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Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells.

Ajay AK, Meena AS, Bhat MK - Cell Biosci (2012)

Bottom Line: However, results presented here for the first time demonstrate that overexpressed p53 is not directly associated with E6 and therefore free, yet it is not functionally active in HPV positive cells.Also, the stability of overexpressed p53 does not seem to be an issue because inhibition of proteasomal degradation did not increase the half-life of overexpressed p53, which is more than endogenous p53.E6 did not decrease p53 protein but phospho-p53 level was significantly reduced.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Centre for Cell Science, NCCS Complex, Pune University Campus, Ganeshkhind, Pune - 411007, India. manojkbhat@nccs.res.in.

ABSTRACT

Background: In HPV infected cells p53 function is abrogated by E6 and even ectopically expressed p53 is unable to perform tumor suppressor functions. In addition to facilitating its degradation, E6 may also inhibit p53 transactivity, though the mechanisms are still poorly understood. It has been reported that inhibition of p300, an acetyltransferase responsible for p53 acetylation is inactivated by E6. Activation of overexpressed p53 to cause cell growth inhibition is facilitated by its phosphorylation. Previously, we reported that non-genotoxically overexpressed p53 in HeLa cells needs to be phosphorylated to perform its cell growth inhibitory functions. Since over expressed p53 by itself was not activated, we hypothesized an inhibitory role for E6.

Results: Majority of reports proposes E6 mediated degradation of p53 as a possible reason for its inactivation. However, results presented here for the first time demonstrate that overexpressed p53 is not directly associated with E6 and therefore free, yet it is not functionally active in HPV positive cells. Also, the stability of overexpressed p53 does not seem to be an issue because inhibition of proteasomal degradation did not increase the half-life of overexpressed p53, which is more than endogenous p53. However, inhibition of proteasomal degradation prevents the degradation of endogenous p53. These findings suggest that overexpressed p53 and endogenous p53 are differentially subjected to proteasomal degradation and the reasons for this discrepancy remain unclear. Our studies demonstrate that p53 over expression has no effect on anchorage independent cell-growth and E6 ifies its cell growth inhibitory effect. E6 overexpression abrogates OA induced p53 occupancy on the p21 promoter and cell death as well. E6 did not decrease p53 protein but phospho-p53 level was significantly reduced.

Conclusion: We report for the first time that E6 de-activates p53 by inhibiting its phosphorylation. This prevents p53 binding to p21 promoter and thereby restraining its cell-growth inhibitory functions. Our study provides new evidence indicating that viral protein E6 inhibits p53 transactivity by mechanism independent of degradation pathway.

No MeSH data available.


Related in: MedlinePlus

Overexpressed p53 is functionally impaired by HPV E6. (A) HTet23p53, HTet26p53 or HTet43GFP cells were transfected with vector or HPV18 E6 plasmid and 18 h post transfection cells were treated with OA 1 h prior to Dox addition. MTT assay was performed after 48 h. Bar represents variations among the wells of an experiment done twice in triplicate. * Indicates P < 0.01 (B) HTet26p53 cells were transfected with HPV18 E6 plasmid and treated as mentioned in A and immunoprecipitation was performed first with E6 antibody (first IP) and secondly by p53 antibody. p53 or E6 was detected in immunoprecipitated complex. (C) p53 post-IP (second IP) following E6 IP was performed and p53 or phospho-p53detection by western blotting was performed. (D) Cells were transfected with p21luciferase construct with or without HPV18 E6 plasmid and treated as mentioned in A. Luciferase assay was performed and luciferase/GFP reading was plotted. Bar represents results from an experiment done in triplicate. (± SE). * Indicates P < 0.05.
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Figure 6: Overexpressed p53 is functionally impaired by HPV E6. (A) HTet23p53, HTet26p53 or HTet43GFP cells were transfected with vector or HPV18 E6 plasmid and 18 h post transfection cells were treated with OA 1 h prior to Dox addition. MTT assay was performed after 48 h. Bar represents variations among the wells of an experiment done twice in triplicate. * Indicates P < 0.01 (B) HTet26p53 cells were transfected with HPV18 E6 plasmid and treated as mentioned in A and immunoprecipitation was performed first with E6 antibody (first IP) and secondly by p53 antibody. p53 or E6 was detected in immunoprecipitated complex. (C) p53 post-IP (second IP) following E6 IP was performed and p53 or phospho-p53detection by western blotting was performed. (D) Cells were transfected with p21luciferase construct with or without HPV18 E6 plasmid and treated as mentioned in A. Luciferase assay was performed and luciferase/GFP reading was plotted. Bar represents results from an experiment done in triplicate. (± SE). * Indicates P < 0.05.

