CoChat
Sign Up
Cited by 99

The DOI matches its Crossref record; 6 of the 6 references it cites were found in a registry or an index.

The E6AP Ubiquitin Ligase Is Required for Transactivation of the hTERT Promoter by the Human Papillomavirus E6 Oncoprotein

Liu, Xuefeng, Yuan, Hang, Fu, Baojin, Disbrow, Gary L., Apolinario, Tania, Tomaić, Vjekoslav, Kelley, Melissa L., Baker, Carl C., Huibregtse, Jon, Schlegel, Richard·2005·Journal of Biological Chemistry
DOI 10.1074/jbc.m410343200
Open this paper in CoChat — free account

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1074/jbc.m410343200

Cites

6 of 6 references verified0 citation errors · 0 not checkable

Statistics

Figures

FIG. 1 Generation and characterization of keratinocyte strains expressing WT and mutant E6 proteins. Primary HFKs were transduced with AU1-tagged WT 16E6A, E6A/8S9A10T, E6A/ 𝚫 9-13, 6bE6A, or empty vector as described. Following antibiotic selection, the cell strains were analyzed for E6 mRNA and protein expression. A , E6 mRNA expression. Total cellular RNA was isolated from the transduced HFK cell strains, and RT-PCR was performed with a set of HPV-16 E6-specific primers described under 'Materials and Methods.' PCR products were analyzed with an Agilent 2100 Bioanalyzer (Agilent Technology). Three species of E6 mRNA were detected in the wild type and mutant E6 transduced cells, with the predominant species being full-length E6. As anticipated, the size of the E6 deletion mutant mRNAs is smaller than the WTorsubstitution mutant. B , E6 protein expression. AU1-tagged E6 proteins were detected in transduced HFKs by immunoprecipitation-Western blot techniques. Total cell lysates were immunoprecipitated with AU1 monoclonal antibody and blotted with the same antibody. All lines expressed similar levels of AU-1-tagged proteins. C , telomerase activity. E6-transduced HFKs were extracted, and a TRAP assay was performed to evaluate endogenous telomerase activity. Typical 'ladder' signals were observed with the WT and substitution mutant E6 proteins. D , quantitative telomerase assay. A quantitative TRAP assay (see 'Materials and Methods') was also performed to quantify the changes observed in C .
FIG. 1 Generation and characterization of keratinocyte strains expressing WT and mutant E6 proteins. Primary HFKs were transduced with AU1-tagged WT 16E6A, E6A/8S9A10T, E6A/ 𝚫 9-13, 6bE6A, or empty vector as described. Following antibiotic selection, the cell strains were analyzed for E6 mRNA and protein expression. A , E6 mRNA expression. Total cellular RNA was isolated from the transduced HFK cell strains, and RT-PCR was performed with a set of HPV-16 E6-specific primers described under 'Materials and Methods.' PCR products were analyzed with an Agilent 2100 Bioanalyzer (Agilent Technology). Three species of E6 mRNA were detected in the wild type and mutant E6 transduced cells, with the predominant species being full-length E6. As anticipated, the size of the E6 deletion mutant mRNAs is smaller than the WTorsubstitution mutant. B , E6 protein expression. AU1-tagged E6 proteins were detected in transduced HFKs by immunoprecipitation-Western blot techniques. Total cell lysates were immunoprecipitated with AU1 monoclonal antibody and blotted with the same antibody. All lines expressed similar levels of AU-1-tagged proteins. C , telomerase activity. E6-transduced HFKs were extracted, and a TRAP assay was performed to evaluate endogenous telomerase activity. Typical 'ladder' signals were observed with the WT and substitution mutant E6 proteins. D , quantitative telomerase assay. A quantitative TRAP assay (see 'Materials and Methods') was also performed to quantify the changes observed in C .CC-BY · From the PDF
Image 2
CC-BY · From the PDF
FIG. 3 E6APbinds to the hTERT promoter in vivo only when E6 is present. E6- or vector-transduced human keratinocytes were analyzed by ChIP assay to determine the presence of E6, E6AP, and Myc proteins on the endogenous hTERT promoter. Following immunoprecipitation ( IP ) of cross-linked cell extracts with the respective antibodies, a 257-kb fragment of the endogenous hTERT promoter was PCR-amplified using sequence-specific primers (see 'Materials and Methods'). Input , PCR signal generated from DNA of E6-transduced HFKs. A positive PCR signal was observed in the E6-transduced cells for Myc, E6, and E6AP. In vector-transduced cells, only the Myc protein was observed on the hTERT promoter.
