Molecular basis of the interactions between the p73 N terminus and p300: Effects on transactivation and modulation by phosphorylation

Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge CB2 0QH, United Kingdom.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2009; 106(9):3142-7. DOI: 10.1073/pnas.0900383106
Source: PubMed


The transcription factor p73 belongs to the p53 family of proteins and can transactivate a number of target genes in common with p53. Here, we characterized the interaction of the p73 N terminus with four domains of the transcriptional coactivator p300 and with the negative regulator Mdm2 by using biophysical and cellular measurements. We found that, like p53, the N terminus of p73 contained two distinct transactivation subdomains, comprising residues 10-30 and residues 46-67. The p73 N terminus bound weakly to the Taz1, Kix, and IBiD domains of p300 but with submicromolar affinity for Taz2, in contrast to previous reports. We found weaker binding of the p73 N terminus to the p300 domains in vitro correlated with a significant decrease in transactivation activity in a cell line for the QS and T14A mutants, and tighter binding of the phosphomimetic T14D in vitro correlated with an increase in vivo. Further, we found that phosphorylation of T14 increased the affinity of the p73 N terminus for Taz2 10-fold. The phosphomimetic p73alpha T14D caused increased levels of transactivation.

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    • "The C/H3 region also contains a unique sub-domain known as TAZ2 that folds independently (53) of, and does not interact with the ZZ domain (54). While the functional significance of the ZZ domain is unclear, TAZ2 serves as docking site for a number of transcription factors (55–59). To identify the precise HBZ-binding sites within the HAT and C/H3 domains, we performed GST pull-down assays with HBZ and various truncations of the HAT and C/H3 domains. "
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    ABSTRACT: The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection.
    Nucleic Acids Research 03/2012; 40(13):5910-25. DOI:10.1093/nar/gks244 · 9.11 Impact Factor
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    • "As shown in Figure 6A, DNp63a is tetrameric demonstrating that the simultaneous presence of both the TA and the TI domains is necessary for the formation of a closed, dimeric conformation. To test whether the OD can interact with the TA domain we titrated the p63 OD with peptides derived from the TA1 (9–32) and TA2 (49–78) regions of the transactivation domain (Burge et al., 2009). While the TA2 peptide did not interact, titrations with the TA1 peptide showed strong chemical shift perturbations (CSP) in the fast exchange regime (Figures 6B, 6C, and 6F). "
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    ABSTRACT: TAp63α, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63α's activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63α inhibition remains unknown. Here, we show that TAp63α is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ∼20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63α is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63α is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes.
    Cell 02/2011; 144(4):566-76. DOI:10.1016/j.cell.2011.01.013 · 32.24 Impact Factor
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    • "Phosphorylation at T17 and T18 increases the helicity much more compared to that seen in unphosphorylated p73 (Figure 9A – 9B). This latter is known to be required for binding of p73 to p300 [62] and our model suggests that the picture is somewhat similar to that seen in p53. Further, our models correlate well with the experimental finding that phosphorylated p73 binds to MDM2 two fold better than does phosphorylated p53 (Compare Figure 8A with Figure 9B). "
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    ABSTRACT: The N terminal transactivation domain of p53 is regulated by ligases and coactivator proteins. The functional conformation of this region appears to be an alpha helix which is necessary for its appropriate interactions with several proteins including MDM2 and p300. Folding simulation studies have been carried out to examine the propensity and stability of this region and are used to understand the differences between the family members with the ease of helix formation following the order p53 > p73 > p63. It is clear that hydrophobic clusters control the kinetics of helix formation, while electrostatic interactions control the thermodynamic stability of the helix. Differences in these interactions between the family members may partially account for the differential binding to, and regulation by, MDM2 (and MDMX). Phosphorylations of the peptides further modulate the stability of the helix and control associations with partner proteins.
    BMC Genomics 02/2010; 11 Suppl 1(Suppl 1):S5. DOI:10.1186/1471-2164-11-S1-S5 · 3.99 Impact Factor
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