Mentions: To determine whether E6 inhibits activity, p53 was first activated by 5 nM OA, a protein phosphatase 2A (PP2A) inhibitor. As a consequence of p53 activation cell growth is retarded in p53 overexpressing HTet23p53 and HTet26p53 cells compared to p53 non-overexpressing HTet23p53, HTet26p53 or HTet43GFP cells. Cell survival inhibitory effect was abolished by ectopic expression of HPV 18 E6 in OA treated p53 overexpressing cells (Figure 6A). Overexpressed p53 is not completely associated with E6 and ectopic expression of E6 does not further enhance this association, though p53 is detected in co-immunoprecipitated complex (Figure 6B). To confirm the presence of free p53 in the lysate immunoprecipitated by E6 antibody (first IP), second immunoprecipitation with p53 (FL-393) was done. Interestingly, ectopic expression of E6 did not affect the p53 protein but it does drastically decrease the level of Ser46 phosphorylated p53 (pSer46p53) (Figure 6C). Further, to confirm that E6 mediated inhibition of p53 phosphorylation actually is responsible for cell growth inhibition, we performed luciferase reporter activation assay for well-known transcriptional target p21. p21 promoter was activated by OA treatment and E6 over expression inhibits promoter activation (Figure 6D).


Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells.

Ajay AK, Meena AS, Bhat MK - Cell Biosci (2012)

Overexpressed p53 is functionally impaired by HPV E6. (A) HTet23p53, HTet26p53 or HTet43GFP cells were transfected with vector or HPV18 E6 plasmid and 18 h post transfection cells were treated with OA 1 h prior to Dox addition. MTT assay was performed after 48 h. Bar represents variations among the wells of an experiment done twice in triplicate. * Indicates P < 0.01 (B) HTet26p53 cells were transfected with HPV18 E6 plasmid and treated as mentioned in A and immunoprecipitation was performed first with E6 antibody (first IP) and secondly by p53 antibody. p53 or E6 was detected in immunoprecipitated complex. (C) p53 post-IP (second IP) following E6 IP was performed and p53 or phospho-p53detection by western blotting was performed. (D) Cells were transfected with p21luciferase construct with or without HPV18 E6 plasmid and treated as mentioned in A. Luciferase assay was performed and luciferase/GFP reading was plotted. Bar represents results from an experiment done in triplicate. (± SE). * Indicates P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285035&req=5

