Whitesell L, Sutphin P D, Pulcini E J, Martinez J D, Cook P H
Department of Pediatrics and Steele Memorial Children's Research Center, University of Arizona, Tucson 85724, USA.
Mol Cell Biol. 1998 Mar;18(3):1517-24. doi: 10.1128/MCB.18.3.1517.
Wild-type p53 is a short-lived protein which turns over very rapidly via selective proteolysis in the ubiquitin-proteasome 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.
野生型p53是一种半衰期短的蛋白质，通过泛素-蛋白酶体途径中的选择性蛋白水解作用，其更新速度非常快。然而，大多数p53突变编码的蛋白质产物在细胞内水平显著增加，并与促进肿瘤的活性相关。突变导致泛素化和蛋白酶体介导的降解受损的机制尚不清楚，但已经注意到许多具有转化活性的p53突变体与hsp70家族的分子伴侣形成稳定的物理结合。为了探究异常的伴侣蛋白相互作用在介导突变型p53功能改变及其细胞内积累中的可能作用，我们研究了与温度敏感型小鼠p53突变体发生物理结合的伴侣蛋白。在突变温度条件下培养的A1-5细胞制备的裂解物中，hsp70与p53Val135共沉淀，正如其他人之前报道的那样，但此外，还检测到细胞伴侣蛋白机制中其他公认的成分，包括hsp90、亲环蛋白40和p23。在有利于野生型p53构象的温度条件下，随着其转录激活活性的恢复，伴侣蛋白与p53的共沉淀消失。在表达两种不同热点突变的人乳腺癌细胞中也检测到了类似于在突变条件下A1-5细胞中所显示的伴侣蛋白相互作用。为了研究直接破坏伴侣蛋白与突变型p53相互作用的影响，我们使用了格尔德霉素（GA），一种选择性的hsp90结合剂，已证明它能改变调节未结合配体的类固醇受体功能的伴侣蛋白结合。GA处理细胞改变了与几种不同突变型p53物种的异源蛋白复合物形成。它增加了p53的更新率，并导致A1-5细胞中该蛋白的核转位。然而，在A1-5细胞或人乳腺癌细胞系中，GA似乎都没有将野生型转录激活活性恢复到突变型p53蛋白。
