USP11 stabilizes HPV-16E7 and further modulates the E7 biological activity - PubMed (original) (raw)

USP11 stabilizes HPV-16E7 and further modulates the E7 biological activity

Ching-Hui Lin et al. J Biol Chem. 2008.

Abstract

HPV-16E7 is a major transforming protein, which has been implicated in the development of cervical cancer. The stability of E7 is thus important to ensure its fully functional status. Using the yeast two-hybrid system, we found that USP11 (ubiquitin-specific protease 11), a member of a protein family that cleaves polyubiquitin chains and/or ubiquitin precursors, interacts and forms a specific complex with HPV-16E7. Our results indicate that the USP11 can greatly increase the steady state level of HPV-16E7 by reducing ubiquitination and attenuating E7 degradation. In contrast, a catalytically inactive mutant of USP11 abolished the deubiquitinating ability and returned E7 to a normal rate of degradation. Moreover, USP11 not only protected E7 from ubiquitination but also influenced E7 function as a modulator of cell growth status. These results suggest that USP11 plays an important role in regulating the levels of E7 protein and subsequently affects the biological function of E7 as well as its contribution to cell transformation by HPV-16E7.

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Figures

FIGURE 1.

FIGURE 1.

Identification of USP11 as a novel interacting protein of HPV-16E7. A, the yeast strain AH109 containing the indicated BD plasmids (16E7, 18E7, p53, Lamin C, and Gal-BD) was co-incubated with Y187 harboring AD plasmids (USP11-C, SV40 LT, and Gal4-AD). After overnight incubation, yeast cultures were streaked on SD medium lacking leucine and tryptophan or grown on a selective minimal plate (SD medium lacking leucine, tryptophan, histidine, and adenine). The Gal4 DNA-binding domain (Gal4 BD) and the Gal4 activation domain (Gal4 AD) were incorporated as negative controls. p53 and simian virus 40 large T-antigen (SV40 LT) were used as positive controls. Laminin C was used to elucidate the specificity of the interaction between 16E7 and USP11-C.B, a schematic summary of A. Yeast colony color was observed after incubation for a standard period of time. The color _blue_represents USP11-C associating with HPV-16E7 and turning on the _lacZ_promoter.

FIGURE 2.

FIGURE 2.

HPV-16E7 associates with USP11 in vivo. A, the_in vivo_ interaction of HPV-16E7 with USP11 by immunoprecipitation assay. FLAG-USP11 and FLAG-16E7 plasmids were transfected into H1299 as indicated. After 48 h of transfection, cell extracts were prepared and subjected to immunoprecipitation (IP) by α-HPV-16E7. The proteins from the immunoprecipitation were separated using 10% SDS-PAGE and analyzed by α-FLAG to detect the associated proteins. Western blotting using α-FLAG indicates the expression of the product. Equal amounts of proteins were loaded as indicated by detection of tubulin. B, the_in vivo_ interaction of HPV-16E7 with USP11 by immunoprecipitation assay in different combinations. An experiment similar to that in _A_was conducted, except that FLAG-USP11 was replaced by HA-USP11, immunoprecipitation was conducted using α-FLAG, and α-HA was used to detect the associated protein. C, to confirm the specific association, HA-USP11 and FLAG-p53 plasmids were transfected into H1299 as indicated. Immunoprecipitations were performed using α-p53, and the precipitates were detected by α-HA antibody. D, the in vivo interaction of USP11 with endogenous E7 in CaSki cells. Immunoprecipitations were performed with cell extracts of CaSki cells using eitherα-HPV-16E7 or normal mouse IgG as a negative control. Precipitates were recognized by α-HPV-16E7, and the association was detected using α-USP11.

FIGURE 3.

FIGURE 3.