FIG. 3 E6APbinds to the hTERT promoter in vivo only when E6 is present. E6- or vector-transduced human keratinocytes were analyzed by ChIP assay to determine the presence of E6, E6AP, and Myc proteins on the endogenous hTERT promoter. Following immunoprecipitation ( IP ) of cross-linked cell extracts with the respective antibodies, a 257-kb fragment of the endogenous hTERT promoter was PCR-amplified using sequence-specific primers (see 'Materials and Methods'). Input , PCR signal generated from DNA of E6-transduced HFKs. A positive PCR signal was observed in the E6-transduced cells for Myc, E6, and E6AP. In vector-transduced cells, only the Myc protein was observed on the hTERT promoter.CC-BY · From the PDF
Image 4
CC-BY · From the PDF
FIG. 5 Knockdown of E6AP inhibits transactivation of the hTERT promoter by E6 in human cells. E6AP-specific duplex siRNA (or control siRNA) was transfected into HeLa cells or E6-transduced keratinocytes as described under 'Materials and Methods.' The cells were analyzed by RT-PCR and Western blotting 24 h later. A , RT-PCR detection of E6AP mRNA ( upper panel ) and GAPDH mRNA ( bottom panel ) in transfected HeLa cells. The PCR signal for E6AP mRNA was reduced in HeLa cells treated with siRNA for E6AP, whereas the expression of GAPDHmRNAwasunaffected. The relative level of mRNA expression was quantified by densitometry and is indicated below the respective lanes . B , Western blot detection of E6AP protein ( upper panel ) and 𝛃 -actin ( bottom panel ) in E6AP-siRNA-treated HeLa cells. HeLa cells were treated identically with siRNA for E6AP as described in A but were then analyzed for E6AP protein expression by Western blot. In parallel to the results with mRNA, E6AP protein expression was reduced in the siRNA-treated cells, whereas actin protein expression was not. Again, densitometric quantification of mRNA expression is indicated below the respective gel lanes. C , hTERT promoter activity in siRNA-transfected HeLa cells. HeLa cells were cotransfected with control or E6AP-specific siRNA duplexes and the hTERT promoter reporter construct. Luciferase activity was assayed 24 h later. The E6AP-siRNA-treated HeLa cells showed an 80% inhibition of promoter activity. D , hTERT promoter activity in siRNA-treated E6-transduced HFKs. Luciferase assays were performed identical to those in C except that E6-transduced keratinocytes were used instead of HeLa cells. There was a similar inhibition (70%) of the hTERT promoter in the presence of the E6AP siRNA. E , Inhibition of telomerase activity in HeLa cells treated with E6AP siRNA. HeLa cells were treated as in C but were then assayed by TRAP assay. The cells treated with E6AP siRNA exhibited reduced telomerase activity, and densitometry techniques were used to quantify the inhibition as previously described (88).
FIG. 5 Knockdown of E6AP inhibits transactivation of the hTERT promoter by E6 in human cells. E6AP-specific duplex siRNA (or control siRNA) was transfected into HeLa cells or E6-transduced keratinocytes as described under 'Materials and Methods.' The cells were analyzed by RT-PCR and Western blotting 24 h later. A , RT-PCR detection of E6AP mRNA ( upper panel ) and GAPDH mRNA ( bottom panel ) in transfected HeLa cells. The PCR signal for E6AP mRNA was reduced in HeLa cells treated with siRNA for E6AP, whereas the expression of GAPDHmRNAwasunaffected. The relative level of mRNA expression was quantified by densitometry and is indicated below the respective lanes . B , Western blot detection of E6AP protein ( upper panel ) and 𝛃 -actin ( bottom panel ) in E6AP-siRNA-treated HeLa cells. HeLa cells were treated identically with siRNA for E6AP as described in A but were then analyzed for E6AP protein expression by Western blot. In parallel to the results with mRNA, E6AP protein expression was reduced in the siRNA-treated cells, whereas actin protein expression was not. Again, densitometric quantification of mRNA expression is indicated below the respective gel lanes. C , hTERT promoter activity in siRNA-transfected HeLa cells. HeLa cells were cotransfected with control or E6AP-specific siRNA duplexes and the hTERT promoter reporter construct. Luciferase activity was assayed 24 h later. The E6AP-siRNA-treated HeLa cells showed an 80% inhibition of promoter activity. D , hTERT promoter activity in siRNA-treated E6-transduced HFKs. Luciferase assays were performed identical to those in C except that E6-transduced keratinocytes were used instead of HeLa cells. There was a similar inhibition (70%) of the hTERT promoter in the presence of the E6AP siRNA. E , Inhibition of telomerase activity in HeLa cells treated with E6AP siRNA. HeLa cells were treated as in C but were then assayed by TRAP assay. The cells treated with E6AP siRNA exhibited reduced telomerase activity, and densitometry techniques were used to quantify the inhibition as previously described (88).CC-BY · From the PDF

