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Synthesis and Phorbol Ester Binding of the Cysteine-rich Domains of Diacylglycerol Kinase (DGK) Isozymes

2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês

10.1074/jbc.m300400200

1083-351X

Mayumi Shindo, Kazuhiro Irie, Akiko Masuda, Hajime Ohigashi, Yasuhito Shirai, Kei Miyasaka, Naoaki Saito,

Peroxisome Proliferator-Activated Receptors

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGKγ-C1A and DGKβ-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGKγ-C1A, human-DGKγ-C1A, and human-DGKβ-C1A gaveKd values of 3.6, 2.8, and 14.6 nm, respectively, suggesting that DGKγ and DGKβ are new targets of phorbol esters. An A12T mutation of human-DGKβ-C1A enhanced the affinity to bind PDBu, indicating that the β-hydroxyl group of Thr-12 significantly contributes to the binding. TheKd value for PDBu of FLAG-tagged whole rat-DGKγ (4.4 nm) was nearly equal to that of rat-DGKγ-C1A (3.6 nm). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGKγ and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGKγ to cause its translocation. Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGKγ-C1A and DGKβ-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGKγ-C1A, human-DGKγ-C1A, and human-DGKβ-C1A gaveKd values of 3.6, 2.8, and 14.6 nm, respectively, suggesting that DGKγ and DGKβ are new targets of phorbol esters. An A12T mutation of human-DGKβ-C1A enhanced the affinity to bind PDBu, indicating that the β-hydroxyl group of Thr-12 significantly contributes to the binding. TheKd value for PDBu of FLAG-tagged whole rat-DGKγ (4.4 nm) was nearly equal to that of rat-DGKγ-C1A (3.6 nm). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGKγ and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGKγ to cause its translocation. diacylglycerol kinase Chinese hamster ovary diacylglycerol green fluorescent protein N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (−)-indolactam-V ingenol 3-benzoate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry 1-oleoyl-2-acetyl-sn-glycerol phorbol 12,13-dibutyrate 1,2-di-(cis-9-octadecenoyl)-sn-glycero-3-phospho-l-serine protein kinase C 12-O-tetradecanoylphorbol 13-acetate high pressure liquid chromatography N-(9-fluorenyl)methoxycarbonyl Diacylglycerol kinase (DGK)1 and protein kinase C (PKC) both interact with the second messenger diacylglycerol (DG) (1Houssa B. van Blitterswijk W.J. Biochem. J. 1998; 331: 677-680Crossref PubMed Scopus (31) Google Scholar, 2van Blitterswijk W.J. Houssa B. Cell. Signal. 2000; 12: 595-605Crossref PubMed Scopus (228) Google Scholar). DGK phosphorylates DG to produce phosphatidic acid, whereas PKC is allosterically activated by DG in the presence of phosphatidylserine. Therefore, DGK may inhibit the activation of PKC by attenuating DG levels, contributing to the regulation of intracellular signal transduction. To date, nine subtypes of mammalian DGKs have been cloned (3–15). All DGK isozymes consist of a conserved catalytic domain and two or three cysteine-rich C1 domains designated as C1A, C1B, and C1C (16Hurley J.H. Newton A.C. Parker P.J. Blumberg P.M. Nishizuka Y. Protein Sci. 1997; 6: 477-480Crossref PubMed Scopus (317) Google Scholar). These isozymes are classified into five classes according to the other functional domains (Fig. 1). The class I isozymes (DGKα, -β, and -γ) have calcium binding domains (EF-hands). The class II isozymes (DGKδ and -η) have a pleckstrin homology