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. 2009 May 21;28(20):2112-8.
doi: 10.1038/onc.2009.71. Epub 2009 Apr 13.

Regulation by phosphorylation of the relative affinities of the N-terminal transactivation domains of p53 for p300 domains and Mdm2

D P Teufel  1 , M Bycroft, A R Fersht
Affiliations

Regulation by phosphorylation of the relative affinities of the N-terminal transactivation domains of p53 for p300 domains and Mdm2

D P Teufel et al. Oncogene. .

Abstract

The transcriptional activity of the tumour suppressor, p53, requires direct binding between its transactivation domain (TAD, 1-57) and the transcriptional coactivator, p300. We systematically assessed the role of TAD phosphorylation on binding of the p300 domains CH3, Taz1, Kix and IBiD. Thr18 phosphorylation increased the affinity up to sevenfold for CH3 and Taz1, with smaller increases from phosphorylation of Ser20, Ser15, Ser37, Ser33, Ser46 and Thr55. Binding of Kix and IBiD was less sensitive to phosphorylation. Strikingly, hepta-phosphorylation of all Ser and Thr residues increased binding 40- and 80-fold with CH3 and Taz1, respectively, but not with Kix or IBiD. Substitution of all phospho-sites with aspartates partially mimicked the effects of hepta-phosphorylation. Mdm2, the main negative regulator of p53, competes with p300 for binding to TAD. Binding of Mdm2 to TAD was reduced significantly only on phosphorylation of Thr18 (sevenfold) or by hepta-phosphorylation (24-fold). The relative affinities of Mdm2 and p300 for p53 TAD can thus be changed by up to three orders of magnitude by phosphorylation. Accordingly, phosphorylation of Thr18 and hepta-phosphorylation dramatically shifts the balance towards favouring the binding of p300 with p53, and is thus likely to be an important factor in its regulation.

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Figures

Figure 1
Figure 1
(a): Sequence alignment of vertebrate p53 N-termini. Serines and threonines that are within the segment of p53 TAD contacted by p300 domains are in bold, and encircled by grey boxes. Kinases involved in their phosphorylation are shown above (Lavin & Gueven, 2006; Toledo & Wahl, 2006; Xu, 2003). p53 L22/W23 and W53/F54, which are required for p53-p300 binding and transcriptional activity, are underlined. The approximate boundaries of p53 transactivation subdomains TAD1 and TAD2 are shown below (dotted boxes), which are compared to the approximate extent of the p300/Mdm2/Mdmx/p62 contact regions (black boxes). (b) Domain structure of p300. Taz1 is often referred to as CH1, PHD as CH2 and ZZ-Taz2 as CH3. Taz2 alone is responsible for the interaction with p53 TAD (Legge et al., 2004; Teufel et al., 2007).
Figure 2
Figure 2
Binding of p53 (phospho-)peptides and derivatives to p300 domains and Mdm2, using fluorescence anisotropy. (a) The very high affinities of phospho-peptide for CH3 had to be determined indirectly by competition. A complex of CH3 (500 nM) and p53 phospho-derivative (400 nM) was dissociated with increasing concentrations of recombinant unlabelled WT p53 (1-93). Data were fit numerically to a 1:1 binding model using software developed by Dmitry Veprintsev (MRC Centre for Protein Engineering), and normalised for the bound fraction of phospho-derivative. The KD of unlabelled 1-93 p53 was 14 nM (personal communication, Jenifer Lum, MRC Centre for Protein Engineering). 7-(P), 7-(Asp) and 7-(Ala) refer to 10-57 Hepta-(P), 10-57 Hepta-(Asp) and 1-57 Hepta-(Ala), respectively. (b), (c), (d), (e): Direct anisotropy titrations, where p300 domains (Taz1, Kix, IBiD) or Mdm2 (296 μL), respectively, are titrated into 1200 μL of 50-200 nM labelled phospho-peptide. All proteins were expressed and purified as previously described. Peptides were synthesised on a CEM Microwave Peptide Synthesiser (Matthews, N.C., USA) and purified by a combination of size exclusion, preparative and analytical HPLC; using acetonitrile/H2O as solvents, and 0.1% trifluoroacetic or phosphoric acid as pH regulators. Titrations were run on a Horiba Jobin Yvon fluorimeter (Longjumeau Cedex, France), using the same physiological ionic strength buffer and extinction coefficients (Teufel et al., 2007).
Figure 3
Figure 3
MALDI TOF mass spectra of kinase-phosphorylated 10-57 S15(P). p53 10-57 S15(P) peptide was successively phosphorylated by casein kinase I, casein kinase II (New England Biolabs, MA, USA) and c-jun N-terminal kinase JNK (Millipore, MA, USA). Each enzyme phosphorylates three, two and one serine or threonine residue, respectively. Repeated rounds of kinase phosphorylations produced the final product, which is phosphorylated on all available seven Ser/Thr on p53 TAD. The presence of the respective mono- to hepta-phosphorylated peptides are colour-coded onto the mass spectra, with molecular weights shown top left. Mass peaks are broad due to the presence of multiple ionic salt species. Kinase reactions were performed for 24 hours at 30 °C according to the manufacturer’s instructions, using (per mL) 200 μM peptide, 100 μL of 100 mM Mg-ATP, 10 μL enzyme, 2 μL EDTA-free protease inhibitor (Roche, Switzerland) and 1 μL β-mercaptoethanol. The final product was purified as above and consisted mostly of hepta-phosphorylated product as confirmed by MALDI-TOF, with small fractions of lower phospho-species.
Figure 4
Figure 4
Comparison of the effect of p53 phospho-derivatives against WT p53 for p300/Mdm2 domain binding. ΔΔG refers to the difference in free binding energy between p53 phospho-derivative and WT p53 peptide for any of the binding proteins. The bars shown therefore represent the free binding energy contributed by a phosphorylation (or Asp, Ala substitution as with 7-(Asp) and 7-(Ala)). ΔG was calculated by -RTlnKD, and ΔΔG was calculated by -RTln(KD,WT/KD,variant). Standard errors were determined as described previously (Teufel et al., 2003).
Figure 5
Figure 5
Overall importance of single phosphorylations on p300 binding. The sum of ΔΔG of a given phospho-peptide for all p300 domains shows the overall importance of a phosphorylation on p300 binding.
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