Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90

The p53 mutant, 143(ala), was translated in vitro in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE). In RRL, p53-143(ala) 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, p53-143(ala) 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 p53-143(ala) was stably transfected into yeast engineered to be defective for HSP90 function, conformational recognition of mutated p53 was impaired. When stable transfectants of p53-143(ala) were prepared in yeast expressing wildtype 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 p53-143(ala). Furthermore, we show that the mutated conformation of p53 can be pharmacologically antagonized by drugs targeting HSP90.