Cell, Vol. 63, 1129-1136,

December

21, 1990, Copyright

0

1990 by Cell Press

The E6 Oncoprotein Encoded by Human Papillomavirus Types 16 and 18 Promotes the Degradation of ~53 Martin Scheffner,” Bruce A. Werness, Jon 54. Huibregtse,‘Arnold J. Levine,t and Peter M. Howley’ * Laboratory of Tumor Virus Biology National Cancer Institute Bethesda, Maryland 20892 t Department of Biology Princeton University Princeton, New Jersey 08540

Summary The E6 protein encoded by the oncogenic human papillomavirus types 16 and 16 is one of two viral products expressed in HPV-associated cancers. E6 is an oncoprotein which cooperates with E7 to immortalize primary human keratinocytes. Insight into the mechanism by which E6 functions in oncogenesis is provided by the observation that the E6 protein encoded by HPV-16 and HPV-16 can complex the wild-type ~53 protein in vitro. Wild-type p53 gene has tumor suppressor properties, and is a target for several of the oncoproteins encoded by DNA tumor viruses. In this study we demonstrate that the E6 proteins of the oncogenic HPVs that bind p53 stimulate the degradation of ~53. The EG-promoted degradation of ~53 is ATP dependent and involves the ubiquitin-dependent protease system. Selective degradation of cellular proteins such as ~53 with negative regulatory functions provides a novel mechanism of action for dominantacting oncoproteins. Introduction The papillomaviruses are small DNA viruses that cause benign squamous epithelial tumors (warts and papillomas) in their natural hosts and have been etiologically implicated in some cancers. There are over 60 different human papillomaviruses (HPVs) associated with a variety of clinical lesions (DeVilliers, 1989) and approximately 20 of these are associated with anogenital tract lesions. Some of these, such as HPV-6 and HPV-11, are considered low risk viruses since they are associated with benign proliferative lesions such as condyloma acuminata that infrequently progress to malignancy. Others, including HPV16, HPV-18, and HW-33, are considered high risk because of their association with potentially preneoplastic lesions and with certain anogenital carcinomas, most notably cancer of the uterine cervix (zur Hausen and Schneider, 1987). Transcriptionally active DNAs of these “high risk” viruses are found in a high percentage of cervical cancers and in cell lines derived from cervical cancer tissues (Schwarz et al., 1985; Yee et al., 1985; Smotkin and Wettstein, 1988). Further support for a causal role of HPV in associated cancers derives from the recognition that the “high risk”

viruses encode oncoproteins. The E6 and E7 proteins of the “high risk” viruses are regularly expressed in cervical cancers and in the derived cell lines. The E8 and E7 genes of these “high risk” viruses have been best studied for HW-18 and HPV-18, and both E6 and E7 have been shown to have transformation properties. E7 is sufficient for the transformation of established rodent cell lines such as NIH 3T3 (Kanda et al., 1988; Phelps et al., 1988; Vousden et al., 1988; Watanabe and Yoshiike, 1988; Bedell et al., 1989; Tanaka et al., 1989) and can cooperate with an activated ras oncogene to transform primary rat cells (Phelps et al., 1988; Storey et al., 1988). The transformation potential of the E8 gene was revealed by studies with primary human keratinocytes and fibroblasts which showed that E8 was required in combination with E7 for efficient immortalization of the cells (Mijnger et al., 1989a; HawleyNelson et al., 1989; Watanabe et al., 1989). An emerging theme among DNA tumor viruses is that the viral-encoded oncoproteins interact specifically with critical cellular regulatory proteins, and that the oncogenic effects of these viruses are at least in part a consequence of these specific interactions. In this regard the oncogenic HPVs show some parallels with the adenoviruses and SV40. All three encode transforming proteins that can complex with pRB (the product of the retinoblastoma tumor susceptibility gene) (Whyte et al., 1988; DeCaprio et al., 1988; Dyson et al., 1989) and with ~53 (Lane and Crawford, 1979; Linzer and Levine, 1979; Sarnow et al., 1982; Werness et al., 1990) which is now also regarded as having properties of a tumor suppressor gene (Finlay et al., 1989; Eliyahu et al., 1989). The E7 proteins encoded by the oncogenic genital HPVs, adenovirus ElA, and the SV40 large T antigen all localize to the nucleus and share certain characteristics. Each can extend the life span of primary cells, cooperate with other cytoplasmic oncoproteins such as ras to fully transform primary rat cells, induce DNA synthesis in growth-arrested cells, transform established rodent cells, and modulate transcription from certain promoters (reviewed by Ruley, 1990). Regions of amino acid sequence similarity exist among the E7, ElA, and large T antigens of these different DNA tumor viruses (Phelps et al., 1988; Figge et al., 1988) and these conserved regions have been shown to participate in the binding to a number of these important cellular proteins, including pRB (DeCaprio et al., 1988; Whyte et al., 1989; Ewen et al., 1989; Mijnger et al., 1989b). Thus the comparable biologic properties of these different oncoproteins may derive from their ability to target a similar set of cellular regulatory proteins. The properties of the E8 proteins encoded by the genital tract papillomaviruses have been less well studied. It has been suggested that the HPV-16 and HW-18 proteins have transcriptional activation properties (Gius et al., 1988; Lamberti et al., 1990). It was the oncogenic properties of the HPV-18 and HPV18 E8 proteins that prompted our studies to investigate whether E6 targeted ~53, similar to SV40 large T antigen and Ad5 ElB. An association of the

