Hsp90 Chaperones Wild-type p53 Tumor Suppressor Protein

Abstract

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.

Hsp90 inhibition has opposing effects on wild-type and mutant p53 and induces p21 expression and cytotoxicity irrespective of p53/ATM status in chronic lymphocytic leukaemia cells

“…Although wt p53 has also been reported as an Hsp90 client protein (Wang and Chen, 2003;Muller et al, 2004;Walerych et al, 2004), our demonstration that GA increased rather than decreased the levels of wt p53 suggests that this is not the case in CLL cells. Instead, our data are more in keeping with the idea that Hsp90 inhibition depletes Akt, which in turn results in loss of MDM2 function and consequent derepression of wt p53.…”

“…Thus in other cell types, wt p53 (Wang and Chen, 2003;Muller et al, 2004;Walerych et al, 2004), mutant p53 (Blagosklonny et al, 1996;Sepehrnia et al, 1996;Whitesell et al, 1998;Nagata et al, 1999) and Akt (Sato et al, 2000;Basso et al, 2002;Fujita et al, 2002) have each been implicated as Hsp90 client proteins. Akt can potentially suppress the function of wt p53 by phosphorylating and activating its inhibitory partner, MDM2 (Vogelstein et al, 2000).…”

A biochemical rationale for the anticancer effects of Hsp90 inhibitors: Slow, tight binding inhibition by geldanamycin and its analogues

“…Protein identity was confirmed by N-terminal sequencing and molecular mass confirmed by liquid chromatography MS. The in vitro chaperone activity of these proteins was characterized by following the method of Walerych et al (20), in which the luciferase is heat-denatured in the presence of Hsp90 and allowed to refold, with addition of Hsp70, Hsp40, and ATP at ambient temperature, followed by quantitation of refolding as a measurement of luciferase activity. By using the purified proteins, the time-dependent refolding of active luciferase was found to be greatly diminished by removing one of the proteins (Hsp70 or Hsp40) or the substrate ATP (Fig.…”

“…The chaperone assay was performed by following the protocol of Walerych et al (20), with modifications described below. Hsp90␣, 2.5 M and luciferase, 0.25 M in denaturation buffer (25 mM Tris, pH 7.5, 8 mM MgSO 4 , 0.01% BGG, and 10% glycerol) were incubated at 50°C for 8 min.…”

Reactivation of mutant p53: molecular mechanisms and therapeutic potential

“…p53 can bind to the chaperone proteins Hsp40, Hsp70 and Hsp90 (Walerych et al, 2004). Unfolded mutant p53 binds Hsp70 with higher affinity than the wild-type protein (Rudiger et al, 2002).…”