title: Molecular mechanism of mutant p53 stabilization: the role of HSP70 and MDM2.
abstract: 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.
authors.affiliation: Department of Molecular Biology, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.
authors.forename: Milena
authors.initials: M
authors.lastname: Wiech
authors.forename: Maciej B
authors.initials: MB
authors.lastname: Olszewski
authors.forename: Zuzanna
authors.initials: Z
authors.lastname: Tracz-Gaszewska
authors.forename: Bartosz
authors.initials: B
authors.lastname: Wawrzynow
authors.forename: Maciej
authors.initials: M
authors.lastname: Zylicz
authors.forename: Alicja
authors.initials: A
authors.lastname: Zylicz
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D059748:Proteolysis; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D050505:Mutant Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C000606460:TP73 protein, human; C000606368:Trp73 protein, mouse; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D051736:Proto-Oncogene Proteins c-mdm2; C492951:Stub1 protein, mouse; D044767:Ubiquitin-Protein Ligases