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Figure 1. The E6 Protein of HPV-1s Induces Degradation of ~53 In Vitro Synthetic RNAs encoding human ~53 or the E6 protein of either HPV11 or HPV-1swere translated in rabbit reticulocyte lysate in the presence of [35S]cysteine.In vitro translated ~53 was incubated either in the absence (lanes a, d, g) or in the presence of approximately equimolar amounts of either HPV-11E6 (lanes b, e, h) or HPV-18E6 (lanes c, f, i) in a total volume of 40 gl containing 10 pl of rabbit reticulocyte lysate, 25 mM R&Cl (pH 7.5), 100 mM NaCI, and 3 mM DTT After a 3 hr incubation at the indicated temperatures, the total reaction mixtures were separated on a 14% SDS-polyacrylamide gel and the proteins visualized by fluorography. The positions of the human p53 and the HPV E6 proteins are indicated on the left, and the positions of molecular weight markers (in kd) are shown on the right.

E6 proteins of the “high risk” HPV types but not of the “low risk” HPV types with ~53 was demonstrated in vitro using programmed rabbit reticulocyte lysates (Werness et al., 1990). It was clear, however, that although the oncoproteins of these different DNA tumor viruses all targeted ~53, the consequence of the protein-protein interaction was likely to,be quite different. In SV40- and AdB-transformed cells, sufficient large T antigen and El6 55 kd protein, respectively, exists to form complexes with ~53; these complexes result in an increased half-life and steady-state levels of the protein in transformed cells (Oren et al., 1961; Reich et al., 1983) and presumably inactivating its normal function as a negative regdator of cell growth. In contrast, levels of ~53 in many cervical carcinoma cell lines and in HPV-16- and HPV-la-transformed cell lines are generally quite low (our unpublished data). For example, in the HeLa cell line, which is an HPV-la-positive human cervical carcinoma cell line, ~53 is reported to be undetectable despite the presence of translatable mRNA (Matlashewski et al., 1986). The studies reported in this article were designed to investigate the functional consequence of the association of E6 and ~53. Results The E6 Proteins Encoded by HPV-16 and HIV-18 Stimulate the Degradation of ~53 The specific association of the E6 proteins encoded by HW-16 and HPV-18 with the cellular ~53 protein has been demonstrated in vitro (Werness et al., 1990). This interaction was shown by the coprecipitation of E6 with wild-type ~53 from mixed lysates of rabbit reticulocyte-translated E6 and ~53 proteins using the p53-specific monoclonal antibody PAb 421 at 4OC. In the course of extending these

