Cross-talk between phosphorylation and SUMOylation regulates transforming activities of an adenoviral oncoprotein

Since the discovery of post-translational modification (PTM) by the small ubiquitin-related modifiers (SUMOs), a multitude of proteins have been described to be reversibly modified, resulting in the alteration of several cellular pathways. Interestingly, various pathogens gain access to this modification system, although the molecular mechanisms and functional consequences are barely understood. We show here that the adenoviral oncoprotein E1B-55K is a substrate of the SUMO conjugation system, which is directly linked to its C-terminal phosphorylation. This regulative connection is indispensable for modulation of the tumor suppressor p53/chromatin-remodeling factor Daxx by E1B-55K and, consequently, its oncogenic potential in primary mammalian cells. In virus infection, E1B-55K PTMs are necessary for localization to viral transcription/replication sites. Furthermore, we identify the E2 enzyme Ubc9 as an interaction partner of E1B-55K, providing a possible molecular explanation for SUMO-dependent modulation of cellular target proteins. In conclusion, these results for the first time provide evidence how E1B-55K PTMs are regulated and subsequently facilitate exploitation of the host cell SUMOylation machinery.

[1]  R. Everett,et al.  The adenovirus type 5 E1b 55K and E4 Orf3 proteins associate in infected cells and affect ND10 components. , 1999, The Journal of general virology.

[2]  Alexandre M. J. J. Bonvin,et al.  Characterizing the N- and C-terminal Small Ubiquitin-like Modifier (SUMO)-interacting Motifs of the Scaffold Protein DAXX* , 2011, The Journal of Biological Chemistry.

[3]  Adenovirus E1B oncoprotein tethers a transcriptional repression domain to p53. , 1994, Genes & development.

[4]  F. Dick,et al.  Identification of a molecular recognition feature in the E1A oncoprotein that binds the SUMO conjugase UBC9 and likely interferes with polySUMOylation , 2010, Oncogene.

[5]  J. Hauber,et al.  Blockage of CRM1-dependent nuclear export of the adenovirus type 5 early region 1B 55-kDa protein augments oncogenic transformation of primary rat cells , 2005, Oncogene.

[6]  A. Levine,et al.  A monoclonal antibody detecting the adenovirus type 5-E1b-58Kd tumor antigen: characterization of the E1b-58Kd tumor antigen in adenovirus-infected and -transformed cells. , 1982, Virology.

[7]  A. Berk,et al.  Corepressor Required for Adenovirus E1B 55,000-Molecular-Weight Protein Repression of Basal Transcription , 1999, Molecular and Cellular Biology.

[8]  R. Everett,et al.  SUMO modification of E1B-55K oncoprotein regulates isoform-specific binding to the tumour suppressor protein PML , 2010, Oncogene.

[9]  K. Leppard.  Regulated RNA Processing and RNA Transport during Adenovirus Infection , 1998 .

[10]  A. Berk,et al.  Recent lessons in gene expression, cell cycle control, and cell biology from adenovirus , 2005, Oncogene.

[11]  G. Demartino,et al.  Intracellular localization of proteasomes. , 2003, The international journal of biochemistry & cell biology.

[12]  L. Hanakahi,et al.  Adenovirus E4 34k and E1b 55k Oncoproteins Target Host DNA Ligase IV for Proteasomal Degradation , 2007, Journal of Virology.

[13]  P. Blanchette,et al.  Control of mRNA Export by Adenovirus E4orf6 and E1B55K Proteins during Productive Infection Requires E4orf6 Ubiquitin Ligase Activity , 2008, Journal of Virology.

[14]  A. Dejean,et al.  Nuclear and unclear functions of SUMO , 2003, Nature Reviews Molecular Cell Biology.

[15]  David C Hay,et al.  Post-translational modification by SUMO. , 2010, Toxicology.

[16]  P. Blanchette,et al.  Adenovirus Type 5 Early Region 1B 55K Oncoprotein-Dependent Degradation of Cellular Factor Daxx Is Required for Efficient Transformation of Primary Rodent Cells , 2011, Journal of Virology.

[17]  F. Melchior,et al.  Concepts in sumoylation: a decade on , 2007, Nature Reviews Molecular Cell Biology.

[18]  A. Sharrocks,et al.  An extended consensus motif enhances the specificity of substrate modification by SUMO , 2006, The EMBO journal.

[19]  P. Branton,et al.  Analysis of Synthesis, Stability, Phosphorylation, and Interacting Polypeptides of the 34-Kilodalton Product of Open Reading Frame 6 of the Early Region 4 Protein of Human Adenovirus Type 5 , 1999, Journal of Virology.

[20]  P. Branton,et al.  Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B protein regulates transforming activity , 1994, Journal of virology.

[21]  A. Levine,et al.  Adenovirus E1b-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells , 1982, Cell.

[22]  V. Wilson,et al.  Bovine Papillomavirus E1 Protein Is Sumoylated by the Host Cell Ubc9 Protein* , 2000, The Journal of Biological Chemistry.

[23]  Structural insights into early events in the conjugation of ubiquitin and ubiquitin-like proteins. , 2007, Molecular cell.

