Nuclear Transportation of Diacylglycerol Kinase γ and Its Possible Function in the Nucleus

Abstract

Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGK␥ came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGK␥ is transported from the cytoplasm to the nucleus. The nuclear transportation of DGK␥ didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGK␥ acts as a nuclear transport signal. Furthermore, to address the function of DGK␥ in the nucleus, we produced stable cell lines of wild-type DGK␥ and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGK␥ were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGK␥ regulates cell cycle. Diacylglycerol (DG)3 is a second messenger regulating various cellular responses (1, 2). One of the important roles of DG is an activating of protein kinase C (PKC) (1,3,4). DG is physiologically produced as a result of the signal-induced hydrolysis of phosphatidylinositol by phospholipase C. The generated DG is phosphorylated to phosphatidic acid by diacylglycerol kinase (DGK) or metabolized by DG lipase (2, 5, 6). Thus, DGK is an important enzyme to inactivate PKC by reducing the DG level, contributing to regulating of the cellular response. In addition, phosphatidic acid itself activates PKC (7), phosphatidylinositol 4-phosphate 5-kinase (8, 9), and mammalian target of rapamycin (10), and modulates Ras GTPase-activating protein (11).Molecular cloning studies revealed that mammalian DGK family consists of at least nine subtypes (2). Although all DGKs have cysteinerich repeats similar to the C1A and C1B domains of PKCs in the N terminus and a catalytic domain in the C terminus, they are divided into five groups on the primary structure of these DGKs. In an effort to elucidate the function of DGK, we unexpectedly found that GFP-fused DGK␥ (GFP-DGK␥) became localized in the nucleus as well as the cytoplasm a few days after transfection but was localized mainly in the cytoplasm just after expressed in CHO-K1 cells. Although nuclear transportation of DGK␥ has never been reported, expression of DGK and DGK in the nucleus has been already described (20,21). In addition, DGK is thought to be involved in the regulation of cell cycle (21). These findings, together with the facts that phosphatidylinositol turnover exists within the nucleus and DG may be involved in the regulation of cell cycle (22-27), suggest that DGK␥ has some physiological function in the nucleus. However, mechanism of the nuclear transportation and physiological functions of DGK␥ are unknown. W...

Cyclic AMP-Stimulated Interaction between Steroidogenic Factor 1 and Diacylglycerol Kinase θ Facilitates Induction of CYP17

“…Many studies have demonstrated roles for nuclear DGK activity in varied cellular processes. For example, DGK-is activated by nerve growth factor in PC12 cells (101) and thrombin in IIC9 fibroblasts (8,107), DGK-promotes myogenesis in C2C12 cells (23), and DGK-␥ plays a role in regulating the cell cycle in CHO-K cells (72). Interestingly, both DGK-(102) and DGK-(23) are localized in the speckle domains of the nucleus.…”

c-Abl Tyrosine Kinase Regulates Serum-induced Nuclear Export of Diacylglycerol Kinase α by Phosphorylation at Tyr-218

“…The phosphorylation at this site may inhibit the nuclear localization signal function of the C1 domain because Tyr-218 is in the C1 domain, because we previously reported that the C1 domain of DGK␥ has an important role in nuclear transport (17). Binding to importin-␣, a carrier protein that transports some proteins to the nucleus (33)(34)(35), or to an as yet unidentified carrier protein may be regulated by this phosphorylation.…”

cAMP-stimulated transcription of DGKθ requires steroidogenic factor 1 and sterol regulatory element binding protein 1

“…DGK isoforms have been implicated in various other cellular processes including inhibition of Rap1 signaling ( 9 ) and retinoblastoma-mediated cell cycle control ( 10 ). DGK is activated by nerve growth factor in PC12 cells ( 11 ) and thrombin in IIC9 fi broblasts ( 12,13 ), whereas DGK promotes myogenesis in C2C12 cells ( 14 ) and DGK ␥ plays a role in regulating the cell cycle in CHO-K cells ( 15 ).…”