studies, we noted that less ~53 was immunoprecipitated from mixed lysates that contained either HPV-16 or -18 E6 than from mixed lysates containing HPV-8 or -11 E6, which do not bind to ~53 (Werness et al., 1990; data not shown). A possible explanation of this phenomenon was that binding of the E6 proteins to ~53 partially masked the epitope recognized by the PAb 421 antibody. Another possibility was that the presence of the E6 proteins capable of complexing ~53 actually led to a degradation of ~53 in the rabbit reticulocyte system. To distinguish between these possibilities, ~53 translated in reticulocyte lysate in the presence of [35S]cysteine was incubated in the absence or presence of labeled HPV-18 or HFV-11 E6 proteins at 4OC. After 3 hr, the total reaction mixtures were processed for SDS-PAGE and the labeled proteins monitored by fluorography. As shown in Figure 1, less ~53 was present after incubation with HPV18 E6 (compare lanes a and c), whereas no difference was detected after incubation with HW-11 E6 (compare lanes a and b). From these results, it was concluded that the presence of HPV-18 E6 leads to the in vitro degradation of p53. Figure 1 also shows the temperature dependency of p53 degradation. Under the conditions used, no significant difference in the amount of ~53 was observed after a 3 hr incubation at any temperature in the absence of E6 proteins (lanes a, d, and g) or in the presence of HPV-11 E8 (lanes b, e, and h). In the presence of HPV-18 E6, however, ~53 was degraded with the highest efficiency at 25OC (about 90% as determined by densitometry) and with reduced efficiencies at rPC (80%-70%) and 4’X (40%). Translation of in vitro synthesized ~53 mRNA in the rabbit reticulocyte system resulted mainly in full-length ~53 protein that comigrated with in vivo labeled ~53 from different cell lines (data not shown), although some minor species of faster migrating ~53 proteins could also be detected (Figure 1). It is not known if these species represent proteins that are generated by premature translation termination or from internal initiation. Interestingly, some of these minor truncated products were degraded while others were not, which may prove useful in defining regions of ~53 necessary for this specific degradation. In the experiment shown in Figure 1, no degradation intermediates were detected after the 3 hr incubation. Therefore, a time course experiment was performed to see if degradation intermediates could be observed at shorter incubation times. As expected, ~53 remained relatively stable throughout the 3 hr incubation at 25°C in the absence of HPV E6 proteins (Figure 2A) or in the presence of HPV-11 E6 (not shown). No degradation was noted after 150 min, although there was a reduction of about 20% of the full-length ~53 protein after 3 hr in the absence of E6 (Figure 2A). In contrast, in the presence of HPV-18 E6 the amount of ~53 decreased rapidly and continuously with time (Figure 26). After 30 min 40% of the input ~53 was degraded, and by 3 hr no full-length ~53 protein was detected. At no time, however, could degradation intermediates of ~53 be detected, suggesting that once ~53 molecules were targeted, degradation occurred rapidly and

HPV E6 Protein 1131

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of p53

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Figure 2. Time Course

of the HPV16

B-Induced

-16

E6

Degradation

of p53

In vitro translated p53 was incubated in the absence (A) or in the presence of HPV-18 E6 (6) at 25% under the conditions described in the legend to Figure 1. The reactions were stopped at different time points and analyzed by gel electrophoresis followed by fluorography. The arrows at the right indicate the positions of migration of p53 and of HPV16 E6.

completely. The level of HPV-18 E8 remained stable throughout the time course (Figure 28) and at the different incubation temperatures (Figure l), consistent with the possibility that HPV-18 E6 may act catalytically for ~53 degradation. To further examine whether the binding of an HPV E6 protein to ~53 correlated with its ability to stimulate the degradation of ~53, the E6 proteins of different HPVs which bind (HPV-16 and HPV-18) or do not bind (HPV-6 and HPV11) to ~53 were incubated with ~53. As demonstrated in Figure 3, ~53 remained stable in the presence of increasing amounts of the E6 proteins of HPV-11 and HPV-6. In the presence of HPV16 or HPV-18, however, ~53 was degraded in a manner dependent on the concentration of the E6 proteins. Interestingly, the HPV-16 E6 was 2- to 3-fold more efficient in promoting degradation than the HPV-18 E6, which correlated with the previous finding that HPV16 E6 has a higher binding affinity for ~53 than HPV18 E6 (Werness et al., 1990). In the experiments described below, the HW-18 E6 was used. Similar results were obtained using HPV16 E6. The Specificity of EG-Stimulated Degradation of p53 Although the EG-dependent degradation of ~53 correlated with the ~53 binding properties of the E6 proteins, the possibility could not be excluded that in the rabbit reticulocyte system, the E6 proteins cause a general degradation of proteins. To control for this, RNA from brome mo.saic virus (BMV) was translated in reticulocyte lysate in the presence of [35S]cysteine and the protein8 were examined for their stability in the presence of HPV18 E6 (Figure 4). The BMV

