C-Terminal Ubiquitination of p53 Contributes to Nuclear Export

The growth inhibitory functions of p53 are controlled in unstressed cells by rapid degradation of the p53 protein. One of the principal regulators of p53 stability is MDM2, a RING finger protein that functions as an E3 ligase to ubiquitinate p53. MDM2 promotes p53 nuclear export, and in this study, we show that ubiquitination of the C terminus of p53 by MDM2 contributes to the efficient export of p53 from the nucleus to the cytoplasm. In contrast, MDM2 did not promote nuclear export of the p53-related protein, p73. p53 nuclear export was enhanced by overexpression of the export receptor CRM1, although no significant relocalization of MDM2 was seen in response to CRM1. However, nuclear export driven by CRM1 overexpression did not result in the degradation of p53, and nuclear export was not essential for p53 degradation. These results indicate that MDM2 mediated ubiquitination of p53 contributes to both nuclear export and degradation of p53 but that these activities are not absolutely dependent on each other.     

Dynamics in the p53-Mdm2 Ubiquitination Pathway

The tumor suppressor p53 is highly regulated under various states of cellular stress. p53 stability is predominantly regulated through the ubiquitin-proteasomal pathway by the E3 ligase Mdm2. p53 ubiquitination is a dynamic process with Mdm2 capable of catalyzing both mono- and polyubiquitination. Additionally, deubiquitination is an important step occurring in p53 and Mdm2 stabilities. Factors such as HAUSP, p14ARF, and MdmX play important regulatory roles in p53 ubiquitination/deubiquitination and their interplay with Mdm2 and p53 compound layers of complexity for regulating this important pathway.     

Protecting p53 from degradation

Inactivation of the p53 function is a common event in cancer. Approx. 50% of human tumours express mutant p53 and there is evidence that in others, including many childhood tumours, p53 function is impaired in other ways. These defects on p53 function may be due to the alteration of cellular factors that modulate p53 or to the expression of viral oncoproteins. Radiotherapy and many of the chemotherapeutic drugs currently used in cancer treatment are potent activators of p53. However, most of these therapies have a serious drawback; that is, the long-term consequences of their DNA-damaging effects. Understanding the mechanisms regulating p53 stability is crucial for the development of new strategies to activate p53 non-genotoxically. Here we describe the effect of a potent activator of the p53 response, the nuclear export inhibitor leptomycin B, on Mdm2 degradation and we provide evidence for the oligomerization of the p14ARF tumour suppressor and Mdm2 inhibitor in response to oxidative stress.     

Ubiquitination of p53 at multiple sites in the DNA-binding domain

The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain.     

突变体p53研究进展

抑癌基因突变是癌症发生过程中一个极为关键的事件。p53作为体内最重要的抑癌基因之一, 在人类癌症中发生突变的频率高达50%。同时, p53突变也是人类遗传病Li-Fraumeni综合征的主要病因。p53最常见的突变形式是错义突变, 所形成的突变体p53不但失去了野生型p53的抑癌功能, 而且还获得了一系列类似于癌基因的功能, 促进了肿瘤的进程。文章拟对突变体p53的结构功能改变, 获得癌基因活性的分子机制, 以及近年来对封闭突变体p53活性所进行的探索等研究方向所取得的进展做一综述。     

Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.     

Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation.

Levels of the tumor suppressor protein p53 are normally quite low due in part to its short half-life. p53 levels increase in cells exposed to DNA-damaging agents, such as radiation, and this increase is thought to be responsible for the radiation-induced G1 cell cycle arrest or delay. The mechanisms by which radiation causes an increase in p53 are currently unknown. The purpose of this study was to compare the effects of gamma and UV radiation on the stability and ubiquitination of p53 in vivo. Ubiquitin-p53 conjugates could be detected in nonirradiated and gamma-irradiated cells but not in cells which were UV treated, despite the fact that both treatments resulted in the stabilization of the p53 protein. These results demonstrate that UV and gamma radiation have different effects on ubiquitinated p53 and suggest that the UV-induced stabilization of p53 results from a loss of p53 ubiquitination. Ubiquitinated forms of p21, an inhibitor of cyclin-dependent kinases, were detected in vivo, demonstrating that p21 is also a target for degradation by the ubiquitin-dependent proteolytic pathway. However, UV and gamma radiation had no effect on the stability or in vivo ubiquitination of p21, indicating that the radiation effects on p53 are specific.     

NIRF泛素化p53的作用过程中NIRF与p53结合域的测定

NIRF(Np95/ICBP90-like RING finger protein)是2002年发现的一种核蛋白,其功能涉及细胞增殖调节、蛋白多聚泛素化降解、细胞癌变进程控制等领域．已有研究报道,NIRF能与p53相互作用, NIRF本身也是一个高度调节蛋白,在细胞正常的生理状态下发挥泛素化E3连接酶的作用,结合p53并将其降解,但NIRF与p53结合的蛋白结合域目前尚不清楚．本文研究证明,NIRF能与p53结合成复合体参与泛素化蛋白降解途径,并测定出NIRF与p53结合的区域．为了检测NIRF的蛋白结合域,将空载体和NIRF缺失突变体质粒分别转染于HEK293细胞,蛋白表达水平通过Western印迹用两种抗体分别检测. 结果显示,所有的突变体都能在细胞中表达,并且两种抗体检测结果完全一致. 同时,免疫共沉淀技术用于进一步分析实验结果. 由于泛素化蛋白通常伴随蛋白酶体通路介导的降解,免疫共沉淀的蛋白纯化过程中用蛋白酶体抑制剂MG-132以抑制蛋白降解. 本研究结果显示,NIRF 通过PHD区域与p53形成复合体. 该复合体可能参与蛋白分选、蛋白降解、DNA修复以及细胞凋亡等一系列重要的细胞活动,从而形成与细胞增殖相关的新的信号通路,在肿瘤的发生发展中可能发挥某种程度的作用．     