USP11 specifically stabilizes HPV-16E7, and the deubiquitinating activity of USP11 is required for this function. A, USP11 stabilized HPV-16E7 but not p53. FLAG-16E7 and FLAG-p53 were transfected into H1299 cells either alone or together with FLAG-USP11 as indicated. At 48 h after transfection, 100 μg of cell extracts from transfected cells were assayed by Western blot using α-FLAG. Approximately equal amounts of proteins were loaded as indicated by the tubulin signal. B, mutant USP11 (USP11 cys mt) lost the ability to stabilize HPV-16E7. The FLAG-USP11 and the mutant form, FLAG-USP11 cys mt, were transfected together with FLAG-16E7 into H1299 cells as indicated. At 48 h after transfection, 100 μg of cell extracts were subjected to Western blot using α-FLAG to detect the levels of FLAG-16E7 and FLAG-USP11 expression. The amount of proteins loaded is indicated by the tubulin signal.

FIGURE 4.

FIGURE 4.

USP11 extends the half-life of both FLAG-16E7 and endogenous HPV-16E7. A, USP11 significantly extends the half-life of HPV-16E7 after cycloheximide treatment. FLAG-16E7 was introduced into H1299 together with pcDNA3-HA empty vector or pcDNA3-HA-USP11. At 48 h after transfection, the cells were treated with 50 g/ml cycloheximide to inhibit protein synthesis. Total cell extracts were collected at each indicated time point and were assayed either with α-FLAG to detect the remaining FLAG-16E7 or with α-HA to indicate HA-USP11 expression. Western blot analysis using α-tubulin showed that an equal amount of protein was loaded onto each lane. B, the same procedure also allowed us to study the ability of USP11 to stabilize endogenous E7 in CaSki cells. The amount of proteins loaded is indicated by the tubulin signals. C, USP11 significantly extends the half-life of endogenous HPV-16E7 after si-USP11 treatment. CaSki (si-) and CaSki (si-USP11) cells were cultured in medium containing 200 μg/ml G418. To assay the endogenous HPV-16E7 protein level, cell extracts were collected at the indicated time points after 50 μg/ml cycloheximide was added. The remaining HPV-16E7 was detected by α-16E7, and α-USP11 was used to detect endogenous USP11 expression.

FIGURE 5.

FIGURE 5.

USP11 deubiquitinates HPV-16E7 both in vivo and in vitro. A, ubiquitination and deubiquitination of HPV-16E7_in vivo_. 293T cells were transfected with expression plasmids for FLAG-16E7, Myc-USP11, and HA-ubiquitin, as indicated. At 20 h after transfection, cells were treated with 20

m

MG132 for 2 h. Total cell extracts were first subjected to immunoprecipitation (IP) using α-FLAG, and then immune complexes were assayed by Western blot, and α-HA was used to detect the ubiquitination levels of FLAG-16E7.mt, FLAG-USP11 cys mt. B, USP11 cys mt has lost its ability to deubiquitinate HPV-16E7 in vitro. The purified polyubiquitinated His-16E7 was incubated with the indicated amounts of purified recombinant GST-USP11 or GST-USP11 cys mt in a deubiquitination buffer at 30 °C for 1 h. Ubiquitinated His-16E7 was detected by Western blot analysis using α-ubiquitin.

FIGURE 6.

FIGURE 6.

Effects of USP11 on subsequent HPV-16E7-mediated protein activation and cell growth activation. A, the USP11 si-RNA leads to reduced HPV-16E7 and increased Rb in CaSki cells. 300-g cell extracts from CaSki (si) and CaSki (si-USP11) cell lines were subjected to Western blot analysis using α-USP11 or α-HPV-16E7 to detect the levels of endogenous HPV-16E7 and USP11 expression, respectively. Tubulin protein was assayed to monitor equal loading between individual lanes. Quantitative measurements of signals are presented on the right. B, colony formation assay. CaSki (si) and CaSki (si-USP11) cells were subcultured and kept in the medium containing G418 (200 μg/ml), and surviving colonies were counted 2 weeks later. Relative cell viability is summarized from the five separate experiments and is presented on the right.

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