domain at the N terminus, and their catalytic region is split into two domains unlike the other DGK isozymes. DGKε has a simple structure and is classified as a class III isozyme. The class IV isozymes DGKζ and ι have a myristoylated alanine-rich C kinase substrate homology domain and four ankyrin repeats. DGKθ, which has three C1 domains unlike other DGK and PKC isozymes, is the only isozyme in class V. The similarity between DGK and PKC isozymes in structure is in the cysteine-rich C1 domains. Recent investigations using NMR spectroscopy and x-ray crystallography have revealed the three-dimensional structure of C1B domains of PKCα, PKCγ, and PKCδ (17Hommel U. Zurini M. Luyten M. Nat. Struct. Biol. 1994; 1: 383-387Crossref PubMed Scopus (137) Google Scholar, 18Ichikawa S. Hatanaka H. Takeuchi Y. Ohno S. Inagaki F. J. Biochem. (Tokyo). 1995; 117: 566-574Crossref PubMed Scopus (61) Google Scholar, 19Xu R.X. Pawelczyk T. Xia T.-H. Brown S.C. Biochemistry. 1997; 36: 10709-10717Crossref PubMed Scopus (121) Google Scholar, 20Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (595) Google Scholar). Each PKC C1 domain has six conserved cysteines and two histidines in the typical core structure HX12CX2CX13–14CX2CX4HX2CX7C (where X is any amino acid) that coordinates two atoms of zinc in a tetrahedral geometry (21Hubbard S.R. Bishop W.R. Kirschmeier P. George S.J. Carmer S.P. Hendrickson W.A. Science. 1991; 254: 1776-1779Crossref PubMed Scopus (161) Google Scholar, 22Quest A.F.G. Bloomenthal J. Bardes E.S.G. Bell R.M. J. Biol. Chem. 1992; 267: 10193-10197Abstract Full Text PDF PubMed Google Scholar). We previously synthesized the 50-mer core structure of the C1 domains of all PKC isozymes and showed that most of the C1 domains of conventional and novel PKC isozymes strongly bind phorbol 12,13-dibutyrate (PDBu) with dissociation constants (Kd) in the nanomolar range (23Irie K. Oie K. Nakahara A. Yanai Y. Ohigashi H. Wender P.A. Fukuda H. Konishi H. Kikkawa U. J. Am. Chem. Soc. 1998; 120: 9159-9167Crossref Scopus (141) Google Scholar, 24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A. Bioorg. Med. Chem. 2001; 9: 2073-2081Crossref PubMed Scopus (75) Google Scholar). The core structure of DGK C1 domains, HX10–12CX2–6CX9–19CX2CX4HX2–4CX5–10C, is slightly different from that of PKC C1 domains, but the six cysteines and two histidines are precisely conserved. In particular, DGKβ-C1A, DGKγ-C1A, DGKδ-C1A, DGKδ-C1B, DGKη-C1A, and DGKθ-C1C have the same core structure as the PKC C1 domains (Fig. 2) and are deduced to show a significant phorbol ester binding affinity. However, there have been no reports that DGK isozymes bind phorbol esters. Ahmed et al. (25Ahmed S. Kozma R. Lee J. Montries C. Harden N. Lim L. Biochem. J. 1991; 280: 233-241Crossref PubMed Scopus (91) Google Scholar) reported that human-DGKα expressed in Escherichia coli or in COS-7 cells did not bind PDBu. Sakane et al. (26Sakane F. Kai M. Wada I. Imai S.-I. Kanoh H. Biochem. J. 1996; 318: 583-590Crossref PubMed Scopus (68) Google Scholar) also detected no specific PDBu binding in either rat-DGKβ or human-DGKγ expressed in COS-7 cells. On the other hand, a recent investigation by Shirai et al.