The Physical Association of Multiple Molecular Chaperone Proteins with Mutant p53 Is Altered by Geldanamycin, an hsp90-Binding Agent

Abstract

Wild-type p53 is a short-lived protein which turns over very rapidly via selective proteolysis in the ubiquitinproteasome pathway. Most p53 mutations, however, encode for protein products which display markedly increased intracellular levels and are associated with positive tumor-promoting activity. The mechanism by which mutation leads to impairment of ubiquitination and proteasome-mediated degradation is unknown, but it has been noted that many transforming p53 mutants are found in stable physical association with molecular chaperones of the hsp70 class. To explore a possible role for aberrant chaperone interactions in mediating the altered function of mutant p53 and its intracellular accumulation, we examined the chaperone proteins which physically associate with a temperature-sensitive murine p53 mutant. In lysate prepared from A1-5 cells grown under mutant temperature conditions, hsp70 coprecipitated with p53Val135 as previously reported by others, but in addition, other well-recognized elements of the cellular chaperone machinery, including hsp90, cyclophilin 40, and p23, were detected. Under temperature conditions favoring wild-type p53 conformation, the coprecipitation of chaperone proteins with p53 was lost in conjunction with the restoration of its transcriptional activating activity. Chaperone interactions similar to those demonstrated in A1-5 cells under mutant conditions were also detected in human breast cancer cells expressing two different hot-spot mutations. To examine the effect of directly disrupting chaperone interactions with mutant p53, we made use of geldanamycin (GA), a selective hsp90-binding agent which has been shown to alter the chaperone associations regulating the function of unliganded steroid receptors. GA treatment of cells altered heteroprotein complex formation with several different mutant p53 species. It increased p53 turnover and resulted in nuclear translocation of the protein in A1-5 cells. GA did not, however, appear to restore wild-type transcriptional activating activity to mutant p53 proteins in either A1-5 cells or human breast cancer cell lines.The wild-type p53 transcription factor is a nuclear tumor suppressor involved in cell cycle regulation, and loss of its normal function through mutation results in genetic instability and abnormalities in the induction of apoptotic cell death (14). Many p53 mutations are also associated with positive tumorpromoting activity, and their protein products are found to display markedly increased intracellular levels. Wild-type p53 is a very short lived protein which turns over rapidly via selective proteolysis in the ubiquitin-proteasome pathway (22). We have recently shown, however, that for several common p53 mutants, the normal processing of the protein is impaired, which results in the marked accumulation of dysfunctional molecules with a prolonged intracellular half-life (40). The mechanism by which mutation leads to impairment of ubiquitination and proteasome-mediated degradation is unknown at this time, but it has been noted...

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

“…Hsp90 inhibition and p53/ATM status in CLL K Lin et al p53 is an Hsp90 client protein (Blagosklonny et al, 1996;Sepehrnia et al, 1996;Whitesell et al, 1998;Nagata et al, 1999). 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.…”

“…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).…”

Wild-type p53 protein shows calcium-dependent binding to F-actin

“…Once in the cytoplasm, p53 may associate with cytoplasmic proteins and such association may play a role in regulation of p53 during the cell cycle. Mutant forms of p53 (mp53) associate with cytoplasmic stress proteins (Fourie et al, 1997;Whitesell et al, 1998), with cytoplasmic anchorage of the stabilized mp53 being dependent on continuous protein synthesis (Gannon and Lane, 1991). In contrast, cytoplasmic wild-type p53 co-locates with cytoskeletal actin ®laments (Katsumoto et al, 1995) and is rapidly turned over by the ubiquitin-proteasome pathway (Maki et al, 1996;Honda et al, 1997).…”

p53-independent association between SV40 large T antigen and the major cytosolic heat shock protein, HSP90

“…In addition to the well-characterized association between LT and p53 (Lane and Crawford, 1979; Linzer and Levine, 1979), HSP90 is also known to be associated with certain mutant forms of p53 (Blagosklonny et al, 1996;Whitesell et al, 1998). Thus, the possibility exists that the observed co-immunoprecipitation of HSP90 with LT could be mediated by p53.…”