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. 1990 Jul;93(3):880–887. doi: 10.1104/pp.93.3.880

Analysis of the State of Posttranslational Calmodulin Methylation in Developing Pea Plants 1

Suk-Heung Oh 1, Daniel M Roberts 1
PMCID: PMC1062604  PMID: 16667596

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. [1986] J Biol Chem 261: 1491-1494) raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson J. M., Charbonneau H., Jones H. P., McCann R. O., Cormier M. J. Characterization of the plant nicotinamide adenine dinucleotide kinase activator protein and its identification as calmodulin. Biochemistry. 1980 Jun 24;19(13):3113–3120. doi: 10.1021/bi00554a043. [DOI] [PubMed] [Google Scholar]
  2. Biro R. L., Daye S., Serlin B. S., Terry M. E., Datta N., Sopory S. K., Roux S. J. Characterization of oat calmodulin and radioimmunoassay of its subcellular distribution. Plant Physiol. 1984 Jun;75(2):382–386. doi: 10.1104/pp.75.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Dauwalder M., Roux S. J., Hardison L. Distribution of calmodulin in pea seedlings: immunocytochemical localization in plumules and root apices. Planta. 1986;168:461–470. [PubMed] [Google Scholar]
  5. Eldik L. J., Grossman A. R., Iverson D. B., Watterson D. M. Isolation and characterization of calmodulin from spinach leaves and in vitro translation mixtures. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1912–1916. doi: 10.1073/pnas.77.4.1912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gregori L., Marriott D., Putkey J. A., Means A. R., Chau V. Bacterially synthesized vertebrate calmodulin is a specific substrate for ubiquitination. J Biol Chem. 1987 Feb 25;262(6):2562–2567. [PubMed] [Google Scholar]
  7. Klee C. B., Krinks M. H. Purification of cyclic 3',5'-nucleotide phosphodiesterase inhibitory protein by affinity chromatography on activator protein coupled to Sepharose. Biochemistry. 1978 Jan 10;17(1):120–126. doi: 10.1021/bi00594a017. [DOI] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Lukas T. J., Iverson D. B., Schleicher M., Watterson D. M. Structural characterization of a higher plant calmodulin : spinacia oleracea. Plant Physiol. 1984 Jul;75(3):788–795. doi: 10.1104/pp.75.3.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lukas T. J., Wallen-Friedman M., Kung C., Watterson D. M. In vivo mutations of calmodulin: a mutant Paramecium with altered ion current regulation has an isoleucine-to-threonine change at residue 136 and an altered methylation state at lysine residue 115. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7331–7335. doi: 10.1073/pnas.86.19.7331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lukas T. J., Wiggins M. E., Watterson D. M. Amino Acid sequence of a novel calmodulin from the unicellular alga chlamydomonas. Plant Physiol. 1985 Jul;78(3):477–483. doi: 10.1104/pp.78.3.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Marshak D. R., Clarke M., Roberts D. M., Watterson D. M. Structural and functional properties of calmodulin from the eukaryotic microorganism Dictyostelium discoideum. Biochemistry. 1984 Jun 19;23(13):2891–2899. doi: 10.1021/bi00308a007. [DOI] [PubMed] [Google Scholar]
  13. Molla A., Kilhoffer M. C., Ferraz C., Audemard E., Walsh M. P., Demaille J. G. Octopus calmodulin. The trimethyllysyl residue is not required for myosin light chain kinase activation. J Biol Chem. 1981 Jan 10;256(1):15–18. [PubMed] [Google Scholar]
  14. Morino H., Kawamoto T., Miyake M., Kakimoto Y. Purification and properties of calmodulin-lysine N-methyltransferase from rat brain cytosol. J Neurochem. 1987 Apr;48(4):1201–1208. doi: 10.1111/j.1471-4159.1987.tb05647.x. [DOI] [PubMed] [Google Scholar]
