Identification of phosphatidylcholine-specifc phospholipase C

• Project/Area Number: 15K14470
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Functional biochemistry
• Research Institution: Chiba University
• Principal Investigator: Sakane Fumio 千葉大学, 大学院理学研究科, 教授 (10183815)
• Co-Investigator(Renkei-kenkyūsha): SAKAI HIROMICHI 島根大学, 総合科学研究支援センター, 助教 (00375255)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2015: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: ホスファチジルコリン-特異的ホスホリパーゼC / ジアシルグリセロールキナーゼ / 生体膜 / 動脈硬化 / ホスファチジルコリン特異的ホスホリパーゼC / Coiled-coil structure / 相互作用 / 液体クロマトグラフィー/質量分析 / PH domain / sterile α motif domain / coiled-coil structure / プレクストリン相同ドメイン

Outline of Final Research Achievements

Although the activity of mammalian phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) was discovered approximately 30 years ago, neither the protein nor its gene has been identified. Because we recently revealed that diacylglycerol kinase (DGK) delta bound to PC-PLC, we attempted to identify PC-PLC using the interaction. In the present study, we found that the coiled-coil region in DGKdelta interacted with PC-PLC. Moreover, we established a new LC-MS-based PC-PLC assay, which has better analytical parameters, such as a lower background (0.02% versus 94%), higher S/B (4242 versus 1.06), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than the conventional, indirect fluorometric assay.

Research Products

• [Int'l Joint Research] Cambridge University(英国)
• [Int'l Joint Research] IGBMC(フランス)
• [Journal Article] Dioleoyl-phosphatidic acid selectively binds to α-synuclein and strongly induces its aggregation (2017)
  • Author(s): Satoru Mizuno, Hirotaka Sasai, Aiko Kume, Mamoru Satoh, Sayaka Kado, Fumio Sakane
  • Journal Title: FEBS Letters Volume: 591 Issue: 5 Pages: 784-791
  • DOI: 10.1002/1873-3468.12592
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Development of a liquid chromatography-mass spectrometry based enzyme activity assay for phosphatidylcholine-specific phospholipase C. (2017)
  • Author(s): Murakami, C., Mizuno, S., Kado, S. and Sakane, F.
  • Journal Title: Anal. Biochem. Volume: 526 Pages: 43-49
  • DOI: 10.1016/j.ab.2017.03.010
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] “Deficiency of diacylglycerol kinase h induces lithium-sensitive mania-like behavior. (2016)
  • Author(s): Isozaki T., Komenoi S., Lu Q., Usuki T., Tomokata S., Usuki T., Tomokata S., Matsutomo D., Sakai H., Bando K., Kiyonari H., Sakane F.
  • Journal Title: Journal of Neurochemistry Volume: 138 Issue: 3 Pages: 448-456
  • DOI: 10.1111/jnc.13661
  • Peer Reviewed / Open Access
• [Journal Article] Myristic acid enhances diacylglycerol kinase δ-dependent glucose uptake in myotubes. (2016)
  • Author(s): Wada, Y., Sakiyama, S., Sakai, H. and Sakane, F.
  • Journal Title: Lipids Volume: 51 Issue: 8 Pages: 897-903
  • DOI: 10.1007/s11745-016-4162-9
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Distinct 1- and 2-monoacylglycerol kinase activities of diacylglycerol kinase isozymes. (2016)
  • Author(s): Sato, Y., Murakami, C., Yamaki, A., Mizuno, S., Sakai, H. and Sakane, F.
  • Journal Title: Biochim. Biophys. Acta - Proteins Proteomics Volume: 1864 Issue: 9 Pages: 1170-1176
  • DOI: 10.1016/j.bbapap.2016.06.012
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Behavioral and pharmacological phenotypes of brain-specific diacylglycerol kinase δ-knockout mice. (2016)
  • Author(s): Usuki, T., Takato, T., Lu Q., Sakai, H., Bando, K., Kiyonari, H. and Sakane, F.
  • Journal Title: Brain Res. Volume: 1648 Pages: 193-201
  • DOI: 10.1016/j.brainres.2016.07.017
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Activation of conventional and novel protein kinase C isozymes by different diacylglycerol molecular species. (2016)
  • Author(s): Kamiya, Y., Mizuno, S., Komenoi, S., Sakai, H., Sakane, F.
  • Journal Title: Biochemistry and Biophysics Reports Volume: 7 Pages: 361-366
  • DOI: 10.1016/j.bbrep.2016.07.017
  • Peer Reviewed / Open Access
• [Journal Article] Cloning and characterization of novel testis-specific diacylglycerol kinase η splice variant 3 and 4. (2016)
  • Author(s): Murakami, E., Shionoya, T. Komenoi, S., Suzuki, Y., Sakane, F.