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Research works printed but not linked: 82

  1. zur Hausen, 2001, J. Natl. Cancer Inst., 93, 252, 10.1093/jnci/93.4.252 · DOI
  2. Munger, 1989, J. Virol., 63, 4417, 10.1128/jvi.63.10.4417-4421.1989 · DOI · Paper page
  3. Munger, 2002, Virus Res., 89, 213, 10.1016/S0168-1702(02)00190-9 · DOI
  4. Scheffner, 1990, Cell, 63, 1129, 10.1016/0092-8674(90)90409-8 · DOI · Paper page
  5. Scheffner, 1993, Cell, 75, 495, 10.1016/0092-8674(93)90384-3 · DOI · Paper page
  6. Werness, 1990, Science, 248, 76, 10.1126/science.2157286 · DOI · Paper page
  7. Rapp, 1998, Biochim. Biophys. Acta, 1378, F1
  8. Chen, 1995, Science, 269, 529, 10.1126/science.7624774 · DOI · Paper page
  9. Tong, 1997, Proc. Natl. Acad. Sci. U. S. A., 94, 4412, 10.1073/pnas.94.9.4412 · DOI
  10. Lee, 1997, Proc. Natl. Acad. Sci. U. S. A., 94, 6670, 10.1073/pnas.94.13.6670 · DOI · Paper page
  11. Kiyono, 1997, Proc. Natl. Acad. Sci. U. S. A., 94, 11612, 10.1073/pnas.94.21.11612 · DOI · Paper page
  12. Kuhne, 1998, J. Biol. Chem., 273, 34302, 10.1074/jbc.273.51.34302 · DOI
  13. Kukimoto, 1998, Biochem. Biophys. Res. Commun., 249, 258, 10.1006/bbrc.1998.9066 · DOI
  14. Ronco, 1998, Genes Dev., 12, 2061, 10.1101/gad.12.13.2061 · DOI · Paper page
  15. Gross-Mesilaty, 1998, Proc. Natl. Acad. Sci. U. S. A., 95, 8058, 10.1073/pnas.95.14.8058 · DOI · Paper page
  16. Veldman, 2003, Proc. Natl. Acad. Sci. U. S. A., 100, 8211, 10.1073/pnas.1435900100 · DOI · Paper page
  17. Thomas, 1998, Oncogene, 17, 2943, 10.1038/sj.onc.1202223 · DOI · Paper page
  18. Thomas, 1999, J. Gen. Virol., 80, 1513, 10.1099/0022-1317-80-6-1513 · DOI · Paper page
  19. Gao, 1999, Mol. Cell. Biol., 19, 733, 10.1128/MCB.19.1.733 · DOI
  20. Gao, 2002, Cancer Res., 62, 3315
  21. Patel, 1999, EMBO J., 18, 5061, 10.1093/emboj/18.18.5061 · DOI · Paper page
  22. Zimmermann, 1999, J. Virol., 73, 6209, 10.1128/JVI.73.8.6209-6219.1999 · DOI · Paper page
  23. Li, 1999, Oncogene, 18, 5727, 10.1038/sj.onc.1202960 · DOI · Paper page
  24. Nakagawa, 2000, Mol. Cell. Biol., 20, 8244, 10.1128/MCB.20.21.8244-8253.2000 · DOI · Paper page
  25. Gao, 2000, J. Biol. Chem., 275, 14824, 10.1074/jbc.275.20.14824 · DOI
  26. Lee, 2000, J. Virol., 74, 9680, 10.1128/JVI.74.20.9680-9693.2000 · DOI
