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.

Numerous p53 missense mutations possess gain-of-function activities. Studies in mouse models have demonstrated that the stabilization of p53 R172H (R175H in human) mutant protein, by currently unknown factors, is a prerequisite for its oncogenic gain-of-function phenotype such as tumour progression and metastasis. Here we show that MDM2-dependent ubiquitination and degradation of p53 R175H mutant protein in mouse embryonic fibroblasts is partially inhibited by increasing concentration of heat shock protein 70 (HSP70/HSPA1-A). These phenomena correlate well with the appearance of HSP70-dependent folding intermediates in the form of dynamic cytoplasmic spots containing aggregate-prone p53 R175H and several molecular chaperones. We propose that a transient but recurrent interaction with HSP70 may lead to an increase in mutant p53 protein half-life. In the presence of MDM2 these pseudoaggregates can form stable amyloid-like structures, which occasionally merge into an aggresome. Interestingly, formation of folding intermediates is not observed in the presence of HSC70/HSPA8, the dominant-negative K71S variant of HSP70 or HSP70 inhibitor. In cancer cells, where endogenous HSP70 levels are already elevated, mutant p53 protein forms nuclear aggregates without the addition of exogenous HSP70. Aggregates containing p53 are also visible under conditions where p53 is partially unfolded: 37°C for temperature-sensitive variant p53 V143A and 42°C for wild-type p53. Refolding kinetics of p53 indicate that HSP70 causes transient exposure of p53 aggregate-prone domain(s). We propose that formation of HSP70- and MDM2-dependent protein coaggregates in tumours with high levels of these two proteins could be one of the mechanisms by which mutant p53 is stabilized. Moreover, sequestration of p73 tumour suppressor protein by these nuclear aggregates may lead to gain-of-function phenotypes.

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin-proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or "lid" in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2(S17D)) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg(97)/Lys(98), and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.

The murine double minute (mdm2) gene encodes an E3 ubiquitin ligase that plays a key role in the degradation of p53 tumor suppressor protein. Nevertheless recent data highlight other p53-independent functions of MDM2. Given that MDM2 protein binds ATP, can interact with the Hsp90 chaperone, plays a role in the modulation of transcription factors and protection and activation of DNA polymerases, and is involved in ribosome assembly and nascent p53 protein biosynthesis, we have evaluated and found MDM2 protein to possess an intrinsic molecular chaperone activity. MDM2 can substitute for the Hsp90 molecular chaperone in promoting binding of p53 to the p21-derived promoter sequence. This reaction is driven by recycling of MDM2 from the p53 complex, triggered by binding of ATP to MDM2. The ATP binding mutant MDM2 protein (K454A) lacks the chaperone activity both in vivo and in vitro. Mdm2 cotransfected in the H1299 cell line with wild-type p53 stimulates efficient p53 folding in vivo but at the same time accelerates the degradation of p53. MDM2 in which one of the Zn 2؉ coordinating residues is mutated (C478S or C464A) blocks degradation but enhances folding of p53. This is the first demonstration that MDM2 possesses an intrinsic molecular chaperone activity, indicating that the ATP binding function of MDM2 can mediate its chaperone function toward the p53 tumor suppressor.The p53 tumor suppressor gene encodes a sequence-specific transcription factor that is mutated in the vast majority of human cancers (1). Two other paralogs of p53, namely p63 and p73, have been identified, but the physiological functions of each member of the p53 family appear to be rather distinct (for review, see Refs. 2 and 3). One of the foremost characterized target genes of p53 is the mdm2 gene. MDM2 protein possesses E3 ubiquitin ligase activity toward p53 that plays a role in the negative regulation of p53 and its degradation by the proteasome (for review, see Ref. 4).Through its ability to ubiquitylate p53 and target it for proteasomal degradation, MDM2 plays a key role in maintaining p53 at very low levels under non-stress conditions. In such circumstances MDM2 and p53 form a negative feedback loop in which p53 induces mdm2 transcription and MDM2 targets p53 for degradation (2). In stress situations, MDM2-dependent degradation of p53 is inhibited by a variety of mechanisms, including p14ARF binding to MDM2, phosphorylation of the C terminus of MDM2 by the ATM kinase, stress-induced phosphorylation of sites in the trans-activation domain of p53, subsequent binding of the p300 coactivator, and further acetylation of p53 in its C-terminal region (5), which results in an increase of the steady-state level of the p53 transcription factor and consequential flux in the expression of more than a hundred genes, including those involved in cell cycle arrest, senescence, and apoptosis (2).Recent reports indicate that apart from its initially discovered RING finger-dependent enzymatic E3 ubiquitin ligase activity, MDM2 has other functions. A hydroph...