Calmodulin-stimulated dephosphorylation of p-nitrophenyl phosphate and free phosphotyrosine by calcineurin.
Calcineurin, a calmodulin-binding protein from brain, has been shown to possess a metal ion-dependent and calmodulin-stimulated phosphatase activity towards phosphorylase kinase and inhibitor-1 (Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Lett. 137, 80-84). In this report, we show that calcineurin can also dephosphorylate p-nitrophenyl phosphate and free phosphotyrosine. However, calcineurin does not show significant activity towards phosphothreonine, phosphoserine, or several other low molecular weight phosphocompounds tested. As we have found with phosphorylase kinase and phosphocasein, the dephosphorylation of p-nitrophenyl phosphate and free phosphotyrosine is stimulated by calmodulin and is metal ion-dependent with the order of efficiency being Mn2+ much greater than Co2+ greater than Ca2+. The dephosphorylation of these substrates appears to be an intrinsic property of calcineurin and is not due to contamination by alkaline phosphatases since the pH optimum for calcineurin activity occurs at a neutral rather than an alkaline pH. The dephosphorylation of p-nitrophenyl phosphate provides an easy, rapid, and accurate method for the quantification of calcineurin activity as well as permitting insight into reaction kinetics. The dephosphorylation of free phosphotyrosine by calcineurin suggests that this compound may be a physiological substrate of calcineurin.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins; D002413:Cations, Divalent; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009596:Nitrophenols / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D019000:Phosphotyrosine; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002413:Cations, Divalent; D009419:Nerve Tissue Proteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D019000:Phosphotyrosine; C008644:nitrophenylphosphate; D014443:Tyrosine; D008274:Magnesium; D002118:Calcium