Isolation and characterisation of calcineurin from adrenal cell cytoskeleton: identification of substrates for Ca2+-calmodulin-dependent phosphatase activity.
Ca2+-calmodulin-dependent protein phosphatase activity is found in cytoskeletons of Y-1 mouse adrenal and bovine fasciculata cells. The activity is inhibited by three inhibitors of calmodulin (trifluoperazine, W-7 and pimozide) with EC50 in the low micromolar range. Protein phosphatase activity is inhibited by vanadate, fluoride, Zn2+ and pyrophosphate, stimulated by Mn2+ and found to be tightly bound to the cytoskeleton. Substrates for endogenous phosphatase activity were defined by one- and two-dimensional polyacrylamide gels. Phosphatase activity was seen with proteins that are substrates for both cyclic AMP-dependent and cyclic AMP-independent kinase enzymes. One specific Ca2+-calmodulin-dependent phosphatase, namely calcineurin, was purified to near homogeneity from cytoskeletons of Y-1 cells. The enzyme was found to be a heterodimer (MW 61,000 and 16,000) and the smaller subunit was shown to cross-react with antibodies raised against calcineurin from bovine brain. The purified enzyme catalyzes dephosphorylation of proteins (phosphorylase kinase and casein), phosphoamino acids (tyr greater than thre greater than ser) and a synthetic substrate (p-nitrophenyl phosphate). In addition, a new application of membrane transfer was devised by which the purified enzyme was incubated with a Western blot of cytoskeleton following incubation with [32P]ATP. This method defined four specific substrates of the enzyme (MW 150,000, 55,000, 35,000 and 30,000). Anti-calcineurin revealed that only a single Ca2+-calmodulin-dependent phosphatase is found in adrenal cell cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)
Papadopoulos
V
V
Department of Endocrinology, Prince of Wales Hospital, Randwick, N.S.W., Australia.
Brown
A S
AS
Hall
P F
PF
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D019703:Calcineurin; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D002417:Cattle; D003599:Cytoskeleton / Q000032:analysis / Q000201:enzymology* / Q000378:metabolism; D011756:Diphosphates / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008815:Mice, Inbred Strains; D010710:Phosphates / Q000037:antagonists & inhibitors / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D010764:Phosphorylase Kinase / Q000378:metabolism; D010766:Phosphorylation; D010868:Pimozide / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology; D015383:Zona Fasciculata / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure
D000906:Antibodies; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D011756:Diphosphates; D010710:Phosphates; D010868:Pimozide; D014268:Trifluoperazine; D014638:Vanadates; D012969:Sodium Fluoride; D010764:Phosphorylase Kinase; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D015032:Zinc; D002118:Calcium