Human p53 Phosphorylation Mimic, S392E, Increases Nonspecific DNA Affinity and Thermal Stability

Nichols, Nicole M.; Matthews, Kathleen S.

doi:10.1021/bi011736r

Cited by 42 publications

(35 citation statements)

References 46 publications

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“…Substitution of one of the phosphorylation sites in the C terminus, S392E, by mutation mimics constitutive phosphorylation and increases DNA binding and stability of the core (Nichols and Matthews, 2002). Binding of the bacterial heat-shock protein DnaK or PAb421 antibody to the C-terminal basic region, and to a lesser extent deletion of this region, increases the resistance of the core domain to thermal denaturation as assessed by the retention of PAb1620 epitope upon heating to 371C (Hansen et al, 1996).…”

Section: Structural Basis For Mutant P53 Reactivationmentioning

confidence: 99%

Reactivation of mutant p53: molecular mechanisms and therapeutic potential

2007

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Section: Structural Basis For Mutant P53 Reactivationmentioning

confidence: 99%

Reactivation of mutant p53: molecular mechanisms and therapeutic potential

2007

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“…All these chaperones tested positive for activity by the luciferase refolding assay (see below). p53 human recombinant protein was purified essentially as described (43).…”

Section: Figmentioning

confidence: 99%

Hsp90 Chaperones Wild-type p53 Tumor Suppressor Protein

Walerych

Kudla

Gutkowska

et al. 2004

Journal of Biological Chemistry

136

129

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Immortalized human fibroblasts were used to investigate the putative interactions of the Hsp90 molecular chaperone with the wild-type p53 tumor suppressor protein. We show that geldanamycin or radicicol, specific inhibitors of Hsp90, diminish specific wild-type p53 binding to the p21 promoter sequence. Consequently, these inhibitors decrease p21 mRNA levels, which lead to a reduction in cellular p21/Waf1 protein, known to induce cell cycle arrest. In control experiments, we show that neither geldanamycin nor radicicol affect p53 mRNA levels. A minor decrease in p53 protein level following the treatment of human fibroblasts with the inhibitors suggests the potential involvement of Hsp90 in the stabilization of wild-type p53. To support our in vivo findings, we used a reconstituted system with highly purified recombinant proteins to examine the effects of Hsp90 on wildtype p53 binding to the p21 promoter sequence. The human recombinant Hsp90 ␣-isoform as well as bovine brain Hsp90 were purified to homogeneity. Both of these molecular chaperones displayed ATPase activity and the ability to refold heat-inactivated luciferase in a geldanamycinand radicicol-sensitive manner, suggesting that posttranslational modifications are not involved in the modulation of Hsp90␣ activity. We show that the incubation of recombinant p53 at 37°C decreases the level of its wildtype conformation and strongly inhibits the in vitro binding of p53 to the p21 promoter sequence. Interestingly, Hsp90 in an ATP-dependent manner can positively modulate p53 DNA binding after incubation at physiological temperature of 37°C. Other recombinant human chaperones from Hsp70 and Hsp40 families were not able to efficiently substitute Hsp90 in this reaction. Consistent with our in vivo results, geldanamycin can suppress Hsp90 ability to regulate in vitro p53 DNA binding to the promoter sequence. In summary, the results presented in this article state that chaperone activity of Hsp90 is important for the transcriptional activity of genotypically wild-type p53.

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“…The molecular basis for p53 protein latency requires the C-terminal domain to destabilize and unfold the core DNA-binding domain by allosteric effects (5), suggesting that p53 is part of a growing group of regulatory proteins that are native and unfolded in the absence of posttranslational modification (13). Phosphorylation of p53 in the C-terminal domain neutralizes the destabilizing effect of the C terminus on the core domain of p53 and maintains p53 conformation and activity (30).…”

Section: Vol 23 2003 Proline Repeat Motif Constrains P53 Acetylatiomentioning

confidence: 99%

The Proline Repeat Domain of p53 Binds Directly to the Transcriptional Coactivator p300 and Allosterically Controls DNA-Dependent Acetylation of p53

Dornan

Shimizu

Burch

et al. 2003

Molecular and Cellular Biology

126

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The transcription coactivator p300 cannot acetylate native p53 tetramers, thus revealing intrinsic conformational constraints on p300-catalyzed acetylation. Consensus site DNA is an allosteric effector that promotes acetylation of p53, suggesting that p300 has an undefined conformationally flexible interface within the p53 tetramer. To identify such conformationally responsive p300-binding sites, p300 was subjected to peptide selection from a phage-peptide display library, a technique that can define novel protein-protein interfaces. The enriched p300-binding peptides contained a proline repeat (PXXP/PXPXP) motif, and five proline repeat motifs actually reside within the p53 transactivation domain, suggesting that this region of p53 may harbor the second p300 contact site. p300 binds in vitro to PXXP-containing peptides derived from the proline repeat domain, and PXXP-containing peptides inhibit sequence-specific DNA-dependent acetylation of p53, indicating that p300 docking to both the LXXLL and contiguous PXXP motif in p53 is required for p53 acetylation. Deletion of the proline repeat motif of p53 prevents DNA-dependent acetylation of p53 by occluding p300 from the p53-DNA complex. Sequence-specific DNA places an absolute requirement for the proline repeat domain to drive p53 acetylation in vivo. Chromatin immunoprecipitation was used to show that the proline repeat deletion mutant p53 is bound to the p21 promoter in vivo, but it is not acetylated, indicating that prolinedirected acetylation of p53 is a post-DNA binding event. The PXXP repeat expands the basic interface of a p300-targeted transactivation domain, and proline-directed acetylation of p53 at promoters indicates that p300-mediated acetylation can be highly constrained by substrate conformation in vivo.

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Human p53 Phosphorylation Mimic, S392E, Increases Nonspecific DNA Affinity and Thermal Stability

Cited by 42 publications

References 46 publications

Reactivation of mutant p53: molecular mechanisms and therapeutic potential

Reactivation of mutant p53: molecular mechanisms and therapeutic potential

Hsp90 Chaperones Wild-type p53 Tumor Suppressor Protein

The Proline Repeat Domain of p53 Binds Directly to the Transcriptional Coactivator p300 and Allosterically Controls DNA-Dependent Acetylation of p53

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