International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy
Correspondence
Lawrence Banks
banks{at}icgeb.org
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ABSTRACT |
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MAIN TEXT |
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Although E6-induced loss of p53 is an important element of E6-induced cellular transformation, recent studies have identified a number of additional cellular targets of E6 that may also play an important role [reviewed by Mantovani & Banks (2001); Thomas et al. (2002a)
]. The discovery that high-risk, but not low-risk, HPV E6 proteins can bind to the PDZ domain-containing proteins and target them for ubiquitin-mediated degradation was particularly interesting, considering that high-risk types cause lesions that can progress to cervical carcinoma, whereas the low-risk types are very rarely associated with malignancies (zur Hausen & Schneider, 1987
). PDZ domains are motifs of 8090 aa, which are present in a variety of proteins that are involved in clustering of ion channels, signalling enzymes and adhesion molecules to specific structures at the membranecytoskeleton interface of polarized cells [reviewed by Kim (1997)
]. Among them is hScribble, a protein that is expressed at epithelial tight junctions, which has recently been shown to be a substrate for ubiquitination by the E6/E6-AP complex in vitro (Nakagawa & Huibregtse, 2000
). E6 oncoproteins were also shown to bind to the human homologue of the Drosophila discs large (Dlg) tumour suppressor (hDIg) and the membrane-associated guanylate kinase (MAGUK) inverted (MAGI) family of proteins and to induce their proteasome-mediated degradation (Gardiol et al., 1999
; Glaunsinger et al., 2000
; Thomas et al., 2001
, 2002b
). However, the mechanisms by which these proteins can be directed for degradation by E6 are still unclear (Pim et al., 2000
, 2002
; Thomas et al., 2001
, 2002b
).
Recently, we showed that a panel of inhibitory peptides exerted different effects on the ability of HPV E6 to direct the degradation of a number of its substrates. In addition, by using an E6-AP immunodepletion assay, we obtained evidence that MAGI-1 degradation was E6-AP-independent (Sterlinko Grm et al., 2004). Therefore, the present study was initiated to investigate whether E6-directed degradation of the PDZ domain-containing proteins Dlg and MAGIs required the E6-AP ubiquitin ligase. We performed in vitro E6-AP immunodepletion assays to determine whether there were any differences in the E6-induced degradation of these targets in the presence or absence of E6-AP. To do this, HPV-16 and -18 E6, p53, hDlg and MAGI proteins were translated in vitro by using the TNT rabbit reticulocyte system (Promega) and incubated with rabbit polyclonal anti-E6-AP antibody (kindly provided by Martin Scheffner, Institute of Biochemistry, University of Cologne, Germany) for 30 min on ice, followed by adsorption of the immune complexes onto protein ASepharose beads. The supernatant fractions were then used as in vitro-translated proteins depleted of E6-AP. Immunodepletion with a pre-immune antibody was included as a negative control. To assess the effectiveness of the depletion, we first performed an anti-E6-AP Western blot on wheat-germ extract (Promega) and on untreated, pre-immune-depleted and E6-AP-depleted rabbit reticulocyte extract (Promega). As shown in Fig. 1(a)
, no E6-AP was detected in wheat-germ extract, in contrast to rabbit reticulocyte extract, where a strong band was detected at 100 kDa, in agreement with previously published data (Huibregtse et al., 1991
). Immunodepletion with anti-E6-AP antibody effectively removed most of the E6-AP protein from the rabbit reticulocyte lysate (Fig. 1a
). In contrast, the control immunodepletion with pre-immune sera had little effect. These data showed that the polyclonal anti-E6-AP antibody raised against human E6-AP efficiently recognized the rabbit E6-AP in the rabbit reticulocyte lysate. This is in agreement with the very high degree of conservation in the E6-AP protein between species, with over 99 % homology between human and mouse E6-AP. Furthermore, one round of immunodepletion was sufficient to remove most of the E6-AP protein present within the lysate. As it has been demonstrated that E6-mediated degradation of p53 is E6-AP-dependent, we used this to test the effectiveness of our immunodepletion. In vitro-translated p53 was immunodepleted of E6-AP and incubated with similarly immunodepleted HPV-16 or -18 E6 for 30 min at 30 °C. The remaining p53 protein was then immunoprecipitated with rabbit polyclonal C4 antibody and the results obtained are shown in Fig. 1(b)
. As can be seen, p53 was effectively degraded by E6 over the course of the assay in the sample that was immunodepleted with the pre-immune antibody (negative control). However, E6-mediated degradation of p53 was blocked in the E6-AP-depleted samples, in agreement with previous publications (Huibregtse et al., 1991
, 1993
; Scheffner et al., 1993
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ciechanover, A., Orian, A. & Schwartz, A. L. (2000). Ubiquitin-mediated proteolysis: biological regulation via destruction. Bioessays 22, 442451.[CrossRef][Medline]
Gardiol, D., Kühne, C., Glaunsinger, B., Lee, S. S., Javier, R. & Banks, L. (1999). Oncogenic human papillomavirus E6 proteins target the discs large tumour suppressor for proteasome-mediated degradation. Oncogene 18, 54875496.[CrossRef][Medline]
Glaunsinger, B. A., Lee, S. S., Thomas, M., Banks, L. & Javier, R. (2000). Interactions of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins. Oncogene 19, 52705280.[CrossRef][Medline]
Haupt, Y., Maya, R., Kazaz, A. & Oren, M. (1997). Mdm2 promotes the rapid degradation of p53. Nature 387, 296299.[CrossRef][Medline]
Hengstermann, A., Linares, L. K., Ciechanover, A., Whitaker, N. J. & Scheffner, M. (2001). Complete switch from Mdm2 to human papillomavirus E6-mediated degradation of p53 in cervical cancer cells. Proc Natl Acad Sci U S A 98, 12181223.