  15. Persechini A., Kretsinger R. H. The central helix of calmodulin functions as a flexible tether. J Biol Chem. 1988 Sep 5;263(25):12175–12178. [PubMed] [Google Scholar]
  16. Putkey J. A., Draetta G. F., Slaughter G. R., Klee C. B., Cohen P., Stull J. T., Means A. R. Genetically engineered calmodulins differentially activate target enzymes. J Biol Chem. 1986 Jul 25;261(21):9896–9903. [PubMed] [Google Scholar]
  17. Putkey J. A., Slaughter G. R., Means A. R. Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene. J Biol Chem. 1985 Apr 25;260(8):4704–4712. [PubMed] [Google Scholar]
  18. Roberts D. M., Burgess W. H., Watterson D. M. Comparison of the NAD Kinase and Myosin Light Chain Kinase Activator Properties of Vertebrate, Higher Plant, and Algal Calmodulins. Plant Physiol. 1984 Jul;75(3):796–798. doi: 10.1104/pp.75.3.796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Roberts D. M., Crea R., Malecha M., Alvarado-Urbina G., Chiarello R. H., Watterson D. M. Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis. Biochemistry. 1985 Sep 10;24(19):5090–5098. doi: 10.1021/bi00340a020. [DOI] [PubMed] [Google Scholar]
  20. Roberts D. M. Detection of a calcium-activated protein kinase in mougeotia by using synthetic Peptide substrates. Plant Physiol. 1989 Dec;91(4):1613–1619. doi: 10.1104/pp.91.4.1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Roberts D. M., Rowe P. M., Siegel F. L., Lukas T. J., Watterson D. M. Trimethyllysine and protein function. Effect of methylation and mutagenesis of lysine 115 of calmodulin on NAD kinase activation. J Biol Chem. 1986 Feb 5;261(4):1491–1494. [PubMed] [Google Scholar]
  22. Roberts D. M., Zimmer W. E., Watterson D. M. The use of synthetic oligodeoxyribonucleotides in the examination of calmodulin gene and protein structure and function. Methods Enzymol. 1987;139:290–303. doi: 10.1016/0076-6879(87)39093-7. [DOI] [PubMed] [Google Scholar]
  23. Rowe P. M., Murtaugh T. J., Bazari W. L., Clarke M., Siegel F. L. Radiometric assay of S-adenosylmethionine:calmodulin(lysine)N-methyltransferase by calcium-dependent hydrophobic interaction chromatography. Anal Biochem. 1983 Sep;133(2):394–400. doi: 10.1016/0003-2697(83)90100-8. [DOI] [PubMed] [Google Scholar]
  24. Rowe P. M., Wright L. S., Siegel F. L. Calmodulin N-methyltransferase. Partial purification and characterization. J Biol Chem. 1986 May 25;261(15):7060–7069. [PubMed] [Google Scholar]
  25. Schleicher M., Lukas T. J., Watterson D. M. Further Characterization of Calmodulin from the Monocotyledon Barley (Hordeum vulgare). Plant Physiol. 1983 Nov;73(3):666–670. doi: 10.1104/pp.73.3.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stinemetz C. L., Kuzmanoff K. M., Evans M. L., Jarrett H. W. Correlation between calmodulin activity and gravitropic sensitivity in primary roots of maize. Plant Physiol. 1987;84:1337–1342. doi: 10.1104/pp.84.4.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Takeda T., Yamamoto M. Analysis and in vivo disruption of the gene coding for calmodulin in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3580–3584. doi: 10.1073/pnas.84.11.3580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Van Eldik L. J., Lukas T. J. Site-directed antibodies to vertebrate and plant calmodulins. Methods Enzymol. 1987;139:393–405. doi: 10.1016/0076-6879(87)39101-3. [DOI] [PubMed] [Google Scholar]
  29. Watterson D. M., Iverson D. B., Van Eldik L. J. Spinach calmodulin: isolation, characterization, and comparison with vertebrate calmodulins. Biochemistry. 1980 Dec 9;19(25):5762–5768. doi: 10.1021/bi00566a015. [DOI] [PubMed] [Google Scholar]

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