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• W2015392890 abstract "The E6 protein encoded by the oncogenic human papillomaviruses (HPVs) targets p53 for ubiquitin-dependent proteolysis. E6-mediated p53 degradation requires the 100-kDa cellular protein E6-associated protein (E6AP). E6AP and E6 together provide the E3-ubiquitin protein ligase activity in the transfer of ubiquitin to p53. In vitro studies have shown that E6AP can form a high energy thiolester bond with ubiquitin and, in the presence of E6, transfer ubiquitin to p53. In this study we have addressed the role of E6AP in vivo in the degradation of p53. Overexpression of wild-type E6AP in HeLa cells, which are HPV18-positive and express E6, resulted in a decreased steady state level of p53 and a decrease in the half-life of p53. Mutant forms of E6AP proteins were identified that were catalytically incapable of participating in E6-dependent ubiquitination of p53 and functioned in a dominant-negative manner in that they inhibited the E6-mediated ubiquitination of p53 by the wild-type E6AP in vitro. Transient transfection of one of these dominant negative (dn) mutants resulted in an increase in both the steady state level and half-life of p53 in vivo in HeLa cells. Consistent with this observation, overexpression of the dn E6AP resulted in a marked G1 shift in the cell cycle profile. In contrast, dn E6AP had no effect on p53 levels in U2OS cells, an HPV-negative cell line that contains wild-type p53. These studies provide evidence for the involvement of E6AP in E6-mediated p53 degradation in vivo and also indicate that E6AP may not be involved in the regulation of p53 ubiquitination in the absence of E6. The E6 protein encoded by the oncogenic human papillomaviruses (HPVs) targets p53 for ubiquitin-dependent proteolysis. E6-mediated p53 degradation requires the 100-kDa cellular protein E6-associated protein (E6AP). E6AP and E6 together provide the E3-ubiquitin protein ligase activity in the transfer of ubiquitin to p53. In vitro studies have shown that E6AP can form a high energy thiolester bond with ubiquitin and, in the presence of E6, transfer ubiquitin to p53. In this study we have addressed the role of E6AP in vivo in the degradation of p53. Overexpression of wild-type E6AP in HeLa cells, which are HPV18-positive and express E6, resulted in a decreased steady state level of p53 and a decrease in the half-life of p53. Mutant forms of E6AP proteins were identified that were catalytically incapable of participating in E6-dependent ubiquitination of p53 and functioned in a dominant-negative manner in that they inhibited the E6-mediated ubiquitination of p53 by the wild-type E6AP in vitro. Transient transfection of one of these dominant negative (dn) mutants resulted in an increase in both the steady state level and half-life of p53 in vivo in HeLa cells. Consistent with this observation, overexpression of the dn E6AP resulted in a marked G1 shift in the cell cycle profile. In contrast, dn E6AP had no effect on p53 levels in U2OS cells, an HPV-negative cell line that contains wild-type p53. These studies provide evidence for the involvement of E6AP in E6-mediated p53 degradation in vivo and also indicate that E6AP may not be involved in the regulation of p53 ubiquitination in the absence of E6. There is compelling evidence associating several specific types of the human papillomaviruses (HPVs) 1The abbreviations used are: HPV, human papillomavirus; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin protein ligase; dn, dominant negative; ATPγS, adenosine 5′-O-(thiotriphosphate); FACS, fluorescence-activated cell sorting. with certain human anogenital cancers (1zur Hausen H. Virology. 1991; 184: 9-13Crossref PubMed Scopus (858) Google Scholar). These “high risk” HPV types such as HPV16 and HPV18 encode two oncoproteins, E6 and E7, which target the important cellular growth regulatory proteins p53 and pRb, respectively (2Scheffner M. Huibregtse J.M. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8797-8801Crossref PubMed Scopus (235) Google Scholar). The E6 proteins of the high risk HPV types but not of the “low risk” HPV types are able to enter into a complex with p53 (3Werness B.A. Levine A.J. Howley P.M. Science. 