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

Abstract

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 tricholoracetic 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, ifany, modification of the B subunit (Mr = 18,000). When carboxyl-methylated calcineurin (r1-2 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.Carboxyl methylation of glutamic and/or aspartic acid residues of proteins by the enzyme protein carboxyl 0-methyltransferase (protein O-methyltransferase; EC 2.1.1.24) is well documented (1-3), although the functional significance of this reversible modification of proteins remains an enigma (4). It has been suggested that carboxyl methylation can influence neurotransmitter synthesis and release (5-7), leukocyte chemotaxis (8, 9), and repair/recognition of Daspartate residues in protein (10)(11)(12)(13)(14)(15) METHODS Materials. S-Adenosyl-L-[methyl-3H]methionine (Ado[3H]-Met) (62 Ci/mmol and 15 Ci/mmol; Ci = 37 GBq) was purchased from Amersham; 2-(N-morpholino)ethanesulfonic acid (Mes) was from Calbiochem; AdoMet and Sadenosylhomocysteine (AdoHcy) were from Sigma; AdoHcy-agarose was from Bethesda Research Laboratories; electrophoresis supplies were from Bio-Rad; and frozen bovine brains were from Hazelton Labs (Denver, PA). Allreagents were of the highest available purity. CalmodulinSepharose was prepared according to the method of Kincaid and Vaughan (25).Enzyme Preparations. Bovine brain protein carboxyl 0-methyltransferase was purified to apparent homogeneity by using minor modifications of previously published techniques (26,27). Bovine brain calcineurin was purified to homogeneity (28) and contained subunits of Mr 61,000 and Mr 18,000 in approximately equal proportions, which accounted for -95% of Coomassie-stained protein after NaDodSO4 gel electrophoresis.Carboxyl Methylation Assays. Reactions were carried out in a fi...

Secretory Proteins from Adrenal Medullary Cells Are Carboxyl‐Methylated In Vivo and Released Under Their Methylated Form by Acetylcholine

“…Recently, a second abnormal amino acid, L-isoaspartic acid, was found to be methylated in synthetic peptides (Murry and Clarke, 1984). The methylation of this abnormal acidic amino acid residue (Aswad, 1984;Johnson et al, 1985) may explain the high methylation rate of some proteins which reaches up to 0.5 mol CH3/mol of protein for calmodulin and up to 2 mol CH3/mol ofprotein for calcineurin (Billingsley et al, 1985).…”

Kinetics of Carboxylmethylation of the Charge Isoforms of Myelin Basic Protein by Protein Methyltransferase II

“…The present report provides evidence that: (a) all MBP charge isoforms are effective substrates for the cytosolic PM I1 enzyme, (b) the kinetics of carboxylmethylation show significant differences among the MBP isofoms assayed, and (c) the carboxylmethylation of MBP proceeds via a mechanism that differs substantially from that described for the carboxylmethylation of two previously examined proteins (Jamaluddin et al, 1975;Billingsley et al, 1985).…”

“…It may be that similar kinetics would have been observed in these systems had the substrate concentrations been taken to lower values. Although deviations from Michaelis-Menten kinetics for the carboxylmethylation of several protein substrates have been noted by others at concentrations near K, (Aswad and Deight, 1983;Billingsley et al, 1985), no kinetic explanation of these findings was suggested. Despite the observed deviations, various rearrangements of the Michaelis-Menten equations into linear forms were used to extrapolate K, and V,,, values (Aswad and Deight, 1983).…”

A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin.

“…These results suggest several (isozymic) species of the catalytic subunit and do not result from treatment with 6 M urea, since native gel isoelectric focusing of calcineurin also showed multiple focused bands of calcineurin. Whether the apparent isozymes of calcineurin are the result of posttranslational differences, such as methylation (33), glycosylation, or differences in the primary protein structure and whether they exhibit unique catalytic or regulatory properties is not known.…”