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¹ Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nadaku, Kobe 657-8501, Japan
² Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe 658-8558, Japan
³ Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan
⁴ Department of Immunology and Oncology, National Center of Biotechnolog, CSIC, Campus de Cantoblanco, Madrid E-28049, Spain

	Abstract

Top Abstract Introduction Results Discussion Experimental procedures References

 
Diacylglycerol kinase (DGK) has been suggested to be a pharmacological target of D--tocopherol for diabetic renal dysfunctions. However, the DGK subtypes involved in the D--tocopherol-induced improvement of diabetic renal dysfunctions and the activation mechanisms of DGK by D--tocopherol are still unknown. Therefore, using GFP-tagged DGK, ß, , and , we analyzed their response to D--tocopherol and its derivatives. Only DGK was translocated from the cytoplasm to the plasma membrane with elevation of kinase activity. In addition, troglitazone and trolox possessing ‘chroman ring’ similarly to D--tocopherol, induced the subtype-specific translocation of DGK. Furthermore, the translocation of DGK was abolished by pretreatment with tyrosine kinase inhibitors or by mutation on Tyr-334 of the kinase (YF mutant). D--tocopheryl succinate enhanced the Src-mediated tyrosine phosphorylation of wild-type DGK but the same reagent did not enhance that of the YF mutant. These results demonstrate that tyrosine phosphorylation on Tyr-334 and chroman ring are important for the D--tocopherol-induced translocation of DGK.

	Introduction

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It is well established that hyperglycemia is responsible for alterations in renal dysfunction, neuropathy and retinopathy in diabetic model animals and diabetic patients. However, the mechanisms of diabetic complications are not cleared well. One of the mechanisms is that hyperglycemia increases diacylglycerol (DG), resulting in the activation of PKC, especially ß subtype (Craven & DeRubertis 1989; Craven et al. 1990; Inoguchi et al. 1992; Ishii et al. 1996; Koya & King 1998). It has been reported that D--tocopherol treatment prevents early changes of diabetic renal dysfunctions by normalizing both DG levels and PKC activity in glomerular cells (Koya et al. 1997; Lee et al. 1999). This improvement by D--tocopherol is likely due to activation of DG kinase (DGK).

The DGK phosphorylates DG, which is generated by phospholipase C or phospholipase D upon the receptor stimulation, resulting in both production phosphatidic acid and decreasing DG level (Topham & Prescott 1999; van Blitterswijk & Houssa 2000; Kanoh et al. 2002). Thus, DGK is an important enzyme for inactivating PKC, contributing to regulate cellular responses. At least nine subtypes of mammalian DGKs have been cloned and divided into five groups based on their structure (Topham & Prescott 1999; van Blitterswijk & Houssa 2000; Kanoh et al. 2002). Generally, all DGKs have cysteine-rich regions homologous to the C1A and C1B motifs of PKCs in the regulatory domain at the N terminus and possess a conserved catalytic domain in the C terminus. Type I DGKs, DGK, ß and , have EF-hand motifs and two cysteine-rich regions in the regulatory domain (Sakane et al. 1990; Goto et al. 1992, 1994; Goto & Kondo 1993). Type II DGKs, DGK and , have a pleckstrin homology domain instead of the EF-hand motif in addition to two cysteine-rich regions. The catalytic domains of DGK and are separated (Klauck et al. 1996; Sakane et al. 1996). Type III, DGK, has only two cysteine-rich regions in the regulatory domain (Tang et al. 1996). Type IV, DGK and , have a motif similar to the myristoylated alanine-rich C-kinase substrate phosphorylation site in the regulatory domain and four ankyrin repeats at its C terminus (Bunting et al. 1996; Goto & Kondo 1996; Ding et al. 1998). Type V, DGK, which has three cysteine-rich regions and a pleckstrin homology domain (Houssa et al. 1997).