  • Journal Title: PLoS One Volume: 11 Issue: 9 Pages: 1-14
  • DOI: 10.1371/journal.pone.0162997
  • Peer Reviewed / Open Access
• [Journal Article] Diacylglycerol kinase ζ generates dipalmitoyl-phosphatidic acid species during neuroblastoma cell differentiation (2016)
  • Author(s): Satoru Mizuno, Sayaka Kado, Kaoru Goto, Daisuke Takahashi, Fumio Sakane
  • Journal Title: Biochemistry and Biophysics Reports Volume: 8 Pages: 352-359
  • DOI: 10.1016/j.bbrep.2016.10.004
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Diacylglycerol Kinases as Emerging Potential Drug Targers for a Variety of Diseases: An Update. (2016)
  • Author(s): Sakane F., Mizuno S., Komenoi S.
  • Journal Title: Frontiers in Cell and Developmental Biology Volume: 4 Pages: 1-8
  • DOI: 10.3389/fcell.2016.00082
  • Peer Reviewed / Open Access
• [Journal Article] The pleckstrin homology domain of diacylglycerol kinase η strongly and selectively binds to phosphatidylinositol 4,5-bisphosphate. (2016)
  • Author(s): Kume, A., Kawase, K., Komenoi, S., Usuki, T., Takeshita, E., Sakai, H. and Sakane, F.
  • Journal Title: J. Biol. Chem. Volume: 291 Issue: 15 Pages: 8150-8161
  • DOI: 10.1074/jbc.m115.648717
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Deficiency of diacylglycerol kinase η induces lithium-sensitive mania-like behaviors. (2016)
  • Author(s): Isozaki, T., Komenoi, S., Lu Q., Usuki, T., Tomokata, S., T., Matsutomo, D., Sakai, H. Bando, K., Kiyonari, H. and Sakane, F.
  • Journal Title: J. Neurochem. Volume: 137
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Diacylglycerol kinases η1 is a high affinity isozyme for diacylglycerol (2015)
  • Author(s): Komenoi, S., Takemura, F., Sakai, H. and Sakane, F.
  • Journal Title: FEBS Lett. Volume: in press Issue: 11 Pages: 1272-1277
  • DOI: 10.1016/j.febslet.2015.03.032
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] A novel diacylglycerol kinase α-selective inhibitor, CU-3, induces cancer cell apoptosis and enhances immune response (2015)
  • Author(s): 1)Lui K, Kunii N, Sakuma M, Yamaki A, Mizuno S, Sato M, Sakai H, Kado S, Kumagai K, Kojima H, Okabe T, Nagano T, Shirai Y. and Sakane F
  • Journal Title: J lipid res Volume: ５７ Issue: 3 Pages: 368-379
  • DOI: 10.1194/jlr.m062794
  • Peer Reviewed / Open Access
• [Presentation] Roles of diacylglycerol kinase isozymes and phosphatidic acid species in neurological functions and disorders. (2017)
  • Author(s): Sakane, F.
  • Organizer: The 7th International Symposium on Diacylglycerol Kinase
  • Place of Presentation: Kobe, , Hyogo, Japan
  • Int'l Joint Research / Invited
• [Presentation] Expression and purification of diacylglycerol kinase α catalytic domain for crystallographic studies. (2017)
  • Author(s): Takahashi, D., Satoh, E. and Sakane, F.
  • Organizer: The 7th International Symposium on Diacylglycerol Kinase
  • Place of Presentation: Kobe, , Hyogo, Japan
  • Int'l Joint Research
• [Presentation] New evaluation method of diacylglycerol-generating activity of phosphatidylcholine-specific phospholipase C using mass spectrometry. (2017)
  • Author(s): Murakami, C. and Sakane, F.
