A novel assay for screening WIP1 phosphatase substrates in nuclear extracts
| Autor: | Kamila Burdova, René H. Medema, Matous Palek, Radka Storchova, Libor Macurek |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
DNA Repair DNA repair DNA damage Phosphatase Bone Neoplasms Biochemistry 03 medical and health sciences 0302 clinical medicine Tumor Cells Cultured Humans Protein Interaction Domains and Motifs Phosphorylation Protein kinase A Molecular Biology Adaptor Proteins Signal Transducing Cell Nucleus Osteosarcoma Chemistry Kinase Acetylation Cell Biology G2-M DNA damage checkpoint Cell cycle Cell biology Protein Phosphatase 2C 030104 developmental biology 030220 oncology & carcinogenesis Biological Assay Tumor Suppressor Protein p53 DNA Damage |
| Zdroj: | The FEBS Journal. 288:6035-6051 |
| ISSN: | 1742-4658 1742-464X |
| Popis: | Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates. |
| Databáze: | OpenAIRE |
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