Calmodulin Antagonists Differentiate between Ni²⁺-and Mn²⁺-Stimulated Phosphatase Activity of Calcineurin

Abstract

The interaction of calmodulin antagonists with a phosphoprotein phosphatase, calcineurin, was investigated using para-nitrophenyl phosphate (pNPP) as a substrate. Calmidazo-lium, a potent calmodulin antagonist, inhibited the Ni²⁺-stimulated calmodulin- indepen-dent phosphatase activity to much the same extent as it did the Ca²⁺/calmodulin-stimulated activity. Other calmodulin antagonists, such as trifluoperazine, thioridazine, and W-7, also inhibited the Ni²⁺-stimulated phosphatase activity. On the other hand, calmidazolium only weakly and partially inhibited the Mn²⁺- stimulated phosphatase activity and the other calmodulin antagonists examined increased the Ni²⁺-stimulated activity, in the absence of calmodulin. With the addition of an equimolar amount, as to the inhibited holoenzyme, of the purified B subunit of calcineurin, the Ni²⁺-stimulated phosphatase activity recovered from 38 to 63% of the control level in the presence of 5μM calmidazolium. When the amount of additional B subunit was increased, the phosphatase activity recovered to 94% of the control level, thereby implying that calmidazolium inhibits the Ni²⁺-stimulated phosphatase activity by interacting with the B subunit, in the absence of calmodulin. The Mn²⁺-stimulated phosphatase activity also recovered from the inhibition by calmidazo-lium, but a much larger amount of the B subunit was necessary for the recovery. These results indicate that the Ni²⁺-and Mn²⁺-stimulated activities of calcineurin are differentially affected by calmodulin antagonists and that the B subunit plays a crucial role in the expression of the Ni²⁺-stimulated phosphatase activity.