Huibregtse, J. M., Scheffner, M. & Howley, P. M. (1991). A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO J 10, 41294135.[Abstract]
Huibregtse, J. M., Scheffner, M. & Howley, P. M. (1993). Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol Cell Biol 13, 775784.[Abstract]
Huibregtse, J. M., Scheffner, M., Beaudenon, S. & Howley, P. M. (1995). A family of proteins structurally and functionally related to the E6-AP ubiquitinprotein ligase. Proc Natl Acad Sci U S A 92, 25632567.[Abstract]
Kim, S. K. (1997). Polarized signaling: basolateral receptor localization in epithelial cells by PDZ-containing proteins. Curr Opin Cell Biol 9, 853859.[CrossRef][Medline]
Kubbutat, M. H., Jones, S. N. & Vousden, K. H. (1997). Regulation of p53 stability by Mdm2. Nature 387, 299303.[CrossRef][Medline]
Li, X. & Coffino, P. (1996). High-risk human papillomavirus E6 protein has two distinct binding sites within p53, of which only one determines degradation. J Virol 70, 45094516.[Abstract]
Mantovani, F. & Banks, L. (2001). The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene 20, 78747887.[CrossRef][Medline]
Mantovani, F., Massimi, P. & Banks, L. (2001). Proteasome-mediated regulation of the hDlg tumour suppressor protein. J Cell Sci 114, 42854292.
Nakagawa, S. & Huibregtse, J. M. (2000). Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase. Mol Cell Biol 20, 82448253.
Pim, D., Thomas, M., Javier, R., Gardiol, D. & Banks, L. (2000). HPV E6 targeted degradation of the discs large protein: evidence for the involvement of a novel ubiquitin ligase. Oncogene 19, 719725.[CrossRef][Medline]
Pim, D., Thomas, M. & Banks, L. (2002). Chimaeric HPV E6 proteins allow dissection of the proteolytic pathways regulating different E6 cellular target proteins. Oncogene 21, 81408148.[CrossRef][Medline]
Rolfe, M., Beer-Romero, P., Glass, S., Eckstein, J., Berdo, I., Theodoras, A., Pagano, M. & Draetta, G. (1995). Reconstitution of p53ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP). Proc Natl Acad Sci U S A 92, 32643268.[Abstract]
Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J. & Howley, P. M. (1990). The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63, 11291136.[Medline]
Scheffner, M., Huibregtse, J. M., Vierstra, R. D. & Howley, P. M. (1993). The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75, 495505.[Medline]
Scheffner, M., Huibregtse, J. M. & Howley, P. M. (1994). Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53. Proc Natl Acad Sci U S A 91, 87978801.[Abstract]
Scheffner, M., Nuber, U. & Huibregtse, J. M. (1995). Protein ubiquitination involving an E1E2E3 enzyme ubiquitin thioester cascade. Nature 373, 8183.[CrossRef][Medline]
Sterlinko Grm, H., Weber, M., Elston, R., McIntosh, P., Griffin, H., Banks, L. & Doorbar, J. (2004). Inhibition of E6-induced degradation of its cellular substrates by novel blocking peptides. J Mol Biol 335, 971985.[CrossRef][Medline]
Thomas, M., Glaunsinger, B., Pim, D., Javier, R. & Banks, L. (2001). HPV E6 and MAGUK protein interactions: determination of the molecular basis for specific protein recognition and degradation. Oncogene 20, 54315439.[CrossRef][Medline]
Thomas, M., Pim, D. & Banks, L. (2002a). Human papillomavirus E6 protein interactions. In Human Papilloma Viruses (Perspectives in Medical Virology 8), pp. 7199. Edited by D. J. McCance. Amsterdam: Elsevier.
Thomas, M., Laura, R., Hepner, K., Guccione, E., Sawyers, C., Lasky, L. & Banks, L. (2002b). Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation. Oncogene 21, 50885096.[CrossRef][Medline]
Werness, B. A., Levine, A. J. & Howley, P. M. (1990). Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 7679.[Medline]
zur Hausen, H. & Schneider, A. (1987). The role of papillomaviruses in human anogenital cancer. In The Papillomaviruses, pp. 245263. Edited by N. P. Salzman & P. M. Howley. New York: Plenum.
Received 13 February 2004;
accepted 18 June 2004.