In spite of much knowledge in the molecular structures of DGKs, subtype-specific functions and mechanisms to regulate each DGK subtype are not well-known. Specifically, which subtype of DGK plays an important role in the D--tocopherol-induced improvement of diabetic renal dysfunctions and how D--tocopherol activates DGK. Therefore, we investigated translocations and activation of each subtype of DGK in response to D--tocopherol and the derivatives, and explored the activation mechanism.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
D--tocopherol-induced subtype-specific translocation of DGK

It is well known that DGK is translocated to the plasma membrane when activated and we have previously reported that DGKs show subtype-specific translocation (Shirai et al. 2000). To identify the DGK subtype involved in the D--tocopherol-induced improvement of renal dysfunction, we generated fusion proteins of DGK, ß, , and with GFP, and investigated their translocations in response to D--tocopherol. The GFP-tagged DGK, and were detected in the cytoplasm of unstimulated cells (Fig. 1). On the other hand, GFP-tagged DGK ß and were localized on the plasma membrane and abundantly in the nucleus, respectively. By the application of 0.2 mM D--tocopherol, only DGK showed significant translocation from the cytoplasm to the plasma membrane (Fig. 1 lower panel). In addition to DGK, some of GFP-DGK translocated at higher concentration of D--tocopherol; one of three cells expressing GFP-DGK showed translocation (Fig. 1 upper panel).

View larger version (52K): [in this window] [in a new window]   Figure 1  Subtype-specific membrane translocation of DGK induced by D--tocopherol. GFP-tagged DGK, ß, , , were expressed in DDT1–MF2 cells and stimulated with D--tocopherol at 1.0 mM (upper) and 0.2 mM (lower). GFP fluorescence was visualized by confocal laser fluorescence scanning microscopy.

Involvement of tyrosine phosphorylation in the D--tocopherol-induced translocation and activation of DGK

Next, we investigated whether the translocation of DGK accompanies the kinase activation using D--tocopheryl succinate because this compound is most effective on the translocation. Five times elevation of DGK activity was induced by D--tocopheryl succinate at 1 min compared to activity at 0 min and reduced to three times elevation at 15 min when the drug was applied to the cells (Fig. 4). However, both D--tocopheryl succinate and D--tocopherol failed to activate directly the purified DGKin vitro (data not shown), suggesting that the elevation of DGK activity was not caused by direct binding of D--tocopherol or D--tocopheryl succinate. Alternatively, the direct binding might be insufficient to activate DGK.

View larger version (10K): [in this window] [in a new window]   Figure 4  Activation of DGK induced by D--tocopheryl succinate in DDT1 cells. The cells expressing GFP-DGK were stimulated by 0.2 mM D--tocopheryl succinate or ethanol as a control. After 0, 1 and 15 min, the cells were collected and measured the activity of DGK as described in the Material and Methods. Results were shown as mean ± SE.

View larger version (33K): [in this window] [in a new window]   Figure 5  Requirement of tyrosine kinase activation for the translocation of DGK induced by D--tocopheryl succinate. Cells were pretreated with U-73122 (10 µM, 30 min), wortmanin (1 µM, 15 min), BAPTA-AM/EGTA (30 µM BAPTA-AM and 2 mM EGTA, 15 min), genistein (200 µM, 30 min), herbimycin (1 µM, 30 min) or PP2 (30 µM, 30 min), and then stimulated by 0.2 mM D--tocopheryl succinate.

View larger version (27K): [in this window] [in a new window]   Figure 6  Critical role of Tyr344 in the D--tocopheryl succinate-induced translocation of DGK. (A) Importance of 344-Tyr on the D--tocopheryl succinate-induced translocation of DGK. DDT1-MF2 cells transfected with GFP- DGKYF mutant were stimulated with 0.2 mM D--tocopheryl succinate (sVtE) or 0.1 mM arachidonic acid (AA). (B) Importance of 344-Tyr on the D--tocopheryl succinate-induced tyrosine phosphorylation on DGK. Wild-type GFP-DGK (GFP-DGKWT) or its mutant (GFP-DGKYF) were co-transfected into DDT-MF1 cells with either FLAG-tagged wild-type Src (FLAG-SrcWT) or kinase-dead one (FLAG-Src KN) and treated by 1 mMD--tocopheryl succinate (sVtE) or vehicle for 1 min. After lysis and immunoprecipitation with anti-GFP antibody, tyrosine phosphorylation of DGK and the YF mutant were detected by anti-phospho-tyrosine (P-Tyr) antibody (upper panel). Western blotting using anti-DGK antibody show comparable amount of DGK and the YF mutant in the immunoprecipitates (middle panel). To show the equal expression of FLAG-SrcWT and FLAG-SrcKN, the homogenate was subjected to immunoblotting using FLAG antibody (bottom panel).