  27. Glaunsinger, 2000, Oncogene, 19, 5270, 10.1038/sj.onc.1203906 · DOI · Paper page
  28. Degenhardt, 2001, J. Virol., 75, 151, 10.1128/JVI.75.1.151-160.2001 · DOI
  29. Kumar, 2002, Mol. Cell. Biol., 22, 5801, 10.1128/MCB.22.16.5801-5812.2002 · DOI · Paper page
  30. Zeng, 2002, J. Biol. Chem., 277, 45611, 10.1074/jbc.M208447200 · DOI
  31. Lu, 2004, J. Biol. Chem., 279, 35664, 10.1074/jbc.M403385200 · DOI · Paper page
  32. Huibregtse, 1993, Mol. Cell. Biol., 13, 4918
  33. Mantovani, 2001, Oncogene, 20, 7874, 10.1038/sj.onc.1204869 · DOI
  34. Chen, 1997, Virology, 236, 30, 10.1006/viro.1997.8725 · DOI
  35. Ned, 1997, J. Virol., 71, 4866, 10.1128/jvi.71.6.4866-4870.1997 · DOI
  36. Gewin, 2001, J. Virol., 75, 7198, 10.1128/JVI.75.15.7198-7201.2001 · DOI · Paper page
  37. Song, 1999, J. Virol., 73, 5887, 10.1128/JVI.73.7.5887-5893.1999 · DOI
  38. Sherman, 1996, J. Virol., 70, 3269, 10.1128/jvi.70.5.3269-3279.1996 · DOI
  39. Sherman, 1997, Virology, 237, 296, 10.1006/viro.1997.8778 · DOI
  40. Huibregtse, 1995, Proc. Natl. Acad. Sci. U. S. A., 92, 2563, 10.1073/pnas.92.7.2563 · DOI
  41. Scheffner, 1995, Nature, 373, 81, 10.1038/373081a0 · DOI
  42. Kishino, 1997, Nat. Genet., 15, 70, 10.1038/ng0197-70 · DOI
  43. Matsuura, 1997, Nat. Genet., 15, 74, 10.1038/ng0197-74 · DOI
  44. Nuber, 1996, J. Biol. Chem., 271, 2795, 10.1074/jbc.271.5.2795 · DOI
  45. Nawaz, 1999, Mol. Cell. Biol., 19, 1182, 10.1128/MCB.19.2.1182 · DOI
  46. Klingelhutz, 1996, Nature, 380, 79, 10.1038/380079a0 · DOI · Paper page
  47. Greider, 1996, Annu. Rev. Biochem., 65, 337, 10.1146/annurev.bi.65.070196.002005 · DOI
  48. Harley, 1990, Nature, 345, 458, 10.1038/345458a0 · DOI · Paper page
  49. Vaziri, 1993, Am. J. Hum. Genet., 52, 661
  50. Nakamura, 1997, Science, 277, 955, 10.1126/science.277.5328.955 · DOI · Paper page
  51. Meyerson, 1997, Cell, 90, 785, 10.1016/S0092-8674(00)80538-3 · DOI · Paper page
  52. Nugent, 1998, Genes Dev., 12, 1073, 10.1101/gad.12.8.1073 · DOI
  53. Avilion, 1996, Cancer Res., 56, 645
  54. Ramakrishnan, 1998, Cancer Res., 58, 622
  55. Takakura, 1998, Cancer Res., 58, 1558
  56. Counter, 1998, Oncogene, 16, 1217, 10.1038/sj.onc.1201882 · DOI
  57. Oh, 2001, J. Virol., 75, 5559, 10.1128/JVI.75.12.5559-5566.2001 · DOI · Paper page
  58. Veldman, 2001, J. Virol., 75, 4467, 10.1128/JVI.75.9.4467-4472.2001 · DOI · Paper page
