Alternative splicing of a novel diacylglycerol kinase in tomato leads to a calmodulin‐binding isoform

Abstract

Summary Calmodulin is a regulatory protein activated during Ca2+ signalling. We have isolated a cDNA, designated LeCBDGK (Lycopersicon esculentum calmodulin‐binding diacylglycerol kinase) encoding a novel calmodulin‐binding protein with sequence similarity to diacylglycerol kinases from animals. Diacylglycerol kinases convert diacylglycerol to phosphatidic acid. We delineated the calmodulin‐binding domain to approximately 25 residues near the C‐terminus of LeCBDGK. We have also isolated a second diacylglycerol kinase cDNA, designated LeDGK1, identical to LeCBDGK, except that it lacks the calmodulin‐binding domain. Both recombinant LeCBDGK and LeDGK1 were catalytically active in vitro. Anti‐DGK antiserum detected two immunoreactive proteins associated with microsomal and plasma membrane fractions from cell suspensions. The higher molecular weight immunoreactive protein was also present in soluble extracts and bound to calmodulin–agarose in the presence of calcium, demonstrating that native LeCBDGK is a calmodulin‐binding protein. In the presence of calcium, LeCBDGK associated with membrane cell fractions in vitro, but calmodulin antagonists disrupted this association, suggesting a possible role of calcium in the recruitment of LeCBDGK from soluble to membrane cell fractions. Native LeCBDGK and calmodulin co‐immunoprecipitated from tomato soluble cell extracts, suggesting their interaction in vivo. The same gene encodes both LeCBDGK and LeDGK1 and the calmodulin‐binding domain of LeCBDGK is encoded by a separate exon. Thus, alternative transcript splicing leads to calmodulin‐binding and non‐binding forms of diacylglycerol kinases in tomato. Possible roles of LeCBDGK and LeDGK1 in calcium and lipid signalling are discussed.

AtDGK2, a Novel Diacylglycerol Kinase from Arabidopsis thaliana, Phosphorylates 1-Stearoyl-2-arachidonoyl-sn-glycerol and 1,2-Dioleoyl-sn-glycerol and Exhibits Cold-inducible Gene Expression

“…Furthermore, biochemical studies are required to demonstrate that the predicted proteins indeed code for enzymatically active DGKs, namely in the absence of a DAG/PE-binding domain. However, as mentioned before, earlier studies have shown that tomato LeDGK1, which lacks such a domain, exhibits diacylglycerol kinase activity upon heterologous expression in E. coli (17).…”

“…Generation of the AtDGK5␤ splice variant is also evident from serial analysis of gene expression (SAGE) experiments, deposited in the NCBI GEO expression data repository (GEO Data Set accession number GDS101). Importantly, splice variants have also been reported for LeDGK1 from tomatoes (17). Whereas LeDGK1 itself does not bind calmodulin, the LeCB-DGK protein, which is encoded by an alternatively spliced transcript, contains a calmodulin-binding domain at its very C terminus.…”

“…However, in the presence of Ca 2ϩ , LeCBDGK associated with membrane cell fractions in vitro. This association was disrupted by the addition of CaM antagonists, suggesting that Ca 2ϩ plays a role in recruiting the enzyme from soluble to membrane cell fractions (17).…”

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

“…Samples were either assayed immediately for NADK activity or were frozen in liquid nitrogen and stored at 280°C. Recombinant NADK2, which is able to bind CaM-agarose, was purified using CaM-affinity chromatography essentially as described (Snedden and Blumwald, 2000). The soluble bacterial extract containing recombinant NADK2 was adjusted to a final concentration of 3.0 mM CaCl 2 and loaded onto a CaM-agarose affinity column (300 mL) preequilibrated with CaM-binding buffer (50 mM Tris-HCl, pH 7.5, 1 mM CaCl 2 , 10% [w/v] glycerol, 1 mM DTT, and 1 mM PMSF).…”

“…CaM81-AF532 interacting proteins were detected by fluorescence imaging using a Typhoon 8600 phosphor imager (Uppsala) at excitation and emission wavelengths of 532 and 555 nm, respectively. A GST-fusion protein containing the CaM-binding domain of tomato diacylglyerol kinase (Snedden and Blumwald, 2000) was used as a positive control for CaM81-AF532 binding.…”