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

 
In this study, we showed for the first time that D--tocopherol induced the subtype-specific translocation of DGK (Fig. 1) and chroman ring is a key structure for the translocation (Figs 1–3). In addition, results shown in Figs 5 and 6 clearly indicate that tyrosine phosphorylation at Tyr-344 by Src family tyrosine kinase is involved in the translocation induced by D--tocopherol and its derivatives; tyrosine phosphorylation of DGK by Src was enhanced with D--tocopheryl succinate treatment and the mutation on the 344-Tyr abolished both the translocation and the enhancement of the phosphorylation. Against our prediction, basal tyrosine phosphorylation was still detected in the YF mutant whether the cells were treated with D--tocopherol or not. These results suggest that DGK has multiple tyrosine phosphorylation sites in addition to Tyr-334 and some of them are constitutively phosphorylated. The importance of tyrosine phosphorylation in the activation and translocation of DGK is supported by recent findings; herbimycin inhibited membrane translocation of DGK in T cells (Sanjuán et al. 2001) and Src binds, phosphorylates, and activates DGK in a HGF and VEGF dependent manner in hepatocytes and HUVEC (Cutrupi et al. 2000; Baldanzi et al. 2004).

However, how the chroman ring structure in D--tocopherol activates Src family tyrosine kinases is still unknown, although we revealed that the anti-oxidation effect appears not to be necessary for at least the translocation of DGK. Recently, much attention has been given to the nonanti-oxidant molecular functions of -tocopherol. For example, D--tocopherol and its non-oxidative derivatives inhibit cellular proliferation, PKC activation and protein expression (Boscoboinik et al. 1991; Azzi et al. 2000, 2002). In these functions, it is considered that the phospholipids-TAP (tocopherol associated protein) complex may be important; formation of the complex of -tocopherol with TAP changes the balance of phospholipids in the plasma membrane, resulting in modulation of several enzyme activities including PKC. Similar mechanism may be involved in the -tocopherol-induced activation of Src family tyrosine kinase. In fact, it has been reported that Src family tyrosine kinase is activated by several phospholipids (Sato et al. 1997).

In addition to the tyrosine phosphorylation, an additional molecular mechanism appears to be necessary for D--tocopherol-induced translocation of DGK because the tyrosine phosphorylation was not sufficient to induce the translocation; C1A or C1B mutants, whose Cys 218 in the C1A or Cys384 in the C1B region was substituted to Gly, lost ability to translocate in response to D--tocopherol (data not shown), although tyrosine at 344 in the both mutants was intact. These results indicate that, in addition to the tyrosine phosphorylation, the C1 domain of DGK also plays an important role in the D--tocopherol-induced translocation of DGK.

In the meantime, our result that D--tocopherol subtype-specifically translocated and activated DGK (Figs 1 and 3) suggest that DGK is a likely candidate to be involved in the D--tocopherol-induced improvement of renal dysfunction. In fact, a considerable amount of DGK, but not DGK, was detected in rat glomeruli (data not shown). A recent report also supports the importance of DGK in the improvement of renal dysfunction; up-regulation of DGK is important to suppress DAG-PKC signaling in the protection of vascular inflammation induced by peroxisome-proliferator-activated nuclear transcription factor (PPAR), which is also known as a master regulator in adipogenesis and glucose homeostasis (Verrier et al. 2004). In addition, DGK activity decreases in the aorta and kidney of diabetic rats (Nobe et al. 1998). Together with these findings, our results suggest that DGK is a potential target of drugs for anti-diabetic renal dysfunction, although further experiments to directly prove the involvement of DGK in the D--tocopherol-induced improvement in renal dysfunction would be necessary.

