Cloning and Characterization of Novel Testis-Specific Diacylglycerol Kinase η Splice Variants 3 and 4

Abstract

Diacylglycerol kinase (DGK) phosphorylates DG to generate phosphatidic acid. Recently, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the secondary spermatocytes and round spermatids of the testis. In this study, we cloned the full length DGKη3 gene and confirmed the endogenous expression of its protein product. During the cloning procedure, we found a new testis-specific alternative splicing product of the DGKη gene, DGKη4, which lacks half of the catalytic domain. We examined the DGK activity and subcellular localization of DGKη3 and η4. DGKη3 had almost the same activity as DGKη1, whereas the activity of DGKη4 was not detectable. In resting NEC8 cells (human testicular germ cell tumor cell line), DGKη1, η3 and η4 were broadly distributed in the cytoplasm. When osmotically shocked, DGKη1 and η4 were distributed in punctate vesicles in the cytoplasm. In contrast, DGKη3 was partly translocated to the plasma membrane and co-localized with the actin cytoskeleton. These results suggest that DGKη3 and η4 have properties different from those of DGKη1 and that they play roles in the testis in a different manner.

Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains

“…The cDNAs encoding human DGK␦2 and human DGK␦2-⌬SAMD that were subcloned into the expression plasmids, p3ϫFLAG-CMV (Sigma, Tokyo, Japan) and pAcGFP-C1 (Clontech-Takara Bio, Kusatsu, Japan), for expression in mammalian cells were generated as described (Fig. 1B) (21,(40)(41)(42). To express 6ϫHis-tagged protein, human DGK␦, human SMSr, and SMSr-⌬SAMD (amino acid residues 79 -413) were subcloned into the pSF-CMV-NH 2 -His-EKT3 vector (OG332, Oxford Genetics, Begbroke, UK).…”

The Plasma Membrane and Mechanoregulation in Cells

“…Force more than 100 pN (below which GFP can withstand by virtue of its stiff β-barrels) loading at the ends of the cassette stretches the helix, increasing the distance and reducing FRET. 101,102 Besides FRET-based techniques, fluorescence-lifetime-based techniques are also in use for quantifying mechanoregulation. Flipper-TR (FliptR) is a molecule composed of two dithienothiophenes (DTTs).…”

“…At rest, stFRET has a robust energy transfer with a strong emission at 527 nm and a quenched donor emission at 475 nm. Force more than 100 pN (below which GFP can withstand by virtue of its stiff β-barrels) loading at the ends of the cassette stretches the helix, increasing the distance and reducing FRET. , …”

“…Force more than 100 pN (below which GFP can withstand by virtue of its stiff β-barrels) loading at the ends of the cassette stretches the helix, increasing the distance and reducing FRET. 102 , 103 …”

Hypervirulent Klebsiella pneumoniae: Characteristic and clinical perspective

“…However, until now the explanation is still limited. The most probable factor is colibactin, which has been shown to be genotoxic, causing DNA damage and cell death [40]. However, there are some strains of hvKP that do not produce colibactin (for example, the non-cg23-K1 capsule type) but can also cause abscesses to form.…”