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 has been the focus of attention during the past year. The calcineurin B-binding domain of calcineurin A has been identified and shown to be required to reconstitute an active enzyme. The residues of calcineurin B which are affected by binding of a synthetic calcineurin A peptide have been determined by multidimensional NMR in collaboration with Jacob Anglister and Ad Bax (NIDDK). Zhong-Hua Gao has constructed and expressed in E. coli calcineurin B mutants deficient in Ca2+ binding to either one of the four Ca2+ sites. The ability of these mutants to interact with and support the Ca2+-dependent activation of the beta-isoform of calcineurin and its calmodulin-independent derivative reconstituted from its subunits expressed in E. coli by Hao Ren is under study. The specific inhibition of calcineurin by the immunosuppressive drug FK506 complexed with its binding protein, FKBP12, was used by Xutong Wang to show that in situ, calcineurin is 10-20 times more active than is purified calcineurin, and is subject to a time- and Ca2+/calmodulin-dependent reversible inactivation facilitated by small, heat stable, inactivator(s). A factor that prevents the inactivation of calcineurin in vitro and in vivo was identified as superoxide dismutase, suggesting that the inactivation is the result of oxidative damage to the Fe-Zn active center of calcineurin. The involvement of the redox state of iron in the regulation of calcineurin activity provides a mechanism to desensitize the enzyme and to couple Ca2+-dependent protein dephosphorylation to the redox state of the cell. The protection of calcineurin against inactivation by superoxide dismutase adds a new aspect to the physiological roles of superoxide dismutase, namely, the modulation of Ca2+-dependent regulation of cellular processes by protein phosphorylation. Lack of this protective effect could be associated with diseases caused by superoxide dismutase mutations as, for example, familial amyotrophic lateral sclerosis.
