Radiometric assay of S-adenosylmethionine:calmodulin(lysine)N-methyltransferase by Calcium-dependent hydrophobic interaction chromatography

Rowe, Paul M; Murtaugh, Timothy J.; Bazari, W L; Clarke, Margaret; Siegel, Frank L.

doi:10.1016/0003-2697(83)90100-8

Cited by 14 publications

(14 citation statements)

References 19 publications

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“…It thus appears as if calmodulin's function may be regulated by posttranslational modification as well as by the intracellular concentration of calcium. We have also found that rat pituitary contains an enzyme activity, designated calmodulin converting enzyme, that alters the electrophoretic mobility of calmodulin (Murtaugh et al, 1983). This report describes regional differences in the modification and POSTTRANSLATIONAL MODlFICATtON OF CALMODULIN I65 processing of calmodulin in brain and pituitary that may be relevant to the actions of calmodulin in these tissues and details the nature of t h e pituitary enzyme that alters calmodulin structure.…”

mentioning

confidence: 70%

“…Cytosolic fractions (100 pg of protein) were incubated for 30 min at 37°C in 0. I M sodium acetate buffer, pH 6.0, containing 5 pCi of [methyl-3H]S-adenosyI-~-methionine (AdoMet; 80 Ciimmol) in a total volume of 50 pl (Murtaugh et al, 1983). In some incubations, beef testis calmodulin (8 pg) was added as indicated in the figure legends.…”

Section: Carboxylmethylationmentioning

confidence: 99%

“…The activity of calmodulin (lysine) N-methyltransferase (CLNMT) was determined by the method of Rowe et al (1983). Tissue cytosolic fractions (100 pg of protein) were incubated in 0.1 M glycylglycine (pH 8.0), 0.1 M NaCI, 2 mM MgCI,, 10 pg of Dicfyosteliurn discoideum calmodulin, and 0.5 pCi of [methyl-3H]AdoMet (0.5 pCi/ nmol) in a total volume of 100 wl.…”

Section: Calmodulin (Lysine) N-methyltransferase Assaymentioning

confidence: 99%

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Posttranslational Modification of Calmodulin in Rat Brain and Pituitary

Murtaugh

Wright

Siegel

1986

Journal of Neurochemistry

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T h e posttranslational modification of ciilmodulin has been studied in six brain regions and the anterior pituitary. Carboxylmethylation. calmoddin converting enzyme, and calmodulin (lysine) N-methyllransferase activities w e r e d e t e r m i n e d . I n c u b a t i o n of calmodulin with cytosolic extracts of these tissues in the presence of the methyl donor [methyl-3H]-S-adenosyl-~methionine and identification of labeled proteins by gel

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mentioning

confidence: 70%

Section: Carboxylmethylationmentioning

confidence: 99%

Section: Calmodulin (Lysine) N-methyltransferase Assaymentioning

confidence: 99%

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Posttranslational Modification of Calmodulin in Rat Brain and Pituitary

Murtaugh

Wright

Siegel

1986

Journal of Neurochemistry

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“…Radiometric calmodulin N-methyltransferase assay procedures were done as previously described (25 dependence of the enzyme, the incubation buffer was modified to contain 25 mm Hepes NaOH (pH 7.5), 2 mm DTT, containing 0.01% (w/v) Triton X-100, and various amounts of MgCl2, CaCl2, and EGTA designed to give a Mg2+ concentration of 5 mm and a free Ca2+ concentration that varied from 10-2 to 10-9 M. Chelex treatment of buffers and quantitation of metal ions by atomic absorption spectroscopy were done as previously described (24).…”

Section: Methodsmentioning

confidence: 99%

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

Oh¹,

Roberts²

1990

Plant Physiol.

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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...

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“…The CaM H6 mutant has three principal changes compared with wild type: the removal of two positive charged residues (Arg-106 and His-107 replaced with Gly and Thr) and the introduction of a bulky, charged arginine for Thr-110. The remaining substitution, an Asn-111 to Ser change has previously been shown not to affect methylation (5,20). To test whether these residues are important in methylation, charge-to-alanine mutants were generated for Arg-106 and His-107, and Thr-110 was changed to an arginine.…”

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confidence: 99%

Structural Requirements for N-Trimethylation of Lysine 115 of Calmodulin

Cobb

Roberts

2000

Journal of Biological Chemistry

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Calmodulin is trimethylated at lysine 115 by a highly specific methyltransferase that utilizes S-adenosylmethionine as a co-substrate. Lysine 115 is found within a highly conserved six-amino acid loop (LGEKLT) that forms a 90°turn between EF-hand III and EF-hand IV in the carboxyl-terminal lobe. In the present work a mutagenesis approach was used to investigate the structural features of the carboxyl-terminal lobe that lead to the specificity of calmodulin methylation. Three structural regions within the carboxyl-terminal lobe appear to be involved in methyltransferase recognition: the highly conserved six-amino acid loop-turn region that contains lysine 115 as well as the adjacent ␣-helices (helix 6 and helix 7) from EF-hands III and IV. Sitedirected mutagenesis of residues in the loop show that three residues, glycine 113, glutamate 114, and leucine 116 are essential for methylation. In addition, subdomain (individual helix or Ca 2؉ binding loop) exchange mutants show that the substitutions of either helix 6 (EF-hand III) with helix 2 (EF-hand I) or helix 7 (EFhand IV) with helix 3 (EF-hand II) compromises methylation. Charge-to-alanine mutations in helix 7 show that substitution of conserved charged residues at positions 118, 120, 122, 126, and 127 reduced lysine 115 methylation rates, suggesting possible electrostatic interactions between this helix and the methyltransferase. Single substitutions in helix 6 did not affect calmodulin methylation, suggesting this region may play a more indirect role in stabilizing the conformation of the methyltransferase recognition sequence.Calmodulin is a highly conserved calcium sensor protein that modulates the activities of multiple enzymes. Calmodulin is a monomer consisting of two structurally similar globular calcium binding lobes (1) connected by a flexible linker region (2, 3). Each lobe consists of two helix-loop-helix EF-hand calcium binding sites, with EF-hand domains I and II constituting the amino-terminal lobe and EF-hands III and IV constituting the carboxyl-terminal lobe. Many naturally occurring calmodulins are posttranslationally trimethylated on a single lysine residue at position 115 (reviewed in Ref. 4). Lysine 115 is a solventexposed residue that is found on a highly conserved six-amino acid loop-turn region (LGEKLT) located between helix 6 of EF-hand III and helix 7 of EF-hand IV (Fig. 1). Trimethylation of calmodulin at lysine 115 is catalyzed by an N-methyltransferase that utilizes S-adenosylmethionine as a co-substrate reviewed in Ref. 4). This enzyme appears to have the dedicated function of trimethylating lysine 115 in calmodulin from a wide variety of species. From a functional perspective, calmodulin methylation selectively affects the regulation of certain enzymes such as NAD kinase (10 -12) and might also influence posttranslational ubiquitination of the protein (13).Previous work shows that the site on calmodulin recognized by the calmodulin N-methyltransferase resides solely on the COOH-terminal lobe (residues 78 -148) (7). Mutations or ch...

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Radiometric assay of S-adenosylmethionine:calmodulin(lysine)N-methyltransferase by Calcium-dependent hydrophobic interaction chromatography

Cited by 14 publications

References 19 publications

Posttranslational Modification of Calmodulin in Rat Brain and Pituitary

Posttranslational Modification of Calmodulin in Rat Brain and Pituitary

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

Structural Requirements for N-Trimethylation of Lysine 115 of Calmodulin

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