Analysis of the State of Posttranslational Calmodulin Methylation in Developing Pea Plants

Abstract

A specific calmodulin-N-methyltransferase was used in a radiometric assay to analyze the degree of methylation of lysine-115 in pea (Pisum sativum) plants. Calmodulin was isolated from dissected segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by incubation with the calmodulin methyltransferase in the presence of [3H]methyl-S-adenosylmethionine. By this approach, the presence of unmethylated calmodulins were demonstrated in pea tissues, and the levels of methylation varied depending on the developmental state of the tissue tested. Calmodulin methylation levels were lower in apical root segments of both etiolated and green plants, and in the young lateral roots compared with the mature, differentiated root tissues. The incorporation of methyl groups into these calmodulin samples appears to be specific for position 115 since site-directed mutants of calmodulin with substitutions at this position competitively inhibited methyl group incorporation. The present findings, combined with previous data showing differences in the ability of methylated and unmethylated calmodulins to activate pea NAD kinase (DM Roberts et al. [19861 J Biol Chem 261: 1491-1494 raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity.Calmodulin is a highly conserved, ubiquitous, calciummodulated protein that interacts with a number of enzymes and stimulates their activities (22). NE-Trimethyllysine is a posttranslational modification that is found at position 115 of many calmodulins. This site is methylated by a S-adenosyl-L-methionine:lysine N-methyltransferase (15, 26). The question of the functional significance of calmodulin methylation is unclear. Although this modification is commonly found in calmodulin, naturally occurring calmodulins that have unmodified lysine at 115 rather than trimethyllysine have been purified and characterized from some organisms such as Chlamydomonas reinhardtii (10), Dictyostelium discoideum (13), and trypanosomes (27). Additionally, active recombinant DNA-derived calmodulins that do not possess trimethyllysine 115 have been expressed in Escherichia coli from cloned calmodulin genes or cDNAs (16,17,20).With respect to the regulatory activities of calmodulin, unmethylated calmodulins are not significantly different from methylated calmodulins in their abilities to bind calcium and activate a number of enzymes (16-18, 20, 26 the case of one calmodulin-dependent enzyme, plant nicotinamide adenine dinucleotide kinase, the level of enzyme activation by unmethylated calmodulins is at least three-fold greater than the activation by methylated calmodulins (13,19,20). Further, this higher level of activation by unmethylated calmodulins can be reversed by enzymatic methylation in vitro (21). The results raise the possibility that selective calmodulin activator activities could be attenuated by posttranslational methylation of lysine 115. For example, the methylation of...

Synthesis and Accumulation of Calmodulin in Suspension Cultures of Carrot (Daucus carota L.)

“…Nevertheless, if the trimethylation state of Lys"5 inversely reflects the rate of CaM turnover, our results predict that the pool of CaM in cells at stationary phase should be more highly trimethylated than is CaM from cells at exponential phase. Consistent with this prediction is the observation that the level of Lys'5 trimethylation in CaM from pea roots varies with the developmental state of the tissue (23). In this system, the levels of CaM trimethylation were higher in mature (nondividing) regions of pea roots compared with younger, more actively growing regions near the root apex.…”

“…In two independent experiments, extracts containing equal amounts of soluble protein (200 ug) were analyzed for their relative CaM content, after ethanol fractionation to enrich for CaM (23,32,33), by SDS-PAGE and western blotting followed by densitometric scanning of the immunostained blots. In a separate experiment, duplicate total soluble protein samples were analyzed by a competition radioimmunoassay for their CaM content.…”

Changes in protein methylation associated with the elicitation response in cell cultures of alfalfa (Medicago sativaL.)

“…The less active PMT catalyses the formation of uncharacterised base-stable methylated proteins and is presumabley an N-methyltransferase analogous to the enzymes present in tobacco [18] and peas [4] which have activities toward the large subunit of ribulose 1,5-bisphosphate carboxylase and calmodulin, respectively. Significantly the large subunit of ribulose 1,5-bisphosphate carboxylase appears to be a substrate for protein methylation in alfalfa leaves.…”

“…The role of methylation of amino and sulfydryl groups in proteins is less well understood but modified proteins may demonstrate altered functional activity, localisation and increased turnover times [1]. Various protein methylation reactions have been demonstrated in plants most notably the N-methylation of lysine residues in cytochrome c [2] and histone [3] in wheat germ, calmodulin [4,5] in carrots and peas, and the large subunit of ribulose-l,5-bisphosphate carboxylase in a number of species [6]. Although in most instances the effects of these post-translational modifications are unknown, in peas the extent of methylation of the lysine-115 residue of calmodulin has been shown to be higher in mature differentiated tissues than in dividing tissues [4].…”

“…Various protein methylation reactions have been demonstrated in plants most notably the N-methylation of lysine residues in cytochrome c [2] and histone [3] in wheat germ, calmodulin [4,5] in carrots and peas, and the large subunit of ribulose-l,5-bisphosphate carboxylase in a number of species [6]. Although in most instances the effects of these post-translational modifications are unknown, in peas the extent of methylation of the lysine-115 residue of calmodulin has been shown to be higher in mature differentiated tissues than in dividing tissues [4]. The importance of calmodulin methylation in plant development has been confirmed by the impaired growth and development observed in tobacco expressing a foreign calmodulin which could not undergo lysine methylation [7].…”