Cancer-derived p53 mutants suppress p53-target gene expression--potential mechanism for gain of function of mutant p53

Tumour-derived p53 mutants are thought to have acquired ‘gain-of-function’ properties that contribute to oncogenicity. We have tested the hypothesis that p53 mutants suppress p53-target gene expression, leading to enhanced cellular growth. Silencing of mutant p53 expression in several human cell lines was found to lead to the upregulation of wild-type p53-target genes such as p21, gadd45, PERP and PTEN. The expression of these genes was also suppressed in H1299-based isogenic cell lines expressing various hot-spot p53 mutants, and silencing of mutant p53, but not TAp73, abrogated the suppression. Consistently, these hot-spot p53 mutants were able to suppress a variety of p53-target gene promoters. Analysis using the proto-type p21 promoter construct indicated that the p53-binding sites are dispensable for mutant p53-mediated suppression. However, treatment with the histone deacetylase inhibitor trichostatin-A resulted in relief of mutant p53-mediated suppression, suggesting that mutant p53 may induce hypo-acetylation of target gene promoters leading to the suppressive effects. Finally, we show that stable down-regulation of mutant p53 expression resulted in reduced cellular colony growth in human cancer cells, which was found to be due to the induction of apoptosis. Together, the results demonstrate another mechanism through which p53 mutants could promote cellular growth.     

Links between mutant p53 and genomic instability

The tumor suppressor p53 has long been known to play a central role in maintaining a stable genome in the face of toxic insults through its role in promoting cell-cycle checkpoints, DNA repair, and apoptosis. However, p53 null cells still retain some function of certain checkpoint and repair processes, reducing the genomic changes that otherwise would occur if these mechanisms were absent. Accumulating evidence suggests that mutant forms of p53 proteins may drastically perturb these residual genome-stabilizing mechanisms through gain-of-function interactions with multiple proteins leading to a higher level of genomic instability than in p53 null cells. This review summarizes the current body of evidence that mutp53 plays a role in promoting various forms of genomic instability and provides an overview of current mechanistic proposals.     

PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1^Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.     

突变型p53与其合成致死基因的研究进展

恶性肿瘤的靶向治疗已经成为现阶段肿瘤治疗的热点。随着人们对癌基因认知的加深,借助合成致死的方法靶向治疗肿瘤已成为针对肿瘤特异性治疗的新策略。p53基因突变在肿瘤的形成和发展过程中具有重要作用。因此,了解肿瘤中与突变型p53基因有合成致死关系的靶基因的作用方式,有助于指导由突变型p53基因诱发肿瘤的个性化治疗。与突变型p53基因具有合成致死关系的靶基因可分为细胞周期调控基因和细胞非周期调控基因,文章综述了这两类靶基因与突变型p53基因如何构成合成致死作用以及此作用的现实意义。     

Cotranslation of activated mutant p53 with wild type drives the wild-type p53 protein into the mutant conformation

Activating mutations of p53 promote tumor progression. The mutant protein adopts a characteristic conformation, which lacks the growth suppressor function of wild-type p53. We show that mutant p53 can drive cotranslated wild-type p53 into the mutant conformation: a similar effect in vivo would block wild-type suppressor function with dominant negative effect. The cotranslational effect of mutant p53 on wild-type conformation depends upon interaction between nascent polypeptides and oligomerization of the full-length proteins. We also show that oligomers of p53 proteins can be induced to change conformation in a cooperative manner. Cell growth stimulation induces a similar conformational change in p53, and our present results indicate that this may involve allosteric regulation.     

Family feud in chemosensitvity: p73 and mutant p53

The importance of p53 in chemotherapy-induced apoptosis of cancer cells is well established. p53 plays a critical role in the cellular response to DNA damage by regulating genes involved in cell cycle progression, apoptosis, and genomic stability. As a result, p53 tumor status is a critical determinant of both responses to anti-cancer treatment and clinical prognosis. Interestingly, tumors expressing certain mutant forms of p53 ("gain of function") are particularly resistant to chemotherapy, even when compared to cells that lack any detectable p53. Until recently, the explanation for this enhanced chemoresistance was not clear. Recent studies have shown that the p53 homologues, p73 and p63, are also activated by chemotherapies, leading to tumor cell death. Now the discovery that mutant p53 interacts with p73, and that regulation of this interaction by a p53 polymorphism can modulate chemosensitvity provide a new model for how p53-family interactions can influence the response of tumors to anti-cancer therapies. Since p53 mutations are found in more than 50% of human tumors, strategies aimed at manipulating these interactions may prove useful in enhancing the chemotherapy response, and perhaps, overcoming chemoresistance.