Calmodulin Isoforms Research Articles

Differential Activation of NAD Kinase by Plant Calmodulin Isoforms THE CRITICAL ROLE OF DOMAIN I

NAD kinase is a Ca2+/calmodulin (CaM)-dependent enzyme capable of converting cellular NAD to NADP. The enzyme purified from pea seedlings can be activated by highly conserved soybean CaM, SCaM-1, but not by the divergent soybean CaM isoform, SCaM-4 (Lee, S. H., Kim, J. C., Lee, M. S., Heo, W. D., Seo, H. Y., Yoon, H. W., Hong, J. C., Lee, S. Y., Bahk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806-21812). To determine which domains were responsible for this differential activation of NAD kinase, a series of chimeric SCaMs were generated by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-4111, a chimeric SCaM-1 that contains the first domain of SCaM-4, was severely impaired (only 40% of maximal) in its ability to activate NAD kinase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of SCaM-1 exhibited nearly full ( approximately 70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than half-maximal activation of NAD kinase. To define the amino acid residue(s) in domain I that were responsible for this differential activation, seven single residue substitution mutants of SCaM-1 were generated and tested for NAD kinase activation. Among these mutants, only K30E and G40D showed greatly reduced NAD kinase activation. Also a double residue substitution mutant, K30E/G40D, containing these two mutations in combination was severely impaired in its NAD kinase-activating potential, reaching only 20% of maximal activation. Furthermore, a triple mutation, K30E/M36I/G40D, completely abolished NAD kinase activation. Thus, our data suggest that domain I of CaM plays a key role in the differential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Further, the residues Lys30 and Glu40 of SCaM-1 are critical for this function.

Characterization of the calmodulin gene family in wheat: structure, chromosomal location, and evolutionary aspects.

Calmodulin is a ubiquitous transducer of calcium signals in eukaryotes. In diploid plant species, several isoforms of calmodulin have been described. Here, we report on the isolation and characterization of calmodulin cDNAs corresponding to 10 genes from hexaploid (bread) wheat (Triticum aestivum). These genes encode three distinct calmodulin isoforms; one isoform is novel in that it lacks a conserved calcium binding site. Based on their nucleotide sequences, the 10 cDNAs were classified into four subfamilies. Using subfamily-specific DNA probes, calmodulin genes were identified and the chromosomal location of each subfamily was determined by Southern analysis of selected aneuploid lines. The data suggest that hexaploid wheat possesses at least 13 calmodulin-related genes. Subfamilies 1 and 2 were both localized to the short arms of homoeologous-group 3 chromosomes; subfamily 2 is located on all three homoeologous short arms (3AS, 3BS and 3DS), whereas subfamily 1 is located only on 3AS and 3BS but not on 3DS. Further analysis revealed that Aegilops tauschii, the presumed diploid donor of the D-genome of hexaploid wheat, lacks a subfamily-1 calmodulin gene homologue, whereas diploid species related to the progenitors of the A and B genomes do contain such genes. Subfamily 3 was localized to the short arm of homoeologous chromosomes 2A, 2B and 2D, and subfamily 4 was mapped to the proximal regions of 4AS, 4BL and 4DL. These findings suggest that the calmodulin genes within each subfamily in hexaploid wheat represent homoeoallelic loci. Furthermore, they also suggest that calmodulin genes diversified into subfamilies before speciation of Triticum and Aegilops diploid species.

Calmodulin Isoforms Differentially Enhance the Binding of Cauliflower Nuclear Proteins and Recombinant TGA3 to a Region Derived from the Arabidopsis Cam-3 Promoter

Calmodulin Isoforms Differentially Enhance the Binding of Cauliflower Nuclear Proteins and Recombinant TGA3 to a Region Derived from the Arabidopsis Cam-3 Promoter

Calmodulin isoforms differentially enhance the binding of cauliflower nuclear proteins and recombinant TGA3 to a region derived from the Arabidopsis Cam-3 promoter.

Many stimuli increase cytoplasmic Ca2+ concentrations as an early signal transduction event and alter the patterns of nuclear gene transcription, but the mechanisms by which Ca2+ signals are transduced to the nucleus are not known. This article shows that at least four DNA binding proteins from cauliflower nuclear extracts are also calmodulin (CaM) binding proteins. CaM enhances the binding of these proteins to a C/G-box sequence element in the Arabidopsis Cam-3 promoter. Binding to the C/G-box is enhanced preferentially by the CaM isoform encoded by Cam-3. However, it is not clear whether the effect is mediated directly by CaM or indirectly through the activity of a CaM-regulated protein phosphatase. CaM also binds recombinant TGA3 and enhances its binding to the same Cam-3 promoter element. These results are consistent with the idea that a Ca(2+)-mediated signalling pathway eliciting some changes in gene expression may consist of CaM, or a structurally related Ca2+ binding protein, and transcription factors.