1990; 248: 76-79Crossref PubMed Scopus (2167) Google Scholar) and interfere with the ability of p53 to transcriptionally activate p53 responsive promoters (4Meitz J.A. Unger T. Huibregtse J.M. Howley P.M. EMBO J. 1992; 11: 5013-5020Crossref PubMed Scopus (352) Google Scholar). The steady state levels of p53 are generally quite low in HPV-positive carcinoma cell lines and in cells immortalized by the HPV oncoproteins (5Scheffner M. Munger K. Byrne J.C. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5523-5527Crossref PubMed Scopus (761) Google Scholar). The observation that E6 proteins of high risk HPV types 16 and 18 promote the ubiquitin-dependent degradation of the p53 protein in vitro (6Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text PDF PubMed Scopus (3464) Google Scholar) led to the hypothesis that E6-mediated ubiquitination of p53 accounted for the low steady state levels of p53. In further studies examining the mechanism of E6-mediated degradation, a 100-kDa cellular protein, E6AP (E6AssociatedProtein), was found to mediate the binding of E6 to p53 (7Huibregtse J.M. Scheffner M. Howley P.M. EMBO J. 1991; 10: 4129-4135Crossref PubMed Scopus (695) Google Scholar). E6AP functions as an E3 ubiquitin protein ligase in the ubiquitination of p53 in vitro (8Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar, 9Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell Biol. 1993; 13: 775-784Crossref PubMed Scopus (468) Google Scholar, 10Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell Biol. 1993; 13: 4918-4927Crossref PubMed Scopus (349) Google Scholar). The functional domains of E6AP are summarized in Fig. 1 (10Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell Biol. 1993; 13: 4918-4927Crossref PubMed Scopus (349) Google Scholar). The 100-kDa E6AP protein contains an 18-amino acid region (amino acids 391–408) that is sufficient for binding E6. The E6-dependent binding of p53 involves amino acids 280–781, a domain that encompasses the E6-binding region. Finally, in addition to the sequences necessary for p53 binding, an intact COOH terminus is necessary for E6-mediated p53 ubiquitination (10Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell Biol. 1993; 13: 4918-4927Crossref PubMed Scopus (349) Google Scholar). The COOH-terminal 350 amino acids comprise the hect(homology to E6AP Cterminus) domain, a region of homology shared by several proteins structurally and functionally related to E6AP (11Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar). The carboxyl-terminal segment that is required for the ubiquitination function of E6AP is highly conserved among the hect family of proteins and contains a conserved cysteine residue at position 833 (11Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar, 12Huibregtse J.M. Scheffner M. Howley P.M. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 237-245Crossref PubMed Scopus (47) Google Scholar). In vitro studies have shown that an E6AP mutant with a cysteine to alanine substitution at position 833 is unable to form a thiolester bond with ubiquitin (11Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar, 13Scheffner M. Nuber U. Huibregtse J. Nature. 1995; 373: 81-83Crossref PubMed Scopus (747) Google Scholar). Another E6AP mutant with a 6-amino acid deletion from the COOH terminus retains the ability to form a thiolester bond with ubiquitin but is incapable of transferring the moiety to p53 (11Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar). The ubiquitination of protein substrates is carried out by a series of cellular enzymes known as E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin protein ligase) that result in polyubiquitination of proteins that are then recognized by the 26 S proteosome and degraded. In this pathway, the 76-amino acid ubiquitin moiety is activated by E1 in an ATP-dependent step, through the formation of a high energy thiolester bond between the active site cysteine on E1 and the COOH-terminal glycine of ubiquitin. This activated ubiquitin moiety is then transferred to one of a family of different E2s characterized by a highly conserved catalytic site. E2 enzymes catalyze the formation