Figure 6: Overexpressed p53 is functionally impaired by HPV E6. (A) HTet23p53, HTet26p53 or HTet43GFP cells were transfected with vector or HPV18 E6 plasmid and 18 h post transfection cells were treated with OA 1 h prior to Dox addition. MTT assay was performed after 48 h. Bar represents variations among the wells of an experiment done twice in triplicate. * Indicates P < 0.01 (B) HTet26p53 cells were transfected with HPV18 E6 plasmid and treated as mentioned in A and immunoprecipitation was performed first with E6 antibody (first IP) and secondly by p53 antibody. p53 or E6 was detected in immunoprecipitated complex. (C) p53 post-IP (second IP) following E6 IP was performed and p53 or phospho-p53detection by western blotting was performed. (D) Cells were transfected with p21luciferase construct with or without HPV18 E6 plasmid and treated as mentioned in A. Luciferase assay was performed and luciferase/GFP reading was plotted. Bar represents results from an experiment done in triplicate. (± SE). * Indicates P < 0.05.
Mentions: To determine whether E6 inhibits activity, p53 was first activated by 5 nM OA, a protein phosphatase 2A (PP2A) inhibitor. As a consequence of p53 activation cell growth is retarded in p53 overexpressing HTet23p53 and HTet26p53 cells compared to p53 non-overexpressing HTet23p53, HTet26p53 or HTet43GFP cells. Cell survival inhibitory effect was abolished by ectopic expression of HPV 18 E6 in OA treated p53 overexpressing cells (Figure 6A). Overexpressed p53 is not completely associated with E6 and ectopic expression of E6 does not further enhance this association, though p53 is detected in co-immunoprecipitated complex (Figure 6B). To confirm the presence of free p53 in the lysate immunoprecipitated by E6 antibody (first IP), second immunoprecipitation with p53 (FL-393) was done. Interestingly, ectopic expression of E6 did not affect the p53 protein but it does drastically decrease the level of Ser46 phosphorylated p53 (pSer46p53) (Figure 6C). Further, to confirm that E6 mediated inhibition of p53 phosphorylation actually is responsible for cell growth inhibition, we performed luciferase reporter activation assay for well-known transcriptional target p21. p21 promoter was activated by OA treatment and E6 over expression inhibits promoter activation (Figure 6D).

Bottom Line: However, results presented here for the first time demonstrate that overexpressed p53 is not directly associated with E6 and therefore free, yet it is not functionally active in HPV positive cells.Also, the stability of overexpressed p53 does not seem to be an issue because inhibition of proteasomal degradation did not increase the half-life of overexpressed p53, which is more than endogenous p53.E6 did not decrease p53 protein but phospho-p53 level was significantly reduced.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Centre for Cell Science, NCCS Complex, Pune University Campus, Ganeshkhind, Pune - 411007, India. manojkbhat@nccs.res.in.

ABSTRACT

Background: In HPV infected cells p53 function is abrogated by E6 and even ectopically expressed p53 is unable to perform tumor suppressor functions. In addition to facilitating its degradation, E6 may also inhibit p53 transactivity, though the mechanisms are still poorly understood. It has been reported that inhibition of p300, an acetyltransferase responsible for p53 acetylation is inactivated by E6. Activation of overexpressed p53 to cause cell growth inhibition is facilitated by its phosphorylation. Previously, we reported that non-genotoxically overexpressed p53 in HeLa cells needs to be phosphorylated to perform its cell growth inhibitory functions. Since over expressed p53 by itself was not activated, we hypothesized an inhibitory role for E6.

Results: Majority of reports proposes E6 mediated degradation of p53 as a possible reason for its inactivation. However, results presented here for the first time demonstrate that overexpressed p53 is not directly associated with E6 and therefore free, yet it is not functionally active in HPV positive cells. Also, the stability of overexpressed p53 does not seem to be an issue because inhibition of proteasomal degradation did not increase the half-life of overexpressed p53, which is more than endogenous p53. However, inhibition of proteasomal degradation prevents the degradation of endogenous p53. These findings suggest that overexpressed p53 and endogenous p53 are differentially subjected to proteasomal degradation and the reasons for this discrepancy remain unclear. Our studies demonstrate that p53 over expression has no effect on anchorage independent cell-growth and E6 ifies its cell growth inhibitory effect. E6 overexpression abrogates OA induced p53 occupancy on the p21 promoter and cell death as well. E6 did not decrease p53 protein but phospho-p53 level was significantly reduced.

Conclusion: We report for the first time that E6 de-activates p53 by inhibiting its phosphorylation. This prevents p53 binding to p21 promoter and thereby restraining its cell-growth inhibitory functions. Our study provides new evidence indicating that viral protein E6 inhibits p53 transactivity by mechanism independent of degradation pathway.

No MeSH data available.


Related in: MedlinePlus