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High-Risk Human Papillomavirus E7 Proteins Target PTPN14 for Degradation

White, Elizabeth A., Münger, Karl, Howley, Peter M.·2016·mBio
DOI 10.1128/mbio.01530-16PMC PMC5030362
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Abstract

The major transformation activity of the high-risk human papillomaviruses (HPV) is associated with the E7 oncoprotein. The interaction of HPV E7 with retinoblastoma family proteins is important for several E7 activities; however, this interaction does not fully account for the high-risk E7-specific cellular immortalization and transformation activities. We have determined that the cellular non-receptor protein tyrosine phosphatase PTPN14 interacts with HPV E7 from many genus alpha and beta HPV types. We find that high-risk genus alpha HPV E7, but not low-risk genus alpha or beta HPV E7, is necessary and sufficient to reduce the steady-state level of PTPN14 in cells. High-risk E7 proteins target PTPN14 for proteasome-mediated degradation, which requires the ubiquitin ligase UBR4, and PTPN14 is degraded by the proteasome in HPV-positive cervical cancer cell lines. Residues in the C terminus of E7 interact with the C-terminal phosphatase domain of PTPN14, and interference with the E7-PTPN14 interaction restores PTPN14 levels in cells. Finally, PTPN14 degradation correlates with the retinoblastoma-independent transforming activity of high-risk HPV E7. IMPORTANCE High-risk human papillomaviruses (HPV) are the cause of cervical cancer, some other anogenital cancers, and a growing fraction of oropharyngeal carcinomas. The high-risk HPV E6 and E7 oncoproteins enable these viruses to cause cancer, and the mechanistic basis of their carcinogenic activity has been the subject of intense study. The high-risk E7 oncoprotein is especially important in the immortalization and transformation of human cells, which makes it a central component of HPV-associated cancer development. E7 oncoproteins interact with retinoblastoma family proteins, but for several decades, it has been recognized that high-risk HPV E7 oncoproteins have additional cancer-associated activities. We have determined that high-risk E7 proteins target the proteolysis of the cellular protein tyrosine phosphatase PTPN14 and find that this activity is correlated with the retinoblastoma-independent transforming activity of E7.

