Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response

• Project/Area Number: 20K06644
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 44010:Cell biology-related
• Research Institution: Juntendo University
• Principal Investigator: Ichimura Yoshinobu 順天堂大学, 医学部, 先任准教授 (80400993)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2022: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2021: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2020: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 選択的オートファジー / p62 / ULK1 / リン酸化 / p62-KEAP1-NRF2経路 / p62/SQSTM1 / NRF2-KEAP1システム / リン酸化酵素 / ULK1キナーゼ / KEAP1-NRF2経路 / 液滴 / 筋萎縮性側索硬化症(ALS) / 前頭側頭骨変性症（FTD） / キナーゼ / p62-Nrf2-Keap1 / 脱リン酸化 / p62-Keap1-Nrf2経路 / ホスファターゼ

Outline of Research at the Start

p62のリン酸化は、選択的オートファジーとKeap1-Nrf2経路を直結させることで強固な生体防御系を構築するが、その破綻は癌の増殖に寄与する。我々は、このp62のリン酸化が可逆的であること、つまり脱リン酸化制御を受ける決定的な証拠を得た（未発表）。そこで本研究課題では、(1) p62のリン酸化に関与するホスファターゼとキナーゼを同定、(2) 同定したホスファターゼとキナーゼの欠損細胞を作製し、選択的オートファジーとKeap1-Nrf2経路の活性化を検証、さらに、(3) がん細胞を用いて、その増殖に与える影響を調査。得られた結果より、p62リン酸化の制御およびその破綻によるがん増殖のメカニズムを明らかにする。

Outline of Final Research Achievements

p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of phosphorylation remain unclear. Herein, we identify ULK1 as a kinase responsible for phosphorylation of p62. ULK1 co-localizes with p62 bodies, and directly interacts with p62. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351 corresponding to human Ser349 is replaced by Glu. These mice, but not phosphodefective p62S351A/S351A mice, exhibit NRF2 hyperactivation and growth retardation, the latter caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insight into the role of phase separation in this process.

Academic Significance and Societal Importance of the Research Achievements

本研究成果は細胞のストレス応答機構や液―液相分離の生理的役割について新たな知見を与えるものである。p62顆粒は肝疾患、神経変性疾患の病変細胞や肝細胞がんの病変部位で過剰に蓄積することが知られており、これら病態においてレドックス非依存性ストレス応答が調整不全となっていることが強く疑われる。本研究で得られた知見をもとに、より詳細な解析を進めることで、p62顆粒が関与する病態発症機序の解明が期待される。

Research Products

• [Int'l Joint Research] Massachusetts General Hospital/Brigham and Women’s Hospital(米国)
• [Int'l Joint Research] University of Helsinki(フィンランド)
• [Int'l Joint Research] Hannam University(韓国)
• [Journal Article] Considering the mechanism by which droplets of ALS-FTD-associated SQSTM1/p62 mutants cause pathology (2022)
  • Author(s): Ichimura Y, Komatsu M
  • Journal Title: Autophagy Reports Volume: 1 Issue: 1 Pages: 9-13
  • DOI: 10.1080/27694127.2022.2031380
  • Peer Reviewed / Open Access
• [Journal Article] Phase-separated protein droplets of amyotrophic lateral sclerosis-associated p62/SQSTM1 mutants show reduced inner fluidity (2021)
  • Author(s): Faruk MO, Ichimura Y, Kageyama S, Komatsu-Hirota S, El-Gowily AH, Sou YS, Koike M, Noda NN, Komatsu M.
  • Journal Title: J Biol Chem Volume: 297 Issue: 6 Pages: 101405-101405
  • DOI: 10.1016/j.jbc.2021.101405
  • Peer Reviewed / Open Access
• [Journal Article] Selective autophagy (2021)
  • Author(s): Faruk Mohammad Omar、Ichimura Yoshinobu、Komatsu Masaaki
  • Journal Title: Cancer Science Volume: 112 Issue: 10 Pages: 3972-3978
  • DOI: 10.1111/cas.15112
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A Description of Novel Variants and Review of Phenotypic Spectrum in UBA5-related Early Epileptic Encephalopathy (2021)
  • Author(s): Briere LC, Walker MA, High FA, Rogers CA, Callahan C, Cooper C, Ishimura R, Ichimura Y, Caruso PA, Sharma N, Brokamp E, Koziura ME, Mohammad SS, Dale RC, Riley LG, Network UD, Phillips JA, Komatsu M, Sweetser DA.
  • Journal Title: Cold Spring Harb Mol Case Stud. Volume: mcs Issue: 3 Pages: a005827-a005827
  • DOI: 10.1101/mcs.a005827
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response (2021)
  • Author(s): Kageyama S, Gudmundsson SR, Sou YS, Ichimura Y, Tamura N, Kazuno S, Ueno T, Miura Y, Noshiro D, Abe M, Mizushima T, Miura N, Okuda S, Motohashi H, Lee JA, Sakimura K, Ohe T, Noda NN, Waguri S, Eskelinen EL, Komatsu M.
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 16-16
  • DOI: 10.1038/s41467-020-20185-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] ULK1によるp62液―液相分離制御 (2022)
  • Author(s): 一村 義信、池田 良、森下 英晃、能代 大輔、野田 展生、小松 雅明
  • Organizer: 日本Cell Death学会学術集会
• [Presentation] ULK1/2-mediated p62-phosphorylation at Ser349 is physiologically important for NRF2 activation (2022)
  • Author(s): Ryo Ikeda, ○Yoshinobu Ichimura, Daisuke Noshiro, Hideaki Morishita, Shuhei Takada, Tomoko Funakoshi, Satoko Komatsu-Hirota, Shun Kageyama, Ritsuko Arai, Manabu Abe, Kenji Sakimura, Arata Horii, Satoshi Waguri, Nobuo N Noda, and Masaaki Komatsu
  • Organizer: The 10th International Symposium on Autophagy
  • Int'l Joint Research
• [Presentation] p62脱リン酸化酵素の同定 (2020)
  • Author(s): 池田良、一村義信、小松聡子、小松雅明
  • Organizer: オートファジー研究会
• [Remarks] オートファジーフォーラム
  • URL: https://proteolysis.jp/a_forum/threads/view/234
• [Remarks] 非膜型細胞小器官p62顆粒の機能を解明
  • URL: https://prtimes.jp/main/html/rd/p/000000252.000021495.html