HPV16 E6 binds to ING4 and hinders its association with p53.

<p>(A) HPV16 E6 does not affect ING4 stability. p53^−/−Mdm2^−/−MEF cells were transfected with ING4 and an increasing amount of either Flag-HPV16 E6 or control vector. The levels of ING4, HPV16 E6 and GAPDH were examined by Western blot. (B) HPV16 E6, ING4 and p53 bind each other. Flag-E6, PCDNA-ING4, and Myc-p53 were transfected into p53^−/−Mdm2^−/−MEF cells. Cell lysates were immunoprecipitated with EzviewTM Red anti-Flag M2 Affinity Gel (lanes 3 and 4), Flag-E6 and the associated proteins were eluted with 3XFlag peptide. Twenty percent of the eluent was subject to Western analysis using indicated antibodies. The remaining eluent was used for secondary immunoprecipitation with anti-myc antibody (lanes 5and 6). (C) Saos2 cells were transfected with PCDNA-ING4, Myc-p53, and either increasing amounts of HPV16 E6 or the vector control. Transfected cells were treated with MG132 for 6 h. The association of PCDNA-ING4 and Myc-p53 was analyzed by immunoprecipitation assay with anti-p53 antibody. (D) Knockdown of HPV16 E6 increases the ING4-p53 interaction. CaSki and SiHa cells with lentivirus-delivered GFP labeled shRNA against HPV16 E6 (shE6) or scramble control (shC) were shown on the left panel. On the other side, the top panel showed the whole cell lysates were immunoprecipitated with anti-p53 antibody and checked ING4 expression by Western blot. Input equivalent to 10% of the whole cell lysates used for immunoprecipitation was subjected to Western blot using the indicated antibody. (E) ING4 mediated p53 acetylation was attenuated by HPV16 E6. Saos2 cells were transfected with PCDNA-ING4, Myc-p53, in the present of either HPV16 E6 or its mutant L50G. At 24 hr post transfection, trichostatin A were added for additional 6 hours, immunoprecipitation and western blot showed that HPV16 E6L50G attenuated binding and acetylation induced by ING4 on p53 without p53 degradation mediated by E6AP. (F) HPV16 E6 attenuates the binding affinity between ING4 and p53 in vitro. Myc-tagged p53 was incubated with bacterially-expressed GST or GST-ING4 in the presence of Flag-tagged HPV16 E6 or control vector for GST-pull down assay. Concentration of each fusion protein used in GST-pull down assays was kept the same. The level of myc-p53 pulled down by GST-ING4 was less in the presence of HPV16 E6.