h

i

Figure 3. Degradation the HFV E6 Proteins

j

k

of ~53 Is Correlated

I

m

with Its Ability to Complex

Unlabeled E6 proteins of HPV-6b or HF’V-11, which do not bind to ~53, or of HPV-16 or HPV18, which do bind to p53 (Werness et al., 1990) were generated by in vitro translation in the absence of radioactively labeled amino acids. The relative amount of each E6 protein was determined in a parallel in vitro translation reaction in the presence of 35Slabeled cysteine (see Experimental Procedures). Approximately equal amounts of the different E6 proteins were then incubated with radioactively labeled p53 under standard reaction conditions (see Figure 1). Lane a, incubation of p53 in the absence of E6 proteins. Lanes b-m, incubation of p53 in the presence of increasing relative amounts (lx, 2x, or 4x) of HPV6b E6 (lanes b-d), HPV11 E6 (lanes e-g), HPV16 E6 (lanes h-i), and HPV18 E6 (lanes k-m).

proteins remained stable in the absence or presence of HPV-18 E6 (lanes a-c), whereas under the same conditions ~53 was degraded, even in the presence of labeled BMV proteins, demonstrating the specificity of the E6induced degradation (lanes d-i). In experiments not shown, in vitro translated SV40 large T antigen and in vitro translated pRB were each shown to be stable in the presence of the HPV-18 E6 protein. Although these results establish specificity for ~53 in this EG-stimulated degradation, they do not exclude the possibility that other specific cellular proteins may also be affected by E8. SV40 Large T Antigen Does Not Stimulate Degradation of p53 Oncoproteins encoded by other DNA tumor viruses, namely the S/40 large T antigen and the Ad5 ElB 55 kd protein, also bind to ~53. In vivo, the binding of ~53 by SV40 large T antigen or by the Ad5 ElB 55 kd protein leads to an increased half-life of ~53 (Oren et al., 1981). To exam-

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Figure 4. The HPV-16 EG-Induced Cannot Be Conferred by Binding

Degradation Is Specific for ~53 and of SV40 Large T Antigen to p53

(A) RNA from BMV was translated in rabbit reticulocyte lysate in the presence of [%]cysteine. The resulting proteins were incubated with unlabeled HFV-18 E6 under standard degradation conditions (see Experimental Procedures). Lanes a-c, incubation of BMV proteins in the absence (lane a) or in the presence of increasing amounts of HPV-18 E6 (lanes band c); lanes d-f, same as lanes a-c but with radioactively labeled ~53 added; lanes g-i, incubation with ~53 in the absence of BMV proteins. The running position of the BMV proteins and ~53 are indicated at left. Molecular size markers are indicated on the right. (B) Increasing amounts of immunopurified SW0 large T antigen (Tag) were incubated with ~53 under standard degradation conditions at the indicated temperatures. Lanes a and e, no T antigen; lanes b and f, 125 ng T antigen; lanes c and g, 250 ng of T antigen; lanes d and h, 500 ng of T antigen; lane i, no T antigen but with HPV-18 E6.

ine whether stimulation of the degradation of reticulocytetranslated ~53 by E6 was specific for E6 or would be a consequence of any ~53 binding protein, the effect of large T antigen on ~53 in vitro was tested. Increasing amounts of immunopurified T antigen were incubated with ~53 under the same conditions used in the experiments with E6 described above (Figure 48). In a parallel immunoprecipitation assay, 60%-70%~ of the input ~53 was bound to T antigen at the highest T antigen concentration used (data not shown). Degradation of ~53, however, could not be detected (Figure 48, lanes a-h), demonstrating that the binding of E6 specifically targets ~53 for degradation in a manner not conferred by binding of SV40 large T antigen. In another experiment (not shown) it was observed that the EG-stimulated degradation of ~53 was not inhibited by SV40 large T antigen. This suggests that E6 and T antigen may bind to different regions on ~53 or that E6 has a higher binding affinity for ~53 than T antigen does. EG-induced Degradation of p53 Is ATP Dependent A property of most protein degradation systems in vivo and in vitro is a requirement for energy in the form of ATP

