Hsp90 chaperones wild-type p53 tumor suppressor protein.
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 wild-type p53 binding to the p21 promoter sequence. The human recombinant Hsp90 alpha-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 geldanamycin- and radicicol-sensitive manner, suggesting that post-translational modifications are not involved in the modulation of Hsp90alpha activity. We show that the incubation of recombinant p53 at 37 degrees C decreases the level of its wild-type 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 degrees 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.
Walerych
Dawid
D
International Institute of Molecular and Cell Biology in Warsaw, 02-109 Warsaw, Poland.
Kudla
Grzegorz
G
Gutkowska
Malgorzata
M
Wawrzynow
Bartosz
B
Muller
Lin
L
King
Frank W
FW
Helwak
Aleksandra
A
Boros
Joanna
J
Zylicz
Alicja
A
Zylicz
Maciej
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016227:Benzoquinones; D015153:Blotting, Western; D001921:Brain / Q000196:embryology / Q000378:metabolism; D002417:Cattle; D002453:Cell Cycle; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D047029:Lactams, Macrocyclic; D007783:Lactones / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D018942:Macrolides; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D011809:Quinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D007783:Lactones; D018942:Macrolides; D020033:Protein Isoforms; D011809:Quinones; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D000255:Adenosine Triphosphate; D008156:Luciferases; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; C035359:monorden; C001277:geldanamycin