Inhibition of p53-dependent transcription by BOX-I phospho-peptide mimetics that bind to p300

Abstract

The N-terminal BOX-I domain of p53 containing a docking site for the negative regulator MDM2 and the positive effector p300, harbours two recently identified phosphorylation sites at Thr18 or Ser20O whose affect on p300 is undefined. Biochemical assays demonstrate that although MDM2 binding is inhibited by these phosphorylations, p300 binding is strikingly stabilized by Thr18 or Ser20 phosphorylation. Introducing EGFP-BOX-I domain peptides with an aspartate substitution at Thr18 or Ser20 induced a significant inhibition of endogenous p53-dependent transcription in cycling cells, in irradiated cells, as well as in cells transiently co-transfected with p300 and p53. In contrast an EGFP-wild-type BOX-I domain peptide stimulated p53 activity via inhibition of MDM2 protein binding. These results suggest that phosphorylation of p53 at Thr18 or Ser20 can activate p53 by stabilizing the p300-p53 complex and also identify a class of small molecular weight ligands capable of selective discrimination between MDM2- and p300-dependent activities.

Fig. 1. Phosphorylation stabilizes p300-p53 BOX-I peptide complexes. The binding of: (A) full-length p300 protein, (B) truncated p300 (1135–2414) and (C) full-length MDM2 protein, to the indicated biotinylated-peptides was analysed by ELISA as described in the Methods. The amount of p300 or MDM2 protein bound are represented as relative light units (RLUs). The amount of biotinylated peptide titrated onto streptavidin-coated ELISA surfaces is indicated in this Figure, and the other Figures where ELISA is used, as follows: dark blue bars, 1 ng; brown bars, 0.1 ng; yellow bars, 0.01 ng and light blue bars, 0 ng.

Fig. 2. In vivo inhibition of endogenous p53-dependent transcription by phospho-peptide mimetics. (A) p300 and (B) MDM2 binding in vitro to aspartate-substituted BOX-I domain peptides. The binding of p300 protein and MDM2 protein, to biotinylated-peptides substituted with aspartate at the indicated positions was as described in Figure 1. (C). EGFP-Asp18 and Asp20 peptide fusion proteins inhibit p53-dependent transactivation in vivo. EGFP-constructs or the mutant p53^HIS175 allele (100 ng) were transiently transfected with 2 µg p21-Luc or 2 µg control-Luc and 1 µg control-β-Gal-reporter into cycling A375 cells, and the cells harvested 24 h post-transfection. p53-dependent activity (RLUs) is expressed as a ratio of p21-luciferase activity (dark blue bars) or control-luciferase activity (brown bars) to the internal transfection control (β-Gal). (D). Expression levels of EGFP-peptide fusion proteins in A375 cells. Lysates from cells transfected with the indicated EGFP-peptide fusion constructs, as described in Figure 2C, were immunoblotted with antibodies to EGFP to determine the relative level of each fusion protein expressed in cells transfected with the 2 µg p21-Luc or control-Luc vectors. (E) p300 can recover p53 activity in cells co-transfected with the inhibitory EGFP-Asp18 and Asp20 peptide fusion proteins. A375 cells were co-transfected with increasing amounts of the p300 gene (dark blue bars, 0 µg; brown bars, 1 µg; yellow bars, 2 µg and light blue bars, 5 µg), and fixed levels of p21-Luc (2 µg), β-Gal-reporter (1 µg) and the EGFP-peptide fusion vectors (100 ng), the cells were then processed for analysis of p53 activity as described in the legend for Figure 2C. (F) EGFP-Asp18 and Asp20 peptide fusion proteins inhibit p53-dependent transactivation in irradiated cells. EGFP-constructs (100 ng) were transiently transfected with 2 µg p21-Luc or 2 µg control-Luc and 1 µg control-β-Gal-reporter into A375 cells that were either untreated (dark blue bars), damaged with 20 J/m² UV-C (brown bars), or with 5 Gy ionizing radiation (yellow bars). The cells were processed for analysis of p53 activity as described in the legend for Figure 2C.

Fig. 3. In vivo inhibition of ectopically expressed p53 from the p21 promoter by phospho-peptide mimetic fusion proteins. (A) Stimulation of p53 activity by co-transfection with p300. Saos-2 cells were transiently co-transfected with 1 µg pCMV-p53, 2 µg p21-Luc, 1 µg pCMVβ-Gal and increasing amounts of pCMV-βp300: brown bars, 0 ng; yellow bars, 1 µg; light blue bars, 2 µg and purple bars, 5 µg. As a negative control, (dark blue bars) 5 µg pCMV-βp300, 2 µg p21-Luc and 1 µg pCMVβ-Gal were co-transfected. The cells were harvested 30 h post-transfection and the relative activity is expressed as a ratio of luciferase activity to β-Gal activity. (B) EGFP-S20D peptide inhibits p300 induction of p53-dependent gene expression. Saos-2 cells were transiently co-transfected with 1 µg pCMV-p53, 2 µg p21-Luc, 5 µg pCMVβp300, 1 µg pCMVβ-Gal and increasing amounts of EGFP-constructs as indicated (yellow bars, 1 µg; light blue bars, 2 µg and purple bars, 5 µg). To function as a critical control, 5 µg of each EGFP construct and 5 µg of pCMVβp300 were co-transfected with 2 µg p21-Luc and pCMVβGal (brown bars), and as a negative control (dark blue bars), 5 µg pCMV-βp300, 2 µg p21-Luc and 1 µg pCMVβ-Gal co-transfected. The cells were processed as described in the legend of Figure 3A. (C) Immunoblots of p53 and EGFP-fusion proteins in transfected Saos-2 cells. Lysates from transfected Saos-2 cells (as described in Figure 3B) were normalized for protein content by Bradford assay and loading for immunoblots was confirmed by Red Ponceau staining. The constructs transfected (in µg) are highlighted by the legend above the Figure (increasing amounts of EGFP fused to NS, BOX-I, S15D, T18D, and S20D BOX-I domain peptides) and are described below the Figure as ‘+’. The levels of p300, p53 and EGFP proteins are in the top, middle or bottom panels, respectively. p53-dependent activity (in RLUs) from Figure 3B is listed below the immunoblots for direct comparison of p53 activity to p53 protein and EGFP protein levels.