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1.
Parc: a cytoplasmic anchor for p53   总被引:18,自引:0,他引:18  
Nikolaev AY  Li M  Puskas N  Qin J  Gu W 《Cell》2003,112(1):29-40
Nuclear localization of p53 is essential for its tumor suppressor function. Here, we have identified Parc, a Parkin-like ubiquitin ligase, as a cytoplasmic anchor protein in p53-associated protein complexes. Parc directly interacts and forms a approximately 1 MDa complex with p53 in the cytoplasm of unstressed cells. In the absence of stress, inactivation of Parc induces nuclear localization of endogenous p53 and activates p53-dependent apoptosis. Overexpression of Parc promotes cytoplasmic sequestration of ectopic p53. Furthermore, abnormal cytoplasmic localization of p53 was observed in a number of neuroblastoma cell lines; RNAi-mediated reduction of endogenous Parc significantly sensitizes these neuroblastoma cells in the DNA damage response. These results reveal that Parc is a critical regulator in controlling p53 subcellular localization and subsequent function.  相似文献   

2.
CUL7, a recently identified member of the cullin family of E3 ubiquitin ligases, forms a unique SCF-like complex and is required for mouse embryonic development. To further investigate CUL7 function, we sought to identify CUL7 binding proteins. The p53-associated, parkin-like cytoplasmic protein (PARC), a homolog of CUL7, was identified as a CUL7-interacting protein by mass spectrometry. The heterodimerization of PARC and CUL7, as well as homodimerization of PARC and CUL7, was confirmed in vivo. To determine the biological role of PARC by itself and in conjunction with CUL7, a targeted deletion of Parc was created in the mouse. In contrast to the neonatal lethality of the Cul7 knockout mice, Parc knockout mice were born at the expected Mendelian ratios and exhibited no apparent phenotype. Additionally, Parc deletion did not appear to affect the stability or function of p53. These results suggest that PARC and CUL7 form an endogenous complex and that PARC and CUL7 functions are at least partially nonoverlapping. In addition, although PARC and p53 form a complex, the absence of effect of Parc deletion on p53 stability, localization, and function suggests that p53 binding to PARC may serve to control PARC function.  相似文献   

3.
p193/CUL7 is an E3 ubiquitin ligase initially identified as an SV40 Large T Antigen binding protein. Expression of a dominant interfering variant of mouse p193/CUL7 (designated 1152stop) conferred resistance to MG132- and etoposide-induced apoptosis in U2OS cells. Immune precipitation/Western analyses revealed that endogenous p193/CUL7 formed a complex with Parc (a recently identified parkin-like ubiquitin ligase) and p53. Apoptosis resistance did not result from 1152stop-mediated disruption of the endogenous p193/CUL7 binding partners. Moreover, 1152stop molecule did not directly bind to endogenous p193/CUL7, Parc or p53. These data suggested a role for p193/CUL7 in the regulation of apoptosis independently of p53 and Parc activity.  相似文献   

4.
Huang J  Xu LG  Liu T  Zhai Z  Shu HB 《FEBS letters》2006,580(3):940-947
Recently, it has been shown that really interesting new gene (RING)-in between ring finger (IBR)-RING domain-containing proteins, such as Parkin and Parc, are E3 ubiquitin ligases and are involved in regulation of apoptosis. In this report, we show that p53-inducible RING-finger protein (p53RFP), a p53-inducible E3 ubiquitin ligase, induces p53-dependent but caspase-independent apoptosis. p53RFP contains an N-terminal RING-IBR-RING domain and an uncharacterized, evolutionally highly conserved C-terminal domain. p53RFP interacts with E2 ubiquitin-conjugating enzymes UbcH7 and UbcH8 but not with UbcH5, and this interaction is mediated through the RING-IBR-RING domain of p53RFP. Interestingly, the conserved C-terminal domain of p53RFP is required and sufficient for p53RFP-mediated apoptosis, suggesting p53RFP-mediated apoptosis does not require its E3 ubiquitin ligase activity. Together with a recent report showing that p53RFP is involved in ubiquitination and degradation of p21, a p53 downstream protein promoting growth arrest and antagonizing apoptosis, our findings suggest that p53RFP is involved in switching a cell from p53-mediated growth arrest to apoptosis.  相似文献   

