Alternative splicing of a novel diacylglycerol kinase in tomato leads to a calmodulin-binding isoform.
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.
Snedden
W A
WA
Department of Botany, University of Toronto, Toronto, Ontario, Canada, M5S 3B2.
Blumwald
E
E
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020013:Calcium Signaling; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D019852:Diacylglycerol Kinase / Q000235:genetics* / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology
D002147:Calmodulin; D018076:DNA, Complementary; D018744:DNA, Plant; D007527:Isoenzymes; D019852:Diacylglycerol Kinase