Cellular memory depending on phosphorylation states of a signaling protein

• Project/Area Number: 15H02394
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Sako Yasushi 国立研究開発法人理化学研究所, 開拓研究本部, 主任研究員 (20215700)
• Co-Investigator(Kenkyū-buntansha): 日比野 佳代 国立遺伝学研究所, 構造遺伝学研究センター, 助教 (40435673)
• Research Collaborator: Okamoto Kenji
• Project Period (FY): 2015-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥40,170,000 (Direct Cost: ¥30,900,000、Indirect Cost: ¥9,270,000); Fiscal Year 2018: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000); Fiscal Year 2017: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000); Fiscal Year 2016: ¥10,920,000 (Direct Cost: ¥8,400,000、Indirect Cost: ¥2,520,000); Fiscal Year 2015: ¥13,650,000 (Direct Cost: ¥10,500,000、Indirect Cost: ¥3,150,000)
• Keywords: 細胞情報・動態

Outline of Final Research Achievements

A cytoplasmic serine-threonine kinase RAF is a key regulator of cell fate decision in animal cells. RAF has a open/close structural dynamics which can be detected by using intramolecular FRET of a probe molecule of RAF conjugated with two fluorophores at the each end. We found that average conformation of RAF has a wide single-cell variation and the variation correlates with RAF response after cell stimulation. RAF has a multiple phosphorylation sites, of which combination regulates RAF conformation. Single-molecule FRET imaging of RAF revealed the pathway of conformational change upon cell stimulation. These results suggest that activation of various intracellular kinase molecules of RAF changes the RAF conformation which regulate cellular response to the next round of stimulation.

Academic Significance and Societal Importance of the Research Achievements

RAFは細胞増殖・分化などの運命決定を制御する分子であり、その異常は遺伝病の発症や発癌に関係している。RAFの特徴は活性と関連する極めて多数のリン酸化部位と、開閉の構造ダイナミクスを持つことである。我々は多数のRAF分子の平均構造が細胞毎に異なっており、構造がリン酸化の組み合わせと相関することを発見し、詳細な解析を行った。本研究は、細胞内情報処理蛋白質の多重リン酸化を利用した新たな細胞記憶の形成・保持機構を研究し、その分子機構に由来する細胞個性の発現や遺伝的変異による発病の理解に繋がるものである。

