Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90.
The p53 mutant, 143ala, was translated in vitro in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE). In RRL, p53-143ala protein of both mutant and wild-type conformation, as detected immunologically with conformation-specific antibodies, was translated. The chaperone protein HSP90, present in RRL, was found to coprecipitate only with the mutated conformation of p53. Geldanamycin, shown previously to bind to HSP90 and destabilize its association with other proteins, decreased the amount of immunologically detectable mutated p53 and increased the amount of detectable wild-type protein, without affecting the total translation of p53. When translated in WGE, known to contain functionally deficient HSP90, p53-143ala produced p53 protein, which was not recognized by a mutated conformation-specific antibody. In contrast, the synthesis of conformationally detectable wild-type p53 in this system was not compromised. Reconstitution of HSP90 function in WGE permitted synthesis of conformationally detectable mutated p53, and this was abrogated by geldanamycin. Finally, when p53-143ala was stably tansfected into yeast engineered to be defective for HSP90 function, conformational recognition of mutated p53 was impaired. When stable transfectants of p53-143ala were prepared in yeast expressing wild-type HSP90, conformational recognition of mutated p53 was antagonized by macbecin I, a geldanamycin analog also known to bind HSP90. Taken together, these data demonstrate a role for HSP90 in the achievement and/or stabilization of the mutated conformation of p53-143ala. Furthermore, we show that the mutated conformation of p53 can be pharmacologically antagonized by drugs targeting HSP90.
Blagosklonny
M V
MV
Clinical Pharmacology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Toretsky
J
J
Bohen
S
S
Neckers
L
L
D000818:Animals; D016227:Benzoquinones; D002474:Cell-Free System; D016158:Genes, p53; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D047029:Lactams, Macrocyclic; D018832:Molecular Chaperones; D017354:Point Mutation; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017510:Protein Folding; D011809:Quinones / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae; D014908:Triticum; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D018832:Molecular Chaperones; D011809:Quinones; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; C001277:geldanamycin