5.
Y Wu  Y Liu  L Lee  Z Miner    M Kulesz-Martin 《The EMBO journal》1994,13(20):4823-4830
A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparison and requires activation. Furthermore, p53as and p53 proteins formed hetero-oligomers when co-translated in vitro, resulting in inactivation of p53as DNA-binding activity. Gel filtration indicated that p53as translated in vitro, like p53, formed tetramers. In support of a functional role of p53as in cells, p53as/p53 hetero-oligomers were coimmunoprecipitated from mouse cells, and both protein forms were detectable in nuclear extracts by electrophoretic mobility shift assays. These results suggest that the biochemical functions of p53 are mediated by interaction between two endogenous protein products of the wild-type p53 gene.  相似文献   

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The steady-state levels of p53 protein and p53 mRNA in transformed and nontransformed cells were examined to elucidate the mechanisms controlling expression of p53. mRNA levels were determined by Northern blot hybridization analysis, employing a p53-specific cDNA clone (M. Oren and A.J. Levine, Proc. Natl. Acad. Sci. U.S.A. 80:56-59, 1983), and protein levels were determined by the Western blotting technique. Analysis of p53 mRNA revealed a single polyadenylated mRNA species migrating at ca. 18S. Levels of p53 mRNA in simian virus 40-transformed cell line (SVT2) and in an homologous nontransformed cell line (3T3) were equivalent, although the steady-state levels of p53 protein were 25- to 100-fold higher in the SVT2 cells than in the 3T3 cells. A study with a non-virus-transformed cell system revealed a different result. Embryonal carcinoma cells (F9) were found to have nearly 20-fold higher levels of p53 mRNA in comparison with differentiated benign progeny cells. In this system the difference in p53 mRNA levels corresponded to the difference in p53 protein levels. Pulse-chase experiments were performed to study the half-life of p53 protein in these four types of cells. The turnover of p53 protein occurred with biphasic kinetics. In addition, it was found that protein synthesis inhibitors placed in the medium during the chase period prevented the turnover of p53 protein in transformed cells, but not in nontransformed (3T3) cells. These results provide evidence that the regulation of p53 expression in cells can occur at the level of p53 mRNA abundancy or p53 protein stability depending upon the experimental system under study, and that a regulated degradation process controls the turnover of p53 protein.  相似文献   

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11.
MdmX protects p53 from Mdm2-mediated degradation   总被引:10,自引:0,他引:10       下载免费PDF全文
The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be examined. We report here that MdmX is capable of associating with p53 yet is unable to facilitate nuclear export or induce p53 degradation. In addition, expression of MdmX can reverse Mdm2-targeted degradation of p53 while maintaining suppression of p53 transactivation. Using a series of MdmX deletions, we have determined that there are two distinct domains of the MdmX protein that can stabilize p53 in the presence of Mdm2. One domain requires MdmX interaction with p53 and results in the retention of both proteins within the nucleus and repression of p53 transactivation. The second domain involves the MdmX ring finger and results in stabilization of p53 and an increase in p53 transactivation. The potential basis for stabilization and increased p53 transactivation by the MdmX ring finger domain is discussed. Based on these observations, we propose that the MdmX protein may function to maintain a nuclear pool of p53 protein in undamaged cells.  相似文献   

12.
The growth suppressor protein p53 and the protein kinase CK2 are both implicated in cellular growth regulation. We previously found that p53 binds to protein kinase CK2 via its regulatory beta-subunit. In the present study, we analyzed the consequences of the binding of p53 to CK2 for the enzymatic activity of CK2 in vitro and in vivo. We found that the carboxy-terminus of p53 which is a potent transforming agent stimulated CK2 activity whereas full length wild-type p53 which is a growth suppressor inhibited the activity of protein kinase CK2. Inhibition of protein kinase CK2 by p53 was dose-dependent and was seen for various CK2 substrates. Experiments with heat-denatured p53 and the conformational mutant p53(R175H) revealed that an intact conformation of p53 seemed to be necessary. Transfection of wild-type and of mutant p53 into p53-/- cells showed that the inhibition of p53 on CK2 activity was also detectable in intact cells and specific for wild-type p53 indicating that the growth suppressing function of p53 might at least be partially achieved by down-regulation of protein kinase CK2.  相似文献   

13.
Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.  相似文献   

14.
Inhibition of stress-inducible kinase pathways by tumorigenic mutant p53   总被引:6,自引:0,他引:6  
More than 50% of human cancers contain p53 gene mutations and as a result accumulate altered forms of the full-length p53 protein. Although certain tumor types expressing mutant p53 protein have a poor prognostic process, the precise role of mutant p53 protein in highly malignant tumor cells is not well defined. Some p53 mutants, but not wild-type p53, are shown here to interact with Daxx, a Fas-binding protein that activates stress-inducible kinase pathways. Interaction of Daxx with p53 is highly dependent upon the specific mutation of p53. Tumorigenic mutants of p53 bind to Daxx and inhibit Daxx-dependent activation of the apoptosis signal-regulating kinase 1 stress-inducible kinases and Jun NH(2)-terminal kinase. Mutant p53 forms complexes with Daxx in cells, and consequently, mutant p53 is able to rescue cells from Daxx-dependent inhibition of proliferation. Thus, the accumulation of mutant p53 in tumor cells may contribute to tumorigenesis by inhibiting stress-inducible kinase pathways.  相似文献   

