The WTX tumor suppressor enhances p53 acetylation by CBP/p300

WTX encodes a tumor suppressor, frequently inactivated in Wilms tumor, with both plasma membrane and nuclear localization. WTX has been implicated in β-catenin turnover, but its effect on nuclear proteins is unknown. We report an interaction between WTX and p53, derived from the unexpected observation of WTX, p53, and E1B 55K colocalization within the characteristic cytoplasmic body of adenovirus-transformed kidney cells. In other cells without adenovirus expression, the C-terminal domain of WTX binds to the DNA-binding domain of p53, enhances its binding to CBP, and increases CBP/p300-mediated acetylation of p53 at Lys 373/382. WTX knockdown accelerates CBP/p300 protein turnover and attenuates this modification of p53. In p53-reconstitution experiments, cell-cycle arrest, apoptosis, and p53 target-gene expression are suppressed by depletion of WTX. Together, these results suggest that WTX modulates p53 function, in part through regulation of its activator CBP/p300.     

p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2

The tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post-translational modification. Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress-inducing agents including hypoxia, anti-metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase-deficient mutant, efficiently induces specific p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBP-mediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19(ARF), indicating that regulation of acetylation is a central target of the p53-MDM2-p19(ARF) feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP-mediated acetylation may be a universal and critical modification for p53 function.     

On the mechanism of sequence-specific DNA-dependent acetylation of p53: the acetylation motif is exposed upon DNA binding

P53 acetylation requires p300-docking to two contiguous sites in the activation domain that in turn mediates DNA-dependent acetylation of the tetramer. In an attempt to further define the mechanism of DNA-dependent acetylation of p53, an in vitro system has been reconstituted with distinct p53 isoforms and has been used to reveal conformational constraints on p53 acetylation. Two native p53 tetrameric isoforms purified from Sf9 cells differing by the extent of phosphorylation within the C-terminal acetylation site are both acetylated in a sequence-specific DNA-dependent manner. By contrast, p53 purified from an Escherichia coli expression system is in a largely denatured conformation and its acetylation is DNA-independent. Heating native p53 to destroy the folded structure restores DNA-independent acetylation similar to that seen with bacterially expressed p53. There are at least two sites of conformational flexibility in the p53 tetramer: the first in the flexible S10 beta-sheet within the MDM2 ubiquitination sequence and the second in the C-terminal regulatory domain. We analysed therefore whether DNA-dependent acetylation correlated with conformational changes in either of these two regions. DNA-dependent acetylation of p53 is maintained in a dose-dependent manner by low concentrations of consensus site DNA under conditions where flexibility in the S10 beta-sheet region is maintained. Oligonucleotide DNAs that promote acetylation stimulate the binding of monoclonal antibodies PAb421 and ICA-9; two antibodies whose contiguous epitopes overlap the C-terminal acetylation motif. By contrast, bent oligonucleotide DNAs that conceal both the S10 beta-sheet from binding of the monoclonal antibody DO-12 and attenuate binding of the monoclonal antibody PAb421 can preclude acetylation. These data suggest that, in the absence of DNA, the acetylation motif of p53 is in a cryptic state, but after DNA binding, allosteric effects mediate an exposure of the acetylation motif to allow DNA-dependent acetylation of the tetramer.     

ING2 regulates the onset of replicative senescence by induction of p300-dependent p53 acetylation

ING2 is a candidate tumor suppressor gene that can activate p53 by enhancing its acetylation. Here, we demonstrate that ING2 is also involved in p53-mediated replicative senescence. ING2 protein expression increased in late-passage human primary cells, and it colocalizes with serine 15-phosphorylated p53. ING2 and p53 also complexed with the histone acetyltransferase p300. ING2 enhanced the interaction between p53 and p300 and acted as a cofactor for p300-mediated p53 acetylation. The level of ING2 expression directly modulated the onset of replicative senescence. While overexpression of ING2 induced senescence in young fibroblasts in a p53-dependent manner, expression of ING2 small interfering RNA delayed the onset of senescence. Hence, ING2 can act as a cofactor of p300 for p53 acetylation and thereby plays a positive regulatory role during p53-mediated replicative senescence.     

Co-condensation between transcription factor and coactivator p300 modulates transcriptional bursting kinetics

1. Download : Download high-res image (212KB)
2. Download : Download full-size image