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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC005231-24
Application #
6100777
Study Section
Special Emphasis Panel (LB)
Project Start
Project End
Budget Start
Budget End
Support Year
24
Fiscal Year
1998
Total Cost
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code