Figure

5. Degradation

of ~53 Is Dependent

on ATP Hydrolysis

(A) Degradation reactions were performed in the presence of increasing concentrations of AMP or of the nonhydrolyzable ATP analog ATPyS, as indicated. ~53 and the high molecular weight forms of ~53 (~53’) are marked on the left; the running positions of molecular weight markers (in kd) are indicated on the right. (8) Radioactively labeled HPV-18 E6 and ~53 were incubated in the absence or in the presence of AMP or of ATPyS under standard degradation conditions at 25%. The amount of HPV18 E6 bound to ~53 was determined bycoimmunoprecipitation using the p53-specific monoclonal antibody PAb 421 followed by SDS-polyacrylamide gel electrophoresis and fluorography. In a parallel control experiment using the SW0 large T antigen-specific monoclonal antibody PAb 419, which does not recognize ~53, no HPV-18 E6 or ~53 could be detected (not shown). Lane a, immunoprecipitation of ~53 in the absence of HPV18 E6; lane b, in the presence of HPV-18 E6; lane c, with 4 mM AMP; lane d, with 2 mM ATP$S; lane e, same as lane b, but incubation was performed at 4%. The high molecular weight forms of ~53 seen in (A) were also detected in this immunoprecipitation analysis; however, in this experiment they were only visible at long exposure times.

Since it is a component of the reticulocyte lysate translation system, ATP was present in all reaction mixtures of the experiments shown above. To determine if ATP was required for the EBinduced degradation of ~53, AMP or ATPYS, a nonhydrolyzable ATP analog, was added to a standard degradation assay (Figure 5A). Both AMP (Figure 5A, lanes c-e) and ATP+ (lanes f-h) inhibited the EG-dependent degradation of ~53, indicating that the degradation is dependent on ATP hydrolysis. The presence of ATPyS not only inhibited the degradation of ~53, but caused much of the labeled ~53 to migrate as high molecular weight forms (marked as ~53’ in Figure 5A). ~53 was the only labeled protein in the reaction mixtures, suggesting that the higher migrating proteins are indeed modified ~53 molecules. This assumption was supported by immunoprecipitation analysis using the p53-specific monoclonal antibody PAb 421, which showed that the higher migrating forms contain ~53 (data not

7;3\

E6 Protein

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Degradation

of p53

shown). Densitometric quantitation revealed that the presence of ATPyS completely inhibited the degradation, since the normal migrating and high migrating forms together contained all of the radioactivity initially added to the reaction. Furthermore, the occurrence of ~53 high molecular weight forms was dependent on the presence of HPV-18 E8 (Figure 5A, lane j), since the higher molecular forms were not observed when HPV-18 E6 was not included in the incubations. Finally, E6 is unlikely to be part of this slower migrating form, since this form was not observed in experiments using unlabeled ~53 and labeled HPV-18 E6 (data not shown). The addition of AMP to the degradation reaction had a similar effect to that of ATPyS, although more of the ~53 proteins migrated at the normal molecular size and less migrated as higher molecular weight forms. In addition, the higher migrating forms had a lower apparent molecular weight compared with the forms observed in the presence of ATP+. To demonstrate that the inhibition of ~53 degradation by AMP and ATPyS was not due to a reduced binding of HPV18 E6 to ~53, a coimmunoprecipitation analysis was performed using PAb 421 (Figure 56). HPV-18 E6 was immunoprecipitable in the presence of AMP (lane c) or ATP$S (lane d). Lane e shows coimmunoprecipitation of E6 under standard degradation conditions at 4X, while lane b again demonstrates that ~53 is nearly totally degraded at 25% in the absence of AMP or ATPyS. These results confirmed that the EG-induced degradation of p53 is dependent on ATP hydrolysis. The Ubiquitin-Dependent Protease System Is Involved in ~53 Degradation A well-characterized eukaryotic ATP-dependent proteolytic pathway is mediated by the ubiquitin-dependent protease system (reviewed in Ciechanover and Schwartz, 1989). Most studies concerning this degradation pathway have been done in reticulocyte lysate, since reticulocytes contain large amounts of factors involved in ubiquitindependent protein degradation. Prior to degradation, multiple moieties of ubiquitin are covalently linked to the target protein by specialized ubiquitin-conjugating enzymes. The ubiquitination of a protein leads to its slower migration in denaturing protein gels. The occurrence of ubiquitinated protein forms as an intermediate step in degradation has generally been established either by using radioactively labeled ubiquitin or by immunoblots with antibodies directed against ubiquitin. Because of the high molecular weight forms of ~53 observed in Figure 5A, it seemed possible that the ubiquitin system may be involved in the EG-induced degradation of ~53. To determine if the higher migrating forms of p53 represented ubiquitinated ~53, unlabeled reticulocyte lysate-translated ~53 and HPV18 E6 were incubated together in the presence of ATP$S. p53 was then immunoprecipitated with PAb 421, and the immunoprecipitated complex was probed with a ubiquitin-specific rabbit polyclonal antibody (Haas and Bright, 1985) in a Western blot (Figure 6). Bound antibodies were visualized by 1251-labeled protein A; therefore, a strong signal, which was not