15.
鼻咽癌是南方常见的恶性肿瘤,NPC与EB病毒关系十分密切。通过免疫亲和层析的方法了解NPC肿瘤标本中p53与病毒或细胞蛋白间潜在的相互作用。这种相互作用可能引起P53在NPC组织中累积。建立单克隆抗体pAb1801,pAb2401免疫亲和层析柱,从NPC转移淋巴结分离p53结合蛋白。  相似文献   

16.
Regulation of Mdm2-Directed Degradation by the C Terminus of p53   总被引:12,自引:6,他引:6       下载免费PDF全文
The stability of the p53 tumor suppressor protein is regulated by interaction with Mdm2, the product of a p53-inducible gene. Mdm2-targeted degradation of p53 depends on the interaction between the two proteins and is mediated by the proteasome. We show here that in addition to the N-terminal Mdm2 binding domain, the C terminus of p53 participates in the ability of p53 to be degraded by Mdm2. In contrast, alterations in the central DNA binding domain of p53, which change the conformation of the p53 protein, do not abrogate the sensitivity of the protein to Mdm2-mediated degradation. The importance of the C-terminal oligomerization domain to Mdm2-targeted degradation of p53 is likely to reflect the importance of oligomerization of the full-length p53 protein for interaction with Mdm2, as previously shown in vitro. Interestingly, the extreme C-terminal region of p53, outside the oligomerization domain, was also shown to be necessary for efficient degradation, and deletion of this region stabilized the protein without abrogating its ability to bind to Mdm2. Mdm2-resistant p53 mutants were not further stabilized following DNA damage, supporting a role for Mdm2 as the principal regulator of p53 stability in cells. The extreme C terminus of the p53 protein has previously been shown to contain several regulatory elements, raising the possibility that either allosteric regulation of p53 by this domain or interaction between this region and a third protein plays a role in determining the sensitivity of p53 to Mdm2-directed degradation.  相似文献   

17.
The p53 tumor suppressor protein plays a key role in maintaining genomic integrity. Enhanced expression of p53 during genotoxic stress is due to both increased protein stability and translational up regulation. Previous reports have shown that p53 mRNA is translated from an alternative initiation codon to produce N-terminal truncated isoform (ΔN-p53) besides full-length p53. We have demonstrated that two internal ribosome entry sites (IRESs) regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. Here, we report that polypyrimidine tract-binding protein (PTB) is a p53 IRES interacting trans-acting factor. PTB protein binds specifically to both the p53 IRESs but with differential affinity. siRNA-mediated knockdown of PTB protein results in reduction of activity of both IRESs and also the levels of both the isoforms. It is well known that DNA-damaging agents such as doxorubicin enhance the expression of p53. Our results indicate that during doxorubicin treatment, PTB protein translocates from nucleus to the cytoplasm, probably to facilitate IRES mediated p53 translation. These observations suggest that the relative cytoplasmic abundance of PTB protein, under DNA-damaging conditions, might contribute to regulating the coordinated expression of the p53 isoforms, owing to the differential affinity of PTB binding to the two p53 IRESs.  相似文献   

18.
Analysis of p53 mutants for transcriptional activity   总被引:21,自引:8,他引:13       下载免费PDF全文
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It has been reported that mammalian cells incubated with excess nitric oxide (NO) accumulate p53 protein but concomitantly this p53 loses its capacity for binding to its DNA consensus sequence. As nitration of tyrosine residues in various proteins has been shown to inhibit their functions, we examined whether NO nitrates tyrosine residues in p53 protein. MCF-7 cells expressing wild-type p53 were incubated with S-nitrosoglutathione for 4 h and cellular extracts were immunoprecipitated with an anti-p53 antibody. Western blot analyses of immunoprecipitates for p53 or for nitrotyrosine revealed low levels of nitrotyrosine in p53 from untreated cells. Incubation with 2 mM S-nitrosoglutathione induced a significant increase in the nitrotyrosine level in p53 protein compared to nontreated cells. These results suggest that excess NO produced in inflamed tissues could nitrate p53 protein, playing a role in carcinogenesis by impairing functions of this tumor-suppressor protein.  相似文献   

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