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 DGKγ 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 DGKγ is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKγ didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKγ acts as a nuclear transport signal. Furthermore, to address the function of DGKγ in the nucleus, we produced stable cell lines of wild-type DGKγ and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKγ were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKγ regulates cell cycle. 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 DGKγ 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 DGKγ is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKγ didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKγ acts as a nuclear transport signal. Furthermore, to address the function of DGKγ in the nucleus, we produced stable cell lines of wild-type DGKγ and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKγ were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKγ regulates cell cycle. Diacylglycerol (DG) 3The abbreviations used are: DG, diacylglycerol; PKC, protein kinase C; DGK, diacylglycerol kinase; CHO, Chinese hamster overly; C1, conserved region 1; C1A, first half of C1 domain; C1B, second half of C1 domain; KN, kinase negative; COS, African green monkey kidney; GFP, green fluorescent protein; NLS, nuclear localization signal; PBS, phosphate-buffered saline; aa, amino acid(s).3The abbreviations used are: DG, diacylglycerol; PKC, protein kinase C; DGK, diacylglycerol kinase; CHO, Chinese hamster overly; C1, conserved region 1; C1A, first half of C1 domain; C1B, second half of C1 domain; KN, kinase negative; COS, African green monkey kidney; GFP, green fluorescent protein; NLS, nuclear localization signal; PBS, phosphate-buffered saline; aa, amino acid(s). is a second messenger regulating various cellular responses (1Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5757) Google Scholar, 2Topham M.K. Prescott S.M. J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). One of the important roles of DG is an activating of protein kinase C (PKC) (1Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5757) Google Scholar, 3Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3531) Google Scholar, 4Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4218) Google Scholar). 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 (2Topham M.K. Prescott S.M. J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 5Bell R.L. Kennerly D.A. Stanford N. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3238-3241Crossref PubMed Scopus (597) Google Scholar, 6Moriyama T. Urade R. Kito M. J. Biochem. (Tokyo). 1999; 125: 1077-1085Crossref PubMed Scopus (46) Google Scholar). 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 ζPKC (7Limatola C. Schaap D. Moolenaar W.H. van Blitterswijk W.J. Biochem. J. 1994; 304: 1001-1008Crossref PubMed Scopus (286) Google Scholar), phosphatidylinositol 4-phosphate 5-kinase (8Jones D.R. Sanjuan M.A. Merida I. FEBS Lett. 2000; 476: 160-165Crossref PubMed Scopus (65) Google Scholar, 9Luo B. Prescott S.M. Topham M.K. Cell. Signal. 2004; 16: 891-897Crossref PubMed Scopus (68) Google Scholar), and mammalian target of rapamycin (10Fang Y. Vilella-Bach M. Bachmann R. Flanigan A. Chen J. Science. 2001; 294: 1942-1945Crossref PubMed Scopus (861) Google Scholar), and modulates Ras GTPase-activating protein (11Tsai M.H. Yu C.L. Stacey D.W. Science. 1990; 250: 982-985Crossref PubMed Scopus (123) Google Scholar). Molecular cloning studies revealed that mammalian DGK family consists of at least nine subtypes (2Topham M.K. Prescott S.M. J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Although all DGKs have cysteine-rich 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 DGKα,-β, and -γ, have EF-hand motifs and two cysteine-rich regions (C1 domain) in the regulatory domain (12Kanoh H. Yamada K. Sakane F. J. Biochem. (Tokyo). 2002; 131: 629-633Crossref PubMed Scopus (114) Google Scholar, 13Jiang Y. Qian W. Hawes J.W. Walsh J.P. J. Biol. Chem. 2000; 275: 34092-34099Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), whereas Type II DGKs, DGKδ and -η, have a pleckstrin homology domain instead of the EF-hand motif in addition to the C1 domain (14Sakane F. Imai S. Kai M. Wada I. Kanoh H. J. Biol. Chem. 1996; 271: 8394-8401Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 15Klauck T.M. Xu X. Mousseau B. Jaken S. J. Biol. Chem. 1996; 271: 19781-19788Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The separated catalytic domains of DGKδ and -η are characteristic of Type II DGK. Type III, DGKϵ, has only one C1 domain in the regulatory domain (16Tang W. Bunting M. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1996; 271: 10237-10241Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Myristoylated alanine-rich protein kinase C substrate phosphorylation site-like region and four ankyrin repeats are unique domains in Type IV DGK (17Bunting M. Tang W. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1996; 271: 10230-10236Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 18Ding L. Traer E. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1998; 273: 32746-32752Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The final group, type V, includes DGKθ, which has three cysteine-rich regions and a pleckstrin homology domain with overlapping Ras-associating domain (19Houssa B. Schaap D. van der Wal J. Goto K. Kondo H. Yamakawa A. Shibata M. Takenawa T. van Blitterswijk W.J. J. Biol. Chem. 1997; 272: 10422-10428Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The DGKs are thought to be involved in development, differentiation, construction of neural network and immunity, etc. However, subtype specific function and regulation mechanisms of DGKs are not clear. Several groups have reported many different localization and translocation of DGKs, possibly contributing to their subtype-specific functions. In an effort to elucidate the function of DGK, we unexpectedly found that GFP-fused DGKγ (GFP-DGKγ) 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 DGKγ has never been reported, expression of DGKθ and DGKζ in the nucleus has been already described (20Bregoli L. Baldassare J.J. Raben D.M. J. Biol. Chem. 2001; 276: 23288-23295Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 21Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (250) Google Scholar). In addition, DGKζ is thought to be involved in the regulation of cell cycle (21Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (250) Google Scholar). These findings, together with the facts that phosphatidylinositol turnover exists within the nucleus and DG may be involved in the regulation of cell cycle (22York J.D. Majerus P.W. J. Biol. Chem. 1994; 269: 7847-7850Abstract Full Text PDF PubMed Google Scholar, 23D'Santos C.S. Clarke J.H. Divecha N. Biochim. Biophys. Acta. 1998; 1436: 201-232Crossref PubMed Scopus (159) Google Scholar, 24Divecha N. Banfic H. Irvine R.F. EMBO J. 1991; 10: 3207-3214Crossref PubMed Scopus (415) Google Scholar, 25Divecha N. Treagus J. Vann L. D'Santos C. Semin. Biol. 1997; PubMed Scopus Google Scholar, H. M. Divecha N. Irvine R.F. Biochem. J. PubMed Scopus (114) Google Scholar, T. H. J. Sci. 2002; Google Scholar), that DGKγ has physiological function in the nucleus. However, of the nuclear transportation and physiological of DGKγ are investigated and physiological of nuclear transportation of cells were from M. of and cells were from the and were from was from Science. was from was from produced Y. T. N. K. K. Y. and N. in was from was from was from cells were in and cells were in cells were in cells were at in and all were with and with and The used was not The cells was with of and The DGKγ at its N terminus (GFP-DGKγ) or at C terminus and of to in the C1A domain or to in the C1B domain were described Y. S. M. Goto K. N. N. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The the the C1A or C1B domain or were described M. K. A. H. Y. K. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mutant of DGKγ was produced by of to the was to the with an The were and The was by its Furthermore, to kinase-negative of or to and the of DGKγ was with and and into the and in the and In addition, to DGKγ a nuclear localization signal of DGKγ with at the N terminus and at the C terminus were produced by a kinase-negative mutant of DGKγ as described The were first into a with and the DGKγ was into the and in the The were and of the C1 of of C1 domain with in the N terminus and in the C terminus were produced by a DGKγ as the K. M. Kondo H. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). The were first into a with and the C1 domain was into the and in the expression or The C1 domain were and The C1A domain were and The C1B domain were and The C1A domain were and The were and The C1B domain were and on were of transfection and of to the cells were at cells as described and transfection were at with acid in and with in at were with or and cells were three with of an to amino of DGKγ M. Sakane F. Imai S. Wada I. Kanoh H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google was used was by that the DGKγ has with or of and were fluorescent The was at with was at with a the of the GFP, which we described The cells in which the of of cytoplasm and nucleus was were as in the and the cells with the of were as in the In the the was the cells were as in the cytoplasm and the and were into cells a or into cells transfection the cells were and at The cells were in and the were to by a were by with in at The was with at with the was with three with the were a the kinase of DGKγ and mutants, of the which of the protein of DGKγ or by were to (14Sakane F. Imai S. Kai M. Wada I. Kanoh H. J. Biol. Chem. 1996; 271: 8394-8401Abstract Full Text Full Text PDF PubMed Scopus (142) Google by was used as The of phosphatidic acid was separated on a acid and by a of the and of the KN, and were into CHO-K1 cells by as described of the cells were by The of the cells was of of KN, and were by transfection to the was to the after the the cells were to at The was by the of cells of stable cell lines were on three and cells were with after and by at The cells were in of with in and The time of the lines were from on the of the cells. of of the lines was by the of of and and were into cells. of both phase and were from to after transfection fluorescent with on the the time of the time the first and the second was cells of stable cell lines were on The cells were at the of S phase by and the cells were with stable or stable the first by an of and the second T. T. Y. T. K. T. N. M. K. H. J. Biol. 2000; PubMed Scopus Google Scholar). the cell the cells were well and in after the cells were with and in a by the cells were by on and by and cycle was by a in of the of CHO-K1 cells expressing we different localization of cells in the nucleus and just after the transfection many of the