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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The E6 protein of the oncogenic human papillomavirus types 16 and 18 facilitates the rapid degradation of the tumor-suppressor protein p53 via the ubiquitin-dependent proteolytic pathway. The E6 protein binds to a cellular protein of 100 kDa termed E6-AP. The complex of E6 and E6-AP specifically interacts with p53 and induces the ubiquitination of p53 in a reaction which requires the ubiquitin-activating enzyme (E1) and a cellular fraction thought to contain a mammalian ubiquitin-conjugating enzyme (E2). This mammalian E2 activity could be replaced with bacterially expressed UBC8 from Arabidopsis thaliana, which belongs to a subfamily of E2s including yeast UBC4 and UBC5 which are highly conserved at the amino acid level. In this paper we describe the cloning of a human cDNA encoding a human E2 that we have designated UbcH5 and that is related to Arabidopsis UBC8 and the other members of this subfamily. We demonstrate that UbcH5 can function in the E6/E6-AP-induced ubiquitination of p53.
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PMID:Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53. 809 Jul 26

The wild-type p53 gene product plays an important role in the control of cell proliferation, differentiation, and survival. Altered function is frequently associated with changes in p53 stability. We have studied the role of the ubiquitination pathway in the degradation of p53, utilizing a temperature-sensitive mutant, ts20, derived from the mouse cell line BALB/c 3T3. We found that wild-type p53 accumulates markedly because of decreased breakdown when cells are shifted to the restrictive temperature. Introduction of sequences encoding the human ubiquitin-activating enzyme E1 corrects the temperature sensitivity defect in ts20 and prevents accumulation of p53. The data therefore strongly indicate that wild-type p53 is degraded intracellularly by the ubiquitin-mediated proteolytic pathway.
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PMID:Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway. 811 31

Ubiquitin-carrier proteins (E2s, ubiquitin-conjugating enzymes, UBCs) participate in proteolysis by catalyzing transfer of activated ubiquitin to the protein substrates, which are bound to specific ubiquitin-protein ligases (E3s). Yeast UBC2 (RAD6) and the mammalian E2(14kDa) bind to the ligase that recognizes and is involved in the degradation of certain free amino-terminal substrates ("N-end rule" substrates). As such proteins are rather scarce, the role of these E2s in general proteolysis is probably limited. Here, we report the purification and characterization of a novel 18-kDa species of E2 from rabbit reticulocytes. Unlike most members of the E2 family, this enzyme does not adsorb to anion exchange resin in neutral pH, and it is purified from the unadsorbed material (Fraction 1). Thus, it is designated E2-F1. Like all members of the E2 family, it generates a thiol ester with ubiquitin that serves as an intermediate in the conjugation reaction. Sequence analysis revealed a significant homology to many known species of E2s. The enzyme generates multiply ubiquitinated proteins in the presence of an E3 that has not been characterized yet. Most importantly, the ubiquitination via this E2 leads to the degradation of certain non-"N-end rule" substrates such as glyceraldehyde-3-phosphate dehydrogenase (Val at the NH2 terminus) and to the ubiquitination and degradation of certain N-alpha-acetylated proteins such as histone H2A, actin, and alpha-crystallin. The enzyme is also involved in the conjugation and degradation of the tumor suppressor protein p53.
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PMID:Purification and characterization of a novel species of ubiquitin-carrier protein, E2, that is involved in degradation of non-"N-end rule" protein substrates. 814 44

The tumor suppressor protein p53 is extremely unstable in most cell lines. In contrast, many mutant and oncogenic species of the protein are stable. The degradation of p53 in vivo requires metabolic energy; however, the proteolytic system(s) involved have not been identified. The ubiquitin system has been implicated in the degradation of p53 in vitro. The degradation is stimulated significantly by the human papillomavirus (HPV) oncoprotein E6 that associates with p53 and facilitates conjugate formation and subsequent degradation. Complex formation between E6 and p53 is promoted by a cellular protein designated E6-associated protein (E6-AP). Initial dissection of the conjugation process have demonstrated a role for the ubiquitin-activating enzyme, E1, but the ubiquitin-carrier protein (E2, UBC) and the ubiquitin protein ligase, E3, have not been identified. In this study, we report that a novel species of ubiquitin-carrier protein designated E2-F1 (Blumenfeld, N., Gonen, H., Mayer, A., Smith, C., Siegel, N.R., Schwartz, A.L., and Ciechanover, A. (1994) J. Biol. Chem. 269, 9574-9581) is involved in the conjugation and degradation of p53. This E2 enzyme recognizes non-"N-end rule" protein substrates and appears to mediate their conjugation via a novel species of E3. The process of recognition appears to be selective; E2-F1 is not required for the conjugation and degradation of human N-myc. The involvement of E2-F1 in the in vitro process appears to be physiologically meaningful and to reproduce the in vivo process; mutant species of p53 that do not interact with E6 and are stable in vivo are not recognized by the cell free system.
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PMID:Degradation of the tumor suppressor protein p53 by the ubiquitin-mediated proteolytic system requires a novel species of ubiquitin-carrier protein, E2. 814 45

