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

Abstract

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.Calmodulin, a highly conserved and ubiquitous protein in eukaryotes, mediates Ca 2ϩ signals to various target proteins (1). A variety of regulatory enzymes and proteins such as protein kinases, ion channels, Ca 2ϩ pumps, nitric oxide synthetase, inositol trisphosphate kinase, cyclic nucleotide phosphodiesterase, and NAD kinase are known to be regulated by Ca 2ϩ and calmodulin (2-4). While a great deal of information has been known for biological roles of calmodulin in animal cells, very little is known about the roles of calmodulin in plant cells. This is mainly due to the absence of purified calmodulindependent enzymes and/or their genes in plants. As an effort to investigate the biological role(s) of calmodulin in plants, calmodulin genes in various plant species have been cloned and characterized recently (4). Interestingly, in Arabidopsis, cDNAs encoding multiple calmodulin isoforms have been isolated although the degree of sequence divergence is minor, only 6 amino acid differences between the two most divergent isoforms among Arabidopsis calmodulins (5-7). This is very notable because, in animal cells, only a single form of calmodulin is produced by a calmodulin multigene family (8, 9). However, it has not been determined whether plant calmodulin isoforms have the same biochemical properties such as calcium-binding abilities and activation of calmodulin-dependent enzymes. Also the biological role of multiple calmodulin isoforms...

Cloning and Characterization of Two NAD Kinases from Arabidopsis. Identification of a Calmodulin Binding Isoform

“…A diversity of Ca 21 sensors, including Ca 21 -dependent protein kinases and calcineurin-like proteins, likely endow plants with a highly specific and complex Ca 21 signaling network (Luan et al, 2002). Consistent with earlier reports (Lee et al, 1995(Lee et al, , 1997Liao et al, 1996), we observed a differential response of native NADK activity to a variety of CaM-related proteins, including seven that had not previously been cloned or studied. Interestingly, CML10, which is about 64% identical to the conserved CaM but possesses a unique 42-residue Figure 6.…”

“…Effect of Various CaM-Related Proteins on Native NADK Activity Previous studies on pea (Pisum sativum) NADK have demonstrated a differential response of the enzyme to various CMLs unique to plants (Lee et al, 1995(Lee et al, , 1997Liao et al, 1996). We examined the responsiveness of Arabidopsis native NADK to seven novel Arabidopsis CML recombinant proteins and a previously described protein, CML10 (originally termed CaBP-22; Ling and Zielinski, 1993).…”

Identification of a Calmodulin-Regulated Soybean Ca2+-ATPase (SCA1) That Is Located in the Plasma Membrane

“…The vanadate-sensitive ATPase activities of vector control (triangles), SCA1p (squares), SCA1p nt52 (diamonds), and SCA1p nt85 (circles) were determined at 5 M free [Ca 2ϩ ]. The concentration of soybean calmodulin isoform 1 (SCaM-1; Lee et al, 1995) was varied from 0 to 100 nM. Data shown are the average of three independent experiments.…”

“…Isolation of total RNA and RNA gel blot hybridization were performed as described (Lee et al, 1995). SB-P cell culture was maintained in KN-1 medium as described (Horn et al, 1989).…”

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

“…Calmodulin was purified from bovine brain as described with a few modifications (Gopalakrishna and Anderson, 1982;Lee et al, 1995) from 350 g of bovine brain. The homogenate of rat brain (SD) was prepared by Warring blender and Potter-Elvehjem homogenizer in one volume of 30 mM Hepes, pH 7.4, 2 mM EDTA, 2 mM EGTA, 1 mM CaCl 2 , 1 mM DTT, 1 mM PMSF, 4 µg/ml aprotinin, 2 µg/ml leupeptin.…”