Identification of an autoinhibitory domain in calcineurin

The hypothesis that Calcineurin, the Ca2+/calmodulin-dependent protein Phosphatase, contains an autoinhibitory domain was tested using synthetic Peptides corresponding to regions of the carboxyl-terminus of Calcineurin. Of the several Peptides analyzed, one, containing residues I-T-S-F-E-E-A-K-G-L-D-R-I-N-E-R-M-P-P-R-R-D-A-M-P, gave complete inhibition of its protein Phosphatase activity. Using [32P]Myosin light chain as substrate an IC50 of about 10 microM was obtained with either native Calcineurin, assayed in the presence of Ca2+/Calmodulin, or with Calcineurin subjected to partial proteolysis which converts it to a fully active Phosphatase when assayed in the presence of [ethylenebis (oxyethylenenitrilo)]tetraacetic acid. With 50 mM p-nitrophenylphosphate as substrate an IC50 of about 40 microM was observed. Studies with overlapping Peptides suggested that the sequence P-P-R-R-D-A-M-P was essential but not sufficient for the observed inhibition. Kinetic analysis indicated that the inhibition of Phosphatase activity was not competitive with respect to [32P]Myosin light chain. This peptide did not show significant inhibition of the catalytic subunits of protein phosphatases type I or type IIA or of Ca2+/calmodulin-dependent protein kinase II. These results indicate that Amino acids within this sequence of Calcineurin constitute a unique autoinhibitory domain which interacts with the active site and is responsible for the low basal Phosphatase activity in the absence of Ca2+/Calmodulin.