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

• Published in: Journal of biochemistry (Tokyo) 1991, Vol.110 (3), p.402-406
• Main Authors: Mukai, Hideyuki, Ito, Akira, Kishima, Koji, Kuno, Takayoshi, Tanaka, Chikako
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cell physiology; Fundamental and applied biological sciences. Psychology; Molecular and cellular biology
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a123593

The interaction of calmodulin antagonists with a phosphoprotein phosphatase, calcineurin, was investigated using para-nitrophenyl phosphate (pNPP) as a substrate. Calmidazolium, a potent calmodulin antagonist, inhibited the Ni2+-stimulated calmodulin-independent 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 Mn2+-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 5 μM 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 calmidazolium, 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.