CHO-K1 cells only in the cytoplasm type but days enzyme was localized in the cytoplasm and nucleus type we considered the that the nuclear localization of was to the itself is localized in the nucleus and the cytoplasm as in in However, of was found at by indicating that the enzyme is transported from the cytoplasm to the nucleus we investigated of that the of the CHO-K1 cells expressing only in the cytoplasm while the of the cells expressing the protein both in the cytoplasm and the nucleus with of the type CHO-K1 cells was at but to at The of type to type cells from at to at to at the cells. In the of the type cells and that of the type cells are similar at but the type cells became the with to at and at Furthermore, we the cell DGKγ are expressed in the In DGKγ showed different of the localization type and type of the type cells was at and was at with the in from to the at These that DGKγ is transported to the nucleus on in all cell lines of the C1 domain and its of DGKγ in CHO-K1 cells. of the C1 domain and its C1 domain is from to aa, C1A domain is from to aa, C1B domain is from to aa, C1A domain is from to aa, is from to and C1B domain is from to of localization of C1 domain and its in CHO-K1 cells. The cells were and The were by and localization of C1 domain in CHO-K1 cells. of domain of DGKγ and was into CHO-K1 cells and after the The was by The are the of and the and of is the nuclear transportation of were generated as in and their and nuclear transportation was revealed that mutant size and that were that kinase activity, whereas DGKγ at the N terminus as reported Y. S. M. Goto K. N. N. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The in the C1A or C1B domains and and the C1A or C1B domain and showed but wild-type in the C1A domain in the C1B domain the kinase the kinase-negative mutants, KN, and in the was to showed kinase kinase both and were localized in the nucleus as well as the cytoplasm of CHO-K1 but the transportation of into the nucleus was slower type and the and not the in the of cells expressing the in the cytoplasm and the nucleus, they kinase and and not their localization and the kinase of the and their transportation to the nucleus. These that is the kinase and the nuclear suggesting that nuclear transportation of DGKγ not its kinase of and and their nuclear localization in CHO-K1 of in the in a the as seen in the of and and and in the C1A or C1B domains the nuclear transportation of suggesting that the C1 domain is important the nuclear transportation of DGKγ and a nuclear transport signal. the function of C1 domain as a nuclear transport signal and which of the C1 domain is important the we generated from the C1 domain with C1 C1A domain C1B C1A and C1B domain and investigated their localization The C1 domain was localized in the nucleus but the not nuclear localization C1 domain was localized in the nucleus, whereas was seen the cells These that the C1 domain is to as a nuclear localization signal at least in of investigate physiological function of DGKγ in the nucleus, we first the size of CHO-K1 cells expressing GFP, or KN, we that the size of cells expressing DGKγ were that of cells expressing wild-type the of the CHO-K1 cells expressing and was whereas that of was suggesting the of DGKγ in cell However, is not the in the cell size was to the of DGKγ the in the nucleus or in the cytoplasm expressed both in the cytoplasm and the nucleus. the size of cells expressing DGK only in the cytoplasm to an of DGK on the cell used the cells expressing the has kinase and only in the the the cells expressing were used as the cells with kinase and were found in the size of cells expressing and the size of cells expressing and were similar to that of or cells Furthermore, we the of the CHO-K1 cells expressing these to investigate of the cells in in cell in the cells expressing were of the cells expressing In addition, of the cells was the of the and of cells were and whereas of the cells were and These that the of DGKγ the cell and we stable CHO-K1 cell lines of GFP, KN, and and the size of the cells. The stable cells expressing in the nucleus and the cytoplasm were the the size of cells expressing GFP, and were These that on the nuclear but not the size and of of the stable cell lines of GFP, KN, and of cell in cytoplasm and in cytoplasm and in in a to elucidate the of DGKγ of we of the stable cell lines that the cells the the and that time of stable cells was which was from its the of GFP, and cells were and These that the of was slower investigate of nuclear DGKγ on the we nuclear localization signal and as and their The cell expressing a to with cells. time of the cells expressing was whereas that of cells was These that nuclear the of cell cycle. we cell cycle of by and with cell lines expressing and the of which is and after the from the of stable cells was different from of and stable indicating that but not DGKγ the cell cycle. we the cell cycle and stable cells at the of S phase by the T. T. Y. T. K. T. N. M. K. H. J. Biol. 2000; PubMed Scopus Google Scholar). to the we of the cells In the of stable cell with in and a stable cell was not at and was these we to at stable cells and cells after of the cells to whereas the cells expressing in phase were only of the cells were in phase at but the of in phase to The stable cells to phase at and the size of of in phase at the the after the of stable cells in and only cells were in a of to phase at was by with the stable