In conclusion, among DGK subtypes tested, DGK are subtype-specifically translocated and activated by D--tocopherol and its derivatives. For the subtype-specific translocation and activation of DGK, tyrosine phosphorylation on Tyr-334 and the chroman ring in the structure are important.

	Experimental procedures

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Materials

L-glutamine was purchased from nacalai tesque (Kyoto, Japan). D--tocopherol, D--tocopheryl succinate, dl--tocopheryl acetate were from Sigma (St. Louis, MO, USA). Troglitazone and ciglitazone were given from Sankyo Co. Ltd. (Tokyo, Japan). U-73122, wortmanin, genistein, PP2, herbimycin, BAPTA-AM were from CALBIOCHEM (San Diego, CA, USA). Trolox, 6-hydoroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, was obtained from BIOMOL (Plymouth Meeting, PA, USA). All other chemicals were of analytical grade.

The plasmids bearing cDNA for ratDGKß, and GFP-ratDGK were donated from Dr Goto (Yamagata University, Japan). cDNAs of wild-type and kinase-dead chicken Src with FLAG tag were kindly given by Dr Fukami (Kobe University, Japan).

Cell culture

DDT1-MF2 cells were obtained from B. B. Fredholm (Karolinska Institute, Sweden). The DDT1-MF2 cells were grown in Dulbecco's Modified Eagle Medium (high glucose) (Gibco BRL, Rockville, MD, USA) supplemented with 5% Fatal Bovine Serum (FBS), 2 mM L-glutamine, 100 unit/mL penicillin G and 100 µg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C.

Constructs of plasmids encoding fusion protein of DGKs and their mutants with GFP

The plasmids encoding GFP-pigDGK and GFP-ratDGK were prepared as previously described (Shirai et al. 2000). The plasmid bearing cDNA for ratDGKß (Goto & Kondo 1993) was used as a template for a PCR using 5'-ATAGGATCCATGGCAAACCAGGAAAAATGG-3' and 5'-ATAGGATCCTCATTCCTTGCTTCGGTTT-3' as the sense and anti-sense primers, respectively. The PCR product of DGKß was subcloned into BamHI site of pEGFPC1 (GFP-DGKß). Similarly, GFP-DGK were made using ratDGK (Goto & Kondo 1996) as a template and 5'-ATAGAATTCATGGAGCCGCGGGAC-3' and 5'-ATAGGATCTCTACACAGCTGTCTCCTG-3' as the sense and anti-sense primers.

The cDNA encoding a YF mutant of DGK was generated replacing Tyr334 in the GFP-DGK by Phe using the Quick site-directed mutagenesis kit (Stratagene, Cedar Creek, TX).

Observation of translocation of GFP-fused protein in living cells

Transfection of plasmids were performed by lipofection using Fugene^TM 6 transfection reagent (Roche diagnostic Co., Indianapolis, IN, USA), according to the manufacturer's standard protocol. The transfected cells were spread on to the glass bottom culture dishes (MatTek Corp., Ashland, MA, USA) and cultured at 37 °C for at least 24 h before the observation. The culture medium was replaced with Ringer's buffer composed of Krebs-Ringer phosphate buffer (120 mM NaCl, 4.8 mM KCl, 1 mM CaCl₂, 1.2 mM MgSO₄, 16.5 mM NaH₂PO₄/Na₂HPO₄, pH 7.4, and 5 mMß-D-glucose). Translocation of fusion proteins was triggered by a direct application of various stimulants at 10 times higher concentration into the buffer to obtain the appropriate final concentration. The fluorescence of the fusion proteins was monitored at 488-nm argon excitation using a 510–535–nm band pass barrier filter under confocal laser scanning fluorescence microscopy (model LSM 510 invert; Carl Zeiss, Jena, Germany). Images were collected sequentially every 3 s.

Data analysis

The time course of translocation was recorded as a time series of 30–50 images for each experiment. Image analysis was performed using the Zeiss LSM 510 software, and the membrane fluorescence ratio was calculated.

