Trimethyllysine and protein function. Effect of methylation and mutagenesis of lysine 115 of calmodulin on NAD kinase activation

Unmethylated calmodulins have been enzymatically methylated at lysine 115, and a direct effect of this methylation on NAD kinase activation has been shown. Similar to naturally occurring calmodulins with trimethyllysine 115, the enzymatically methylated calmodulins activated an NAD kinase preparation to a maximal level that was at least 3-fold lower than the level of activation obtained with the corresponding unmethylated calmodulins. Methylation did not alter the cyclic nucleotide phosphodiesterase activator properties of these calmodulins. A genetically engineered calmodulin containing an arginine at position 115 instead of a lysine was produced by site-specific mutagenesis of a cloned synthetic calmodulin gene. The arginine derivative retained the higher maximal NAD kinase activator properties of the unmethylated calmodulins but was no longer susceptible to the effects of the methyltransferase. The data indicate that the reduction in the level of NAD kinase activation is the direct result of trimethylation of lysine 115 of calmodulin, provide a precedent for a functional effect of trimethyllysine in a protein, and raise the possibility that some of calmodulin's physiological activities may be affected by lysine methylation.     

Calmodulin-Mediated Signal Transduction Pathways in Arabidopsis Are Fine-Tuned by Methylation

Calmodulin N-methyltransferase (CaM KMT) is an evolutionarily conserved enzyme in eukaryotes that transfers three methyl groups to a highly conserved lysyl residue at position 115 in calmodulin (CaM). We sought to elucidate whether the methylation status of CaM plays a role in CaM-mediated signaling pathways by gene expression analyses of CaM KMT and phenotypic characterization of Arabidopsis thaliana lines wherein CaM KMT was overexpressed (OX), partially silenced, or knocked out. CaM KMT was expressed in discreet spatial and tissue-specific patterns, most notably in root tips, floral buds, stamens, apical meristems, and germinating seeds. Analysis of transgenic plants with genetic dysfunction in CaM KMT revealed a link between the methylation status of CaM and root length. Plants with suppressed CaM methylation had longer roots and CaM KMT OX lines had shorter roots than wild type (Columbia-0). CaM KMT was also found to influence the root radial developmental program. Protein microarray analyses revealed a number of proteins with specificity for methylated forms of CaM, providing candidate functional intermediates between the observed phenotypes and the target pathways. This work demonstrates that the functionality of the large CaM family in plants is fine-tuned by an overarching methylation mechanism.     

Calmodulin N-methyltransferase. Partial purification and characterization

The distribution, properties, and substrate specificity of S-adenosylmethionine:calmodulin (lysine) N-methyltransferse (EC 2.1.1.60, calmodulin N-methyltransferase) of the rat have been studied. This enzyme is cytosolic and is found at high levels in tissues with high levels of calmodulin and at low levels in tissues with little calmodulin. In liver, heart, and skeletal muscle, which have low levels of calmodulin and very low calmodulin N-methyltransferase activity (a low ratio of calmodulin N-methyltransferase to calmodulin), calmodulin was found to be incompletely methylated, as judged by its ability to act as a substrate for purified calmodulin N-methyltransferase. Calmodulin N-methyltransferase was purified 470-fold with a 33% yield from rat testis cytosol, using ammonium sulfate precipitation and chromatography on DEAE-cellulose, CM-Sepharose, and Sephadex G-100. At pH 7.4, calmodulin N-methyltransferase did not bind to DEAE-cellulose, but bound strongly to CM-Sepharose. The enzyme eluted from Sephadex G-100 with an apparent molecular weight of 55,000. Purified calmodulin N-methyltransferase was incubated with extracts of rat tissues, and [methyl-3H]AdoMet and methylated proteins were resolved by electrophoresis in an attempt to discover substances other than calmodulin, but this enzyme only catalyzed the methylation of calmodulin, indicating a high degree of substrate specificity. Conditions were established for the in vitro preparative methylation of des(methyl)-calmodulin from Dictyostelium discoideum. Three moles of methyl/mol of calmodulin were incorporated into lysine 115 of des(methyl)calmodulin, resulting in the formation of 1 mol of trimethyllysine at the site normally methylated in calmodulins from most species. Activation of cyclic nucleotide phosphodiesterase by des(methyl)calmodulin was indistinguishable from activation by in vitro methylated or sham methylated Dictyostelium calmodulin, indicating that methylation does not affect the ability of calmodulin to activate this enzyme.     