Research Products

• [Int'l Joint Research] Ulsan University(韓国)
• [Int'l Joint Research] University Colledge Dublin(アイルランド)
• [Int'l Joint Research] McGill University(Canada)
• [Int'l Joint Research] National University of Singapore(Singapore)
• [Journal Article] In-cell single-molecule FRET measurements reveal three conformational state changes in RAF protein (2019)
  • Author(s): Okamoto Kenji、Hibino Kayo、Sako Yasushi
  • Journal Title: Biochimica et Biophysica Acta (BBA) - General Subjects Volume: in press Pages: 1-42
  • DOI: 10.1016/j.bbagen.2019.04.022
  • Peer Reviewed / Open Access
• [Journal Article] Inferring a nonlinear biochemical network model from a heterogeneous single-cell time course data (2018)
  • Author(s): Shindo Yuki、Kondo Yohei、Sako Yasushi
  • Journal Title: Scientific Reports Volume: 8 Pages: 1-10
  • DOI: 10.1038/s41598-018-25064-w
  • NAID: 120006502573
  • Peer Reviewed / Open Access
• [Journal Article] Automated single-molecule imaging in living cells (2018)
  • Author(s): Yasui Masato、Hiroshima Michio、Kozuka Jun、Sako Yasushi、Ueda Masahiro
  • Journal Title: Nature Communications Volume: 9 Pages: 1-11
  • DOI: 10.1038/s41467-018-05524-7
  • Peer Reviewed / Open Access
• [Journal Article] Single-molecule diffusion-based estimation of ligand effects on G protein coupled receptors (2018)
  • Author(s): Yanagawa Masataka、Hiroshima Michio、Togashi Yuichi、Abe Mitsuhiro、Yamashita Takahiro、Shichida Yoshinori、Murata Masayuki、Ueda Masahiro、Sako Yasushi
  • Journal Title: Sci. Signal. Volume: 11 Pages: 1-16
  • DOI: 10.1126/scisignal.aao1917
  • Peer Reviewed / Open Access
• [Journal Article] Single-Molecule Foerster Resonance Energy Transfer Measurement Reveals the Dynamic Partially Ordered Structure of the Epidermal Growth Factor Receptor C-Tail Domain (2018)
  • Author(s): Okamoto Kenji、Sako Yasushi
  • Journal Title: The Journal of Physical Chemistry B Volume: 123 Pages: 571-581
  • DOI: 10.1021/acs.jpcb.8b10066
  • Peer Reviewed / Open Access
• [Journal Article] Single-molecule imaging and manipulation of biomolecular machines and systems. (2018)
  • Author(s): Iino Ryota、Iida Tatsuya、Nakamura Akihiko、Saita Ei-ichiro、You Huijuan、Sako Yasushi
  • Journal Title: BBA General Subjects Volume: 1862 Pages: 241-252
  • DOI: 10.1016/j.bbagen.2017.08.008
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Transient Acceleration of Epidermal Growth Factor Receptor Dynamics Produces Higher-Order Signaling Clusters (2018)
  • Author(s): Hiroshima Michio、Pack Chan-gi、Kaizu Kazunari、Takahashi Koichi、Ueda Masahiro、Sako Yasushi
  • Journal Title: Journal of Molecular Biology Volume: 430 Pages: 1386-1401
  • DOI: 10.1016/j.jmb.2018.02.018
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Lipid-Protein Interplay in Dimerization of Juxtamembrane Domains of Epidermal Growth Factor Receptor (2018)
  • Author(s): Maeda Ryo、Sato Takeshi、Okamoto Kenji、Yanagawa Masataka、Sako Yasushi
  • Journal Title: Biophysical Journal Volume: 114 Pages: 893-903
  • DOI: 10.1016/j.bpj.2017.12.029
  • Peer Reviewed / Open Access
• [Journal Article] Single-molecule fluorescence-based analysis of protein conformation, interaction, and oligomerization in cellular systems (2017)
  • Author(s): Okamoto Kenji、Hiroshima Michio、Sako Yasushi
  • Journal Title: Biophysical Reviews Volume: 10 Pages: 317-326
  • DOI: 10.1007/s12551-017-0366-3
  • Peer Reviewed / Open Access
• [Journal Article] A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C. (2017)
  • Author(s): Makino, A., Sako, Y. et al.
  • Journal Title: FASEB Journal Volume: 31 Pages: 1301-1322
  • DOI: 10.1096/fj.201500075r
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Recent advances in FRET for the study of protein interactions and dynamics (2017)
