh demonstrated that a preparation of purified calcineurin that contains 0.7 mol of Fe3+, determined by electron paramagnetic resonance, and 0.8 mol of Zn2+ per mol of enzyme was inactive but was activated to a high specific activity, similar to that of the crude enzyme, upon treatment with ascorbate in the absence of added metal. The activation was accompanied by a conversion of Fe3+ to Fe2+. Thus, as shown by Xutong Wang, the displacement of the autoinhibitory domain upon exposure to calmodulin at concentrations of Ca2+ found in stimulated cells (5 x 10-7M) exposes Fe2+, in the catalytic center, to the oxidative damage of the superoxide anion. The involvement of the redox state of iron in the regulation of calcineurin activity provides a mechanism to desensitize calcineurin and to couple Ca2+-dependent protein dephosphorylation to the redox state of the cell.