of an isopeptide bond between the COOH-terminal glycine of ubiquitin and the ε-amino group on a lysine residue of the protein substrate, either directly or in conjunction with an E3. Ubiquitin protein ligases comprise the third and least well characterized group of enzymes involved in the ubiquitination of substrates destined for degradation by the ubiquitin proteolytic pathway (14Hochstrasser M. Curr. Opin. Biol. 1995; 7: 215-223Crossref PubMed Scopus (784) Google Scholar). Previously, although few E3 proteins were well characterized, the accepted model suggested that E3 ubiquitin ligases functioned as “docking proteins” that served to bring a specific E2 and a substrate into close proximity, enabling the E2 to transfer ubiquitin to a lysine residue on the substrate. This was based on studies of the yeast E3, Ubr-1, which was shown to bind to the E2 Rad 6 (Ubc2) as well as to the substrate (15Reiss Y. Heller H. Hershko A. J. Biol. Chem. 1989; 264: 10378-10383Abstract Full Text PDF PubMed Google Scholar, 16Bartel B. Wunning I. Varshavsky A. EMBO J. 1990; 9: 3179-3189Crossref PubMed Scopus (301) Google Scholar, 17Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar). Studies with E6AP provided an alternative model in which an E3 participates directly in the ubiquitination of substrates. E6AP forms a thiolester bond with ubiquitin and in association with E6 is capable of transferring ubiquitin to the p53 target substrate (8Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar). Thus some E3 enzymes participate directly in the “cascade” of ubiquitin thiolester transfers ultimately resulting in the ubiquitination of substrates, leading to their degradation by the 26 S proteasome (11Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar, 13Scheffner M. Nuber U. Huibregtse J. Nature. 1995; 373: 81-83Crossref PubMed Scopus (747) Google Scholar). E6AP is a member of a family of proteins that share COOH-terminal sequence homology. Such proteins have been found in yeast, Drosophila, Caenorhabditis elegans as well as higher vertebrates. This growing family of hect proteins now has several members that have been shown to function as E3 ubiquitin ligase enzymes through their ability to form thiolester bonds with ubiquitin (11Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar, 12Huibregtse J.M. Scheffner M. Howley P.M. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 237-245Crossref PubMed Scopus (47) Google Scholar). E6AP has also recently been implicated in the human genetic disorder “Angelman syndrome” (18Matsukura T. Sutcliffe J.S. Fang P. Galjaard R.J. Jiang Y.H. Benton C.S. Rommens J.M. Beaudet A.L. Nat. Genet. 1997; 15: 1-5Crossref PubMed Scopus (4) Google Scholar, 19Kishino T. Lalande M. Wagstaff J. Nat. Genet. 1997; 15: 70-73Crossref PubMed Scopus (1023) Google Scholar), suggesting a role for E6AP in brain development. The normal substrate(s) and regulation of E6AP in the absence of HPV E6 are currently under study in our laboratory (20Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). For other hect E3 enzymes, some cellular substrates have now been defined. Both GAP1 and Fur4 permeases were identified as substrates of an essential yeast E3 hectprotein, Rsp5/Npi1 (21Hein C. Springael J. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (298) Google Scholar). Rpb1, the largest subunit of RNA polymerase II has also been identified as a substrate of Rsp5/Npi1 (22Huibregtse J.M. Yang J.C. Beaudenon S.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 8: 3656-3661Crossref Scopus (182) Google Scholar). Pub1, the Schizosaccharomyces pombe homologue of Rsp5/Npi1 has been demonstrated to target cdc25 for degradation (23Nefsky B. Beach D. EMBO J. 1996; 15: 1301-1312Crossref PubMed Scopus (97) Google Scholar). Further studies on E3 enzymes for which cellular substrates are defined may elucidate how the specificity of protein substrates is determined and how degradation of target substrates is regulated by the cell. The role of E6AP in E6-mediated p53 degradation in vitro has been well characterized. The current studies were undertaken to investigate the role of E6AP in p53 degradation in vivo in HPV-positive E6 expressing cells, as well as