  • Author(s): Okamoto, K. and Sako, Y.
  • Journal Title: Curr. Opin. Strct. Biol. Volume: 46 Pages: 16-23
  • DOI: 10.1016/j.sbi.2017.03.010
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A role for anti-viral host defense mechanism in the phylogenetic divergence in baculovirus evolution (2016)
  • Author(s): Nagamine, T. and Sako, Y.
  • Journal Title: PLoS ONE Volume: 11
  • Peer Reviewed / Open Access
• [Journal Article] Cortical Polarity of the RING Protein PAR-2 Is Maintained by Exchange Rate Kinetics at the Cortical-Cytoplasmic Boundary. (2016)
  • Author(s): Arata, Y., Hiroshima, M., Pack, C.-G., Ramanujam, R., Motegi, F., Nakazato, K., Wiseman, P. W., Sawa, H., Kobayashi, T. J., Brandao, H. B., Shibata, T., and Sako, Y.
  • Journal Title: Cell Reports Volume: 16 Pages: 2156-2165
  • DOI: 10.1016/j.celrep.2016.07.047
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] 細胞内シグナルのアナログ・デジタル変換 (2016)
  • Author(s): 新土優樹、小迫英尊、佐甲靖志、高橋恒一
  • Journal Title: 生物物理 Volume: 56 Pages: 334-336
  • NAID: 130005170660
  • Peer Reviewed / Open Access
• [Journal Article] Interaction of a novel fluorescent GTP analogue with the small G-protein K-Ras. (2016)
  • Author(s): Seigo IWATA, Kaori MASUHARA, Nobuhisa UMEKI, Yasushi SAKO and Shinsaku MARUTA
  • Journal Title: J. Biochem. Volume: 159 Pages: 41-48
  • DOI: 10.1093/jb/mvv071
  • NAID: 40020725621
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] State transition analysis of spontaneous branch migration of the Holliday junction by photon-based single-molecule fluorescence resonance energy transfer (2016)
  • Author(s): Okamoto, K. and Sako, Y.
  • Journal Title: Biophysical Chemistry Volume: 209 Pages: 21-27
  • DOI: 10.1016/j.bpc.2015.11.004
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Conversion of graded phosphorylation into switch-like nuclear translocation via autoregulatory mechanisms in ERK signalling (2016)
  • Author(s): Y. Shindo, K. Iwamoto, K. Mouri, K. Hibino, M. Tomita, H. Kosako, Y. Sako and K. Takahashi.
  • Journal Title: Nat. Comm. Volume: 7 Pages: 10485-10485
  • DOI: 10.1038/ncomms10485
  • NAID: 120006535049
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains. (2016)
  • Author(s): Nakamura, Y., Hibino, K., Yanagida, T., and Sako, Y.
  • Journal Title: Biophys. Physicobiol. Volume: 13 Pages: 1-11
  • NAID: 130005121334
  • Peer Reviewed
• [Presentation] Single-molecule pharmacology of G protein-coupled receptors (2019)
  • Author(s): Sako, Y.
  • Organizer: 1st international symposium on Interdisciplinary Approaches to Integrative Understanding of Biological Signaling Networks
  • Int'l Joint Research / Invited
• [Presentation] In cell automated single-molecule analysis and its extensive applications (2018)
  • Author(s): Hiroshima, M., Yasui, M., Kozuka, J., Sako, Y., and Ueda, M.
  • Organizer: 日本生物物理学会第56回年会
  • Int'l Joint Research / Invited
• [Presentation] Single-molecule analysis of a cell signaling protein, SOS (2018)
  • Author(s): Nakamura, Y., Hibino, K., Yanagida, T., and Sako, Y.
  • Organizer: 日本生物物理学会第56回年会
  • Int'l Joint Research / Invited
• [Presentation] Sinlgle-molecular pharmacology of GPCRs (2018)
  • Author(s): Sako, Y.
  • Organizer: The 3rd Biosignal Research Center International Symposium
  • Int'l Joint Research / Invited
• [Presentation] Lateral mobility of GPCRs reflects their activation kinetics (2018)
  • Author(s): Sako, Y.
  • Organizer: The 91st Annual Meeting of the Japanese Biochemical Society Symposium
  • Int'l Joint Research / Invited
• [Presentation] 細胞膜受容体の１分子動態、生理学から薬理学へ (2018)
  • Author(s): 佐甲靖志
  • Organizer: 日本顕微鏡学会 第42回関東支部講演会
  • Invited
• [Presentation] Lipid-protein cooperativity in the regulation of juxtamembrane domain dimer formation in epidermal growth factor receptor (2017)
