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 hydrolysis of carboxyl methylester groups on calcineurin increased rapidly above pH 6.5 with those on native carboxyl-methylated calcineurin substantially more labile than for trichloracetic acid-precipitated calcineurin. Polyacrylamide gel electrophoresis in the presence of NaDodSO4 (pH 2.4) confirmed that the A subunit of calcineurin (Mr = 61,000) was the primary site of carboxyl methylation with little, if any, modification of the B subunit (Mr = 18,000). When carboxyl-methylated calcineurin (approximately 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.
Billingsley
M L
ML
Kincaid
R L
RL
Lovenberg
W
W
D000818:Animals; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004789:Enzyme Activation / Q000187:drug effects; D008345:Manganese / Q000494:pharmacology; D008745:Methylation; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011496:Protein Methyltransferases / Q000378:metabolism*; D011497:Protein O-Methyltransferase / Q000378:metabolism*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008345:Manganese; D011496:Protein Methyltransferases; D011497:Protein O-Methyltransferase; D010749:Phosphoprotein Phosphatases; D010744:Phosphoric Monoester Hydrolases