(27Shirai Y. Segawa S. Kuriyama M. Goto K. Sakai N. Saito N. J. Biol. Chem. 2000; 275: 24760-24766Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) clearly showed that DGKγ expressed in Chinese hamster ovary-K1 (CHO-K1) cells was translocated from the cytoplasm to the plasma membrane irreversibly following 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment. They also suggested that the C1A domain of DGKγ is responsible for this translocation based on the point mutation of each C1 domain. These results prompted us to examine the phorbol ester binding ability of each DGKγ C1 domain, and we have reported recently (28Shindo M. Irie K. Ohigashi H. Kuriyama M. Saito N. Biochem. Biophys. Res. Commun. 2001; 289: 451-456Crossref PubMed Scopus (47) Google Scholar) that rat-DGKγ binds PDBu with high affinity as a preliminary communication. Further detailed studies on the C1 domains of several DGK isozymes other than those of rat-DGKγ have been carried out along with the C1A peptide of human-DGKγ which Sakaneet al. (26Sakane F. Kai M. Wada I. Imai S.-I. Kanoh H. Biochem. J. 1996; 318: 583-590Crossref PubMed Scopus (68) Google Scholar) employed. These additional data have been compiled to give this full report. The following spectroscopic and analytical instruments were used: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), Applied Biosystems Voyager-DETM STR (20 kV); peptide synthesizer, PioneerTM peptide synthesizer model 9030 (Applied Biosystems); and HPLC, Waters model 600E with model 2487UV detector. MALDI-TOF-MS measurements were carried out as follows; each peptide dissolved in 0.1% trifluoroacetic acid aqueous solution (50 pmol/μl) was mixed with saturated α-cyano-4-hydroxycinnamic acid in 50% CH3CN containing 0.1% trifluoroacetic acid at a ratio of 1:1. One microliter of the resultant solution was subjected to the measurement. Angiotensin I and ACTH-(7–38) were used as external references. HPLC was carried out on a YMC-packed SH-342-5 (ODS, 20 mm inner diameter × 150 mm) column (Yamamura Chemical Laboratory) for preparative purposes. [3H]PDBu (17.0 Ci/mmol) was purchased from PerkinElmer Life Science. COS-7 cells were obtained from the Riken Cell Bank (Tsukuba, Japan). Unless otherwise noted, reagents were purchased from Sigma, Allexis, Wako Pure Chemical Industries, or Nacalai Tesque. The 49–52-mer peptides corresponding to the cysteine-rich sequences of DGK isozyme C1 domains (Table I and Fig. 2) were synthesized in a stepwise fashion on 0.1 mmol of preloaded Fmoc-Gly-PEG-PS resin (Applied Biosystems) by PioneerTMusing the Fmoc method as reported previously (23Irie K. Oie K. Nakahara A. Yanai Y. Ohigashi H. Wender P.A. Fukuda H. Konishi H. Kikkawa U. J. Am. Chem. Soc. 1998; 120: 9159-9167Crossref Scopus (141) Google Scholar, 24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A. Bioorg. Med. Chem. 2001; 9: 2073-2081Crossref PubMed Scopus (75) Google Scholar). The coupling reaction was carried out using each Fmoc amino acid (0.4 mmol),N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (0.4 mmol) (29Carpino L.A. J. Am. Chem. Soc. 1993; 115: 4397-4398Crossref Scopus (1380) Google Scholar), andN,N-diisopropylethylamine (0.8 mmol) inN,N-dimethylformamide for 30 min (flow rate, 30 ml/min). After completion of the chain assembly, each peptide resin was cleaved, and the resultant crude peptide was precipitated by diethyl ether. The crude peptide was purified by gel filtration, followed by HPLC as reported previously (23Irie K. Oie K. Nakahara A. Yanai Y. Ohigashi H. Wender P.A. Fukuda H. Konishi H. Kikkawa U. J. Am. Chem. Soc. 1998; 120: 9159-9167Crossref Scopus (141) Google Scholar, 24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A. Bioorg. Med. Chem. 