in HPV-negative cells. A previously described in vitro p53 ubiquitination assay (8Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar) was used to identify catalytically impaired mutant forms of E6AP, which interfered with the ability of the wild-type E6AP protein to target p53 for ubiquitination in the presence of E6. These dominant negative (dn) mutants retained the ability to enter into ternary complexes with E6 and p53 but were incapable either of forming a thiolester bond with ubiquitin (cysteine 833 to alanine substitution) or of transferring the ubiquitin moiety to p53 (a COOH-terminal 6-amino acid deletion) (11Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar). The C833A mutant form of E6AP could be stably expressed in mammalian cells, permitting us to test the role of E6AP in mediating p53 degradation in vivo in HPV-positive cervical cancer cells as well as in human cells not expressing E6. The p53 plasmid used for in vitrotranscription and translation has been previously described (3Werness B.A. Levine A.J. Howley P.M. Science. 1990; 248: 76-79Crossref PubMed Scopus (2167) Google Scholar), as have E6AP wild-type and mutant in vitro expression plasmids (10Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell Biol. 1993; 13: 4918-4927Crossref PubMed Scopus (349) Google Scholar, 11Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar). Baculovirus pVL1393 (Pharmingen) constructs containing either wild-type or mutant E6AP cDNAs for expression in SF9 insect cells have also previously been described (10Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell Biol. 1993; 13: 4918-4927Crossref PubMed Scopus (349) Google Scholar). Mammalian E6AP expression vectors were constructed by subcloning the above cDNAs into pCMV4 (24Andersson S. Davis D.L. Dahlback H. Jornrak H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar) utilizing the BglII and HindIII sites in the polylinker. HPV16 E6 expression constructs were kindly made available by Dr. Karl Munger, with HPV16 E6 and E7 genes directed from the human CMV IE promoter. Plasmid DNA for mammalian cell transfection was propagated in DH5α and purified by standard cesium chloride gradient methods (25Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). In vitroassays were performed as described previously (8Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar) using bacterially expressed wheat E1 and Arabidopsis thaliana UBC8 E2 (26Hatfield P.M. Vierstra R.D. J. Biol. Chem. 1992; 267: 14799-14803Abstract Full Text PDF PubMed Google Scholar,27Sullivan M.L. Vierstra R.D. J. Biol. Chem. 1991; 266: 23878-23885Abstract Full Text PDF PubMed Google Scholar). p53 was translated in TNT wheat germ extract as per the manufacturer's instructions (Promega) in the presence of radiolabeled methionine. Wild-type E6AP was translated in TNT wheat germ extract (Promega) with unlabeled amino acid components. Mutant E6AP and HPV16 E6 proteins were expressed and partially purified from SF9 insect cells as described previously (10Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell Biol. 1993; 13: 4918-4927Crossref PubMed Scopus (349) Google Scholar) using DEAE-Sephacel (Pharmacia Biotech Inc.) or Bio-Rad S, respectively. Ubiquitination reactions containing 5 μl of [35S]methionine-labeled p53 (from 100 μl of total wheat germ extract translation reaction) were incubated for 3 h at 25 °C along with 1 μl of E1, 1 μl of E2, 2 mmATPγS, 1 mm MgCl2, 6 μg of ubiquitin (Sigma), 5 μl of HPV16 E6 (a 1:100 dilution of partially purified baculovirus expressed protein) and 5 μl of E6AP (from a 100-μl total translation in wheat germ extract). Competition assays for the identification of dominant mutant forms of E6AP utilized Sf9 insect cell-derived mutant E6AP proteins, with wild-type E6AP protein or wild-type baculovirus SF9 cell fractions of equal volume as negative controls. These were added in 5-μl volumes containing 1 μl of baculovirus-derived protein (or corresponding wild-type baculovirus fraction), and 5 μl of a 1:10 dilution, a 1:100 dilution, a 1:1000 dilution, or a 1:10,000 dilution were added to the reaction described above. DEAE-purified baculovirus-derived protein preparation concentrations were quantitated against bovine serum albumin standards. 