  • Author(s): Maeda, R., Sato, T., Okamoto, K., and Sako, Y.
  • Organizer: 第55回日本生物物理学会年会
  • Invited
• [Presentation] Single-molecule FRET measurement for EGFR-RAS-MAPK signal transduction pathway (2017)
  • Author(s): Okamoto, K., Maeda, R., and Sako, Y.
  • Organizer: “Deciphering complex energy landscape and kinetic network from single molecules to cells: a new challenge to make theories meet experiments”
  • Int'l Joint Research / Invited
• [Presentation] 細胞内情報伝達のイメージング (2017)
  • Author(s): 佐甲靖志
  • Organizer: 理研公開シンポジウム「観る・測る・解く」４次元細胞計測の現状と未来
  • Invited
• [Presentation] 細胞膜受容体の運動・会合体形成による情報伝達 (2017)
  • Author(s): 佐甲靖志
  • Organizer: 第69回日本細胞生物学会大会
  • Invited
• [Presentation] 細胞内の１分子イメージング：生理学から薬理学へ (2016)
  • Author(s): 佐甲靖志
  • Organizer: 東京理科大学研究推進機構総合研究院 イメージングフロンティアセンターシンポジウム
  • Place of Presentation: 東京理科大学（野田）
  • Year and Date: 2016-12-10
  • Invited
• [Presentation] Motional dynamics of cell membrane receptors (2016)
  • Author(s): Sako, Y.
  • Organizer: Pioneering New Fields: Forefront of RIKEN’s Science and Beyond
  • Place of Presentation: RIKEN (Wako)
  • Year and Date: 2016-11-21
  • Int'l Joint Research / Invited
• [Presentation] 細胞膜受容体の１分子可視化 (2016)
  • Author(s): 佐甲靖志
  • Organizer: 第25回日本バイオイメージング学会学術集会
  • Place of Presentation: 名古屋市立大学（名古屋）
  • Year and Date: 2016-09-05
  • Invited
• [Presentation] 細胞内の確率過程 (2016)
  • Author(s): 佐甲靖志
  • Organizer: 研究会「異常拡散をめぐる最近の進展」
  • Place of Presentation: 慶應義塾大学（横浜）
  • Year and Date: 2016-09-01
  • Invited
• [Presentation] 増殖因子刺激に対するswitch-likeなERKの核移行様式 (2016)
  • Author(s): 新土優樹 他
  • Organizer: 第68回日本細胞生物学会大会、日本ケミカルバイオロジー学会合第11回年会合同大会
  • Place of Presentation: 京都テルサ（京都）
  • Year and Date: 2016-06-15
  • Invited
• [Presentation] １分子計測で見るEGF受容体の細胞膜動態 (2016)
  • Author(s): 佐甲靖志
  • Organizer: 蛋白研セミナー
  • Place of Presentation: 大阪大学、吹田
  • Year and Date: 2016-03-04
  • Invited
• [Presentation] 細胞内情報処理反応の１分子可視化計測 (2016)
  • Author(s): 佐甲靖志
  • Organizer: CBI学会 第370回研究講演会
  • Place of Presentation: 東京大学、東京
  • Year and Date: 2016-03-03
  • Invited
• [Presentation] 細胞膜受容体の動的会合体形成と機能 (2015)
  • Author(s): 佐甲靖志
  • Organizer: 第13回JCGGシンポジウム
  • Place of Presentation: ウインク愛知、名古屋
  • Year and Date: 2015-10-01
  • Invited
• [Presentation] Single-molecule analysis of cell signaling reactions (2015)
  • Author(s): 佐甲靖志
  • Organizer: 第3回 少数性成物学トレーニングコース
  • Place of Presentation: 大阪大学、吹田
  • Year and Date: 2015-08-01
  • Invited
• [Presentation] 情報伝達蛋白質RAFの疾病変異体のコンフォメーションと機能のイメージング計測 (2015)
  • Author(s): 日比野佳代、上田昌宏、佐甲靖志
  • Organizer: 第67回 日本細胞生物学会大会
  • Place of Presentation: タワーホール船堀、東京
  • Year and Date: 2015-07-01
  • Invited
• [Presentation] RAS-MAPK systemの可視化反応解析 １分子病理・薬理学の可能性 (2015)
  • Author(s): 佐甲靖志
  • Organizer: 岡山大学大学院薬学研究科セミナー
  • Place of Presentation: 岡山大学、岡山
  • Year and Date: 2015-06-26
  • Invited
• [Presentation] １分子計測で細胞内反応システムを理解する (2015)
  • Author(s): 佐甲靖志
  • Organizer: 第12回 原子・分子・光科学 (AMO) 討論会
  • Place of Presentation: 東京大学、東京
  • Year and Date: 2015-06-20
  • Invited
• [Remarks] http://www.riken.go.jp/cell-info/
• [Remarks] 佐甲細胞情報研究室Webpage
  • URL: http://www.riken.go.jp/cell-info/
• [Patent(Industrial Property Rights)] Method for evaluating activity of G protein-coupled receptor (GPCR) (2018)
  • Inventor(s): Masataka Yanagawa, et al.
  • Industrial Property Rights Holder: 理化学研究所
  • Industrial Property Rights Type: 特許
  • Filing Date: 2018
  • Overseas
• [Patent(Industrial Property Rights)] Gタンパク質共役型受容体(GPCR)の活性評価方法 (2017)
  • Inventor(s): 柳川正隆、佐甲靖志、廣島通夫、安井真人、上田昌宏、冨樫祐一
  • Industrial Property Rights Holder: 柳川正隆、佐甲靖志、廣島通夫、安井真人、上田昌宏、冨樫祐一
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2017-084803
  • Filing Date: 2017