2001; 9: 2073-2081Crossref PubMed Scopus (75) Google Scholar). Lyophilization gave a corresponding pure C1 peptide, the purity of which was confirmed by HPLC (>98%). Each purified peptide exhibited satisfactory mass spectrometric data. The yields and mass data of the C1 peptides synthesized in this study are summarized in Table I.Table IYields and MALDI-TOF-MS data of the C1 peptides of DGK isozymesC1 peptidesYieldObserved massCalculated mass (MH+)%Rat-DGKβ-C1A1-aRat-DGKβ-(244–292).0.35656.655655.86Human-DGKβ-C1A1-bHuman-DGKβ-(245–294).6.35755.985755.01Rat-DGKγ-C1A1-cRat-DGKγ-(269–318).22.45876.555877.20Human-DGKγ-C1A1-dHuman-DGKγ-(272–321).5.45956.195955.28Rat-DGKγ-C1B1-eRat-DGKγ-(334–383).5.95774.065774.71Human-DGKδ-C1A1-fHuman-DGKδ-(120–169).0.55642.785642.52Human-DGKδ-C1B1-gHuman-DGKδ-(192–242).0.95868.915869.06Hamster-DGKη-C1A1-hHamster-DGKη-(170–219).3.15641.935642.52Hamster-DGKη-C1B1-iHamster-DGKη-(242–291).2.15710.535710.91Human-DGKθ-C1A1-jHuman-DGKθ-(60–107).7.75629.565628.81Human-DGKθ-C1C1-kHuman-DGKθ-(183–233).2.15499.475500.32A12T h-DGKβ-C1A1-lThe A12T mutant of human-DGKβ-C1A. To prevent racemization and oxidation during the synthesis, glycine was added to the C-terminal cysteine in each peptide. For example, rat-DGKβ-C1A means rat-DGKβ-(244–292) + Gly (50-mer peptide). All these sequences derive from Refs. 3-15.4.95785.745785.041-a Rat-DGKβ-(244–292).1-b Human-DGKβ-(245–294).1-c Rat-DGKγ-(269–318).1-d Human-DGKγ-(272–321).1-e Rat-DGKγ-(334–383).1-f Human-DGKδ-(120–169).1-g Human-DGKδ-(192–242).1-h Hamster-DGKη-(170–219).1-i Hamster-DGKη-(242–291).1-j Human-DGKθ-(60–107).1-k Human-DGKθ-(183–233).1-l The A12T mutant of human-DGKβ-C1A. To prevent racemization and oxidation during the synthesis, glycine was added to the C-terminal cysteine in each peptide. For example, rat-DGKβ-C1A means rat-DGKβ-(244–292) + Gly (50-mer peptide). All these sequences derive from Refs. 3Sakane F. Yamada K. Kanoh H. Yokoyama C. Tanabe T. Nature. 1990; 344: 345-348Crossref PubMed Scopus (176) Google Scholar, 4Schaap D. de Widt J. van der Wal J. Vandekerckhove J. van Damme J. Gussow D. Ploegh H.L. van Blitterwijk W.J. van der Bendl R.L. FEBS Lett. 1990; 275: 151-158Crossref PubMed Scopus (88) Google Scholar, 5Goto K. Watanabe M. Kondo H. Yuasa H. Sakane F. Kanoh H. Brain Res. Mol. Brain Res. 1992; 16: 75-87Crossref PubMed Scopus (105) Google Scholar, 6Goto K. Kondo H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7598-7602Crossref PubMed Scopus (119) Google Scholar, 7Goto K. Funayama M. Kondo H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13042-13046Crossref PubMed Scopus (100) Google Scholar, 8Sakane F. Imai S.-I. Kai M. Wada I. Kanoh H. J. Biol. Chem. 1996; 271: 8394-8401Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 9Tang 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, 10Klauck 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, 11Bunting 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, 12Houssa 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, 13Ding 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, 14Topham M.K. Prescott S.M. J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 15Caricasole A. Bettini E. Sala C. Roncarati R. Kobayashi N. Caldara F. Goto K. Terstappen G.C. J. Biol. Chem. 2002; 277: 4790-4796Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar. Open table in a new tab The plasmid for FLAG-tagged DGKγ-(1–789) (FLAG-DGKγ) was generated by PCR using BS412 (rat-DGKγ) as a template as reported previously (28Shindo M. Irie K. Ohigashi H. Kuriyama M. Saito N. Biochem. Biophys. Res. Commun. 