Amino Acid sequence of a novel calmodulin from the unicellular alga chlamydomonas

An amino acid sequence for a Chlamydomonas calmodulin has been elucidated with emphasis on the characterization of differences that are unique to Chlamydomonas and Dictyostelium calmodulin. While the concentration of calmodulin required for half-maximal activation of plant NAD kinase varies among vertebrate, higher plant, algal, and slime mold calmodulins, only calmodulins from the unicellular alga Chlamydomonas and the slime mold Dictyostelium show increased maximal activation of NAD kinase (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798; Marshak, Clarke, Roberts, Watterson 1984 Biochemistry 23: 2891-2899). The same preparations of calmodulin do not show major differences in phosphodiesterase or myosin light chain kinase activator activity.

We report here that a Chlamydomonas calmodulin has four primary structural features similar to Dictyostelium that are not found in other calmodulins characterized to date: an altered carboxy terminus including a novel 11-residue extension for Chlamydomonas calmodulin, unique residues at positions 81 and 118, and an unmethylated lysine at position 115. The only amino acid sequence identity unique to Chlamydomonas and Dictyostelium calmodulin is the presence of a lysine at position 115 instead of a trimethyllysine. These studies indicate that the methylation state of lysine 115 may be important in the maximal NAD kinase activator activity of calmodulin and support the concept that calmodulin has multiple functional domains in addition to multiple structural domains.

     

Comparison of the NAD Kinase and Myosin Light Chain Kinase Activator Properties of Vertebrate, Higher Plant, and Algal Calmodulins

In the preceding paper (Lukas, Iverson, Schleicher, Watterson 1984 Plant Physiol 75: 788-795), we reported that the amino acid sequence of spinach calmodulin has at least 13 amino acid sequence differences from vertebrate calmodulin. In the present study, we investigated the effect of these amino acid sequence substitutions on the enzyme activator properties of vertebrate and plant calmodulins. Calmodulins from spinach and the green alga Chlamydomonas reinhardtii activate chicken gizzard myosin light chain kinase in a manner similar but not identical to chicken calmodulin. In contrast, these calmodulins have very different NAD kinase activator properties. The concentration required for half-maximal activation of pea seedling NAD kinase by spinach calmodulin (3-4 nanomolar) is lower than the corresponding concentrations of chicken (20 nanomolar) and Chlamydomonas (40 nanomolar) calmodulins. However, the maximum level of activation obtained with Chlamydomonas calmodulin is 4- to 6-fold higher than spinach or chicken calmodulin. These data indicate that the limited structural heterogeneity among calmodulins have differential effects on their biochemical activities.     

Novel eye-specific calmodulin methylation characterized by protein mapping in Drosophila melanogaster

Post-translational methylation of the epsilon-amino group of lysine residues regulates a number of protein functions. Calmodulin, a key modulator of intracellular calcium signaling, is methylated on lysine 115 in many species. Although the amino acid sequence of calmodulin is highly conserved in eukaryotes, it has been shown that lysine 115 is not methylated in Drosophila calmodulin and no other methylation site has been reported. In this study, we characterized in vivo modification states of Drosophila calmodulin using proteomic methodology involving the protein mapping of microdissected Drosophila tissues on 2-D gels. We found that Drosophila calmodulin was highly expressed in methylated forms in the compound eye, whereas its methylation was hardly detected in other tissues. We identified that lysine 94 located in an EF-hand III is the methylation site in Drosophila calmodulin. The predominance of methylated calmodulin in the compound eye may imply the involvement of calmodulin in photoreceptor-specific functions through methylation.     

Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis

A gene coding for a calmodulin was synthesized and expressed in Escherichia coli. The gene was produced by the enzymatic ligation of 61 chemically synthesized DNA fragments. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis by the replacement of specific gene segments with synthetic double-stranded DNA. An expression vector containing the calmodulin gene was used to transform E. coli. Purification and characterization of calmodulin (VU-1 calmodulin) expressed by these transformants showed that it lacks two posttranslational modifications: an amino-terminal blocking group and N epsilon, N epsilon, N epsilon-trimethyllysine at position 115. The cyclic nucleotide phosphodiesterase activator properties of VU-1, higher plant, and vertebrate calmodulins were not statistically different. However, VU-1 calmodulin was found to activate nicotinamide adenine dinucleotide (NAD) kinase to a maximal level that was at least 3-fold higher than that found with higher plant and vertebrate calmodulins. This higher level of activation is also characteristic of calmodulins from Dictyostelium discoideum and Chlamydomonas reinhardtii [Roberts, D. M., Burgess, W. H., & Watterson, D. M. (1984) Plant Physiol. 75, 796-798; Marshak, D. R., Clarke, M., Roberts, D. M., & Watterson, D. M. (1984) Biochemistry 23, 2891-2899]. The only common feature among Dictyostelium, Chlamydomonas, and VU-1 calmodulins not found in higher plant and vertebrate calmodulins is an unmethylated lysine at position 115. The results indicate that the lack of methylation of lysine-115 may contribute to the maximal level of NAD kinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.     

Structural and functional properties of calmodulin from the eukaryotic microorganism Dictyostelium discoideum

Calmodulin was purified from the eukaryotic microorganism Dictyostelium discoideum and characterized in terms of its nearly complete primary structure and quantitative activator activity. The strategy for amino acid sequence analysis took advantage of the highly conserved structure of calmodulin and employed a new procedure for limited cleavage of calmodulin that uses a protease from mouse submaxillary gland. Fourteen amino acid sequence differences between Dictyostelium and bovine calmodulin were identified unequivocally, as well as an unmethylated lysine at residue 115 instead of N epsilon, N epsilon, N epsilon-trimethyllysine. Seven of the amino acid substitutions in Dictyostelium calmodulin are novel in that the residues at these positions are invariant in all calmodulin sequences previously examined, most notably an additional residue at the carboxy terminus. Comparison of the Dictyostelium calmodulin sequence with other calmodulin sequences shows that the region with the greatest extended sequence identity includes parts of the first and second structural domains and the interdomain region between domains 1 and 2. Dictyostelium calmodulin activated bovine brain cyclic nucleotide phosphodiesterase in a manner indistinguishable from that of bovine brain calmodulin. However, Dictyostelium calmodulin activated pea NAD kinase to a maximal level 4.6-fold greater than that produced by bovine brain calmodulin. This functional difference demonstrates the potential biological importance of the limited number of amino acid sequence differences between Dictyostelium calmodulin and other calmodulins and provides further insight into the structure, function, and evolution of the calmodulin family of proteins.     

Calmodulin during development and metamorphosis in urodelan amphibians

Calmodulin isolated and purified to homogeneity from young larvae is very similar to that obtained from adult Pleurodeles waltlii and these proteins are almost identical to previously described vertebrate calmodulins. During P. waltlii development, an increase in total individual calmodulin content is observed after the heart beating stage. In dorsal axial muscle, calmodulin level which is very high at the beginning of larval life (premetamorphosis) decreases strikingly in the first part of prometamorphosis. Such an evolution is observed in Ambystoma mexicanum too. Then, a significant increase occurs during metamorphosis. In contrast, calmodulin level in P. waltlii cardiac ventricular muscle increases continuously from hatching to the end of metamorphic climax. Thyroxine treatment which promotes precocious metamorphosis in P. waltlii and experimental metamorphosis in neotenic A. mexicanum, induces a rapid and significant increase in muscle calmodulin concentration.     

Loss of recovery capacity of plasmalemma k influx after cutting in chlorsulfuron pretreated maize roots

Active K⁺ influx was studied in apical segments from maize (Zea mays L., hybrid lines XL 342) and pea (Pisum sativum L. var Laxton superbo) seedlings pretreated with the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl) aminocarbonyl]benzenesulfonamide).

