Instituto de Biologia Molecular y Celular de Rosario (IBR-CONICET), Departamento de Microbiologia, Facultad de Ciencias Bioquimicas, Suipacha 531, 2000 Rosario, Argentina1
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy2
Author for correspondence: Daniela Gardiol. Fax +54 341 4804598. e-mail dgardiol{at}fbioyf.unr.edu.ar
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Abstract |
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Introduction |
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The human discs large tumour suppressor protein h-Dlg/SAP97 (Dlg) (Lue et al., 1994 ; Müller et al., 1995
), homologous to Drosophila tumour suppressor Dlg-A (Woods & Bryant, 1991
), is a member of the MAGUK (membrane-associated guanylate kinase homologous) family of proteins. These proteins are characterized by having specific protein recognition domains, including SH3, PDZ and guanylate kinase homologous (GuK) regions (Anderson, 1996
). PDZ domains are specific modules for proteinprotein interactions (Ponting & Phillips, 1995
; Fanning & Anderson, 1999
) that allow the clustering of proteins and the formation of multiprotein signalling complexes at specialized sites in the membrane (Kim, 1997
). For protein interaction with PDZ domains, a carboxy-terminal S/TXV motif is required in the partner molecules (Songyang et al., 1997
). In epithelial cells, Dlg is co-localized with E-cadherin at sites of cellcell interaction (Reuver & Garner, 1998
), where it is thought to have both structural and signalling roles. Epithelia form structures of polarized cells with the apical and basolateral sides separated by cell junctions and Dlg is required for this organization (Kim, 1997
). Deregulation of this system leads to defective cellcell adhesion, loss of cell polarity, unregulated proliferation and alteration of the pattern of cell differentiation (Woods et al., 1996
). Dlg has been shown to bind via its PDZ domains to the carboxy terminus of the APC (adenomatous polyposis coli) tumour suppressor (Matsumine et al., 1996
) and it was shown that the APCDlg complex is important for APC-mediated growth suppression (Ishidate et al., 2000
; Suzuki et al., 1999
). Recent studies have shown that in Drosophila, Dlg and two other tumour suppressors, scribble and lgl, act cooperatively to regulate cell polarity and proliferation (Bilder et al., 2000
). This finding suggests an important connection between epithelial organization and cellular growth control and points out the critical role of these oncosuppressors in this regulatory system (Bilder et al., 2000
). In addition, Dlg is a target for several viral oncoproteins, including human T cell leukaemia virus type 1 Tax, high-risk HPV E6 proteins and adenovirus type 9 E4-ORF1 protein (Lee et al., 1997
; Kiyono et al., 1997
). In all cases, the viral proteins bind Dlg through its PDZ domains and inhibit Dlg activity, albeit through different mechanisms. Therefore, the experimental evidence suggests a tumour suppressor role for h-Dlg/SAP97, similar to its Drosophila homologue Dlg-A (Woods et al., 1996
; Goode & Perrimon, 1997
).
We have shown previously that the binding of HPV-16 and -18 E6 proteins to Dlg results in a dramatic reduction in the levels of Dlg both in vivo and in vitro and this is mediated by the ubiquitin proteolytic pathway (Gardiol et al., 1999 ). We have shown also that this activity of E6 is regulated specifically by protein kinase A phosphorylation of the HPV E6 carboxy-terminal motif, which is involved in the binding to the PDZ domains (Kühne et al., 2000
). To investigate further this regulation of Dlg by ubiquitinylation, we constructed a series of Dlg mutants and investigated their susceptibility to proteasome-mediated degradation in the presence and absence of HPV E6. We show that PDZ domain 2 (PDZ2) of Dlg is necessary for the ability of E6 to target Dlg for degradation and sequences within the extreme amino-terminal region of Dlg (NT), prior to the first PDZ domain, are also required for optimal degradation efficiency. In the absence of E6, Dlg is also subjected to ubiquitin-mediated degradation, but, in this case, all of the sequences required would appear to reside within PDZ2. Sequences within the carboxy-terminal SH3 and GuK domains do not appear to be involved in proteasome-mediated regulation of Dlg.
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Methods |
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The cloning of HPV-18 E6 into pSP64 for in vitro translation, pCDNA-3 for in vivo expression and pGEX-2T for the expression of glutathione S-transferase (GST) protein fusion in bacteria has been described previously (Thomas et al., 1995 ).
Cells and tissue culture.