1 μl of baculovirus-derived wild-type or mutant E6AP contained approximately 150 ng of protein, with subsequent dilutions corresponding to 15, 1.5, 0.15, and 0.015 ng of protein. T25N50 buffer (25 mm Tris, pH 7.4, 50 mm NaCl) was used to bring the reaction to a 50-μl volume. Reactions were stopped by the addition of 50 mmTris-HCl, pH 7.6, 100 mm dithiothreitol, 2% SDS, 10% glycerol. Samples were boiled at 97 °C for 5–10 min and were then electrophoresed on 10% SDS-polyacrylamide gels. Radioactively labeled p53 protein as well as polyubiquitinated p53 protein forms migrating as higher molecular weight species were detected by autoradiography. HeLa cells were grown in Dulbecco's modified medium containing 10% fetal bovine serum in 5% CO2 at 37 °C. 60-mm diameter tissue culture plates were seeded with 0.5 × 106 cells 1 day prior to transfection. The following day, cells were fed with prewarmed growth medium several hours prior to transfection and were transiently transfected with 8–16 μg of circular plasmid DNA using standard CaCl2 methods (25Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). DNA precipitates were applied to the cell monolayer and were incubated at 37 °C overnight. 12–14 h later, cells were rinsed with prewarmed growth medium to remove precipitate, fed, and incubated at 37 °C in 5% CO2 for approximately 48 h. To determine transfection efficiency, a control plate for each experiment was transfected with a β-galactosidase expression plasmid and stained with 5-bromo-4-chloro-3-indoyl β-d-galactoside at 24 h post transfection following standard procedures. At 48 h, cell monolayers were rinsed with phosphate-buffered saline, scraped and pelleted in phosphate-buffered saline at 4 °C, and lysed in 0.5 ml of lysis buffer (50 mm Tris, pH 8.0, 5 mm NaCl, 0.5% Nonidet P-40, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin/leupeptin, 1 mm dithiothreitol). Lysates were then incubated on ice for 15–30 min and centrifuged in an Eppendorf microcentrifuge at maximum speed for 10 min. Bradford assays were performed as per the manufacturer's directions (Bio-Rad) to determine protein concentration, and 100 μg of clarified cell lysate was diluted in 2× loading buffer, boiled 5–7 min, and resolved by SDS-polyacrylamide gel electrophoresis. 10% polyacrylamide gels were transferred to Immobilon-p membranes (Millipore) following standard methods at 40 V for 2.5 h at room temperature and were blocked using 5% nonfat powdered milk in TNET buffer (200 mm Tris 7.5, 1 m NaCl, 50 mm EDTA, 2% Tween 20). Blots were rinsed 3 × 10 min in TNET, incubated with anti-p53 antibody 6 (Oncogene Science) at a 1:1000 dilution in TNET for 90 min of shaking, rinsed as above, and incubated for 1 h in horseradish peroxidase-conjugated anti-mouse secondary antibody for 90 min of shaking. Following a final rinse, p53 was detected by enhanced chemiluminescence reagent (NEN Life Science Products). p53 steady state and half-life were quantified by densitometric measurement of Western blot intensity using NIH Image 1.6 graphic software. For p53 half-life experiments identically transfected HeLa cell monolayers were treated with 1 μl of a 20 mg/ml cycloheximide stock solution (in 100% ETOH stored at −20 °C) per ml of medium at approximately 48 h post transfection. Zero time points were harvested immediately as described above, and subsequent time points were incubated in medium containing cycloheximide at 37 °C for 15, 30, or 70 min as indicated. Cells were then processed as described above for lysates, Western blotting, and quantitation by densitometric measurement. p21 immunoprecipitation/Westerns were carried out using CaCl2 transfected HeLa cells at 48 h post transfection. 