2001; 289: 451-456Crossref PubMed Scopus (47) Google Scholar). The cDNA of DGKγ-(1–789) was digested with MunI andBamHI and then subcloned into the EcoRI andBglII sites of pTB701-FL, a mammalian expression vector, to express fusion protein with the N-terminal FLAG epitope. Transient transfection into COS-7 cells was performed by electroporation as reported previously (28Shindo M. Irie K. Ohigashi H. Kuriyama M. Saito N. Biochem. Biophys. Res. Commun. 2001; 289: 451-456Crossref PubMed Scopus (47) Google Scholar). After transfection, the cells were cultured at 37 °C for 48 h and were harvested with phosphate-buffered saline(−), followed by centrifugation at 600 × g for 5 min at 4 °C. The cells were resuspended in 300 μl of homogenization buffer (250 mm sucrose, 10 mm EGTA, 2 mm EDTA, 50 mm Tris/HCl, 20 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 0.5% Triton X-100, pH 7.4) and sonicated on ice. The homogenate was centrifuged at 10,000 × g for 30 min at 4 °C, and the resultant supernatant was saved for the PDBu binding assay described below. The PDBu binding assay was carried out using the procedure of Sharkey and Blumberg (30Sharkey N.A. Blumberg P.M. Cancer Res. 1985; 45: 19-24PubMed Google Scholar) with slight modification (23Irie K. Oie K. Nakahara A. Yanai Y. Ohigashi H. Wender P.A. Fukuda H. Konishi H. Kikkawa U. J. Am. Chem. Soc. 1998; 120: 9159-9167Crossref Scopus (141) Google Scholar,24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A. Bioorg. Med. Chem. 2001; 9: 2073-2081Crossref PubMed Scopus (75) Google Scholar). The standard assay mixture (250 μl) in 1.5-ml Eppendorf tubes contained 50 mm Tris maleate, pH 7.4, 50 μg/ml 1,2-di-(cis-9-octadecenoyl)-sn-glycero-3-phospho-l-serine (phosphatidylserine dioleoyl), 3 mg/ml bovine γ-globulin, [3H]PDBu (17.0 Ci/mmol), and each DGKγ C1 peptide or FLAG-DGKγ. For determination of PDBu saturation curves for Scatchard analysis, concentrations of free [3H]PDBu between 1 and 50 nm were used. For chemically synthesized DGK C1 peptides, metal coordination was carried out in a helium-purged distilled water solution (pH 5.5–6.0) of each C1 peptide. Five mol eq of 10 mm ZnCl2in helium-purged distilled water was added to the peptide solution, and the solution (174 μm) was allowed to stand at 4 °C for 10 min. After 10 μl of the peptide solution was diluted with 990 μl of helium-purged distilled water, the resultant solution (1.5–2.9 μl) was added to the standard assay mixture described above (247.1 μl), and the solution was incubated at 4 °C for 10 min. For FLAG-DGKγ, 5 μl of the enzyme solution after homogenization was similarly added and incubated at 30 °C for 20 min. To the tubes was added 187 μl of 35% (w/w) poly(ethylene glycol) (average molecular weight, 8000), and the mixture was vigorously stirred. The tubes were allowed to stand at 4 °C for 10 min and then centrifuged for 10 min at 12,000 rpm in an Eppendorf microcentrifuge at 4 °C. A 50-μl aliquot of the supernatant of each tube was removed, and its radioactivity was measured to determine the free [3H]PDBu concentration. The remainder of the supernatant of each tube was removed by aspiration. The tips of the tubes were cut off, and the radioactivity in the pellets was measured to determine the bound [3H]PDBu. Specific binding