Human 293 and U2OS cells were grown in DMEM supplemented with 10% foetal calf serum. Transient transfections were carried out using the calciumphosphate precipitation method described previously (Matlashewski et al., 1987 ). Transfection efficiencies were tested by co-transfecting the Escherichia coli
-galactosidase-expressing plasmid pCH100 in parallel and assaying for
-galactosidase activity.
U2OS cells stably expressing HA-tagged Dlg proteins were selected and maintained in culture medium supplemented with geneticin antibiotic (G418) at a concentration of 200 µg/ml.
GST fusion protein expression and binding assays.
GSTHPV-18 E6 fusion protein was expressed in E. coli and purified on glutathioneagarose beads. GST pull-down assays were performed as described previously (Thomas et al., 1995 ).
In vitro and in vivo degradation assays.
In vitro degradation assays were performed as described previously (Pim et al., 1994 ). Briefly, in vitro-translated HA-tagged Dlg proteins were mixed with in vitro-translated wild-type HPV-18 E6 or water-primed lysates as a control at 30 °C. At the indicated time-points, reactions were immunoprecipitated using an anti-HA antibody (Boehringer Mannheim) and the remaining Dlg proteins were visualized by autoradiography after SDSPAGE resolution.
For in vivo degradation experiments, cells were harvested in extraction buffer (250 mM NaCl, 0·1% Nonidet P-40, 50 mM HEPES pH 7·0 and 1% aprotinin) 24 h after transfection. Equal amounts of protein were separated by SDSPAGE and transferred to nitrocellulose membranes. Levels of recovered Dlg protein were determined by immunoblotting using an anti-HA monoclonal antibody (mAb) and the blots were developed using ECL, according to the manufacturers instructions (Amersham).
For proteasome inhibitor protection assays, cells stably expressing the different Dlg mutants were treated with either 50 µM NCBZLEULEULEUAL proteasome inhibitor or an equal amount of DMSO as a control 2 h prior to protein extraction. Levels of Dlg protein were then ascertained by immunoblotting, as described above.
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Results |
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Discussion |
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Having determined which regions of Dlg were required for E6-mediated degradation, we were next interested in investigating whether those same regions were involved in regulating Dlg protein stability in the absence of E6. This was particularly interesting, since we had shown previously that wild-type Dlg was regulated intrinsically by the proteasome in the absence of E6 (Gardiol et al., 1999 ). To address this question, we generated a series of cell lines stably expressing some of the Dlg deletion mutant proteins and then asked whether they could be stabilized following treatment of the cells with proteasome inhibitors. The reasoning being, that if any mutant was processed by the proteasome, then blocking its activity should give rise to an increase in the steady-state levels of the mutant protein. Interestingly, DLG-NTPDZ1-2 and DLG-3PDZ both showed strong increases in protein levels following proteasome inhibition, suggesting that both proteins were being degraded by the proteasome. In contrast, DLG-NTPDZ1 was largely unaffected by this treatment and was only weakly stabilized when compared with the other mutants, suggesting that the protein levels of this mutant Dlg are probably not being regulated by the proteasome. Unfortunately, this mutant is expressed at quite low levels within the stable cell lines, which runs counter to the above argument. However, it is possible that within the context of a stable cell line, this mutant may have inhibitory effects on cell growth and, hence, only be tolerated at low levels. Further studies will be required to clarify this issue. The above results raise a number of interesting points. As in the case of E6, the contribution of PDZ2 to the regulation of Dlg levels in the absence of E6 appears to be essential. However, unlike the case with E6, the amino-terminal region of Dlg does not appear to be important for this regulation in the absence of E6. Therefore, it seems that, as for other cellular targets of E6, different proteins or pathways are involved in the proteolytic degradation of Dlg in the absence or presence of E6. Considering the central role of PDZ2 in regulating Dlg protein stability in the absence of E6, it is interesting also to note that the APC tumour suppressor has been shown to interact with this domain of Dlg and this raises the intriguing possibility that occupation of PDZ2 by APC may contribute to the regulation of Dlg levels and activity. A recent study reported the finding of a PDZ-binding kinase (PBK) which is cell cycle regulated by phosphorylation at mitosis. PBK binds to PDZ2 of Dlg and could probably link Dlg to signal transduction pathways regulating cell cycle and proliferation (Gaudet et al., 2000
). This finding emphasizes the importance of PDZ2 in the modulation of Dlg activities and it is striking that it is this domain that is targeted by the high-risk HPV E6 proteins.
