Isolation and characterisation of calcineurin from adrenal cell cytoskeleton: identification of substrates for Ca 2+-calmodulin-dependent phosphatase activity

Abstract

Ca 2+-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 EC 50 in the low micromolar range. Protein phosphatase activity is inhibited by vanadate, fluoride, Zn 2+ and pyrophosphate, stimulated by Mn 2+ 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 Ca 2+-calmodulin-dependent phosphatase, namely calcineurin, was purified to near homogeneity from cytoskeletons of Y-l cells. The enzyme was found to be a heterodimer (MW 61000 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 > thre > 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 150000, 55000, 35000 and 30000). Anti-calcineurin revealed that only a single Ca 2+-calmodulin-dependent phosphatase is found in adrenal cell cytoskeleton. It is proposed that a highly active Ca 2+-calmodulin-dependent protein phosphatase is closely associated with adrenal cell cytoskeletons (adrenal calcineurin), that this enzyme is capable of rapidly dephosphorylating specific substrates and may in this way influence the function of adrenal cell cytoskeletons and hence steroid synthesis.