represents the difference between the total and nonspecific binding. The nonspecific binding for each tube was calculated from its measured free [3H]PDBu concentration and its partition coefficient to the pellet (about 3%). In competition experiments using rat-DGKγ-C1A, various concentrations of an inhibitor in ethanol solution were added to the reaction mixture mentioned above. The effective concentration of [3H]PDBu and rat-DGKγ-C1A was 20 and 5 nm, respectively. The final ethanol concentration of the mixture was less than 2%. Binding affinity was evaluated by the concentration required to cause 50% inhibition of the specific [3H]PDBu binding, IC50, that was calculated by a computer program (Statistical Analysis System) with a probability unit procedure (31James H.G. Council K.A. Helwig J.T. SAS User's Guide. SAS Institute, Cary, NC1979: 357-360Google Scholar). The binding constant (Ki) was calculated from the IC50 values and Kd for PDBu of rat-DGKγ-C1A by the method of Sharkey and Blumberg (30Sharkey N.A. Blumberg P.M. Cancer Res. 1985; 45: 19-24PubMed Google Scholar). Domain deleted mutants of DGKγ were produced using an ExSite PCR-based Site-directed Mutagenesis kit (Stratagene, La Jolla, CA). The plasmid (BS465) encoding rat-DGKγ (27Shirai Y. Segawa S. Kuriyama M. Goto K. Sakai N. Saito N. J. Biol. Chem. 2000; 275: 24760-24766Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) with anXhoI site in the 5′ terminus and a SmaI site in the 3′ terminus was used as a template. The sense and antisense primers for producing a C1A-deleted mutant (ΔC1A-DGKγ) were 5′-GTGAAAACATACTCCAAAGCCAAAAGG-3′ and 5′-GCGTCCATCCCCCTTGGAG-3′, respectively. The primers for a mutant DGKγ lacking the C1B region (ΔC1B-DGKγ) were 5′-GATGGTGGGGAGCTCAAAGAC-3′ and 5′-CTGCATCACCTCACCGCTC-3′. The sequence was confirmed by verifying sequences. Each C1A- or C1B-deleted mutant of DGKγ cDNA was subcloned into SalI and SmaI sites in pEGFPC1. Plasmids (∼5.5 μg) were transfected into 1.0 × 106 cells by lipofection using FuGENE 6 transfection reagent (Roche Applied Science), according to the manufacturer's standard protocol. After being cultured at 37 °C for 16–24 h, the cells were spread onto glass bottom culture dishes (MatTek Corp., Ashland, MA). Experiments were performed 16–48 h after the transfection. The culture medium was replaced with HEPES buffer composed of 135 mm NaCl, 5.4 mm KCl, 1 mmMgCl2, 1.8 mm CaCl2, 5 mm HEPES, and 10 mm glucose at pH 7.3 (Ringer's solution). Translocation of the GFP fusion protein was triggered by a direct application of a 10 times higher concentration of TPA into the Ringer's solution to obtain the appropriate final concentration. The fluorescence of the fusion protein was monitored with a confocal laser scanning fluorescent microscope (LSM 410 invert, Carl Zeiss, Jena, Germany) at 488 nm argon excitation using a 515–535 nm bandpass barrier filter. All experiments were performed at 37 °C. DGK isozymes have cysteine-rich C1 domains quite similar to those of PKC isozymes (1Houssa B. van Blitterswijk W.J. Biochem. J. 1998; 331: 677-680Crossref PubMed Scopus (31) Google Scholar, 2van Blitterswijk W.J. Houssa B. Cell. Signal. 