Calcineurin is a Ca - and calmodulin-regulated protein serine/threonine phosphatase and an important component of eukaryotic signal transduction pathways, e.g., the T-cell receptor signal transduction pathway that leads to IL-2 gene transcription. Calcineurin is the target of the immunosuppressant agents, cyclosporin A and FK506, important organ transplantation drugs that inhibit calcineurin phosphatase activity in the presence of the cytoplasmic proteins cyclophililin and FK506- binding protein (FKBP), respectively. Calcineurin is a member of the metallophosphatase superfamily of enzymes that accommodate active site dinuclear metal cofactors. In calcineurin, an Fe-Zn dinuclear metal center has been characterized by x-ray diffraction and spectroscopic techniques. Another member of this family, lambda protein phosphatase, has also been characterized by biochemical and spectroscopic techniques and shown to accommodate a dinuclear metal center. The broad, long-terms goals of this proposal will be to characterize structure/function aspects of calcineurin and lambda protein phosphatase related to the active site dinuclear metal centers and their roles in catalysis and metabolic regulation. In vitro studies have demonstrated that the phosphatase activity of calcineurin is sensitive to the redox state of the bound Fe ion. One of the specific aims will be to extend the link between calcineurin activity and the oxidation state of bound metal ions using different substrates. Recent work suggests that calcineurin phosphatase activity may also be regulated by the cellular redox state. We hypothesize that the phosphatase activity of calcineurin is coupled to the intracellular redox state and that this involves the oxidation state of the active site Fe ion.
The specific aims i n this regard are to explore redox regulation of calcineurin in vivo. Further specific aims will focus on lambda protein phosphatase as a model for calcineurin and other protein serine/threonine phosphatases to explore the role of the metal ions in this enzyme and their role in the catalytic mechanism. The three dimensional structure of lambda protein phosphatase will be determined by x-ray diffraction methods, and the catalytic mechanism explored using biochemical and kinetic isotope effect studies. Recent studies have implicated oxidative stress in many cellular processes and disease states including apoptosis and cardiovascular disease. Calcineurin has been implicated as a key signaling component in apoptosis and cardiac hypertrophy. The possibility that calcineurin phosphatase activity may be regulated by the cellular redox provides a link between oxidative stress and calcium- dependent signal transduction pathways.
