title: DNA damage-induced regulatory interplay between DAXX, p53, ATM kinase and Wip1 phosphatase.
abstract: Death domain-associated protein 6 (DAXX) is a histone chaperone, putative regulator of apoptosis and transcription, and candidate modulator of p53-mediated gene expression following DNA damage. DAXX becomes phosphorylated upon DNA damage, however regulation of this modification, and its relationship to p53 remain unclear. Here we show that in human cells exposed to ionizing radiation or genotoxic drugs etoposide and neocarzinostatin, DAXX became rapidly phosphorylated in an ATM kinase-dependent manner. Our deletion and site-directed mutagenesis experiments identified Serine 564 (S564) as the dominant ATM-targeted site of DAXX, and immunofluorescence experiments revealed localization of S564-phosphorylated DAXX to PML nuclear bodies. Furthermore, using a panel of human cell types, we identified the p53-regulated Wip1 protein phosphatase as a key negative regulator of DAXX phosphorylation at S564, both in vitro and in cells. Consistent with the emerging oncogenic role of Wip1, its DAXX-dephosphorylating impact was most apparent in cancer cell lines harboring gain-of-function mutant and/or overexpressed Wip1. Unexpectedly, while Wip1 depletion increased DAXX phosphorylation both before and after DNA damage and increased p53 stability and transcriptional activity, knock-down of DAXX impacted neither p53 stabilization nor p53-mediated expression of Gadd45a, Noxa, Mdm2, p21, Puma, Sesn2, Tigar or Wip1. Consistently, analyses of cells with genetic, TALEN-mediated DAXX deletion corroborated the notion that neither phosphorylated nor non-phosphorylated DAXX is required for p53-mediated gene expression upon DNA damage. Overall, we identify ATM kinase and Wip1 phosphatase as opposing regulators of DAXX-S564 phosphorylation, and propose that the role of DAXX phosphorylation and DAXX itself are independent of p53-mediated gene expression.
authors: a Department of Cell Signaling and Apoptosis.
authors: Jan
authors: J
authors: Brazina
authors: Jan
authors: J
authors: Svadlenka
authors: Libor
authors: L
authors: Macurek
authors: Ladislav
authors: L
authors: Andera
authors: Zdenek
authors: Z
authors: Hodny
authors: Jiri
authors: J
authors: Bartek
authors: Hana
authors: H
authors: Hanzlikova
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism*; D002460:Cell Line; D056970:Co-Repressor Proteins; D004249:DNA Damage; D005786:Gene Expression Regulation; D006801:Humans; D018832:Molecular Chaperones; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D000071636:Protein Phosphatase 2C; D055550:Protein Stability; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D056970:Co-Repressor Proteins; C106985:DAXX protein, human; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D016159:Tumor Suppressor Protein p53; D010768:Phosphoserine; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000607458:PPM1D protein, human; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C
keywords: ATM; ATM, ataxia telangiectasia mutated; CHX, cycloheximide; DAXX, Death domain-associated protein 6; IR, ionizing radiation; NCS, neocarzinostatin; p21, p21WAF1/Cip1 cyclin-dependent kinase inhibitor 1; PML, promyelocytic leukemia protein; VP16, etoposide; Wi; DAXX; DNA damage; Wip1; p53