The mechanism of stimulus-response coupling mediated by Ca2+ and calmodulin was studied at three levels: the modulation of the Ca2+ signal by calmodulin, the regulation of the calmodulin-stimulated protein phosphatase, calcineurin, and the role of protein dephosphorylation in the control of mitosis. 1) Using limited proteolysis as a probe for calmodulin structure, we identified domains I and III and the central helix of calmodulin as domains whose conformation is specifically altered upon the stepwise titration of the calcium sites. A synthetic peptide corresponding to the calmodulin-binding domain of calcineurin increases the affinity of calmodulin for Ca2+ 100-fold and modifies the binding pathway. 2) The high degree of structure conservation of the functional domains of the two subunits of calcineurin has been demonstrated by a comparison of the sequences of the mammalian proteins and the sequences of the two subunits of Drosophila melanogaster calcineurin predicted from the DNA sequences of the recently isolated cDNA and genomic clones. Synthetic peptides and site directed mutagenesis are being used to study the mechanism of action of the calmodulin-binding, inhibitory and catalytic domains of calcineurin which had previously been identified by limited proteolysis. 3) The involvement of Ca2+-regulated protein dephosphorylation, possibly mediated by the calmodulin/calcineurin complex, in the control of the progression of mitotic cells from metaphase to telophase is being investigated by Frank Suprynowicz.