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Figures

FIG 1 HPV E7 proteins bind to PTPN14, and high-risk HPV E7 proteins reduce PTPN14 levels. (A) Lysates from N/Tert-1 cells expressing E7-Flag-HA were used in immunoprecipitations with HA antibody. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to HA, PTPN14, and actin. (Top panels) Protein levels in input lysates. Band intensity was quantified with ImageJ, and the numbers indicate the intensity of the PTPN14 band in each lysate lane relative to the actin intensity, normalized to the amount of PTPN14 in the empty vector cells. (Bottom panels) PTPN14 recovered after immunoprecipitation with anti-HA antibody. HA and PTPN14 were detected on film, and actin was detected using the LI-COR imaging system. Band intensity was quantified with ImageJ, and numbers indicate the amount of PTPN14 recovered in the immunoprecipitation relative to the amount of PTPN14 in the lysates, normalized to the amount recovered in the HPV17a E7 IP. (B) N/Tert-1 cells were transduced with a retrovirus encoding HPV18 E7 or with an empty vector control retrovirus or mock transduced and harvested at indicated time points. Steady-state levels of PTPN14, HPV18-Flag-HA, and actin were measured by Western blotting.
FIG 1 HPV E7 proteins bind to PTPN14, and high-risk HPV E7 proteins reduce PTPN14 levels. (A) Lysates from N/Tert-1 cells expressing E7-Flag-HA were used in immunoprecipitations with HA antibody. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to HA, PTPN14, and actin. (Top panels) Protein levels in input lysates. Band intensity was quantified with ImageJ, and the numbers indicate the intensity of the PTPN14 band in each lysate lane relative to the actin intensity, normalized to the amount of PTPN14 in the empty vector cells. (Bottom panels) PTPN14 recovered after immunoprecipitation with anti-HA antibody. HA and PTPN14 were detected on film, and actin was detected using the LI-COR imaging system. Band intensity was quantified with ImageJ, and numbers indicate the amount of PTPN14 recovered in the immunoprecipitation relative to the amount of PTPN14 in the lysates, normalized to the amount recovered in the HPV17a E7 IP. (B) N/Tert-1 cells were transduced with a retrovirus encoding HPV18 E7 or with an empty vector control retrovirus or mock transduced and harvested at indicated time points. Steady-state levels of PTPN14, HPV18-Flag-HA, and actin were measured by Western blotting.CC BY · PubMed Central
FIG 2 PTPN14 protein levels in cervical cancer cells depend on HPV oncoprotein expression. Cells of the HPV18-positive HeLa, HPV16-positive Caski, and HPV-negative C33A cervical cancer cell lines were transfected with siRNAs targeting the HPV16 E6/E7 and/or HPV18 E6/E7 transcripts at a final concentration of 40 nM. Cells were harvested 72 h posttransfection, and lysates were used for Western blotting with antibodies to PTPN14 and actin.
FIG 2 PTPN14 protein levels in cervical cancer cells depend on HPV oncoprotein expression. Cells of the HPV18-positive HeLa, HPV16-positive Caski, and HPV-negative C33A cervical cancer cell lines were transfected with siRNAs targeting the HPV16 E6/E7 and/or HPV18 E6/E7 transcripts at a final concentration of 40 nM. Cells were harvested 72 h posttransfection, and lysates were used for Western blotting with antibodies to PTPN14 and actin.CC BY · PubMed Central
FIG 3 Identification of protein binding sites on HPV16 E7 and on PTPN14. (A) Schematic of HPV16 and PTPN14 domain organization and mutants used in the study (39). (B) N/Tert-1 cells expressing wild-type (wt) HPV16 E7-Flag-HA, one of several mutants of HPV16 (E7), or the empty vector were subjected to immunoprecipitation with HA antibody. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to HA, PTPN14, UBR4, RB1, and actin. (Left panels) Protein levels in input lysates. (Right panels) Proteins recovered after immunoprecipitation with anti-HA antibody. (C) N/Tert-1 cells expressing wild-type HPV16 E7-Flag-HA were transduced with retroviruses expressing V5-tagged PTPN14 or one of several deletion mutant forms of PTPN14 and were harvested 48 h posttransduction. Cell lysates were subjected to immunoprecipitation with HA antibody. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to HA, V5, and actin. (Top panels) Protein levels in input lysates. (Bottom panel) V5-tagged PTPN14 recovered after immunoprecipitation with anti-HA antibody.