Pull down assay

Plasmids encoding GFP-DGK or GFP-DGK YF mutant (15 µg each) was co-transfected with FLAG-tagged Src or kinase-dead Src (8.5 µg each) into DDT1-MF2 cells by lipofection using Fugene as described above. The cells were harvested 24 h after the transfection, and lyzed in homogenate buffer containing 1% Triton-X 100, 1 mM NaF, 1 mM vanadate 1 mM PMSF, 20 µg/mL leupeptin. After centrifugation (10 000 g for 5 min), the lysates were incubated with GFP antibody for 16 h at 4 °C. The GFP-DGK or GFP-DGK YF mutant bound to the GFP antibody was precipitated by protein G sepharose and subjected to SDS-PAGE and immunoblotting. Amount of GFP-DGK or the mutant and degree of tyrosine phosphorylation of them were checked by anti-DGK and phosphotyrosine antibody (clone 4G10, Upstate, Lake Placid, NY, USA), respectively. Expression of Src and kinase-dead Src were confirmed by anti-FLAG antibody (SIGMA-ALDRICH, St. Louis, MO, USA).

Assay of DGK activity

DDT1-MF2 cells were transfected with GFP-DGK and stimulated with 0.2 mM d- tocopheryl succinate or ethanol as control. The cells were harvested before and after stimulation, and lyzed in a buffer containing 25 mM Tris-HCl (pH 7.4), 0.25 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 20 µg/mL leupeptin, 20 µg/mL aprotinin, and 1 mM polymethylsulfonyl fluoride (PMSF). After sonication at power 5 for 30 s on ice (TOMY SEIKO Co. Ltd, Tokyo, Japan), the homogenate was centrifuged at 800 x g for 10 min, and the supernatant was used for the DGK assay. The DGK activity was measured by the octyl-glucoside mixed micelle assay as described by Sakane et al. (1996). In brief, the reaction was initiated by addition of enzyme in mixture containing 50 mM 3-(N-Morpholino) propanesulfonic acid (MOPS; pH 7.2), 50 mM octyl-glucoside, 100 mM NaCl, 1 mM dithiothreitol (DTT), 20 mM NaF, 2.1 mM CaCl₂, 2 mM EGTA, 0.8 mM EDTA, 10 mM MgCl₂, 6.7 mM phosphatidylserine, and 1 mM[-³²P]-ATP (10 000 cpm/mmol, Sigma, St. Louis, MO, USA) in the presence of 1 mM 1,2-didecanoyl-sn-glycerol (Avanti Polar Lipids, Alabaster, AL, USA) and incubated for 10 min at 30 °C. Lipids were extracted from the mixture, and counted by a liquid scintillation counter.

View larger version (33K): [in this window] [in a new window]   Figure 2  Effects of two non-oxidative derivatives on the translocation of DGK. (A) Chemical structure of D--tocopherol, d--tocopheryl succinate and dl--tocopheryl acetate. (B) Translocation of GFP-DGK in response to D--tocopherol derivatives. DDT1-MF2 cells expressing GFP-DGK were stimulated with 0.2 mM D--tocopherol, 0.2 mM D--tocopheryl succinate and 1.0 mM dl--tocopheryl acetate. (C) Dose-dependent effects of D--tocopherol and its derivatives on the translocation of GFP-DGK. Translocation efficiency was represented as percentage of the cells showing translocation at different concentration of the drugs. Each datum point shows mean ± SE in 3–5 independent experiments using more than 50 cells in total.

View larger version (29K): [in this window] [in a new window]   Figure 3  Improtance of the chroman ring to induced the translocation of DGK. DDT1-MF2 cells transfected with GFP-DGK were stimulated with 10 µM troglitazone, 10 µM ciglitazone, or 500 µM trolox. GFP fluorescence was visualized by confocal laser fluorescence scanning microscopy. Chemical structure of troglitazone, ciglitazone and trolox are also shown.

	Acknowledgements

	Footnotes

 
Communicated by: Kozo Kaibuchi

^* Correspondence: E-mail: shirai{at}kobe-u.ac.jp

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Received: 20 December 2004
Accepted: 13 January 2004

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