[24]  R. Grand,et al.  Adenovirus E1B 55-Kilodalton Protein: Multiple Roles in Viral Infection and Cell Transformation , 2009, Journal of Virology.

[25]  T. Dobner,et al.  Adenovirus Type 5 Early Region 1B 156R Protein Promotes Cell Transformation Independently of Repression of p53-Stimulated Transcription , 2006, Journal of Virology.

[26]  M. Dasso,et al.  Modification in reverse: the SUMO proteases. , 2007, Trends in biochemical sciences.

[27]  L. Latonen,et al.  Nucleolar aggresomes as counterparts of cytoplasmic aggresomes in proteotoxic stress , 2011, BioEssays : news and reviews in molecular, cellular and developmental biology.

[28]  P. Branton,et al.  Regulation of p53-dependent apoptosis, transcriptional repression, and cell transformation by phosphorylation of the 55-kilodalton E1B protein of human adenovirus type 5 , 1997, Journal of virology.

[29]  C. Lima,et al.  A molecular basis for phosphorylation-dependent SUMO conjugation by the E2 Ubc9 , 2009, Nature Structural &Molecular Biology.

[30]  K. Leppard.  Selective effects on adenovirus late gene expression of deleting the E1b 55K protein. , 1993, The Journal of general virology.

[31]  M. Hochstrasser,et al.  Modification of proteins by ubiquitin and ubiquitin-like proteins. , 2006, Annual review of cell and developmental biology.

[32]  T. Shenk,et al.  The adenovirus E1B 55 kd protein influences mRNA transport via an intranuclear effect on RNA metabolism. , 1989, The EMBO journal.

[33]  E. Kremmer,et al.  A 49-Kilodalton Isoform of the Adenovirus Type 5 Early Region 1B 55-Kilodalton Protein Is Sufficient To Support Virus Replication , 2009, Journal of Virology.

[34]  K. Leppard,et al.  Nuclear matrix localization and SUMO-1 modification of adenovirus type 5 E1b 55K protein are controlled by E4 Orf6 protein. , 2003, The Journal of general virology.

[35]  D. Beach,et al.  Cyclin G is a transcriptional target of the p53 tumor suppressor protein. , 1994, The EMBO journal.

[36]  A. Levine,et al.  Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein. , 1983, Virology.

[37]  S. Jentsch,et al.  Ubiquitin and proteasomes: Sumo, ubiquitin's mysterious cousin , 2001, Nature Reviews Molecular Cell Biology.

[38]  T. Dobner,et al.  Nuclear export of adenovirus RNA. , 2001, Current topics in microbiology and immunology.

[39]  M. Rosa-Calatrava,et al.  Adenovirus infection targets the cellular protein kinase CK2 and RNA‐activated protein kinase (PKR) into viral inclusions of the cell nucleus , 2002, Microscopy research and technique.

[40]  S. J. Flint,et al.  Effects of Mutations in the Adenoviral E1B 55-Kilodalton Protein Coding Sequence on Viral Late mRNA Metabolism , 2002, Journal of Virology.

[41]  M. Weitzman,et al.  Inactivating intracellular antiviral responses during adenovirus infection , 2005, Oncogene.

[42]  T. Dobner,et al.  The Human Adenovirus Type 5 E1B 55-Kilodalton Protein Is Phosphorylated by Protein Kinase CK2 , 2011, Journal of Virology.

[43]  T. Shenk,et al.  Localization of the adenovirus early region 1B 55-kilodalton protein during lytic infection: association with nuclear viral inclusions requires the early region 4 34-kilodalton protein , 1991, Journal of virology.

[44]  A. Berk,et al.  Domains required for in vitro association between the cellular p53 and the adenovirus 2 E1B 55K proteins. , 1990, Virology.

[45]  J. Hauber,et al.  The adenovirus type 5 E1B-55K oncoprotein is a highly active shuttle protein and shuttling is independent of E4orf6, p53 and Mdm2 , 2000, Oncogene.

[46]  J. Henley,et al.  Mechanisms, regulation and consequences of protein SUMOylation. , 2010, The Biochemical journal.

[47]  A. Berk,et al.  Adenovirus E1B 55K Represses p53 Activation In Vitro , 1998, Journal of Virology.

[48]  Jianmei Zhu,et al.  Small Ubiquitin-related Modifier (SUMO) Binding Determines Substrate Recognition and Paralog-selective SUMO Modification* , 2008, Journal of Biological Chemistry.

[49]  E. White.  Regulation of the cell cycle and apoptosis by the oncogenes of adenovirus , 2001, Oncogene.

[50]  H. Shih,et al.  SUMOylation of the Transcriptional Co-Repressor KAP1 Is Regulated by the Serine and Threonine Phosphatase PP1 , 2010, Science Signaling.

[51]  T. Dobner,et al.  Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex , 1997, Journal of virology.

[52]  V. Wilson,et al.  Viral interaction with the host cell sumoylation system. , 2001, Virus research.

[53]  S. J. Flint,et al.  Regulation of mRNA production by the adenoviral E1B 55-kDa and E4 Orf6 proteins. , 2003, Current topics in microbiology and immunology.