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Acknowledgments |
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References |
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Androphy, E. J., Hubbert, N. L., Schiller, J. T. & Lowy, D. R. (1987). Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines. EMBO Journal 6, 989-992.[Abstract]
Banks, L., Spence, P., Androphy, E., Hubbert, N., Matlashewski, G., Murray, A. & Crawford, L. (1987). Identification of human papillomavirus type 18 E6 polypeptides in cells derived from human cervical carcinoma. Journal of General Virology 68, 1351-1359.[Abstract]
Bilder, D., Li, M. & Perrimon, N. (2000). Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113-116.
Chen, J. J., Reid, C. E., Band, V. & Androphy, E. J. (1995). Interaction of papillomavirus E6 oncoproteins with a putative calcium-binding protein. Science 269, 529-531.[Medline]
Fanning, A. S. & Anderson, J. M. (1999). PDZ domains: fundamental building blocks in the organization of protein complexes at the plasma membrane. Journal Clinical Investigation 103, 767-772.
Foster, S. A., Demers, G. W., Etscheid, B. G. & Galloway, D. A. (1994). The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest. Journal of Virology 68, 5698-5705.[Abstract]
Gardiol, D. & Banks, L. (1998). Comparison of human papillomavirus type 18 (HPV-18) E6-mediated degradation of p53 in vitro and in vivo reveals significant differences based on p53 structure and cell type but little difference with respect to mutants of HPV-18 E6. Journal of General Virology 79, 1963-1970.[Abstract]
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, 5487-5496.[Medline]
Gaudet, S., Branton, D. & Lue, R. A. (2000). Characterization of PDZ-binding kinase, a mitotic kinase. Proceedings of the National Academy of Sciences, USA 97, 5167-5172.
Glaunsinger, B. A., Lee, S. S., Thomas, M., Banks, L. & Javier, R. (2000). Interaction of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins. Oncogene 19, 5270-5280.[Medline]
Goode, S. & Perrimon, N. (1997). Inhibition of patterned cell shape change and cell invasion by Discs large during Drosophila oogenesis. Genes & Development 11, 2532-2544.
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 Journal 10, 4129-4135.[Abstract]
Huibregtse, J. M., Scheffner, M. & Howley, P. M. (1993). Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins. Molecular and Cellular Biology 13, 4918-4927.[Abstract]
Inoue, T., Oka, K., Yong-Il, h., Vousden, K. H., Kyo, S., Jing, P., Hakura, A. & Yutsudo, M. (1998). Dispensability of p53 degradation for tumorigenicity and decreased serum requirement of human papillomavirus type 16 E6. Molecular Carcinogenesis 21, 215-222.[Medline]
Ishidate, T., Matsumine, A., Toyoshima, K. & Akiyama, T. (2000). The APChDLG complex negatively regulates cell cycle progression from the G0/G1 to S phase. Oncogene 19, 365-372.[Medline]
Ishiwatari, H., Hayasaka, N., Inoue, H., Yutsudo, M. & Hakura, A. (1994). Degradation of p53 only is not sufficient in the growth stimulatory effect of human papillomavirus 16 E6 oncoprotein in human embryonic fibroblasts. Journal of Medical Virology 44, 243-249.[Medline]
Kim, S. K. (1997). Polarized signalling: basolateral receptor localization in epithelial cells by PDZ-containing proteins. Current Opinion in Cell Biology 9, 853-859.[Medline]
Kiyono, T., Hiraiwa, A., Fujita, M., Hayashi, Y., Akiyama, T. & Ishibashi, M. (1997). Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein. Proceedings of the National Academy of Sciences, USA 94, 11612-11616.
Kühne, C. & Banks, L. (1998). E3-ubiquitin ligase/E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G box. Journal of Biological Chemistry 273, 34302-34309.
Kühne, C., Gardiol, D., Guarnaccia, C., Amenitsch, H. & Banks, L. (2000). Differential regulation of human papillomavirus E6 by protein kinase A: conditional degradation of human discs large protein by oncogenic E6. Oncogene 19, 5884-5891.[Medline]
Lee, S. S., Weiss, R. S. & Javier, R. T. (1997). Binding of human virus oncoproteins to hDlg/SAP97, a mammalian homolog of the Drosophila discs large tumor suppressor protein. Proceedings of the National Academy of Sciences, USA 94, 6670-6675.
Lee, S. S., Glaunsinger, B., Mantovani, F., Banks, L. & Javier, R. T. (2000). Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins. Journal of Virology 74, 9680-9693.
Liu, Y., Chen, J. J., Gao, Q., Dalal, S., Hong, Y., Mansur, C. P., Band, V. & Androphy, E. J. (1999). Multiple functions of human papillomavirus type 16 E6 contribute to the immortalization of mammary epithelial cells. Journal of Virology 73, 7297-7307.