  • Organizer: The 7th International Symposium on Diacylglycerol Kinase
  • Place of Presentation: Kobe, , Hyogo, Japan
  • Int'l Joint Research
• [Presentation] 神経分化誘導時に産生されるジアシルグリセロール及びホスファチジン酸分子種の解析 (2016)
  • Author(s): 水野悟，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 宮城・仙台
  • Year and Date: 2016-09-25
• [Presentation] ジアシルグリセロールキナーゼηノックアウトマウスのそう様行動惹起の分子メカニズムアシルグリセロールキナーゼηノックアウトマウスのそう様行動惹起の分子メカニズム (2016)
  • Author(s): 米野井優，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 宮城・仙台
  • Year and Date: 2016-09-25
• [Presentation] 質量分析法を用いた新規ホスファチジルコリン特異的ホスホリパーゼC活性測定法の開発 (2016)
  • Author(s): 村上千明，水野悟，坂根郁夫上千明，水野悟，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 宮城・仙台
  • Year and Date: 2016-09-25
• [Presentation] ジアシルグリセロールキナーゼη欠損によるリチウム感受性躁様行動の誘導 (2016)
  • Author(s): 陸強，磯崎丈志，米野井優，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 仙台
  • Year and Date: 2016-09-25
• [Presentation] ジアシルグリセロールキナーゼηのプレクストリン相同ドメインの精製とその性質アシルグリセロールキナーゼηのプレクストリン相同ドメインの精製とその性質 (2016)
  • Author(s): 川瀬功暉，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 宮城・仙台
  • Year and Date: 2016-09-25
• [Presentation] メラノーマ細胞に対するジアシルグリセロールキナーゼα選択的活性制御化合物の細胞死誘導効果 (2016)
  • Author(s): 八巻篤実，劉可，国井奈央子，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 宮城・仙台
  • Year and Date: 2016-09-25
• [Presentation] C2C12筋芽細胞の筋分化におけるジアシルグリセロールキナーゼδの機能 (2016)
  • Author(s): 堺弘道，松本健一，坂根郁夫
  • Organizer: 第58回日本脂質生化学会
  • Place of Presentation: 秋田・秋田
  • Year and Date: 2016-06-09
• [Presentation] 神経分化誘導時に産生されるジアシルグリセロール及びホスファチジン酸分子種の解析 (2016)
  • Author(s): 水野悟，坂根郁夫
  • Organizer: 第58回日本脂質生化学会
  • Place of Presentation: 秋田・秋田
  • Year and Date: 2016-06-09
• [Presentation] ジアシルグリセロールキナーゼη欠損によるリチウム感受性躁様行動の誘導 (2016)
  • Author(s): 陸強，磯崎丈志，米野井優，坂根郁夫
  • Organizer: 第58回日本脂質生化学会
  • Place of Presentation: 秋田・秋田
  • Year and Date: 2016-06-09
• [Presentation] Diacylglycerol kinase ζ produces 16:0-containing phosphatidic acid molecular species during neuroblastoma cell differentiation. (2016)
  • Author(s): Mizuno, S. and Sakane, F.
  • Organizer: 2016 Annual Meeting of the American Society for Biochemistry and Molecular Biology at Experimental Biology 2016
  • Place of Presentation: San Diego, California, USA
  • Year and Date: 2016-04-02
  • Int'l Joint Research
• [Presentation] A novel diacylglycerol kinase α-selective inhibitor, CU-3, induces cancer cell apoptosis and enhances immune response. (2016)
  • Author(s): Yamaki, A., Liu, Ke., Kunii, N., Mizuno, S. and Sakane, F.
  • Organizer: Joint Workshop on Chirality in Chiba University and on Soft-Molecule Activation
  • Place of Presentation: 千葉大学（千葉県・千葉市）
  • Year and Date: 2016-03-17
  • Int'l Joint Research
• [Presentation] インスリン抵抗性とジアシルグリセロールキナーゼδ (2015)
  • Author(s): 坂根郁夫
  • Organizer: Biochemistry and Molecular Biology 2015（第88回日本生化学会大会・第38回日本分子生物学会年会） ワークショップ「DGシグナリングと糖尿病関連疾患」
  • Place of Presentation: 神戸コンベンションセンター（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] 発達中マウスの脳におけるdiacylglycerol kinase δの発現とその脳特異的欠損マウスの表現型解析 (2015)
  • Author(s): 臼木貴子，高戸珠恵，堺弘道，坂根郁夫
  • Organizer: Biochemistry and Molecular Biology 2015（第88回日本生化学会大会・第38回日本分子生物学会年会）
  • Place of Presentation: 神戸コンベンションセンター（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] 脂質代謝酵素ジアシルグリセロールキナーゼの多様なモノアシルグリセロールキナーゼ活性 (2015)
  • Author(s): 佐藤優里子，坂根郁夫
  • Organizer: Biochemistry and Molecular Biology 2015（第88回日本生化学会大会・第38回日本分子生物学会年会）
