Calmodulin antagonists differentiate between Ni(2+)- and Mn(2+)-stimulated phosphatase activity of calcineurin.
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 Ni(2+)-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 Ni(2+)-stimulated phosphatase activity. On the other hand, calmidazolium only weakly and partially inhibited the Mn(2+)-stimulated phosphatase activity and the other calmodulin antagonists examined increased the Mn(2+)-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 Ni(2+)-stimulated phosphatase activity recovered from 38 to 63% of the control level in the presence of 5 microM 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 Ni(2+)-stimulated phosphatase activity by interacting with the B subunit, in the absence of calmodulin. The Mn(2+)-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 Ni(2+)- and Mn(2+)-stimulated activities of calcineurin are differentially affected by calmodulin antagonists and that the B subunit plays a crucial role in the expression of the Ni(2+)-stimulated phosphatase activity.
Mukai
H
H
Department of Pharmacology, Kobe University School of Medicine, Hyogo.
Ito
A
A
Kishima
K
K
Kuno
T
T
Tanaka
C
C
D009597:4-Nitrophenylphosphatase / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology*; D009532:Nickel / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology*; D013881:Thioridazine / Q000494:pharmacology*; D014268:Trifluoperazine / Q000494:pharmacology*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007093:Imidazoles; D013449:Sulfonamides; D014268:Trifluoperazine; D008345:Manganese; C031938:calmidazolium; C017967:W 7; D009532:Nickel; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D009597:4-Nitrophenylphosphatase; D013881:Thioridazine