Lue, R., Marfatia, S., Branton, D. & Chishti, A. (1994). Cloning and characterization of hdlg: the human homologue of the Drosophila discs large tumor suppressor binds to protein 4.1. Proceedings of the National Academy of Sciences, USA 91, 9818-9822.
Matlashewski, G., Schneider, J., Banks, L., Jones, N., Murray, A. & Crawford, L. (1987). Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells. EMBO Journal 6, 1741-1746.[Abstract]
Matsumine, A., Ogai, A., Senda, T., Okumura, N., Satoh, K., Baeg, G., Kawahara, T., Kobayashi, S., Okada, M., Toyoshima, K. & Akiyama, T. (1996). Binding of APC to the human homolog of the Drosophila discs large tumor suppressor protein. Science 272, 1020-1023.[Abstract]
Müller, B. M., Kistner, U., Veh, R. W., Cases-Langhoff, C., Becker, B., Gundelfinger, E. D. & Garner, C. C. (1995). Molecular characterization and spatial distribution of SAP97, a novel presynaptic protein homologous to SAP90 and the Drosophila discs-large tumor suppressor protein. Journal of Neuroscience 15, 2354-2366.[Abstract]
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. Molecular and Cellular Biology 20, 8244-8253.
Patel, D., Huang, S. M., Baglia, L. & McCance, D. J. (1999). The E6 protein of human papillomavirus type 16 binds to and inhibits co-activation by CBP and p300. EMBO Journal 18, 5061-5072.
Pim, D., Storey, A., Thomas, M., Massimi, P. & Banks, L. (1994). Mutational analysis of HPV-18 E6 identifies domains required for p53 degradation in vitro, abolition of p53 transactivation in vivo and immortalisation of primary BMK cells. Oncogene 9, 1869-1876.[Medline]
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, 719-725.[Medline]
Ponting, C. P. & Phillips, C. (1995). DHR domains in syntrophins, neuronal NO synthases and other intracellular proteins. Trends in Biochemical Sciences 20, 102-103.[Medline]
Reuver, S. M. & Garner, C. C. (1998). E-cadherin mediated cell adhesion recruits SAP97 into the cortical cytoskeleton. Journal of Cell Science 111, 1071-1080.
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, 1129-1136.[Medline]
Schwarz, E., Freese, U., Gissman, L., Mayer, W., Roggenbuck, B., Stremlau, A. & zur Hausen, H. (1985). Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314, 111-114.[Medline]
Smotkin, D. & Wettstein, F. O. (1986). Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. Proceedings of the National Academy of Sciences, USA 83, 4680-4684.[Abstract]
Songyang, Z., Fanning, A. S., Fu, C., Xu, J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan, A. C., Anderson, J. M. & Cantley, L. C. (1997). Recognition of unique carboxy-terminal motifs by distinct PDZ domains. Science 275, 73-77.
Suzuki, T., Ohsugi, Y., Uchida-Toita, M., Akiyama, T. & Yoshida, M. (1999). Tax oncoprotein of HTLV-1 binds to the human homologue of Drosophila discs large suppressor protein, hDLG, and perturbs its function in cell growth control. Oncogene 18, 5967-5972.[Medline]
Thomas, M. & Banks, L. (1998). Inhibition of Bak-induced apoptosis by HPV-18 E6. Oncogene 17, 2943-2954.[Medline]
Thomas, M., Massimi, P., Jenkins, J. & Banks, L. (1995). HPV-18 E6 mediated inhibition of p53 DNA binding activity is independent of E6 induced degradation. Oncogene 10, 261-268.[Medline]
Vousden, K. (1994). Interactions between papillomavirus proteins and tumor suppressor gene products. Advances in Cancer Research 64, 1-24.[Medline]
Woods, D. F. & Bryant, P. J. (1991). The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell 66, 451-464.[Medline]
Woods, D. F., Hough, C., Peel, D., Callaini, G. & Bryant, P. J. (1996). Dlg protein is required for junction structures, cell polarity, and proliferation control in Drosophila epithelia. Journal of Cell Biology 134, 1469-1482.[Abstract]
Wu, H., Reuver, S. M., Kuhlendahl, S., Chung, W. J. & Garner, C. C. (1998). Subcellular targeting and cytoskeletal attachment of SAP97 to the epithelial lateral membrane. Journal of Cell Science 111, 2365-2376.
zur Hausen, H. (1996). Papillomavirus infections: a major cause of human cancers. Biochimica et Biophysica Acta 1288, F55-F78.[Medline]
Received 17 July 2001;
accepted 11 October 2001.