  59. McMurray, 2003, J. Virol., 77, 9852, 10.1128/JVI.77.18.9852-9861.2003 · DOI · Paper page
  60. Schlegel, 1988, EMBO J., 7, 3181, 10.1002/j.1460-2075.1988.tb03185.x · DOI · Paper page
  61. Stoppler, 1997, J. Biol. Chem., 272, 13332, 10.1074/jbc.272.20.13332 · DOI
  62. Cooper, 2003, Virology, 306, 87, 10.1016/S0042-6822(02)00012-0 · DOI
  63. Jiang, 1998, Neuron, 21, 799, 10.1016/S0896-6273(00)80596-6 · DOI · Paper page
  64. Suprynowicz, 2000, J. Biol. Chem., 275, 5111, 10.1074/jbc.275.7.5111 · DOI
  65. Yuan, 2002, J. Virol., 76, 10685, 10.1128/JVI.76.21.10685-10691.2002 · DOI
  66. Baege, 2002, Am. J. Pathol., 160, 1251, 10.1016/S0002-9440(10)62552-7 · DOI · Paper page
  67. Fu, 2003, Cancer Res., 63, 7815
  68. Takakura, 1999, Cancer Res., 59, 551
  69. Conaway, 2002, Science, 296, 1254, 10.1126/science.1067466 · DOI
  70. Muratani, 2003, Nat. Rev. Mol. Cell. Biol., 4, 192, 10.1038/nrm1049 · DOI · Paper page
  71. Xiong, 2003, Mol. Cell. Biol., 23, 8668, 10.1128/MCB.23.23.8668-8690.2003 · DOI
  72. Amati, 2004, Proc. Natl. Acad. Sci. U. S. A., 101, 8843, 10.1073/pnas.0403046101 · DOI
  73. Kim, 2003, Mol. Cell, 11, 1177, 10.1016/S1097-2765(03)00173-4 · DOI · Paper page
  74. Yada, 2004, EMBO J., 23, 2116, 10.1038/sj.emboj.7600217 · DOI
  75. Welcker, 2004, Proc. Natl. Acad. Sci. U. S. A., 101, 9085, 10.1073/pnas.0402770101 · DOI
  76. von der Lehr, 2003, Mol. Cell, 11, 1189, 10.1016/S1097-2765(03)00193-X · DOI · Paper page
  77. Chen, 1998, J. Biol. Chem., 273, 13537, 10.1074/jbc.273.22.13537 · DOI · Paper page
  78. Be, 2001, Biochemistry, 40, 1293, 10.1021/bi0019592 · DOI
  79. Elston, 1998, J. Gen. Virol., 79, 371, 10.1099/0022-1317-79-2-371 · DOI
  80. Xu, 2000, Oncogene, 19, 5123, 10.1038/sj.onc.1203890 · DOI · Paper page
  81. Li, 1999, Oncogene, 18, 6785, 10.1038/sj.onc.1203061 · DOI
  82. Hamad, 2002, Oncogene, 21, 7121, 10.1038/sj.onc.1205860 · DOI

Cited by

See all 99

Metadata

Authors
Liu, X., Yuan, H., Fu, B., Disbrow, G. L., Apolinario, T., Tomaić, V., Kelley, M. L., Baker, C. C., Huibregtse, J., & Schlegel, R.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2005-03
Volume
280
Issue
11
Pages
10807-10816
ISSN
0021-9258
DOI
10.1074/jbc.m410343200
Cited by
99
References
6
Language
en
Licence

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review