60-mm plates were harvested into 500 μl of RIPA buffer (20 mm Tris 7.5, 2 mm EDTA, 150 mm NaCl, 0.25% SDS, 1% Nonidet P-40, 1% deoxycholic acid) on ice. Lysates were then precleared using 20 μl of protein A and protein G-coupled agarose beads for 45 min. 0.5 μl of “15431E” anti-rabbit p21 antibody (Pharmingen) was added to clarified lysates, which were then incubated overnight at 4 °C on a rotater. Protein A- and G-coupled agarose beads were added, and lysates continued rotating for 2 h at 4 °C. Beads were pelleted at maximal speed on the Eppendorf centrifuge and were rinsed four times with RIPA buffer. After the final spin the pellet was resuspended in loading buffer and boiled for 5–7 min prior to loading on 12% polyacrylamide protein gels. These were run and transferred as described previously and stained as per Western blotting procedure using anti-p21 monoclonal antibody 15091A (Pharmingen). Cell Cycle analysis was carried out following the methods of Van den Heuvel (28Zhu L. Van den Heuvel S. Helin K. Fattaey A. Ewen M. Livingston D. Dyson N. Harlow E. Genes Dev. 1993; 7: 1111-1125Crossref PubMed Scopus (470) Google Scholar) using a CD20 expression plasmid and fluorescein isothiocyanate-conjugated anti-CD20 antibody in conjunction with propidium iodide staining. Cell cycle analysis was performed on FACS FACScan at the Core Flow Cytometry Facility at the Dana Farber Cancer Institute, Boston, Massachusetts. U2OS cells co-transfected with E6AP constructs and a CD20 expression plasmid (or green lantern fluorescent protein construct) were harvested live at 48 h post-transfection with 0.1% EDTA, rinsed with Dulbecco's modified Eagle's medium 10% fetal bovine serum, and stained with fluorescein isothiocyanate-conjugated anti-CD20 antibody on ice following standard procedures. Cells were sorted on either a Becton Dickinson FACS Vantage or a Coulter EPICS 750 Series Sorter at the Core Flow Cytometry Facility at the Dana Farber Cancer Institute, Boston, MA. Dominant negative forms of E6AP were identified in an in vitro p53 ubiquitination assay using bacterially derived E1 and E2 (AtUBC8) proteins and ubiquitin as described previously (8Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar, 26Hatfield P.M. Vierstra R.D. J. Biol. Chem. 1992; 267: 14799-14803Abstract Full Text PDF PubMed Google Scholar, 27Sullivan M.L. Vierstra R.D. J. Biol. Chem. 1991; 266: 23878-23885Abstract Full Text PDF PubMed Google Scholar). The p53 protein utilized was translated in vitro in wheat germ extract, and E6 or competing E6AP mutants were partially purified from SF9 insect cell fractions, which contain no detectable endogenous E6AP activity. In this reconstituted p53 degradation assay, p53 is efficiently ubiquitinated and degraded in the presence of ATP (8Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar). However, in the presence of the nonhydrolyzable ATP analogue, ATPγS, p53 can be ubiquitinated but not subsequently degraded by the proteasome. Under these conditions, polyubiquitinated p53 is readily observed as a higher molecular weight, slowly migrating species by SDS-polyacrylamide gel electrophoresis (6Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text PDF PubMed Scopus (3464) Google Scholar, 8Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar). Two mutant forms of E6AP were tested for their ability to compete with the wild-type protein and prevent the E6-dependent ubiquitination of p53 in vitro (Fig. 1). E6AP C833A contains a cysteine to alanine substitution at the active site cysteine, rendering it incapable of forming a thiolester with ubiquitin. The last six amino acids of E6AP Δ6 are deleted, and E6AP Δ6 can form a thiolester bond with ubiquitin. However, it is unable to transfer the ubiquitin moiety to the p53 substrate in vitro (11Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar). In agreement with previously published data from our laboratory, the in vitroubiquitination of p53 is dependent upon the presence of E6 and E6AP (Fig. 2 A). The ubiquitination of p53 by wild-type E6AP synthesized in wheat germ could successfully be competed by either the C833A or Δ6 mutant forms of E6AP. In this experiment, a titration ranging from 0.015 to 150 ng of the E6AP wild-type or mutant protein was used (Fig. 2 B). Therefore each of these mutants functioned in a dn manner in vitro. Furthermore, the competition observed with these dn E6AP mutants was specific and not due to the depletion of the biochemical components required for protein ubiquitination, because p53 ubiquitination was unaffected by the addition of identical quantities of either wild-type E6AP or baculovirus-infected Sf9 fraction controls. Because