2000; 12: 595-605Crossref PubMed Scopus (228) Google Scholar). The zinc finger-like sequences of the C1 domains of conventional and novel PKC isozymes specifically bind phorbol esters (16Hurley J.H. Newton A.C. Parker P.J. Blumberg P.M. Nishizuka Y. Protein Sci. 1997; 6: 477-480Crossref PubMed Scopus (317) Google Scholar, 23Irie K. Oie K. Nakahara A. Yanai Y. Ohigashi H. Wender P.A. Fukuda H. Konishi H. Kikkawa U. J. Am. Chem. Soc. 1998; 120: 9159-9167Crossref Scopus (141) Google Scholar, 24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A. Bioorg. Med. Chem. 2001; 9: 2073-2081Crossref PubMed Scopus (75) Google Scholar). Although DGK binds zinc like PKC isozymes (25Ahmed S. Kozma R. Lee J. Montries C. Harden N. Lim L. Biochem. J. 1991; 280: 233-241Crossref PubMed Scopus (91) Google Scholar), there has been no direct evidence that phorbol esters bind to DGK. We carefully analyzed the cysteine-rich sequences of the C1 domains of all DGK isozymes on the basis of the 20 residues (Fig.2) critical to PDBu binding (16Hurley J.H. Newton A.C. Parker P.J. Blumberg P.M. Nishizuka Y. Protein Sci. 1997; 6: 477-480Crossref PubMed Scopus (317) Google Scholar, 24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A. Bioorg. Med. Chem. 2001; 9: 2073-2081Crossref PubMed Scopus (75) Google Scholar, 32Kazanietz M.G. Wang S. Milne G.W.A. Lewin N.E. Liu H.L. Blumberg P.M. J. Biol. Chem. 1995; 270: 21852-21859Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). DGKβ-C1A, DGKγ-C1A, DGKδ-C1A, DGKδ-C1B, DGKη-C1A, and DGKθ-C1C have the same core structure as the PKC C1 domains, where six cysteines and two histidines are conserved in the pattern HX12CX2CX13–14CX2CX4HX2CX7C. Table II summarizes the percentages of sequence homology of all DGK C1 domains to the 20 residues. Human-DGKβ-C1A, rat-DGKγ-C1A, and human-DGKγ-C1A have perfect sequence homology to all conventional and novel PKC isozyme C1 domains. The C1 domains, which show more than 50% sequence homology, are rat-DGKβ-C1A, human-DGKβ-C1A, rat-DGKγ-C1A, human-DGKγ-C1A, human-DGKδ-C1A, human-DGKδ-C1B, hamster-DGKη-C1A, hamster-DGKη-C1B, human-DGKθ-C1A, and human-DGKθ-C1C that might bind PDBu. We have thus synthesized these cysteine-rich sequences of ∼50 amino acid residues with a PioneerTM peptide synthesizer using HATU as an activator for Fmoc chemistry by a method reported previously (23Irie K. Oie K. Nakahara A. Yanai Y. Ohigashi H. Wender P.A. Fukuda H. Konishi H. Kikkawa U. J. Am. Chem. Soc. 1998; 120: 9159-9167Crossref Scopus (141) Google Scholar) (Table I). To prevent racemization and oxidation during the synthesis, the C terminus of each peptide was extended from the final cysteine to a glycine. These peptides exhibited satisfactory mass spectrometric data (Table I), and their purity was confirmed by HPLC analysis (>98%).Table IISequence homology (%) of the cysteine-rich sequences of all DGK C1 domains to the 20 residues that play an important role in PDBu binding of PKC isozymesDGK isozymesC1AC1BC1C%%%Rat-DGKα3545Rat-DGKβ952-cThe boldface indicates the C1 domains that show more than 50% sequence homology.15Human-DGKβ10015Rat-DGKγ10015Human-DGKγ10015Human-DGKδ8060Hamster-DGKη8055Human-DGKɛ2545Human-DGKζ2515Human-DGKι2015Human-DGKθ502050Conventional PKC2-aConventional PKC: α, βI, βII, and γ.100100Novel PKC2-bNovel PKC: δ, ɛ, η, and θ.100100The 20 residues are shown in Fig. 2.2-a Conventional PKC: α, βI, βII, and γ.2-b Novel PKC: δ, ɛ, η, and θ.2-c The boldface indicates the C1 domains that show more than 50% sequence homology. Open table in a new tab The 20 residues are shown in Fig. 2. These peptides were folded using zinc chloride by a method reported previously (23Irie K. Oie K. Nakahara A. Yanai Y. Ohigashi H. Wender P.A. Fukuda