Stimulus-response coupling mediated by changes in intracellular Ca2+ involves the participation of a family of structurally related Ca2+-binding proteins, which act as sensors and modulators of Ca2+ transients. These proteins undergo a conformational change upon binding Ca2+ that enables them to activate target enzymes. Calmodulin is a unique member of this protein family because of its ability to activate a large number of enzymes, in particular protein kinases and phosphatases. Our goal is to further our understanding the mechanism of action of calmodulin using as a model system the calmodulin stimulation of calcineurin, the only protein phosphatase under the direct control of calmodulin. Calcineurin, the target of the immunosuppressive drugs, FK506 and Cyclosporin A, plays a key role in the regulation of gene expression. Calcineurin plays an important role in the regulation of cellular processes as diverse as immune responses, cell growth and differentiation, ion homeostasis, embryogenesis, neurosecretion and long term memory. Alteration of its activity has recently been implicated in the effects of the immunosuppressive drugs and the pathogeny of an ever increasing number of diseases. In collaboration with Jay Zweier at Johns Hopkins University, we previously showed that calcineurin is an (Fe2+-Zn2+) enzyme which is inactivated by oxidation of Fe2+ to Fe3+. The inactivation of the enzyme is dependent on Ca2+/calmodulin binding to calcineurin and exposure of the active site metal ions. This calmodulin-dependent inactivation, prevented by superoxide dismutase and reversed by ascorbate, provides a mechanism for the temporal regulation of calcineurin in response to Ca2+ transients and the coupling of protein phosphorylation and oxidative stress. An expression system developed by Ren Hao to express (Fe2+-Zn2+) calcineurin in E. coli is being used by Xiaoyun Yang to prepare calcineurin mutants resistant to oxidative stress with which to test the redox sensitivity of calcineurin in vivo . Limited proteolysis of calcineurin in the absence of Ca2+ was used by Seun-Ah Yang to identify the nature of the conformational change induced by Ca2+ binding to the regulatory subunit of calcineurin, calcineurin B. This conformational change is required for interaction and activation by calmodulin and for the interaction and inhibition by the immunosuppressive drugs. This work has been submitted for publication. On the basis of these studies Seun-Ah Yang is now designing a calcineurin A derivative that no longer requires Ca2+ binding to calcineurin B for calmodulin activation or inhibition by the immunosuppressive drugs. In collaboration with Jill Trewhella (Los Alamos National Laboratory), calcineurin B mutants deficient in Ca2+ binding to either one of the four Ca2+-binding sites have been used to demonstrate that, like troponin C, calcineurin B contains two classes of sites. The C-terminal pair of sites, with a very high affinity for Ca2+ (Kd<10-7M) play a structural role, anchoring calcineurin B to calcineurin A, while the moderate affinity sites with Kds in the micromolar range, are the regulatory sites involved in the Ca2+-induced conformational change described above. This work is being prepared for publication. A third project initiated by Sergei Ruvinov is devoted to the identification of the NF-AT (Nuclear Factor of Activated T cells) binding site on calcineurin A in a attempt to examine the specificity of this site for NF-AT and to possibly develop less toxic calcineurin inhibitors specific for a given substrate.