Differential Stimulation of NAD Kinase and Binding of Peptide Substrates by Wild-Type and Mutant Plant Calmodulin Isoforms

Calmodulin fromArabidopsis thalianaconsists of at least four isoforms, which differ in their deduced amino acid sequences by as many as six conservative substitutions. To determine whether these differences are biochemically significant, cDNAs encoding three of the four isoforms were engineered to produce recombinant proteins inEscherischia coli,purified to apparent homogeneity, and assayed for their abilities to activate pea leaf NAD kinasein vitro.The CaM-2 isoform was a significantly more efficient activator of NAD kinase compared with the CaM-4 and -6 isoforms based on both the apparentVmaxit elicited and theK0.5activation. These results are consistent with the hypothesis that theArabidopsisCaM isoforms have evolved to optimize the protein's interaction with different Ca2+/CaM-regulated target enzymes. The ability to activate NAD kinase was also investigated for a carboxy-terminal nonsense mutant of CaM-6 (CaM-6M), which substituted 14 hydrophilic amino acids for a region of seven amino acids that normally form an exposed hydrophobic surface whenwtCaM-6 binds Ca2+. CaM-6M-activated NAD kinase displayed an apparentVmaxthat was reduced 40% and aK0.5that was an order of magnitude greater than the CaM-6-activated enzyme. The Ca2+-dependence of CaM-6M to activate NAD kinase was identical to that of CaM-6, but CaM-6M bound synthetic peptide substrates with lower apparent affinity than did CaM-6. Thus, the carboxy-terminal hydrophobic domain of CaM appears to be critical for its interaction with NAD kinase. In contrast, amino-terminal fusions of a hydrophilic, α-helical 12-residuec-mycepitope tag to CaM-2 and -4 yielded proteins that activated NAD kinase to apparentVmaxvalues within 10% of those obtained with the wild-type CaM proteins.

Regulated expression of a calmodulin Isoform alters growth and development in potato

A transgene approach was taken to study the consequences of altered expression of a calmodulin isoform on plant growth and development. Eight genomic clones of potato calmodulin (PCM1 to 8) have been isolated and characterized (Takezawa et al., 1995). Among the potato calmodulin isoforms studied, PCM1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM1 fused to the CaMV 35S promoter. Transgenic plants showing a moderate increase in PCM1 mRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM1 mRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM1 mRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM1 protein in transgenic plants, indicating that the expression of both mRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM1 alters growth and development in potato plants.

Identification of a Novel Divergent Calmodulin Isoform from Soybean Which Has Differential Ability to Activate Calmodulin-dependent Enzymes

Calmodulin plays pivotal roles in the transduction of various Ca(2+)-mediated signals and is one of the most highly conserved proteins in eukaryotic cells. In plants, multiple calmodulin isoforms with minor amino acid sequence differences were identified but their functional significances are unknown. To investigate the biological function of calmodulins in the regulation of calmodulin-dependent enzymes, we cloned cDNAs encoding calmodulins in soybean. Among the five cDNAs isolated from soybean, designated as SCaM-1 to -5, SCaM-4 and -5 encoded very divergent calmodulin isoforms which have 32 amino acid substitutions from the highly conserved calmodulin, SCaM-1 encoded by SCaM-1 and SCaM-3. SCaM-4 protein produced in Escherichia coli showed typical characteristics of calmodulin such as Ca(2+)-dependent electrophoretic mobility shift and the ability to activate phosphodiesterase. However, the extent of mobility shift and antigenicity of SCaM-4 were different from those of SCaM-1. Moreover, SCaM-4 did not activate NAD kinase at all in contrast to SCaM-1. Also there were differences in the expression pattern of SCaM-1 and SCaM-4. Expression levels of SCaM-4 were approximately 5-fold lower than those of SCaM-1 in apical and elongating regions of hypocotyls. In addition, SCaM-4 transcripts were barely detectable in root whereas SCaM-1 transcripts were as abundant as in apical and elongating regions of hypocotyls. In conclusion, the different biochemical properties together with differential expression of SCaM-4 suggest that this novel calmodulin may have different functions in plant cells.

Insulin-dependent phosphorylation of calmodulin in rat hepatocytes.

Insulin-stimulated phosphorylation of calmodulin in vivo was examined using a highly specific anti-calmodulin monoclonal antibody combined with high resolution two-dimensional gel electrophoresis. The two major isoforms of calmodulin immunoprecipitated from insulin-treated hepatocytes migrated on two-dimensional gel electrophoresis to the same position as nonphosphorylated calmodulin and calmodulin phosphorylated in vitro. Immunoblotting verified the identity of calmodulin. Insulin enhanced the phosphorylation of calmodulin 3.1 +/- 0.4-fold (mean +/- S.E., n = 10), with a stoichiometry in insulin-treated hepatocytes of 0.47 +/- 0.06 (mean +/- S.E., n = 3) mol of phosphate/mol of calmodulin. Two-dimensional phosphopeptide mapping of calmodulin immunoprecipitated from rat hepatocytes and calmodulin phosphorylated in vitro by the insulin receptor kinase or casein kinase II revealed several common phosphopeptides. The common phosphopeptides that appeared insulin-sensitive in intact cells comprised 61 and 40% of casein kinase II- and insulin receptor-catalyzed 32P incorporation into calmodulin in vitro, respectively. This suggests that casein kinase II and the insulin receptor kinase are, at least in part, responsible for insulin-stimulated phosphorylation of calmodulin in rat hepatocytes. These data indicate that phosphorylation of calmodulin in intact hepatocytes is significantly enhanced by insulin, supporting a critical role for calmodulin in insulin signal transduction.

