Stoichiometric methylation of calcineurin by protein carboxyl O-methyltransferase and its effects on calmodulin-stimulated phosphatase activity

Calcineurin, a calmodulin-stimulated protein phosphatase, was a substrate for purified bovine brain protein carboxyl O-methyltransferase (protein O-methyltransferase; EC 2.1.1.24) and incorporated up to 2 mol of CH₃ per mol of calcineurin. Carboxyl methylation was dependent on the concentrations of S-adenosyl-L-(methyl-³H]methionine and was prevented by addition of the carboxyl methylation inhibitor S-adenosylhomocysteine. The stoichiometry of methyl group incorporation was related to the ratio of methyltransferase/calcineurin. The rate of spontaneous hydrolyis of carboxyl methylester groups on calcineurin increased rapidly above pH 6.5 with those on native carboxyl-methylated calcineurin substantially more labile than for tricholoracetic acid-precipitated calcineurin. Polyacrylamide gel electrophoresis in the presence of NaDodSO₄ (pH 2.4) confirmed that the A subunit of calcineurin (M(r) = 61,000) was the primary site of carboxyl methylation with little, if any, modification of the B subunit (M(r) = 18,000). When carboxyl-methylated calcineurin (~ 1-2 mol of CH₃ per mol of protein) was assayed for p-nitrophenyl phosphatase activity at pH 6.5, a marked inhibition of calmodulin-stimulated activity was observed while there was little effect on Mn²⁺-stimulated phosphatase activity. Thus, calcineurin appears to be an excellent substrate for protein carboxyl O-methylation and this modification, which impairs calmodulin stimulation of phosphatase activity, may be of functional significance.