cells. These that the S phase of was suggesting that the of the The of cell cycle by nuclear expression of DGKγ that of the cell cycle the localization of we the localization of in the stable cells after the of the stable cells expressing both in the cytoplasm and the nucleus was and that expressing only in the cytoplasm was of all cells The of the cells expressing both in the cytoplasm and the nucleus from to by In the of the cells expressing the protein only in the cytoplasm from to The of type to type from to These that is transported from the cytoplasm to the nucleus However, these were expressed to nuclear transportation of we In many DGKγ was localized in the cytoplasm but not in the nucleus DGKγ localized in the nucleus as as in the cytoplasm indicating as well as DGKγ be transported to the nucleus, and the nuclear transportation is by These together with the of on cell an important physiological function of nuclear DGKγ in the cell cycle In we showed the first time that DGKγ is transported from the cytoplasm to the nucleus The nuclear transportation of DGKγ was of kinase of DGKγ and but the mutant kinase showed slower nuclear transportation that of The kinase of or a in the of the cells expressing in the cytoplasm and the nucleus or in These that the kinase activity, not may have roles in the nuclear transportation of of kinase activity, the C1 domain of DGKγ was the nuclear transportation of of the C1 domain and or and showed transportation from the cytoplasm to the nucleus and In addition, and the C1 domain were localized only in the nucleus, and the C1 domain was the nuclear localization These at least in the C1 domain acts as a nuclear localization signal. However, the C1 domain of DGKγ a well known NLS, and is not acts as a nuclear transport signal. has been already reported that C1 domains of PKC, DGK, and to lipids M. K. A. H. Y. K. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. K. A. H. H. U. H. Chem. 2001; PubMed Scopus Google Scholar, K. K. A. Y. H. H. H. U. J. Chem. 1998; Scopus Google Scholar). Thus, lipids including DG may be involved in the nuclear transportation of In the in the C1 which are to or their lipid on the that in the C1 domain the S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the nuclear as M.A. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, D. Biol. PubMed Scopus Google may in the nuclear transportation by with C1 the C1 domain of DGK with H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of or to DGKγ C1 domain be to the of C1 domain as Furthermore, is to C1 domains in DGKs and PKCs are important nuclear localization or In addition to the nuclear transportation DGKγ to have Although the C1 domain of DGKγ was localized in the nucleus and DGKγ was expressed in the cytoplasm and the nucleus and not the cells that expressed DGKγ in the nucleus the In addition, the of of the cells expressing a in the nuclear within and the a to that of the nucleus not These that DGK has not only nuclear transport but and that the DGK in the nucleus is as as that in the the of DGKγ the cytoplasm and the nucleus is the C1 domain and investigated physiological function of nuclear DGKγ by expressing kinase DGKγ in the nucleus, which is to the nuclear DGKγ by The cells expressing were larger in the size and and their S phase was suggesting that of cell size is by protein S In addition, nuclear transportation of DGKγ and expressing in the CHO-K1 cells These implicate that DGKγ in the nucleus is involved in regulation of cell cycle. of DGK in regulation is by as of DGK from to S phase I. D.R. A. E. Sanjuan M.A. Merida I. J. 1999; Google Scholar). the of DGK subtypes as DGKζ in the cell cycle has been reported, are cells expressing DGKζ showed the in the size of cell and in that of cell but of DGKζ on the cell cycle were (21Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (250) Google Scholar). The may be to subtype different cell DGKγ and DGKζ are expressed in the K. Kondo H. Chem. 1999; PubMed Scopus Google Scholar), is their as a of the cell cycle in the However, expression of DGKγ has been reported in and cells K. Sakane F. Imai S. S. T. Kanoh H. Biochem. Biophys. PubMed Scopus Google Scholar, S. Kai M. Yamada K. Imai S. K. Kanoh H. Sakane F. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Cell. Biochem. PubMed Scopus Google Scholar). the expression of DGKγ in various including and in addition to at least in these cells and is that nuclear DGKγ is involved in In regulation of by DGKγ in cells has been K. Sakane F. Imai S. S. T. Kanoh H. Biochem. Biophys. PubMed Scopus Google Scholar, M. J. Cell. Biochem. PubMed Scopus Google Scholar). However, remains to be DGK the cell cycle. The of nuclear including phosphatidylinositol the cell cycle (22York J.D. Majerus P.W. J. Biol. Chem. 1994; 269: 7847-7850Abstract Full Text PDF PubMed Google Scholar), and the DG in the nucleus in phase T. H. J. Sci. 2002; Google Scholar). Furthermore, into the nucleus and in the phase J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). These that the DG from phosphatidylinositol PKC to In regulation of nuclear DG is important and DGK be involved in the of nuclear produced by DGK may have function in In the of in the nucleus by the of which has a L. B. Raben D.M. 2002; PubMed Scopus Google Scholar). the of regulation by DGKγ is an important to be In the that DGKγ is transported from the cytoplasm to the nucleus its C1 domain and that DGKγ is involved in with and of and of and on with