The Adenoviral E1B-55k Protein Present in HEK293 Cells Mediates Abnormal Accumulation of Key WNT Signaling Proteins in Large Cytoplasmic Aggregates
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.2. Manipulation of WNT Signaling Activity by Wnt3A CM and G007-LK Treatment
2.3. Immunofluorescence Staining and Microscopy
2.4. Co-Immunoprecipitation (Co-IP)
2.5. LS-MS/MS Analysis
2.6. siRNA Transfection
2.7. Quantification of Spherical TNKS Bodies Number in Cells
2.8. Western Blot Analysis
2.9. Supertop Flash (STF) Luciferase Reporter Gene Assay
2.10. Statistical Analysis
3. Results and Discussion
3.1. Key WNT Signaling Components Are Localized in Large Cytoplasmic Aggregates in HEK293 Cells
3.2. The Cytoplasmic Aggregates Are Organized in Spherical Structures Enclosed by a Narrow Layer of the Adenoviral E1B-55k Protein, which Is Required for Aggregate Formation
3.3. Analysis of E1B-55k Interaction Partners in HEK293 Cells
3.4. Reduction of E1B-55k Protein Levels in HEK293 Cells Causes Decreased WNT/β-Catenin Mediated Transcriptional Activation upon Wnt3A Agonist Treatment
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
- Clevers, H.; Nusse, R. Wnt/β-catenin signaling and disease. Cell 2012, 149, 1192–1205. [Google Scholar] [CrossRef] [Green Version]
- MacDonald, B.T.; Tamai, K.; He, X. Wnt/β-catenin signaling: Components, mechanisms, and diseases. Dev. Cell 2009, 17, 9–26. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Valenta, T.; Hausmann, G.; Basler, K. The many faces and functions of β-catenin. EMBO J. 2012, 31, 2714–2736. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Stamos, J.L.; Weis, W.I. The β-catenin destruction complex. Cold Spring Harb. Perspect. Biol. 2013, 5, a007898. [Google Scholar] [CrossRef]
- Kamal, A.; Riyaz, S.; Srivastava, A.K.; Rahim, A. Tankyrase inhibitors as therapeutic targets for cancer. Curr. Top. Med. Chem. 2014, 14, 1967–1976. [Google Scholar] [CrossRef]
- Haikarainen, T.; Krauss, S.; Lehtio, L. Tankyrases: Structure, function and therapeutic implications in cancer. Curr. Pharm. Des. 2014, 20, 6472–6488. [Google Scholar] [CrossRef] [Green Version]
- DeBruine, Z.J.; Xu, H.E.; Melcher, K. Assembly and architecture of the Wnt/β-catenin signalosome at the membrane. Br. J. Pharmacol. 2017, 174, 4564–4574. [Google Scholar] [CrossRef]
- Gammons, M.V.; Renko, M.; Johnson, C.M.; Rutherford, T.J.; Bienz, M. Wnt Signalosome Assembly by DEP Domain Swapping of Dishevelled. Mol. Cell 2016, 64, 92–104. [Google Scholar] [CrossRef] [Green Version]
- Schwarz-Romond, T.; Fiedler, M.; Shibata, N.; Butler, P.J.; Kikuchi, A.; Higuchi, Y.; Bienz, M. The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization. Nat. Struct. Mol. Biol. 2007, 14, 484–492. [Google Scholar] [CrossRef] [PubMed]
- Lin, Y.C.; Boone, M.; Meuris, L.; Lemmens, I.; Van Roy, N.; Soete, A.; Reumers, J.; Moisse, M.; Plaisance, S.; Drmanac, R.; et al. Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat. Commun. 2014, 5, 4767. [Google Scholar] [CrossRef] [Green Version]
- Graham, F.L.; Smiley, J.; Russell, W.C.; Nairn, R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 1977, 36, 59–74. [Google Scholar] [CrossRef] [PubMed]
- Louis, N.; Evelegh, C.; Graham, F.L. Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line. Virology 1997, 233, 423–429. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Jia, L.; Miao, C.; Cao, Y.; Duan, E.K. Effects of Wnt proteins on cell proliferation and apoptosis in HEK293 cells. Cell Biol. Int. 2008, 32, 807–813. [Google Scholar] [CrossRef] [PubMed]
