We have developed a scintillation proximity assay (SPA) that allows the Ca2+/calmodulin (CaM)-dependent Serine/threoine (Ser/Thr) phosphoprotein phosphatase 2B (calcineurin) activity to be analyzed. A [33P] labeled and biotinylated peptide containing a partial sequence of the regulatory subunit (Rn) of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase was synthesized and used as a synthetic substrate for calcineurin. Following incubation of the peptide with calcineurin, which removes the [33P] label, streptavidin-coated SPA beads were added to capture the biotinylated peptide (the level of the signal detected is inversely proportional to that of the calcineurin activity). Sensitivity is increased in this system by settling or centrifuging the streptavidin-coated SPA beads after binding has occurred. This method allows calcineurin phosphatase assays to be carried out in a 96-well format that is amenable to screening large numbers of compounds.
References
1.
Liu, J., Farmer, J.D., Lane, W.S., Friedman, J., Weissman, I., Schreiber, S. (1991). Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell66:807.
2.
O'Keefe, S.J., Tamura, J., Kincaid, R.L., Tocci, M., O'Neill, E.A. (1992). FK-506 and CsA-sensitive activation of the interleukin-2 promoter by calcineurin. Nature (London)357:692.
3.
Blumenthal, D.K., Takio, K., Hansen, R.S., Krebs, E.G. (1986). Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. J. Biol. Chem.261:8140.
4.
Perrino, B.A., Fong, Y.-L., Brickey, D.A., Saitoh, Y., Ushio, Y., Fukunaga, K., Miyamoto, E., Soderling, T.R. (1992). Characterisation of the phosphatase activity of a baculovirus-expressed calcineurin A isofonn. J. Biol. Chem.267:15965.
5.
Enz, A., Shapiro, G., Chappuis, A., Dattler, A. (1994). Nonradioactive assay for protein phosphatase 2B (calcineurin) activity using a partial sequence of the subunit of cAMP-dependent protein kinase as substrate. Anal. Biochem.216:147.
6.
Cook, N.D. (1996). Scintillation proximity assay: A versatile high-throughput screening technology. Drug Discovery Today1:287.