H. Konishi H. Kikkawa U. J. Am. Chem. Soc. 1998; 120: 9159-9167Crossref Scopus (141) Google Scholar, 24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A. Bioorg. Med. Chem. 2001; 9: 2073-2081Crossref PubMed Scopus (75) Google Scholar) and subjected to a PDBu binding assay. Because our recent investigation found that some C1 domain fragments of PKC isozymes suffered from temperature-dependent inactivation (24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A. Bioorg. Med. Chem. 2001; 9: 2073-2081Crossref PubMed Scopus (75) Google Scholar, 33Irie K. Nakahara A. Ohigashi H. Fukuda H. Wender P.A. Konishi H. Kikkawa U. Bioorg. Med. Chem. Lett. 1999; 9: 2487-2490Crossref PubMed Scopus (16) Google Scholar), the incubation temperature of the binding assay was set at 4 °C for the DGK isozyme C1 peptides. The specific binding to PDBu of the DGK C1 peptides is summarized in Fig.3. Only the DGKβ-C1A and DGKγ-C1A peptides showed significant binding at 20 nm, whereas other DGK C1 peptides with over 50% sequence homology to PKC C1 domains were completely inactive even at 100 and 500 nm (data not shown). Rat-DGKγ-C1A and human-DGKγ-C1A showed quite similar PDBu binding although their sequences were different. In a control experiment, rat-DGKγ-C1B did not show any binding. Scatchard analyses of rat-DGKγ-C1A and human-DGKγ-C1A for PDBu binding gave Kd values of 3.6 ± 0.5 and 2.8 ± 0.1 nm with Bmax values of 31.2 ± 4.5 and 44.1 ± 7.8%, respectively (Fig.4, a and b). These values are close to those of most PKC C1 peptides (0.45–7.4 nm) (24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A. Bioorg. Med. Chem. 2001; 9: 2073-2081Crossref PubMed Scopus (75) Google Scholar), suggesting that whole DGKγ shows strong PDBu binding affinity. The binding affinity of human-DGKβ-C1A was also great (Kd = 14.6 ± 1.7 nm;Bmax = 42.9 ± 1.3%). However, theKd of rat-DGKβ-C1A (202 ± 13 nm) with a Bmax value of 8.3 ± 1.0% was considerably larger than that of human-DGKβ-C1A (Fig. 4, cand d). The weak PDBu binding affinity of human-DGKβ-C1A (Kd = 14.6 nm) compared with human-DGKγ-C1A (Kd = 2.8 nm) suggests that the Ala-12 residue is responsible for the decrease in binding affinity because the Thr-12 residue is strictly conserved in the conventional and novel PKC isozymes that show strong PDBu binding (24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A. Bioorg. Med. Chem. 2001; 9: 2073-2081Crossref PubMed Scopus (75) Google Scholar). The Thr-12 residue along with the Leu-21 and Gly-23 residues of PKCδ-C1B is shown to be involved in the phorbol ester binding in the x-ray structure (20Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (595) Google Scholar). To prove this, the A12T mutant of human-DGKβ-C1A was synthesized, and its PDBu binding affinity was tested. The Kd value for PDBu of the A12T mutant was 4.9 ± 1.0 nm with a Bmax value of 42.4 ± 2.8% (Fig. 4e), suggesting that the Thr-12 residue plays a significant role in the PDBu binding of DGKβ-C1A. FLAG-tagged whole rat-DGKγ (FLAG-DGKγ) was expressed in COS-7 cells to investigate whether whole rat-DGKγ as well as rat-DGKγ-C1A peptide binds PDBu. After homogenization and centrifugation of the COS-7 cells, the supernatant was saved for PDBu binding assay. Scatchard analysis of FLAG-DGKγ in the PDBu binding was performed with the incubation temperature set at 30 °C as for whole PKC isozymes (24Shindo M. Irie K. Nakahara A. Ohigashi H. Konishi H. Kikkawa U. Fukuda H. Wender P.A.