  • Place of Presentation: 神戸コンベンションセンター（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] ジアシルグリセロールキナーゼによって代謝されるジアシルグリセロール分子種のプロテインキナーゼC活性化能 (2015)
  • Author(s): 神谷侑那, 水野悟, 堺弘道, 坂根郁夫
  • Organizer: Biochemistry and Molecular Biology 2015（第88回日本生化学会大会・第38回日本分子生物学会年会）
  • Place of Presentation: 神戸コンベンションセンター（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] ジアシルグリセロールキナーゼηはジアシルグリセロール高親和性のアイソザイムである (2015)
  • Author(s): 米野井優，堺弘道，坂根郁夫
  • Organizer: Biochemistry and Molecular Biology 2015（第88回日本生化学会大会・第38回日本分子生物学会年会）
  • Place of Presentation: 神戸コンベンションセンター（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] ミリスチン酸は筋管細胞のジアシルグリセロールキナーゼδの発現とグルコース取り込み能を亢進する (2015)
  • Author(s): 和田祐子，堺弘道, 崎山静花，坂根郁夫
  • Organizer: Biochemistry and Molecular Biology 2015（第88回日本生化学会大会・第38回日本分子生物学会年会）
  • Place of Presentation: 神戸コンベンションセンター（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] 神経分化時に産生されるホスファチジン酸分子種とその産生酵素の同定 (2015)
  • Author(s): 水野悟，坂根郁夫
  • Organizer: Biochemistry and Molecular Biology 2015（第88回日本生化学会大会・第38回日本分子生物学会年会）
  • Place of Presentation: 神戸コンベンションセンター（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] 精巣に特異的に発現する新規DGKηスプライシングバリアントDGKη3遺伝子のクローニング (2015)
  • Author(s): 村上絵梨，塩谷貴生，坂根郁夫
  • Organizer: Biochemistry and Molecular Biology 2015（第88回日本生化学会大会・第38回日本分子生物学会年会）
  • Place of Presentation: 神戸コンベンションセンター（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] Expression and localization of diacylglycerol kinase δ in the developing mouse brain and phenotype of its brain-specific knockout mice. (2015)
  • Author(s): Usuki, T. and Sakane, F.
  • Organizer: 14th International Conference on Bioactive Lipids in Cancer, Inflammation and Related Diseases.
  • Place of Presentation: Budapest, Hungary
  • Year and Date: 2015-07-12
  • Int'l Joint Research
• [Presentation] Diacylglycerol kinase η is a high affinity isozyme for diacylglycerol. (2015)
  • Author(s): Komenoi, S., Sakai, H. and Sakane, F.
  • Organizer: 14th International Conference on Bioactive Lipids in Cancer, Inflammation and Related Diseases.
  • Place of Presentation: Budapest, Hungary
  • Year and Date: 2015-07-12
  • Int'l Joint Research
• [Presentation] ジアシルグリセロールキナーゼηはジアシルグリセロール高親和性のアイソザイムである (2015)
  • Author(s): 米野井優，堺弘道，坂根郁夫
  • Organizer: 第57回日本脂質生化学会
  • Place of Presentation: 一橋大学一橋講堂（東京都・千代田区）
  • Year and Date: 2015-05-28
• [Presentation] 神経分化時に産生されるホスファチジン酸分子種とその産生酵素の同定 (2015)
  • Author(s): 水野 悟，坂根郁夫
  • Organizer: 第57回日本脂質生化学会
  • Place of Presentation: 一橋大学一橋講堂（東京都・千代田区）
  • Year and Date: 2015-05-28
• [Presentation] ジアシルグリセロールキナーゼαの選択的活性阻害化合物の同定 (2015)
  • Author(s): 劉可，国井奈央子，佐藤麻由，白井康仁，坂根郁夫
  • Organizer: 第57回日本脂質生化学会
  • Place of Presentation: 一橋大学一橋講堂（東京都・千代田区）
  • Year and Date: 2015-05-28
• [Remarks] 千葉大学大学院理学研究科 基盤理学専攻 生体機能化学研究室
  • URL: http://sakane32.wixsite.com/biofunctionchemistry
• [Remarks] 千葉大学大学院理学研究科 基盤理学専攻 生体機能化学研究室
  • URL: http://sakane32.wix.com/biofunctionchemistry
• [Patent(Industrial Property Rights)] ＤＧＫδノックアウトマウス及びこれを用いた方法 (2017)
  • Inventor(s): 坂根郁夫
  • Industrial Property Rights Holder: 坂根郁夫
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2017-004729
  • Filing Date: 2017-01-13
• [Patent(Industrial Property Rights)] ＤＧＫηノックアウトマウス及びこれを用いた方法 (2016)
  • Inventor(s): 坂根郁夫
  • Industrial Property Rights Holder: 坂根郁夫
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2016-220080
  • Filing Date: 2016-11-10