+ p53, + PAb 421

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+ p53, + PAb 419

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b

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e

f Weight

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chain

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Forms of ~53 Are Ubiquitinated

Unlabeled ~53 and HPV-16 E6 were incubated in the absence or in the presence of 2 mM ATPrS. After the reaction, p53 was immunoprecipitated as described in Experimental Procedures using PAb 421 or PAb 419, as indicated. A Western blot was then performed using a ubiquitinspecific polyclonal antibody (Haas and Bright, 1965). Bound antiubiquitin antibodies were detected by 1251-labeled protein A followed by autoradiography.

dependent on the addition of the monoclonal ubiquitin antibody, was observed in all reactions at the size of the heavy chain of the antibodies used in the immunoprecipitation (PAb 421 or PAb 419). In the presence of ATPyS and HPV18 E6, a signal could be detected that correlated well in size with the high molecular weight forms of ~53 apparent in Figure 5 (compare Figure 6, lane c, with Figure 5, lanes e-g). This signal was dependent on the presence of ~53 (Figure 6, lanes d-f) and could not be detected when a control antibody (PAb 419) was used in the immunoprecipitation (lanes g-i). These results strongly suggest that the ubiquitin-dependent protease system is involved in the EG-induced degradation of ~53. Discussion The ~53 protein was initially identified as a cellular protein that binds to SV40 large T antigen (Linzer et al., 1979; Lane et al., 1979). Until approximately two years ago, ~53 was thought to be an oncogene, based on early studies that used a clone of the cellular ~53 gene that contained a mutation (Hinds et al., 1989). Mutant forms of ~53 can immortalize primary rat embryo fibroblasts and cooperate with an activated fas oncogene to transform primary cells (Jenkins et al., 1984; Eliyahu et al., 1984; Parada et al., 1984; Finlay et al. 1988, 1989; Eliyahu et al., 1988; Hinds et al., 1989). The wild-type ~53 gene, however, appears to have tumor suppressor properties. In transfection studies of rodent cells, wild-type ~53 has been shown to reduce the efficiency of transformation by certain oncogenes, while the mutated ~53 augments transformation (Finlay et al., 1989; Eliyahu et al.,1989). Furthermore, in keeping with its proposed role as a tumor suppressor, the ~53 gene has been found to be mutated in Friend virus-induced leukemias (Mowat et al., 1985) in a high percentage of human colon carcinomas (Vogelstein et al., 1989; Baker et al., 1989) and in human lung cancers (Takahashi et al., 1989). Finally, expression of the wild-type p53 gene can specifically suppress the growth of human colon carcinoma cells in vitro (Baker et al., 1990) and is antiprolifera-