Transformation by the human papillomavirus (HPV) early gene products, E6 and E7, involves their interaction with cellular proteins p53 and Rb. Using glutathione S-transferase (GST) fusion proteins, we found that HPV E6 bound human p53 and that the relative efficiency of binding varied such that the GST-HPV type 16 E6 (16E6) protein bound p53 with highest affinity, followed by GST-31E6, GST-18E6, and GST-11E6. The GST-E6 fusion proteins were sufficient for binding p53 purified from a baculovirus expression system as well as in vitro translation sources, while no association was observed with GST-18E7 or a GST-16E6 mutant bearing a five-amino-acid deletion in E6. When the site-specific DNA binding activity of p53 was examined in the presence of GST-E6 proteins, an inhibition of DNA binding was observed. The degree of inhibition correlated with the relative affinity of different E6 proteins for p53; thus, GST-16E6 was the most potent inhibitor of p53 DNA binding activity, and GST-11E6 was the least effective. Prevention of p53 DNA binding is likely to play a role in the abrogation of the transcriptional activity of p53 by HPV E6 and provides a further mechanism for E6 disruption of p53 growth suppressor function in addition to its role in directing specific degradation of p53 through the ubiquitin-mediated pathway. The variation in inhibition of DNA binding seen with the various E6 proteins may thus contribute to the differences in oncogenic potential seen among the HPV types.
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PMID:Inhibition of p53 DNA binding by human papillomavirus E6 proteins. 820 1

The ubiquitin-dependent proteolytic pathway plays a major role in selective protein degradation. Ubiquitination of proteins requires the sequential action of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and in some cases ubiquitin-protein ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor protein p53. The HPV E6 oncoprotein binds to a cellular protein of 100 kd, termed E6-associated protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated ubiquitination of p53. The ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have ubiquitin-protein ligase activity in the absence of E6.
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PMID:The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. 822 89

The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro.
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PMID:Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. 838 Aug 95

The transcription factor c-Fos is a short-lived cellular protein. The levels of the protein fluctuate significantly and abruptly during changing pathophysiological conditions. Thus, it is clear that degradation of the protein plays an important role in its tightly regulated activity. We examined the involvement of the ubiquitin pathway in c-Fos breakdown. Using a mutant cell line, ts20, that harbors a thermolabile ubiquitin-activating enzyme, E1, we demonstrate that impaired function of the ubiquitin system stabilizes c-Fos in vivo. In vitro, we reconstituted a cell-free system and demonstrated that the protein is multiply ubiquitinated. The adducts serve as essential intermediates for degradation by the 26S proteasome. We show that both conjugation and degradation are significantly stimulated by c-Jun, with which c-Fos forms the active heterodimeric transcriptional activator AP-1. Analysis of the enzymatic cascade involved in the conjugation process reveals that the ubiquitin-carrier protein E2-F1 and its human homolog UbcH5, which target the tumor suppressor p53 for degradation, are also involved in c-Fos recognition. The E2 enzyme acts along with a novel species of ubiquitin-protein ligase, E3. This enzyme is distinct from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. We have purified the novel enzyme approximately 350-fold and demonstrated that it is a homodimer with an apparent molecular mass of approximately 280 kDa. It contains a sulfhydryl group that is essential for its activity, presumably for anchoring activated ubiquitin as an intermediate thioester prior to its transfer to the substrate. Taken together, our in vivo and in vitro studies strongly suggest that c-Fos is degraded in the cell by the ubiquitin-proteasome proteolytic pathway in a process that requires a novel recognition enzyme.
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PMID:Degradation of the proto-oncogene product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: identification and characterization of the conjugating enzymes. 852 78

E6-AP, a 100-kDa cellular protein, was originally identified through its interaction with the E6 protein of the oncogenic human papillomavirus types 16 and 18. The complex of E6-AP and E6 specifically interacts with p53 and mediates ubiquitination of p53 in concert with the E1 ubiquitin-activating enzyme and the E2 ubiquitin-conjugating enzyme UbcH5. Recent results suggest that E6-AP is representative of a family of putative ubiquitin-protein ligases. Members of this family are characterized by a conserved C-terminal region, termed hect domain. In this paper, we describe the isolation of two human E2s, designated as UbcH6 and UbcH7, that in addition to UbcH5 can interact with E6-AP. UbcH6 is a novel member of an evolutionally conserved subfamily of E2s that includes UbcH5 and Saccharomyces cerevisiae UBC4. Although UbcH7 does not appear to be a member of this subfamily, UbcH7 efficiently substitutes for UbcH5 in E6-AP-dependent ubiquitination. Surprisingly, UbcH6 was only weakly active in this particular assay. In addition, UbcH5 but not UbcH6 or UbcH7 efficiently interacts with the heet protein RSP5. These results indicate that E6-AP can interact with at least two species of E2 and that different hect proteins may interact with different E2s.
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PMID:Cloning of human ubiquitin-conjugating enzymes UbcH6 and UbcH7 (E2-F1) and characterization of their interaction with E6-AP and RSP5. 857 57

The activity of the p53 tumor suppressor protein is regulated, at least in part, through the stability of the protein. p53 degradation in normal cells is controlled by ubiquitin-dependent proteolysis, and activation of p53 following DNA damage is associated with an increase in the stability of the protein. The human papillomavirus-encoded E6 protein abrogates p53 function by targeting it for rapid degradation, also through the ubiquitin pathway. Although the p53 protein is ubiquitinated following interaction with E6, we show here that none of the lysine residues within p53 are specifically required for E6-targeted degradation. Mutation of lysine residues within the C-terminus of p53 resulted in resistance to E6-mediated degradation in vitro, although the ability of the two proteins to form a complex was not affected. The same mutant was efficiently targeted for degradation in cells, however, illustrating a lack of correlation between the in vitro and the in vivo assays.
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PMID:Sensitivity of p53 lysine mutants to ubiquitin-directed degradation targeted by human papillomavirus E6. 859 13


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