Cloning and characterization of diacylglycerol kinase iota splice variants in rat brain.
Diacylglycerol kinase (DGK) catalyzes phosphorylation of a second messenger diacylglycerol (DG) to phosphatidic acid in cellular signal transduction. Previous studies have revealed that DGK consists of a family of isozymes including our rat clones. In this study we isolated from rat brain cDNA library the cDNA clones for a rat homologue of DGKiota (rDGKiota-1) that contains two zinc finger-like sequences, the highly conserved DGK catalytic domain, a bipartite nuclear localization signal, and four ankyrin repeats at the carboxyl terminus. In addition, we found novel splice variants, which contain either insertion 1 (71 bp) or insertion 2 (19 bp) or both in the carboxyl-terminal portion. Each of the insertions causes a frameshift, and the resultant premature stop codons produce two truncated forms (termed rDGKiota-2 and -iota-3), the former lacking the ankyrin repeats at the carboxyl terminus and the latter lacking a part of the catalytic domain and the ankyrin repeats. Truncation of the carboxyl-terminal portion clearly exerts effects on the detergent solubility and enzymatic activity of the splice variants, although all three variants showed similar cytoplasmic localization in cDNA-transfected cultured neurons despite the continued presence of the nuclear localization signal sequence. Immunoblot analysis using anti-rDGKiota antibody raised against the common amino-terminal portion clearly shows that these rDGKiota variants are indeed expressed in the brain. These results suggest that the carboxyl-terminal truncated forms of rDGKiota-2 and -iota-3 that exhibit reduced enzymatic activities might show a dominant negative effect against the intact rDGKiota-1, and that the modulation of signal transduction by the splice variants may play some roles in the physiologic and/or pathologic conditions of neurons.
Ito
Tsukasa
T
Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Japan.
Hozumi
Yasukazu
Y
Sakane
Fumio
F
Saino-Saito
Sachiko
S
Kanoh
Hideo
H
Aoyagi
Masaru
M
Kondo
Hisatake
H
Goto
Kaoru
K
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D019852:Diacylglycerol Kinase / Q000032:analysis / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D009928:Organ Specificity; D051381:Rats
D007527:Isoenzymes; D019852:Diacylglycerol Kinase