FIG 3 Identification of protein binding sites on HPV16 E7 and on PTPN14. (A) Schematic of HPV16 and PTPN14 domain organization and mutants used in the study (39). (B) N/Tert-1 cells expressing wild-type (wt) HPV16 E7-Flag-HA, one of several mutants of HPV16 (E7), or the empty vector were subjected to immunoprecipitation with HA antibody. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to HA, PTPN14, UBR4, RB1, and actin. (Left panels) Protein levels in input lysates. (Right panels) Proteins recovered after immunoprecipitation with anti-HA antibody. (C) N/Tert-1 cells expressing wild-type HPV16 E7-Flag-HA were transduced with retroviruses expressing V5-tagged PTPN14 or one of several deletion mutant forms of PTPN14 and were harvested 48 h posttransduction. Cell lysates were subjected to immunoprecipitation with HA antibody. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to HA, V5, and actin. (Top panels) Protein levels in input lysates. (Bottom panel) V5-tagged PTPN14 recovered after immunoprecipitation with anti-HA antibody.CC BY · PubMed Central
FIG 4 Overexpression of the PTPN14 PTP domain can block the interaction of E7 with full-length PTPN14 and restore PTPN14 levels. (A) N/Tert-1 cells expressing wild-type (wt) HPV16 E7-Flag-HA or the empty vector were mock transduced, transduced with a retrovirus packaged with the empty vector, or transduced with retroviruses expressing wild-type V5-tagged PTPN14, the PTPN14 L2 domain, or the PTPN14 PTP domain and were harvested 48 h posttransduction. Steady-state levels of full-length PTPN14, V5-tagged PTPN14 fragments, HA-tagged E7, and actin were measured by Western blotting. (B) N/Tert-1 cells expressing wild-type HPV18 E7-Flag-HA were transduced with retroviruses expressing wild-type V5-tagged PTPN14, the PTPN14 L2 domain, the PTPN14 PTP domain, or the empty vector and were harvested 48 h posttransduction. Cell lysates were subjected to immunoprecipitation with HA antibody. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to PTPN14, HA, V5, and actin. (Left panels) Protein levels in input lysates. (Right panels) Proteins recovered after immunoprecipitation with anti-HA antibody. HC, antibody heavy chain.
FIG 4 Overexpression of the PTPN14 PTP domain can block the interaction of E7 with full-length PTPN14 and restore PTPN14 levels. (A) N/Tert-1 cells expressing wild-type (wt) HPV16 E7-Flag-HA or the empty vector were mock transduced, transduced with a retrovirus packaged with the empty vector, or transduced with retroviruses expressing wild-type V5-tagged PTPN14, the PTPN14 L2 domain, or the PTPN14 PTP domain and were harvested 48 h posttransduction. Steady-state levels of full-length PTPN14, V5-tagged PTPN14 fragments, HA-tagged E7, and actin were measured by Western blotting. (B) N/Tert-1 cells expressing wild-type HPV18 E7-Flag-HA were transduced with retroviruses expressing wild-type V5-tagged PTPN14, the PTPN14 L2 domain, the PTPN14 PTP domain, or the empty vector and were harvested 48 h posttransduction. Cell lysates were subjected to immunoprecipitation with HA antibody. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to PTPN14, HA, V5, and actin. (Left panels) Protein levels in input lysates. (Right panels) Proteins recovered after immunoprecipitation with anti-HA antibody. HC, antibody heavy chain.CC BY · PubMed Central
FIG 5 E7 interacts with UBR4 and PTPN14 in a complex distinct from the E7-RB1 complex. (A) Lysates from N/Tert-1 cells expressing wild-type (wt) HPV16 (E7), HPV16 E7 H2A, or HPV16 E7 L82R L83R were fractionated on a Superose 6 column using a GE Akta Pure high-performance liquid chromatography (HPLC) column. Five hundred-microliter fractions were collected, and alternate fractions were used for anti-HA immunoprecipitation. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to PTPN14, HA, UBR4, and RB1. Unfractionated lysate (input) is included on each gel as a control. (B) Schematic of E7/PTPN14/UBR4 complex and approximate interaction sites.
FIG 5 E7 interacts with UBR4 and PTPN14 in a complex distinct from the E7-RB1 complex. (A) Lysates from N/Tert-1 cells expressing wild-type (wt) HPV16 (E7), HPV16 E7 H2A, or HPV16 E7 L82R L83R were fractionated on a Superose 6 column using a GE Akta Pure high-performance liquid chromatography (HPLC) column. Five hundred-microliter fractions were collected, and alternate fractions were used for anti-HA immunoprecipitation. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to PTPN14, HA, UBR4, and RB1. Unfractionated lysate (input) is included on each gel as a control. (B) Schematic of E7/PTPN14/UBR4 complex and approximate interaction sites.CC BY · PubMed Central
FIG 6 PTPN14 is degraded by the proteasome in HPV-positive cervical cancer cells. HPV18-positive HeLa cells and HPV-negative C33A cervical cancer cells were treated for 8 h with 30 µM MG132, 10 mM NH4Cl, or DMSO as a control. Lysates were separated by SDS-PAGE and Western blotted using antibodies to PTPN14, p53, and actin.
FIG 6 PTPN14 is degraded by the proteasome in HPV-positive cervical cancer cells. HPV18-positive HeLa cells and HPV-negative C33A cervical cancer cells were treated for 8 h with 30 µM MG132, 10 mM NH4Cl, or DMSO as a control. Lysates were separated by SDS-PAGE and Western blotted using antibodies to PTPN14, p53, and actin.CC BY · PubMed Central
