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.p53 transactivation domain ͉ phosphorylation ͉ protein-protein interaction ͉ transcriptional coactivator ͉ tumor suppressor T he p53 tumor suppressor is activated as a transcriptional regulator in response to DNA damage, leading to the arrest of cell growth and apoptosis. p53 is a modular protein that binds DNA as a tetramer; each subunit contains an N-terminal transactivation domain (TAD), proline-rich domain, core DNA binding domain, tetramerization domain, and C-terminal regulatory domain. In the absence of cellular stress, p53 binds target promoters in an inactive latent state and recruits HDM2 (the human homolog of mouse double minute 2, MDM2) to chromatin (1, 2). HDM2 functions as a ubiquitin E3 ligase that maintains p53 at low levels by continuous proteasomal degradation (3). DNA damage initiates a cascade of phosphorylation and acetylation events at multiple sites on p53 (Fig. 1A), resulting in stabilization and enhancement of p53 transcriptional activity (4-7). In particular, phosphorylation at threonine-18 (T18) helps stabilize p53 by inhibiting binding to HDM2 (8, 9), whereas phosphorylation of serines 15 and 20 (S15, S20) enhances recruitment of the general transcriptional coactivators and acetylases, CREB binding protein (CBP) and p300 (10-12). S15 must be phosphorylated before phosphorylation can occur at T18 and S20 (13).CBP and p300 play a central role in regulation of p53 stability and the response to genotoxic stress (14-16). In unstres...

Objective: The objective of this study was to investigate the association among adiposity, insulin resistance, and inflammatory markers [high‐sensitivity C‐reactive protein (hs‐CRP), interleukin (IL)‐6, and tumor necrosis factor (TNF)‐α] and adiponectin and to study the effects of exercise training on adiposity, insulin resistance, and inflammatory markers among obese male Korean adolescents. Research Methods and Procedures: Twenty‐six obese and 14 lean age‐matched male adolescents were studied. We divided the obese subjects into two groups: obese exercise group (N = 14) and obese control group (N = 12). The obese exercise group underwent 6 weeks of jump rope exercise training (40 min/d, 5 d/wk). Adiposity, insulin resistance, lipid profile, hs‐CRP, IL‐6, TNF‐α, and adiponectin were measured before and after the completion of exercise training. Results: The current study demonstrated higher insulin resistance, total cholesterol, LDL‐C levels, triglyceride, and inflammatory markers and lower adiponectin and HDL‐C in obese Korean male adolescents. Six weeks of increased physical activity improved body composition, insulin sensitivity, and adiponectin levels in obese Korean male adolescents without changes in TNF‐α, IL‐6, and hs‐CRP. Discussion: Obese Korean male adolescents showed reduced adiponectin levels and increased inflammatory cytokines. Six weeks of jump rope exercise improved triglyceride and insulin sensitivity and increased adiponectin levels.

The activity and stability of the tumor suppressor p53 is regulated by interactions with key cellular proteins such as MDM2 and CBP/p300. The transactivation domain (TAD) of p53 contains two subdomains (AD1 and AD2) and interacts directly with the N-terminal domain of MDM2 and with several domains of CBP/p300. Here we report the NMR structure of the full-length p53 TAD in complex with the nuclear coactivator binding domain (NCBD) of CBP. Both the p53 TAD and NCBD are intrinsically disordered and fold synergistically upon binding, as evidenced by the observed increase in helicity and increased dispersion of the amide proton resonances. The p53 TAD folds to form a pair of helices (denoted Pα1 and Pα2), which extend from Phe19 to Leu25 and Pro47 to Trp53, respectively. In the complex, the NCBD forms a bundle of three helices (Cα1: residues 2066-2075, Cα2: residues 2081-2092, and Cα3: residues 2095-2105) with a hydrophobic groove into which the p53 helices Pα1 and Pα2 dock. The polypeptide chain between the p53 helices remains flexible and makes no detectable intermolecular contacts with the NCBD. Complex formation is driven largely by hydrophobic contacts that form a stable intermolecular hydrophobic core. A salt bridge between D49 of p53 and R2105 of NCBD may contribute to the binding specificity. The structure provides the first insights into simultaneous binding of the AD1 and AD2 motifs to a target protein.The p53 tumor suppressor acts as a hub in signal transduction networks that mediate the cellular response to stress, leading to cell-cycle arrest, senescence, or apoptosis (1,2). Due to its role in determining cell fate, p53 is tightly controlled by numerous regulatory proteins that include MDM2, MDMX, CBP/p300, and various kinases. In unstressed cells, p53 is maintained at extremely low levels through interactions with the ubiquitin E3 ligase MDM2 (3,4). This interaction results in ubiquitination and proteasomal degradation of p53 and also blocks interactions with the basal transcriptional proteins (5,6). MDMX, which is highly homologous to MDM2 but lacks ubiquitin ligase activity, also negatively regulates p53 and inhibits its transactivation function (7,8). Upon cellular stress, specific kinases are activated which phosphorylate the N-terminal region of p53. Phosphorylation facilitates release from MDM2 and enhances binding to the general transcriptional coactivators CBP and p300 (9-13).CBP and p300 function as scaffolds for the recruitment and assembly of the transcriptional machinery and modify both chromatin and transcription factors through their intrinsic † This work was supported by grant CA96865 from the National Institutes of Health. C.W.L. was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2004-214-C00207 (Figure S1), CD spectra of the free proteins and the complexes ( Figure S2), graphs of Cα secondary chemical shifts for each component of the complex ( Figure S3) and a graph showing the locations of paramagne...