Phosphorylation of the 61-kDa calmodulin-stimulated cyclic nucleotide phosphodiesterase at serine 120 reduces its affinity for calmodulin.

Phosphorylation of the 61-kDa isoform of bovine calmodulin (CaM)-stimulated cyclic nucleotide phosphodiesterase (CaM-PDE) by the catalytic subunit of cyclic AMP-dependent protein kinase A (PKA) results in a decrease in the affinity of the enzyme for calmodulin [Sharma, R. K., & Wang, J. H. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 2603-2607]. In the present study, purified 61-kDa CaM-PDE was phosphorylated in the presence of [gamma-32P]ATP and cleaved with a Lys-C endoproteinase. The resultant phosphopeptides were resolved by reverse-phase HPLC and analyzed by electrospray mass spectrometry and Edman sequencing. Serine residues 120 and 138 were identified as the principal sites of phosphorylation. A cDNA encoding the 61-kDa CaM-PDE [Sonnenburg, W. K., Seger, D., & Beavo, J. A. (1993) J. Biol. Chem. 268, 645-652] was used to generate point mutants in which either or both of these serines were replaced with alanine. The mutants were expressed in COS-7 cells, purified, and phosphorylated. Phosphorylation of the mutant Ser 138-->Ala resulted in a decrease in affinity for CaM that was comparable to that seen with the wild-type enzyme. In contrast, phosphorylation of the mutant Ser 120-->Ala had virtually no effect on CaM affinity. We conclude that phosphorylation of serine 120 by PKA is responsible for the reduction in affinity of the 61-kDa CaM-PDE for CaM.

Characterization of the human calmodulin-like protein expressed in Escherichia coli.

The protein-coding region of an intronless human calmodulin-like gene [Koller, M., & Strehler, E. E. (1988) FEBS Lett. 239, 121-128] has been inserted into a pKK233-2 expression vector, and the 148-residue, M(r) = 16,800 human protein was purified to apparent homogeneity by phenyl-Sepharose affinity chromatography from cultures of Escherichia coli JM105 transformed with the recombinant vector. Several milligrams of the purified protein were obtained from 1 L of bacterial culture. A number of properties of human CLP were compared to those of bacterially expressed human calmodulin (CaM) and of bovine brain CaM. CLP showed a characteristic Ca(2+)-dependent electrophoretic mobility shift on SDS-polyacrylamide gels, although the magnitude of this shift was smaller than that observed with CaM. CLP was able to activate the 3',5'-cyclic nucleotide phosphodiesterase to the same Vmax as normal CaM, albeit with a 7-fold higher Kact. In contrast, the erythrocyte plasma membrane Ca(2+)-ATPase could only be stimulated to 62% of its maximal CaM-dependent activity by CLP. CLP was found to contain four Ca(2+)-binding sites with a mean affinity constant of 10(5) M-1, a value about 10-fold lower than that for CaM under comparable conditions. The highly tissue-specifically-expressed CLP represents a novel human Ca(2+)-binding protein showing characteristics of a CaM isoform.

Primary Structures of Arabidopsis Calmodulin Isoforms Deduced from the Sequences of cDNA Clones

Complementary DNA (cDNA) clones encoding calmodulin isoforms were isolated from an Arabidopsis leaf lambdagt10 library by screening with cloned barley calmodulin cDNA probes. Two cDNAs, one a 626-base pair partial-length clone (ACaM-1) and one a 1400-base pair full-length clone (ACaM-2), encode calmodulin polypeptides that differ by four conservative amino acid substitutions. None of the amino acid sequence differences occur within the four Ca(2+)-binding domains of the proteins. Whereas the deduced amino acid sequences of the two Arabidopsis calmodulin isoforms share 97% identity, the nucleotide sequences encoding the two isoforms share 87% sequence identity. Most of these nucleotide sequence differences (80%) occur in codon wobble positions. ACaM-1 and ACaM-2 both hybridize with a distinct set of restriction fragments of Arabidopsis total DNA, indicating that they were derived from transcripts of separate genes; these genes are single- or very low-copy in the Arabidopsis genome. Both cDNAs hybridize to messenger RNA (mRNA) species of 0.8 kilobases that are expressed to a greater extent in developing siliques compared with leaves, flowers, and stems. Northern blot and polymerase chain reaction assays both indicate that ACaM-1 mRNA is more highly expressed than ACaM-2 mRNA in developing siliques. The steady-state levels of both isoform mRNAs increase as a result of touch stimulation; the kinetics and extent of increase are comparable for the two mRNAs.