Even though both plants were sensitive to chlorsulfuron, a strong inhibition of K⁺ uptake only was evident in maize root segments after 12 hours pretreatment with 10 micromolar chlorsulfuron. The inhibition was revealed only when maize root segments were washed for 2 hours before uptake measurements. This was done in order to recover K⁺ influx inhibited by cutting injury. Consequently, we demonstrated that roots from chlorsulfuron pretreated maize seedlings lost the capacity to recover from cutting injury by washing. By contrast, K⁺ influx in pea roots was not inhibited by chlorsulfuron because pea roots notoriously do not exhibit the `washing' effect.

     

Initiation of nitric oxide (NO) synthesis in roots of etiolated seedlings of pea (Pisum sativum L.) under the influence of nitrogen-containing compounds

The level of nitric oxide (NO) in roots of 2-day-old etiolated pea (Pisum sativum L.) seedlings was investigated by fluorescence microscopy using the fluorescent probe 4,5-diaminofluorescein diacetate. Segments representing transversal (cross) cuts of the roots having thickness of 100 to 150 μm (a segment of the root located 10 to 15 mm from the apex) were analyzed. A substantial concentration of NO in the roots was registered when the seedlings were grown in water (control). Addition of 4 mM sodium nitroprusside, 20 mM KNO₃, 2 mM NaNO₂, 2 mM L-arginine into the growth medium increased NO concentration with respect to the control by 1.7- to 2.3-fold. Inhibitors of animal NO-synthase — 1 mM Nω-nitro-L-arginine methyl ester hydrochloride and 1 mM aminoguanidine hydrochloride — reduced the intensity of fluorescence in the root segments in the presence of all the studied compounds. In medium with KNO₃, the inhibitor of nitrate reductase ?150 μM sodium tungstate -lowered the fluorescence intensity by 60%. Scavengers of nitric oxide — 100 μM 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and 4 μM hemoglobin — lowered NO concentration in all the studied variants. Potassium ferrocyanide (4 mM) as the inactive analog of sodium nitroprusside inhibited generation of NO. These results are discussed regarding possible pathways of NO synthesis in plants.     

Calmodulin mRNA in Barley (Hordeum vulgare L.) : Apparent Regulation by Cell Proliferation and Light

Calmodulin is encoded by a 650-nucleotide mRNA in higher plants. This messenger was identified in barley and pea by a combination of in vitro translation and blot hybridization experiments using anti-sense RNA produced from an eel calmodulin cDNA probe. In all plant tissues tested, calmodulin mRNA represents between 0.01 and 0.1% of the total translatable mRNA population. Calmodulin mRNA levels are three- to fourfold higher in the meristematic zone of the first leaf of barley. At all other stages of leaf cell differentiation, calmodulin mRNA levels are nearly identical. During light-induced development in barley leaves, the relative proportion of translatable calmodulin mRNA declines about twofold. Cytoplasmic mRNAs that may encode calmodulin-like proteins were also detected. The levels of several of these putative Ca²⁺-binding protein mRNAs are modulated during the course of light-induced barley leaf cell development.     

Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae

Calmodulin from the yeast Saccharomyces cerevisiae was purified to complete homogeneity by hydrophobic interaction chromatography and HPLC gel filtration. The biochemical properties of the purified protein as calmodulin were examined under various criteria and its similarity and dissimilarity to other calmodulins have been described. Like other calmodulins, yeast calmodulin activated bovine phosphodiesterase and pea NAD kinase in a Ca2+-dependent manner, but its concentration for half-maximal activation was 8-10 times that of bovine calmodulin. The amino acid composition of yeast calmodulin was different from those of calmodulins from other lower eukaryotes in that it contained no tyrosine, but more leucine and had a high ratio of serine to threonine. Yeast calmodulin did not contain tryptophanyl or tyrosyl residues, so its ultraviolet spectrum reflected the absorbance of phenylalanyl residues, and had a molar absorption coefficient at 259 nm of 1900 M-1 cm-1. Ca2+ ions changed the secondary structure of yeast calmodulin, causing a 3% decrease in the alpha-helical content, unlike its effect on other calmodulins. Antibody against yeast calmodulin did not cross-react with bovine calmodulin, and antibody against bovine calmodulin did not cross-react with yeast calmodulin, presumably due to differences in the amino acid sequences of the antigenic sites. It is concluded that the molecular structure of yeast calmodulin differs from those of calmodulins from other sources, but that its Ca2+-dependent regulatory functions are highly conserved and essentially similar to those of calmodulins of higher eukaryotes.     