The transcriptional activity of p53 is regulated by a cascade of posttranslational modifications. Although acetylation of p53 by CREB-binding protein (CBP)/p300 is known to be indispensable for p53 activation, the role of phosphorylation, and in particular multisite phosphorylation, in activation of CBP/p300-dependent p53 transcriptional pathways remains unclear. We investigated the role of single site and multiple site phosphorylation of the p53 transactivation domain in mediating its interaction with CBP and with the ubiquitin ligase HDM2. Phosphorylation at Thr18 functions as an on/off switch to regulate binding to the N-terminal domain of HDM2. In contrast, binding to CBP is modulated by the extent of p53 phosphorylation; addition of successive phosphoryl groups enhances the affinity for the TAZ1, TAZ2, and KIX domains of CBP in an additive manner. Activation of p53-dependent transcriptional pathways requires that p53 compete with numerous cellular transcription factors for binding to limiting amounts of CBP/p300. Multisite phosphorylation represents a mechanism for a graded p53 response, with each successive phosphorylation event resulting in increasingly efficient recruitment of CBP/p300 to p53-regulated transcriptional programs, in the face of competition from cellular transcription factors. Multisite phosphorylation thus acts as a rheostat to enhance binding to CBP/p300 and provides a plausible mechanistic explanation for the gradually increasing p53 response observed following prolonged or severe genotoxic stress.competitive binding | protein-protein interaction | transcriptional coactivator | tumor suppressor

Molecular interactions between the tumor suppressor p53 and the transcriptional coactivators CBP/p300 are critical for the regulation of p53 transactivation and stability. The transactivation domain (TAD) of p53 binds directly to several CBP/p300 domains (TAZ1, TAZ2, NCBD, and KIX). Here we map the interaction between the p53 TAD and the CBP KIX domain using isothermal titration calorimetry and NMR spectroscopy. KIX is a structural domain in CBP/p300 that can simultaneously bind two polypeptide ligands, such as the activation domain of MLL and the kinase inducible activation domain (pKID) of CREB, using distinct interaction surfaces. The p53 TAD consists of two subdomains (AD1 and AD2); peptides corresponding to the isolated AD1 and AD2 subdomains interact with KIX with relatively low affinity, but a longer peptide containing both subdomains binds KIX tightly. In the context of the full-length p53 TAD, AD1 and AD2 bind synergistically to KIX. Mapping of the chemical shift perturbations onto the structure of KIX shows that isolated AD1 and AD2 peptides bind to both the MLL and pKID sites. Spin labeling experiments show that the complex of the full-length p53 TAD with KIX is disordered, with the AD1 and AD2 subdomains each interacting with both the MLL and pKID binding surfaces. Phosphorylation of the p53 TAD at Thr18 or Ser20 increases the KIX binding affinity. The affinity is further enhanced by simultaneous phosphorylation of Thr18 and Ser20 and the specificity of the interaction is increased. The p53 TAD simultaneously occupies the two distinct sites that have been identified on the CBP KIX domain and efficiently competes for these sites with other known KIX-binding transcription factors.