Cdl 1134

tive in SV40-transformed hamster cells (Mercer et al., 1990). SV40 large T antigen and Ad5 ElB 55 kd protein bind to ~53 in the cell. This association results in an increased half-life and increased steady-state levels of ~53, and presumably inactivates its function as a negative regulator of cell proliferation. This model may not accommodate the HPV E6 proteins, which also bind ~53 in vitro but do not cause an increase in the steady-state level of p53 in immortalized cells or in cancers (our unpublished data). The in vitro evidence presented in this article provides a model whereby the interaction of E6 with ~53 leads to a rapid and specific degradation of ~53. From a functional standpoint, the consequence of this interaction would be equivalent to the stoichiometrically binding and inactivating an intracellular negative growth regulator, leading to unrestricted cellular proliferation. The targeted degradation of ~53 by the E6 oncoproteins encoded by the “high risk” HPVs would account for the lowered levels of ~53 protein found in some of the cervical carcinoma cell lines and HPV-immortalized squamous epithelial cell lines (Matlashewski, 1986; our unpublished data). The elimination of wild-type p53 would be predicted to provide a growth advantage to those cells expressing E6 and could account for at least part of the function of this oncoprotein in its role of cooperating with E7 to immortalize primary human cells (Munger et al., 1989a; HawleyNelson et al., 1989; Watanabe et al., 1989). Sufficiently high levels of mutant forms of ~53 can act in a &ens-dominant fashion to abrogate the growth-suppressive properties of wild-type p53 (Finlay et al., 1989; Vogelstein et al., 1989; Baker et al., 1989). These mutant forms of ~53, which have an activated oncogene function, have an altered conformation with a prolonged half-life. The frans-dominant nature of the mutated ~53 can be explained by its ability to oligomerize the wild-type ~53 protein, drawing it into an inactive complex (Eliyahu et al., 1988; Rovinski and Benchimol, 1988; Finlay et al., 1989). There is some evidence, however, that mutated forms of ~53 may have transforming activity even in the absence of wild-type p53 (Wolf et al., 1984). If this is the case, one might anticipate that mutations in the p53 gene could provide a further growth advantage in HPV-associated carcinomas even in the presence of active viral E6 expression. Such a growth advantage in the tumor could only occur, however, if the mutated ~53 proteins were not substrates for EG-catalyzed degradation. Experiments examining these possibilities are in progress. In this study we have shown that the EG-facilitated degradation of p53 is specific and not a general degradation of all proteins in the rabbit reticulocyte lysate. It seems likely that other cellular proteins in addition to ~53 may also be targeted for this degradation. We have not yet examined whether other normally short-lived nuclear regulatory factors such as the myc, fos, or jun proteins may also be specifically degraded in the presence of E6. E6 has been reported to have transcriptional activation properties (Gius et al., 1988; Lamberti et al., 1990). It is certainly possible that these characteristics may result from the specific degradation of cellular regulatory proteins

that modulate transcription. Indeed, the loss of p53 itself through E6 mediated degradation may result in an altered transcriptional milieu within the cell, since recent studies have suggested that the wild-type p53 has transcriptional modulatory activity (Fields and Jang, 1990; Raycroft et al., 1990). The ubiquitin-dependent protease system is likely to be involved in the EG-stimulated degradation of ~53, since slower migrating forms of ~53 as ubiquitinated intermediates were observed in the presence of inhibitors of ATP hydrolysis. The Western blot results depicted in Figure 6 using a polyclonal antibody to ubiquitin establish that ubiquitinated forms of p53 are generated in the presence of HPV-18 E6 after incubation in the rabbit reticulocyte lysate. The mechanism by which E6 stimulates the degradation of ~53 is not yet clear. SV40 large T antigen, which also complexes with ~53, does not lead to its in vitro degradation, indicating that the observed proteolysis of ~53 is not simply due to the fact that it is in a complex in the rabbit reticulocyte lysate. The specific mechanism for the EG-targeted, ubiquitindependent degradation of p53 in reticulocyte extracts has not yet been determined. The accelerated degradation of ~53 does appear to require formation of a complex involving p53 and E6, however, and as such has some similarities with a degradation pathway recently described by Johnson et al. (1990). In this system a determinant for ubiquitin-dependent degradation can be provided in Pans by one protein component to a second in an oligomeric complex, resulting in the targeted degradation of that protein. Whether or not such a mechanism underlies the E6facilitated proteolysis of p53 is being investigated. The selective degradation of important cellular regulatory proteihs with tumor suppressor activity provides a new mechanism whereby dominant-acting oncoproteins may function. The general model based on the interactions of pRB with viral oncoproteins is that the normal regulatory function of the tumor suppressor gene product is abrogated by its specific association with the viral oncoprotein. The association of E6 with ~53, however, may lead to a loss of function for p53 by eliminating the gene product. The finding that E6 leads to the selective degradation of a tumor suppressor gene product may have more general implications for carcinogenesis in that it suggests a possible role for cellular factors that may be involved in affecting the stability of important regulatory proteins involved in controlling cellular proliferation and differentiation. Experimental

