Cooperative regulation of p53 by modulation of ternary complex formation with CBP/p300 and HDM2

Abstract

The tumor suppressor activity of p53 is regulated by interactions with the ubiquitin ligase HDM2 and the general transcriptional coactivators CBP and p300. Using NMR spectroscopy and isothermal titration calorimetry, we have dissected the binding interactions between the N-terminal transactivation domain (TAD) of p53, the TAZ1, TAZ2, KIX, and nuclear receptor coactivator binding domains of CBP, and the p53-binding domain of HDM2. The p53 TAD contains amphipathic binding motifs within the AD1 and AD2 regions that mediate interactions with CBP and HDM2. Binding of the p53 TAD to CBP domains is dominated by interactions with AD2, although the affinity is enhanced by additional interactions with AD1. In contrast, binding of p53 TAD to HDM2 is mediated primarily by AD1. The p53 TAD can bind simultaneously to HDM2 (through AD1) and to any one of the CBP domains (through AD2) to form a ternary complex. Phosphorylation of p53 at T18 impairs binding to HDM2 and enhances affinity for the CBP KIX domain. Multisite phosphorylation of the p53 TAD at S15, T18, and S20 leads to increased affinity for the TAZ1 and KIX domains of CBP. These observations suggest a mechanism whereby HDM2 and CBP/p300 function synergistically to regulate the p53 response. In unstressed cells, CBP/p300, HDM2 and p53 form a ternary complex that promotes polyubiquitination and degradation of p53. After cellular stress and DNA damage, p53 becomes phosphorylated at T18 and other residues in the AD1 region, releases HDM2 and binds preferentially to CBP/p300, leading to stabilization and activation of p53.

Fig. 1.

Domain organization of p53 and CBP/p300. (A) Domains of p53. TAD (N-terminal transactivation domain), P-rich (proline-rich), DBD (DNA-binding domain), TD (tetramerization domain), and REG (C-terminal regulatory domain). The location of the AD1 and AD2 motifs is indicated on a partial amino acid sequence; known sites of phosphorylation are indicated by dots. (B) Domains of CBP/p300. Domains that interact with the p53 transactivation domain, TAZ1 (residues 340–439), KIX (586–672), TAZ2 (1764–1855), NCBD (2059–2117) are shown in black.

Fig. 2.

Addition of p53 TAD to TAZ2. (A) Portion of the ¹H-¹⁵N HSQC spectrum of TAZ2 (black) showing chemical shift changes upon titration with p53 (13–61) at p53:TAZ2 mole ratios of 0.6:1 (red), 1:1 (green), 1.5:1 (yellow), and 2:1 (blue). The curvature in the titrations with excess p53 indicates the presence of a secondary binding site. (B) ¹⁵N chemical shift titration curves for a subset of TAZ2 resonances (colored points corresponding to residues according to the legend) upon titration with increasing amounts of p53 (13–61). The lines represent a global fit to the titration data, using a 2-site binding model with K_d1 = 0.026 μM and K_d2 = 30 μM.

Fig. 3.

Addition of CBP domains to p53 TAD. (A) Chemical shift changes for Trp-23 and Trp-53 Nε cross peaks upon titration of ¹⁵N p53-TAD (13–61) with unlabeled NCBD. Mole ratio p53:NCBD = 1:0 (black), 1:0.5 (red), 1:1 (green), 1:2 (blue). (B) Histogram showing weighted average chemical shift changes Δδ(N,H)_av (= $\sqrt{{\left(\Delta {\delta }_{\text{HN}}\right)}^2+{\left(\Delta {\delta }_{\text{N}}/5\right)}^2}$, where Δδ_HN and Δδ_N correspond to the differences in amide ¹H and ¹⁵N chemical shifts between the free and bound states) for p53 amide resonances caused by binding to TAZ1 (black), KIX (green) and NCBD (red). (C) Histogram showing changes in ¹³Cα chemical shifts upon binding of ¹⁵N,¹³C p53-TAD (13–61) to NCBD (black) or to HDM2 (6–125) (red).

Fig. 4.

Ternary complex formation between CBP, p53, and HDM2. (A) HSQC spectra of ¹⁵N p53 (13–61) free (black), bound to equimolar HDM2 (red), and in 1:1:1 ternary complex with TAZ2 and HDM2 (green). (B) Tryptophan side chain resonances of ¹⁵N p53 (13–61) free (black), bound to equimolar HDM2 (red), and after addition of equimolar TAZ1 to the HDM2 complex (green). For reference, the positions of the Trp Nε cross peaks in the binary p53 (13–61):TAZ1 complex are shown in blue.

Fig. 5.

Model for the molecular events involved in the p53 response mediated by synergistic interactions with CBP/p300 and HDM2. (A) In unstressed cells, AD1 binds strongly to HDM2, whereas AD2 interacts with the TAZ1, KIX, TAZ2, and NCBD domains of CBP/p300, promoting polyubiquitination and degradation of p53. (B) After genotoxic stress, S15, T18, and S20 of the p53 TAD become phosphorylated, lowering the affinity of AD1 for HDM2 and increasing its affinity for the CBP TAZ1 and KIX domains, promoting acetylation of the C terminus of p53 and its activation and stabilization.