- Gujral, T.S.; MacBeath, G. A system-wide investigation of the dynamics of Wnt signaling reveals novel phases of transcriptional regulation. PLoS ONE 2010, 5, e10024. [Google Scholar] [CrossRef] [Green Version]
- Li, V.S.; Ng, S.S.; Boersema, P.J.; Low, T.Y.; Karthaus, W.R.; Gerlach, J.P.; Mohammed, S.; Heck, A.J.; Maurice, M.M.; Mahmoudi, T.; et al. Wnt signaling through inhibition of β-catenin degradation in an intact Axin1 complex. Cell 2012, 149, 1245–1256. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Voronkov, A.; Holsworth, D.D.; Waaler, J.; Wilson, S.R.; Ekblad, B.; Perdreau-Dahl, H.; Dinh, H.; Drewes, G.; Hopf, C.; Morth, J.P.; et al. Structural basis and SAR for G007-LK, a lead stage 1,2,4-triazole based specific tankyrase 1/2 inhibitor. J. Med. Chem. 2013, 56, 3012–3023. [Google Scholar] [CrossRef]
- Querido, E.; Blanchette, P.; Yan, Q.; Kamura, T.; Morrison, M.; Boivin, D.; Kaelin, W.G.; Conaway, R.C.; Conaway, J.W.; Branton, P.E. Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex. Genes Dev. 2001, 15, 3104–3117. [Google Scholar] [CrossRef] [Green Version]
- Lehtio, L.; Chi, N.W.; Krauss, S. Tankyrases as drug targets. FEBS J. 2013, 280, 3576–3593. [Google Scholar] [CrossRef]
- Voronkov, A.; Krauss, S. Wnt/β-catenin signaling and small molecule inhibitors. Curr. Pharm. Des. 2013, 19, 634–664. [Google Scholar] [CrossRef] [Green Version]
- Lau, T.; Chan, E.; Callow, M.; Waaler, J.; Boggs, J.; Blake, R.A.; Magnuson, S.; Sambrone, A.; Schutten, M.; Firestein, R.; et al. A Novel Tankyrase Small-molecule Inhibitor Suppresses APC Mutation-driven Colorectal Tumor Growth. Cancer Res. 2013, 73, 3132–3144. [Google Scholar] [CrossRef] [Green Version]
- Thul, P.J.; Akesson, L.; Wiking, M.; Mahdessian, D.; Geladaki, A.; Ait Blal, H.; Alm, T.; Asplund, A.; Bjork, L.; Breckels, L.M.; et al. A subcellular map of the human proteome. Science 2017, 356. [Google Scholar] [CrossRef] [PubMed]
- Malm, M.; Saghaleyni, R.; Lundqvist, M.; Giudici, M.; Chotteau, V.; Field, R.; Varley, P.G.; Hatton, D.; Grassi, L.; Svensson, T.; et al. Evolution from adherent to suspension: Systems biology of HEK293 cell line development. Sci. Rep. 2020, 10, 18996. [Google Scholar] [CrossRef] [PubMed]
- Huang, S.M.; Mishina, Y.M.; Liu, S.; Cheung, A.; Stegmeier, F.; Michaud, G.A.; Charlat, O.; Wiellette, E.; Zhang, Y.; Wiessner, S.; et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 2009, 461, 614–620. [Google Scholar] [CrossRef]
- Waaler, J.; Machon, O.; Tumova, L.; Dinh, H.; Korinek, V.; Wilson, S.R.; Paulsen, J.E.; Pedersen, N.M.; Eide, T.J.; Machonova, O.; et al. A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice. Cancer Res. 2012, 72, 2822–2832. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Haikarainen, T.; Waaler, J.; Ignatev, A.; Nkizinkiko, Y.; Venkannagari, H.; Obaji, E.; Krauss, S.; Lehtio, L. Development and structural analysis of adenosine site binding tankyrase inhibitors. Bioorg. Med. Chem. Lett. 2016, 26, 328–333. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Li, N.; Wang, Y.; Neri, S.; Zhen, Y.; Fong, L.W.R.; Qiao, Y.; Li, X.; Chen, Z.; Stephan, C.; Deng, W.; et al. Tankyrase disrupts metabolic homeostasis and promotes tumorigenesis by inhibiting LKB1-AMPK signalling. Nat. Commun. 2019, 10, 4363. [Google Scholar] [CrossRef] [Green Version]
