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

Takehiro Matsubara,Yasuhito Shirai,Kei Miyasaka,Takuya Murakami,Yasuto Yamaguchi,Takehiko Ueyama,Masahiro Kai,Fumio Sakane,Hideo Kanoh,Toshiaki Hashimoto,Shinji Kamada,Ushio Kikkawa,Naoaki Saito

doi:10.1074/jbc.m509873200

Takehiro Matsubara, Yasuhito Shirai + Show 11 more

Open Access

https://doi.org/10.1074/jbc.m509873200

Copy DOI

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 DGKgamma 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 DGKgamma is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKgamma didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKgamma acts as a nuclear transport signal. Furthermore, to address the function of DGKgamma in the nucleus, we produced stable cell lines of wild-type DGKgamma and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKgamma were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKgamma regulates cell cycle.

Highlights

Diacylglycerol (DG)3 is a second messenger regulating various cellular responses [1, 2]
In an effort to investigate one of nine subtypes, we found that diacylglycerol kinase (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
We showed for the first time that DGK␥ is transported from the cytoplasm to the nucleus (Fig. 1)

Summary

Molecular cloning studies revealed that mammalian DGK family

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. Type I DGKs, including DGK␣, -␤, and -␥, have EF-hand motifs and two cysteine-rich regions (C1 domain) in the regulatory domain [12, 13], whereas Type II DGKs, DGK␦ and -␩, have a pleckstrin homology domain instead of the EF-hand motif in addition to the C1 domain [14, 15]. The final group, type V, includes DGK␪, which has three cysteine-rich regions and a pleckstrin homology domain with overlapping Ras-associating domain [19]. Subtype specific function and regulation mechanisms of DGKs are not clear. Nuclear transportation of DGK␥ has never been reported, expression of DGK␪ and DGK␨ in the nucleus has been already described [20, 21]. Mechanism of the nuclear transportation and physiological functions of DGK␥ are unknown. We investigated molecular mechanism and physiological significance of nuclear transportation of DGK␥

EXPERIMENTAL PROCEDURES

RESULTS

Localization in the nucleus

Average of cell size

DISCUSSION