[54]  T. Dobner,et al.  Construction of adenovirus type 5 early region 1 and 4 virus mutants. , 2007, Methods in molecular medicine.

[55]  A. Hengstermann,et al.  Activation of p53 by conjugation to the ubiquitin‐like protein SUMO‐1 , 1999, The EMBO journal.

[56]  T. Dobner,et al.  The adenovirus E1B-55K oncoprotein induces SUMO modification of p53 , 2008, Cell cycle.

[57]  R. Hay,et al.  SUMO: a history of modification. , 2005, Molecular cell.

[58]  W. Kaelin,et al.  Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex. , 2001, Genes & development.

[59]  A. Berk,et al.  Adenovirus E1B 55-Kilodalton Protein Is a p53-SUMO1 E3 Ligase That Represses p53 and Stimulates Its Nuclear Export through Interactions with Promyelocytic Leukemia Nuclear Bodies , 2010, Journal of Virology.

[60]  A. Berk,et al.  Adenovirus Ubiquitin-Protein Ligase Stimulates Viral Late mRNA NuclearExport , 2007, Journal of Virology.

[61]  S. Chiocca,et al.  Viruses and sumoylation: recent highlights , 2006, Current Opinion in Microbiology.

[62]  Erica S. Johnson,et al.  Protein modification by SUMO. , 2004, Annual review of biochemistry.

[63]  F. Graham,et al.  Characteristics of a human cell line transformed by DNA from human adenovirus type 5. , 1977, The Journal of general virology.

[64]  C. O'Shea,et al.  Heterochromatin silencing at p53 target genes by a small viral protein , 2010, Nature.

[65]  J. Hauber,et al.  The Adenovirus Type 5 E1B-55K Oncoprotein Actively Shuttles in Virus-Infected Cells, Whereas Transport of E4orf6 Is Mediated by a CRM1-Independent Mechanism , 2001, Journal of Virology.

[66]  Yuan Chen,et al.  Phosphorylation-Dependent Interaction of SATB1 and PIAS1 Directs SUMO-Regulated Caspase Cleavage of SATB1 , 2010, Molecular and Cellular Biology.

[67]  H. Ulrich,et al.  The SUMO system: an overview. , 2009, Methods in molecular biology.

[68]  F. Melchior,et al.  SUMO--nonclassical ubiquitin. , 2000, Annual review of cell and developmental biology.

[69]  Per Stehmeier,et al.  Phospho-regulated SUMO interaction modules connect the SUMO system to CK2 signaling. , 2009, Molecular cell.

[70]  J. Hauber,et al.  Intranuclear targeting and nuclear export of the adenovirus E1B-55K protein are regulated by SUMO1 conjugation , 2007, Proceedings of the National Academy of Sciences.

[71]  T. Dobner,et al.  SUMO-1 modification required for transformation by adenovirus type 5 early region 1B 55-kDa oncoprotein , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[72]  Jaclyn R. Gareau,et al.  The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition , 2010, Nature Reviews Molecular Cell Biology.

[73]  P. Blanchette,et al.  Proteasome-Dependent Degradation of Daxx by the Viral E1B-55K Protein in Human Adenovirus-Infected Cells , 2010, Journal of Virology.

[74]  F. Melchior,et al.  SUMO: regulating the regulator , 2006, Cell Division.

[75]  N. Horikoshi,et al.  Blockage by Adenovirus E4orf6 of Transcriptional Activation by the p53 Tumor Suppressor , 1996, Science.

[76]  A. Shevchenko,et al.  Adenovirus Exploits the Cellular Aggresome Response To Accelerate Inactivation of the MRN Complex , 2005, Journal of Virology.

[77]  A. Berk,et al.  Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein , 1992, Nature.

[78]  M. Weitzman,et al.  Distinct Requirements of Adenovirus E1b55K Protein for Degradation of Cellular Substrates , 2008, Journal of Virology.

[79]  T. Dobner,et al.  Human Pathogens and the Host Cell SUMOylation System , 2011, Journal of Virology.

[80]  A. Santiago,et al.  Identification of two independent SUMO-interacting motifs in Daxx: Evolutionary conservation from Drosophila to humans and their biochemical functions , 2009, Cell cycle.

[81]  E. Kremmer,et al.  Adenovirus type 5 early region 1B 55-kDa oncoprotein can promote cell transformation by a mechanism independent from blocking p53-activated transcription , 2008, Oncogene.

[82]  P. Blanchette,et al.  Identification of Integrin α3 as a New Substrate of the Adenovirus E4orf6/E1B 55-Kilodalton E3 Ubiquitin Ligase Complex , 2009, Journal of Virology.

[83]  M. Weitzman,et al.  Adenovirus oncoproteins inactivate the Mre11–Rad50–NBS1 DNA repair complex , 2002, Nature.

[84]  S. Steinberg,et al.  p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features. , 1992, Oncogene.

[85]  M. Matunis,et al.  SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis. , 2008, Molecules and Cells.

[86]  G. Blobel,et al.  A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex , 1996, The Journal of cell biology.