- Blackford, A.N.; Grand, R.J. Adenovirus E1B 55-kilodalton protein: Multiple roles in viral infection and cell transformation. J. Virol. 2009, 83, 4000–4012. [Google Scholar] [CrossRef] [Green Version]
- Zantema, A.; Fransen, J.A.; Davis-Olivier, A.; Ramaekers, F.C.; Vooijs, G.P.; DeLeys, B.; Van der Eb, A.J. Localization of the E1B proteins of adenovirus 5 in transformed cells, as revealed by interaction with monoclonal antibodies. Virology 1985, 142, 44–58. [Google Scholar] [CrossRef]
- Brown, C.R.; Doxsey, S.J.; White, E.; Welch, W.J. Both viral (adenovirus E1B) and cellular (hsp 70, p53) components interact with centrosomes. J. Cell. Physiol. 1994, 160, 47–60. [Google Scholar] [CrossRef]
- Horwitz, G.A.; Zhang, K.; McBrian, M.A.; Grunstein, M.; Kurdistani, S.K.; Berk, A.J. Adenovirus small e1a alters global patterns of histone modification. Science 2008, 321, 1084–1085. [Google Scholar] [CrossRef] [Green Version]
- Madison, D.L.; Yaciuk, P.; Kwok, R.P.; Lundblad, J.R. Acetylation of the adenovirus-transforming protein E1A determines nuclear localization by disrupting association with importin-α. J. Biol. Chem. 2002, 277, 38755–38763. [Google Scholar] [CrossRef] [Green Version]
- Fleisig, H.B.; Orazio, N.I.; Liang, H.; Tyler, A.F.; Adams, H.P.; Weitzman, M.D.; Nagarajan, L. Adenoviral E1B55K oncoprotein sequesters candidate leukemia suppressor sequence-specific single-stranded DNA-binding protein 2 into aggresomes. Oncogene 2007, 26, 4797–4805. [Google Scholar] [CrossRef]
- Liu, Y.; Shevchenko, A.; Shevchenko, A.; Berk, A.J. Adenovirus exploits the cellular aggresome response to accelerate inactivation of the MRN complex. J. Virol. 2005, 79, 14004–14016. [Google Scholar] [CrossRef] [Green Version]
- Maheswaran, S.; Englert, C.; Lee, S.B.; Ezzel, R.M.; Settleman, J.; Haber, D.A. E1B 55K sequesters WT1 along with p53 within a cytoplasmic body in adenovirus-transformed kidney cells. Oncogene 1998, 16, 2041–2050. [Google Scholar] [CrossRef] [Green Version]
- DuBridge, R.B.; Tang, P.; Hsia, H.C.; Leong, P.M.; Miller, J.H.; Calos, M.P. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell. Biol. 1987, 7, 379–387. [Google Scholar] [CrossRef]
- Gustafsson, M.G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 2000, 198, 82–87. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hung, G.; Flint, S.J. Normal human cell proteins that interact with the adenovirus type 5 E1B 55kDa protein. Virology 2017, 504, 12–24. [Google Scholar] [CrossRef] [PubMed]
- Hidalgo, P.; Ip, W.H.; Dobner, T.; Gonzalez, R.A. The biology of the adenovirus E1B 55K protein. FEBS Lett. 2019, 593, 3504–3517. [Google Scholar] [CrossRef] [PubMed]
- Zhao, L.Y.; Liao, D. Sequestration of p53 in the cytoplasm by adenovirus type 12 E1B 55-kilodalton oncoprotein is required for inhibition of p53-mediated apoptosis. J. Virol. 2003, 77, 13171–13181. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rui, Y.; Xu, Z.; Lin, S.; Li, Q.; Rui, H.; Luo, W.; Zhou, H.M.; Cheung, P.Y.; Wu, Z.; Ye, Z.; et al. Axin stimulates p53 functions by activation of HIPK2 kinase through multimeric complex formation. EMBO J. 2004, 23, 4583–4594. [Google Scholar] [CrossRef] [Green Version]