Procedures

Proteins As a source of the human wild-type ~53 and HPV E6 proteins, we used a combined in vitro transcription/translation system. pGEM (Promega) clones containing the E6 open reading frame of HPV type 6b, 11, 16, or 16, or a cDNA encoding the wild-type human ~63 cDNA (Zakut-Houri et al., 1966) was transcribed under recommended conditions using T7 or SP6 RNA polymerase (Promega). The construction of these different pGEM clones has been described previously (Werness et al., 1990). The resulting mRNAs (from 3 wg of template DNA) were used in a 100 pl translation reaction containing 70 ~1 of pretreated rabbit reticulocyte lysate (Promega). To generate radioactively labeled proteins, translations were performed in the presence of [L-%]cysteine

tir3\E6

Protein

Promotes

Degradation

of p53

(X00 Cilmmol; Amersham). Nonradioactively labeled proteins were generated in a parallel reaction in the presence of unlabeled cysteine. Approximate molar ratios of synthesized proteins were determined by densitometry of fluorographs of SDS-polyacrylamide gels. lmmunopurified SV40 large T antigen was purchased from MBR, Inc. BMV proteins were generated by in vitro translation using RNA supplied by Promega. Drgradatlon Aeeay The degradation-stimulating function of the HPV E6 proteins was assayed in 40 ul volumes containing 2 pl of radioactively labeled p53 (iO,OOO-20,000 cpm) and 0.5-6.0 pl of radioactively labeled or unlabeled HPV E6 protein. The reactions contained 25 mM Tris-Cl (pH 7.5) 100 mM NaCI, and 3 mM DTT, unless stated otherwise. The total amount of rabbit reticulocyte lysate was adjusted in each reaction to 10 PI using reticulocyte lysate that was not programmed with exogenous RNA. Unless otherwise indicated, the reactions were performed at 25OC and were stopped after 3 hr by the addition of 1 vol of 100 mM Tris-HCI (pH 6.6) 200 mM DTT, 4% SDS, 20% glycerol, and boiling for 5-10 min at gPC. Total reaction mixtures were then electrophoresed on SDS-polyacrylamide gels and the radioactively labeled proteins were visualized by fluorography. Quantitation of signals was done by densitometry of the fluorographs. Immunopreclpltetlon and Western Blot For immunoprecipitations, 200 ul reactions were performed as described above using a molar ratio of p53 to HPV18 E6 of approximately 2:l. The mixtures were precleared by incubating with 50 pl of 3% protein A-Sepharose (in 20 mM Tris-Cl [pH 8.01, 100 mM NaCI, 2 mM EDTA, 1% NP40) and then immunoprecipitated with the p53-specific monoclonal antibody PAb 421 as described previously (Werness et al., 1990). In control reactions, the SV40 large T antigen-specific monoclonal PAb 419 was used. The immunoprecipitated proteins were separated on SDS-polyacrylamide gels and either visualized by fluorography or further processed for Western blot analysis. For Western blot analysis of the high molecular forms of ~53, proteins were transferred for 4 hr (6 V/cm) from the polyacrylamide gel to a nitrocellulose membrane (Schleicher 8 Schuell). The proteins were then probed with a polyclonal anti-ubiquitin antibody (Haas and Bright, 1965) and bound antibodies monitored by 1251-labeled protein A (Amersham). Acknowledgments We are grateful to Drs. Joe Bolen and Karl Miinger for helpful discussions during the course of this work, and for a critical reading of the manuscript. We are grateful to Dr. A. Haas for providing the rabbit polyclonal antibodies to ubiquitin. M. S. was supported by the Deutsche Forschungsgemsinschaft, and B. A. W. was supported by a National Research Council-NIH Research Associateship. We are grateful to Carol Comlish for excellent editorial assistance in the preparation of this manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

14, 1990; revised

October

10, 1990

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