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 calcineurin, the only protein phosphatase under the control of calmodulin, is used as a model system. Calcineurin, the target of immunosuppressive drugs, plays a critical role in the regulation of genes involved in cellular processes as diverse as T cell activation, cell differentiation, cardiac hypertrophy, and long term memory. Calcineurin activity is regulated by two, structurallysimilar, but functionally distinct Ca2+-binding proteins: calcineurin B, an integral subunit of the enzyme, and calmodulin. Thedisplacement of an inhibitory domain during the Ca2+-dependent binding of calmodulin to calcineurin results in a large activation ofcalcineurin affecting the Vmax of the enzyme, which is followed by a Ca2+/calmodulin- and time-dependent reversible inactivation. The identification of calcineurin as an [Fe2+-Zn2+] protein phosphatase by M. Ghosh in collaboration with J. Zweier at Johns Hopkins University provided a mechanism for this inactivation that is the result of exposure of Fe2+ at the active site facilitating its oxidation by superoxide anion. Thus, we have uncovered a novel coupling of the regulation of protein phosphorylation by Ca2+ and oxidative stress. The nature of the conformational changes that accompany Ca2+ binding to calmodulin and allow it to activate calcineurin are presently under study in collaboration with Ad Bax (NIDDK). In contrast to calmodulin, calcineurin B is tightly bound to calcineurin A even in the absence of Ca2+ (Kd=10-14M-1), but Ca2+ binding to its three moderate affinity sites is required for activity and calmodulin binding. The Ca2+-binding properties of calcineurin B and the identification of the high and moderate affinity Ca2+ sites has been analyzed by flow dialysis (X. Wang and Z. Gao) and Fourier Transformed Infrared spectroscopy in collaboration with Jill Trewhella (Los Alamos National Laboratory) using calcineurin B mutants deficient in Ca2+ binding to each one of the four Ca2+ sites. A conformational change of the regulatory domain of calcineurin A that accompanies Ca2+ binding to calcineurin B has been identified by Seun-Ah Yang. It provides a mechanism to explain the previously reported dependence of Ca2+ binding to calcineurin B for calmodulin activation and the large increase in Ca2+ affinity of calcineurin for Ca2+ upon removal of the calmodulin- binding domain (Stemmer and Klee, Biochemistry 33: 6859-66, 1994). - Calcineurin, Calcium, Calmodulin, Immunosuppression, Iron, Oxidation, Superoxide dismutase, Protein phosphatases, - Neither Human Subjects nor Human Tissues
