Interaction amongst calcineurin subunits: Stimulatory and inhibitory effects of subunit B on calmodulin stimulation of subunit A phosphatase activity depend on Mn 2+ exposure of the holoenzyme prior to its dissociation by urea

Abstract

Calcineurin was dissociated into subunits A and B by 6 M urea in the presence (method A) and absence (method B) of MnCI 2 and dissociated subunits were isolated by gel filtration in urea in the absence (method B) or presence (method A) of MnCI 2. Phosphatase activity was associated with the A subunit isolated by either method. The phosphatase activity ( nmol mg ) of subunit A isolated by method A was greater (2-5-fold) than by method B. Mn 2+ increased subunit A phosphatase and calmodulin further increased the enzyme activity. Subunit B isolated by method A or B increased Mn 2+ + calmodulin stimulated subunit A phosphatase prepared by method B but interestingly and unexpectedly inhibited such stimulated activity of the subunit A prepared by method A. These results imply the tightly bound cation (in our case, most likely Mn 2+) with subunit A dramatically and differentially influences the effects of two Ca 2+-binding proteins, calmodulin and subunit B, on the subunit A phosphatase.