the competition of wild-type E6AP by the two dn E6AP mutants observed in vitro occurred using protein quantities that might be attainable by transient expression in mammalian cells, we next tested the effect of the dn E6AP mutants upon E6-dependent p53 degradation in vivo. Genes encoding the wild-type and dn E6AP mutant proteins were subcloned into pCMV4 mammalian expression vectors. Transfection of plasmid DNA was performed using standard calcium phosphate methods that had been optimized for HeLa cells, permitting a transfection efficiency of approximately 85%. At 48 h, the cells were analyzed for E6AP and p53 levels by Western blot analysis. As shown in Fig. 3, the levels of expression achieved for transfected wild-type E6AP and for E6AP C833A were high relative to the endogenous protein levels in the vector controls. These high protein levels also resulted in the appearance of a band in the transfected cell lysates that is absent in vector controls and migrates below the endogenous protein on Western blots (Fig. 3). This may represent a breakdown product of the full-length E6AP. Protein expression was also confirmed with hemaglutinnin-tagged versions of the constructs in Western blots using anti-hemaglutinnin antibody (data not shown). E6AP Δ6, however, was not expressed at appreciable levels or was unstable in HeLa cells (data not shown) and was therefore not utilized in subsequent experiments. The transient overexpression of wild-type E6AP in HeLa cells resulted in a decrease in steady state p53 levels, whereas expression of the C833A mutant exhibited an elevated level of p53 relative to cells transfected with vector alone (Fig. 3, A and B). To determine whether higher levels of p53 corresponded to an increase in the ability of p53 to transactivate a p53-responsive promoter, we examined the levels of p21, a protein known to be induced by the p53 protein at the transcriptional level. The increased level of p53 in HeLa cells expressing E6AP C833A resulted in an induction of p21 detected by immunoprecipitation/Western analysis (Fig. 3 C). We also noted that expression of wild-type E6AP, which led to a decrease in p53 level, also resulted in a lower level of p21 protein expression. These data indicate that E6AP is involved in the E6-dependent regulation of p53 protein levels. To determine whether the altered levels of p53 in HeLa cells induced by overexpression of wild-type or dn E6AP were due to changes in protein stability, the half-life of p53 was determined in cells transiently transfected with the above constructs. Because cycloheximide inhibits de novo protein synthesis, the half-life of p53 can be determined by Western blot analysis in cells treated with the drug (29Maki C.G. Huibregtse J.M. Howley P.M. Cancer Res. 1996; 56: 2649-2654Pu" @default.
• W2015392890 created "2016-06-24" @default.
• W2015392890 creator A5030554897 @default.
• W2015392890 creator A5049150854 @default.
• W2015392890 creator A5089706444 @default.
• W2015392890 date "1998-03-01" @default.
• W2015392890 modified "2023-10-06" @default.
• W2015392890 title "The Role of E6AP in the Regulation of p53 Protein Levels in Human Papillomavirus (HPV)-positive and HPV-negative Cells" @default.
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• W2015392890 cites W2008044453 @default.
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• W2015392890 cites W2039382131 @default.
• W2015392890 cites W2046085471 @default.
• W2015392890 cites W2047050750 @default.
• W2015392890 cites W2056530510 @default.
• W2015392890 cites W2060786724 @default.
• W2015392890 cites W2064850779 @default.
• W2015392890 cites W2072280454 @default.
• W2015392890 cites W2075739751 @default.
• W2015392890 cites W2082979850 @default.
• W2015392890 cites W2098598252 @default.
• W2015392890 cites W2106038494 @default.
• W2015392890 cites W2132024812 @default.
• W2015392890 cites W2150525034 @default.
• W2015392890 cites W2164186781 @default.
• W2015392890 cites W2170939558 @default.
• W2015392890 cites W2312929829 @default.
• W2015392890 cites W2326229326 @default.
• W2015392890 cites W276042688 @default.
• W2015392890 cites W4234681287 @default.
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