FIG 7 HPV18 E7-mediated PTPN14 degradation requires the E3 ubiquitin ligase UBR4. (A) N/Tert cells were transfected with siRNAs targeting cullin 3, UBR4, or HPV18 E7 or with a nontargeting control (si Control). Twenty-four hours posttransfection, cells were transduced with a retrovirus expressing HPV18 E7 or with the empty vector control retrovirus. Forty-eight hours post-retrovirus transduction, cells were treated with 40 µg/ml cycloheximide (CHX) and harvested at indicated time points post-cycloheximide treatment. Equal amounts of protein lysates were separated by SDS-PAGE and Western blotted using antibodies to PTPN14, cullin 3, HA, and actin. (B) N/Tert cells were transfected with siRNA targeting UBR4 or with a nontargeting control (si Control). Twenty-four hours posttransfection, cells were transduced with an empty vector control retrovirus. Forty-eight hours post-retrovirus transduction, cells were treated with 40 µg/ml cycloheximide and harvested at indicated time points post-cycloheximide treatment. Lysates were separated by SDS-PAGE and Western blotted using antibodies to PTPN14, UBR4, and actin. The same lysates were run in the first five lanes of the gels in panels A and B. The experiment was repeated four times, and a representative experiment is shown.
FIG 7 HPV18 E7-mediated PTPN14 degradation requires the E3 ubiquitin ligase UBR4. (A) N/Tert cells were transfected with siRNAs targeting cullin 3, UBR4, or HPV18 E7 or with a nontargeting control (si Control). Twenty-four hours posttransfection, cells were transduced with a retrovirus expressing HPV18 E7 or with the empty vector control retrovirus. Forty-eight hours post-retrovirus transduction, cells were treated with 40 µg/ml cycloheximide (CHX) and harvested at indicated time points post-cycloheximide treatment. Equal amounts of protein lysates were separated by SDS-PAGE and Western blotted using antibodies to PTPN14, cullin 3, HA, and actin. (B) N/Tert cells were transfected with siRNA targeting UBR4 or with a nontargeting control (si Control). Twenty-four hours posttransfection, cells were transduced with an empty vector control retrovirus. Forty-eight hours post-retrovirus transduction, cells were treated with 40 µg/ml cycloheximide and harvested at indicated time points post-cycloheximide treatment. Lysates were separated by SDS-PAGE and Western blotted using antibodies to PTPN14, UBR4, and actin. The same lysates were run in the first five lanes of the gels in panels A and B. The experiment was repeated four times, and a representative experiment is shown.CC BY · PubMed Central
FIG 8 Papillomavirus E7 proteins vary in their ability to bind PTPN14 and target PTPN14 for degradation. (A) N/Tert-1 cells expressing one of several HPV E7 proteins or BPV1 E7 protein tagged with Flag and HA were subjected to immunoprecipitation with HA antibody. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to HA, PTPN14, UBR4, RB1, and actin. (Left panels) Protein levels in input lysates. (Right panels) Proteins recovered after immunoprecipitation with anti-HA antibody. PTPN14 and HA protein amounts were quantified using ImageJ. The ratio of PTPN14 protein amount relative to E7-HA protein amount in the E7-expressing cell lines is indicated. (B) PTPN14 and actin protein levels in C127 and ID13 cell lysates were measured by Western blotting.
FIG 8 Papillomavirus E7 proteins vary in their ability to bind PTPN14 and target PTPN14 for degradation. (A) N/Tert-1 cells expressing one of several HPV E7 proteins or BPV1 E7 protein tagged with Flag and HA were subjected to immunoprecipitation with HA antibody. Immunoprecipitates were separated by SDS-PAGE and Western blotted using antibodies to HA, PTPN14, UBR4, RB1, and actin. (Left panels) Protein levels in input lysates. (Right panels) Proteins recovered after immunoprecipitation with anti-HA antibody. PTPN14 and HA protein amounts were quantified using ImageJ. The ratio of PTPN14 protein amount relative to E7-HA protein amount in the E7-expressing cell lines is indicated. (B) PTPN14 and actin protein levels in C127 and ID13 cell lysates were measured by Western blotting.CC BY · PubMed Central

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Metadata

Authors
White, E. A., Münger, K., & Howley, P. M.
Affiliations
  • Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA
  • Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts, USA
Venue
mBio
Publisher
American Society for Microbiology
Published
2016-11-02
Volume
7
Issue
5
ISSN
  • 2161-2129
  • 2150-7511
DOI
10.1128/mbio.01530-16
PMC
PMC5030362
Cited by
101
References
4
Language
en
Licence
Funders
  • HHS | NIH | National Cancer Institute (R35CA197262)
  • Alexander and Margaret Stewart Trust

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