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The luciferase-based in vivo protein-protein interaction assay revealed that CHK1 promotes PP2A and PME-1 interaction.
Ando, Sana; Tanaka, Keiko; Matsumoto, Maharu; Oyama, Yuki; Tomabechi, Yuri; Yamagata, Atsushi; Shirouzu, Mikako; Nakagawa, Reiko; Okimoto, Noriaki; Taiji, Makoto; Sato, Koichi; Ohama, Takashi.
Afiliação
  • Ando S; Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
  • Tanaka K; Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
  • Matsumoto M; Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
  • Oyama Y; Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
  • Tomabechi Y; Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Kanagawa, Japan.
  • Yamagata A; Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Kanagawa, Japan.
  • Shirouzu M; Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Kanagawa, Japan.
  • Nakagawa R; Laboratory for Cell-Free Protein Synthesis, RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo, Japan.
  • Okimoto N; Laboratory for Computational Molecular Design, RIKEN Center for Biosystems Dynamics Research (BDR), Osaka, Japan; Drug Discovery Molecular Simulation Platform Unit, RIKEN Center for Biosystems Dynamics Research (BDR), Osaka, Japan.
  • Taiji M; Laboratory for Computational Molecular Design, RIKEN Center for Biosystems Dynamics Research (BDR), Osaka, Japan; Drug Discovery Molecular Simulation Platform Unit, RIKEN Center for Biosystems Dynamics Research (BDR), Osaka, Japan.
  • Sato K; Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan; Research Institute for Cell Design Medical Science, Yamaguchi University, Yamaguchi, Japan.
  • Ohama T; Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan; Research Institute for Cell Design Medical Science, Yamaguchi University, Yamaguchi, Japan. Electronic address: t.ohama@yamaguchi-u.ac.jp.
J Biol Chem ; 300(5): 107277, 2024 May.
Article em En | MEDLINE | ID: mdl-38588804
ABSTRACT
Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase, and its dysfunction is involved in the onset of cancer and neurodegenerative disorders. PP2A functions as a trimeric holoenzyme whose composition is regulated by the methyl-esterification (methylation) of the PP2A catalytic subunit (PP2Ac). Protein phosphatase methylesterase-1 (PME-1) is the sole PP2Ac methylesterase, and the higher PME-1 expression is observed in various cancer and neurodegenerative diseases. Apart from serving as a methylesterase, PME-1 acts as a PP2A inhibitory protein, binding directly to PP2Ac and suppressing its activity. The intricate function of PME-1 hinders drug development by targeting the PME-1/PP2Ac axis. This study applied the NanoBiT system, a bioluminescence-based protein interaction assay, to elucidate the molecular mechanism that modulates unknown PME-1/PP2Ac protein-protein interaction (PPI). Compound screening identified that the CHK1 inhibitors inhibited PME-1/PP2Ac association without affecting PP2Ac methylation levels. CHK1 directly phosphorylates PP2Ac to promote PME-1 association. Phospho-mass spectrometry identified multiple phospho-sites on PP2Ac, including the Thr219, that affect PME-1 interaction. An anti-phospho-Thr219 PP2Ac antibody was generated and showed that CHK1 regulates the phosphorylation levels of this site in cells. On the contrary, in vitro phosphatase assay showed that CHK1 is the substrate of PP2A, and PME-1 hindered PP2A-mediated dephosphorylation of CHK1. Our data provides novel insights into the molecular mechanisms governing the PME-1/PP2Ac PPI and the triad relationship between PP2A, PME-1, and CHK1.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Hidrolases de Éster Carboxílico / Proteína Fosfatase 2 / Quinase 1 do Ponto de Checagem Limite: Humans Idioma: En Revista: J Biol Chem Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Japão

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Hidrolases de Éster Carboxílico / Proteína Fosfatase 2 / Quinase 1 do Ponto de Checagem Limite: Humans Idioma: En Revista: J Biol Chem Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Japão