A novel assay for screening WIP1 phosphatase substrates in nuclear extracts

Abstract

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.

Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres

“…Expression and purification of human His-PPM1D was described previously ( 52 ). EGFP-TRF2 was purified from transiently transfected U2OS cells using GFP trap and a high salt IP buffer (50 mM Tris pH 8.0, 1 M NaCl, 1% Tween20, 0.1% NP-40, 10% glycerol, 2 mM EDTA, 3 mM EGTA, 10 mM MgCl 2 ) supplemented with PhosSTOP and protease inhibitors.…”

Endoplasmic reticulum stress‑induced cell death as a potential mechanism for targeted therapy in glioblastoma (Review)

“…p53 is a key player in the DNA damage response (DDR), and its expression is specifically induced by the PERK kinase during the UPR following ER stress (25). p53 activation during the DDR has been well studied (26,27). Once activated, p53 will stimulate and suppress different gene products, which aim to either prevent abnormal proliferation by a reversible arrest of the cell cycle to facilitate the repair processes, or to induce irreversible outcomes, including apoptosis or senescence (25).…”