- Rual, J.F.; Venkatesan, K.; Hao, T.; Hirozane-Kishikawa, T.; Dricot, A.; Li, N.; Berriz, G.F.; Gibbons, F.D.; Dreze, M.; Ayivi-Guedehoussou, N.; et al. Towards a proteome-scale map of the human protein-protein interaction network. Nature 2005, 437, 1173–1178. [Google Scholar] [CrossRef] [PubMed]
- Watcharasit, P.; Bijur, G.N.; Zmijewski, J.W.; Song, L.; Zmijewska, A.; Chen, X.; Johnson, G.V.; Jope, R.S. Direct, activating interaction between glycogen synthase kinase-3beta and p53 after DNA damage. Proc. Natl. Acad. Sci. USA 2002, 99, 7951–7955. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yachie, N.; Petsalaki, E.; Mellor, J.C.; Weile, J.; Jacob, Y.; Verby, M.; Ozturk, S.B.; Li, S.; Cote, A.G.; Mosca, R.; et al. Pooled-matrix protein interaction screens using Barcode Fusion Genetics. Mol. Syst. Biol. 2016, 12, 863. [Google Scholar] [CrossRef]
- Li, Q.; He, Y.; Wei, L.; Wu, X.; Wu, D.; Lin, S.; Wang, Z.; Ye, Z.; Lin, S.C. AXIN is an essential co-activator for the promyelocytic leukemia protein in p53 activation. Oncogene 2011, 30, 1194–1204. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Vinayagam, A.; Stelzl, U.; Foulle, R.; Plassmann, S.; Zenkner, M.; Timm, J.; Assmus, H.E.; Andrade-Navarro, M.A.; Wanker, E.E. A directed protein interaction network for investigating intracellular signal transduction. Sci. Signal 2011, 4, rs8. [Google Scholar] [CrossRef]
- Nelson, S.; Nathke, I.S. Interactions and functions of the adenomatous polyposis coli (APC) protein at a glance. J. Cell Sci. 2013, 126, 873–877. [Google Scholar] [CrossRef] [Green Version]
- Major, M.B.; Camp, N.D.; Berndt, J.D.; Yi, X.; Goldenberg, S.J.; Hubbert, C.; Biechele, T.L.; Gingras, A.C.; Zheng, N.; Maccoss, M.J.; et al. Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling. Science 2007, 316, 1043–1046. [Google Scholar] [CrossRef]
- Grohmann, A.; Tanneberger, K.; Alzner, A.; Schneikert, J.; Behrens, J. AMER1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane. J. Cell Sci. 2007, 120, 3738–3747. [Google Scholar] [CrossRef] [Green Version]
- Kim, W.J.; Rivera, M.N.; Coffman, E.J.; Haber, D.A. The WTX tumor suppressor enhances p53 acetylation by CBP/p300. Mol. Cell 2012, 45, 587–597. [Google Scholar] [CrossRef] [Green Version]
- Tanneberger, K.; Pfister, A.S.; Kriz, V.; Bryja, V.; Schambony, A.; Behrens, J. Structural and functional characterization of the Wnt inhibitor APC membrane recruitment 1 (Amer1). J. Biol. Chem. 2011, 286, 19204–19214. [Google Scholar] [CrossRef] [Green Version]
- Veeman, M.T.; Slusarski, D.C.; Kaykas, A.; Louie, S.H.; Moon, R.T. Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr. Biol. 2003, 13, 680–685. [Google Scholar] [CrossRef] [Green Version]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Olsen, P.A.; Krauss, S. The Adenoviral E1B-55k Protein Present in HEK293 Cells Mediates Abnormal Accumulation of Key WNT Signaling Proteins in Large Cytoplasmic Aggregates. Genes 2021, 12, 1920. https://doi.org/10.3390/genes12121920
Olsen PA, Krauss S. The Adenoviral E1B-55k Protein Present in HEK293 Cells Mediates Abnormal Accumulation of Key WNT Signaling Proteins in Large Cytoplasmic Aggregates. Genes. 2021; 12(12):1920. https://doi.org/10.3390/genes12121920
Chicago/Turabian StyleOlsen, Petter Angell, and Stefan Krauss. 2021. "The Adenoviral E1B-55k Protein Present in HEK293 Cells Mediates Abnormal Accumulation of Key WNT Signaling Proteins in Large Cytoplasmic Aggregates" Genes 12, no. 12: 1920. https://doi.org/10.3390/genes12121920