Correlation between Calmodulin Activity and Gravitropic Sensitivity in Primary Roots of Maize

Recent evidence indicates a role for calcium and calmodulin in the gravitropic response of primary roots of maize (Zea mays, L.). We examined this possibility by testing the relationship between calmodulin activity and gravitropic sensitivity in roots of the maize cultivars Merit and B73 × Missouri 17. Roots of the Merit cultivar require light to be gravitropically competent. The gravitropic response of the Missouri cultivar is independent of light. The occurrence of calmodulin in primary roots of these maize cultivars was tested by affinity gel chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with bovine brain calmodulin as standard. The distribution of calmodulin activity was measured using both the phosphodiesterase and NAD kinase assays for calmodulin. These assays were performed on whole tissue segments, crude extracts, and purified extracts. In light-grown seedlings of the Merit cultivar or in either dark- or light-grown seedlings of the Missouri cultivar, calmodulin activity per millimeter of root tissue was about 4-fold higher in the apical millimeter than in the subtending 3 millimeters. Calmodulin activity was very low in the apical millimeter of roots of dark-grown (gravitropically nonresponsive) seedlings of the Merit cultivar. Upon illumination, the calmodulin activity in the apical millimeter increased to a level comparable to that of light-grown seedlings and the roots became gravitropically competent. The time course of the development of gravitropic sensitivity following illumination paralleled the time course of the increase in calmodulin activity in the apical millimeter of the root. The results are consistent with the suggestion that calmodulin plays an important role in the gravitropic response of roots.     

Isolation and characterization of calmodulin from the motile green alga Chlamydomonas reinhardtii

Calmodulin, a calcium-binding protein with no known enzymatic activity but multiple, in vitro effector activities, has been purified to apparent homogeneity from the unicellular green alga Chlamydomonas reinhardtii and compared to calmodulin from vertebrates and higher plants. Chlamydomonas calmodulin was characterized in terms of electrophoretic mobility, amino acid composition, limited amino acid sequence analysis, immunoreactivity, and phosphodiesterase activation. Chlamydomonas calmodulin has two histidine residues similar to calmodulin from the protozoan Tetrahymena. However, unlike the protozoan calmodulin, only one of the histidinyl residues of Chlamydomonas calmodulin is found in the COOH-terminal third of the molecule. Chlamydomonas calmodulin lacks trimethyllysine but does have a lysine residue at the amino acid sequence position corresponding to the trimethyllysine residue in bovine brain and spinach calmodulins. The lack of this post-translational modification does not prevent Chlamydomonas calmodulin from quantitatively activating bovine brain phosphodiesterase. These studies also demonstrate that this unique calmodulin from a phylogenetically earlier eukaryote may be as similar to vertebrate calmodulin as it is to higher plant calmodulins, and suggest that Chlamydomonas calmodulin may more closely approximate the characteristics of a putative precursor of the calmodulin family than any calmodulin characterized to date.     

DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development

We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated.     

Further Characterization of Calmodulin from the Monocotyledon Barley (Hordeum vulgare)

We report here that calmodulin isolated from the monocotyledon barley is indistinguishable by a variety of criteria from calmodulin isolated from the dicotyledon spinach. In contrast to previous reports, we find that barley (Hordeum vulgare) calmodulin has an amino acid composition similar to that of vertebrate and spinach calmodulins, including the presence of a single trimethyllysinyl residue, and that barley calmodulin quantitatively activates cyclic nucleotide phosphodiesterase. Furthermore, spinach and barley calmodulins are similar in terms of tryptic peptide maps and immunoreactivity with various antisera that differ in their molecular specificities for calmodulins. Limited amino acid sequence analysis demonstrates that the region around the single histidinyl and trimethyllysinyl residues is identical among barley, spinach, and vertebrate calmodulins and that barley calmodulin, like spinach calmodulin, has a novel glutamine residue at position 96. We conclude that calmodulin is highly conserved among higher plants and that detailed sequence analysis is required before significant differences, if any, can be assigned to barley or other higher plant calmodulins. These studies suggest that calmodulin's fundamental importance to the eukaryotic cell may have been established prior to the evolutionary emergence of higher plants.