Article

Viral oncoproteins E1A and E7 and cellular LxCxE proteins repress SUMO modification of the retinoblastoma tumor suppressor.

Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Oncogene (Impact Factor: 8.56). 06/2005; 24(23):3810-8. DOI: 10.1038/sj.onc.1208539
Source: PubMed

ABSTRACT The retinoblastoma tumor suppressor protein (pRB) is a major regulator of cell-cycle progression and cellular differentiation. Central to pRB function is the pocket domain, which serves as the main binding region for cellular regulators. In tumors pRB is frequently inactivated by mutations in the pocket domain or by binding of viral oncoproteins to this region. A characteristic feature of these viral oncoproteins and many cellular pRB-binding partners is an LxCxE sequence motif, which interacts with pRB's pocket domain. Here, we show that the ubiquitin-like modifier SUMO is covalently attached to a distinct residue (K720) of pRB within the B-box of the pocket region that binds LxCxE-motif proteins. We provide evidence that SUMO preferentially targets the active, hypophosphorylated form of pRB and show that tumorigenic mutations of pRB in the pocket domain lead to a loss of SUMOylation. Notably, the level of pRB SUMOylation is controlled by the interaction of pRB with viral and cellular LxCxE-motif proteins. Inhibitors of pRB function, including the viral oncoproteins E1A and E7 and the cellular E1A-like inhibitor of differentiation EID-1, completely abolish SUMO modification of pRB. Conversely, pRB mutants deficient in binding of LxCxE-motif proteins exhibit a drastically enhanced modification by SUMO. Finally, we provide evidence that SUMOylation can influence pRB function, as the SUMO-deficient pRB(K720R) mutant exerts a slightly higher repressive potential on an E2F-responsive reporter gene than wild-type pRB. Taken together, these data identify SUMO modification as a novel post-translational modification of pRB that may control pRB activity by modulating LxCxE-pocket interactions.

0 Bookmarks
 · 
63 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Tumor suppressor Rb is an important cell cycle regulator, arresting cells in early G1. It is commonly inactivated in cancers and its level is maintained during the cell cycle. Rb is regulated by various post-translational modifications such as phosphorylation, acetylation, ubiquitylation and so on. Several E3 ligases including MDM2 promote the degradation of Rb. This study focuses on the role of HAUSP (Herpes-virus associated ubiquitin-specific protease) on Rb. Here, we show that HAUSP colocalizes and interacts with Rb to stabilize it from proteasomal degradation by removing wild type (WT) and K48-linked ubiquitin chains in HEK293 cells. HAUSP deubiquitinates Rb in vivo and in vitro, leading to increased cell population in the G1 phase. Hence, HAUSP is a novel deubiquitinase for Rb. Immunohistochemistry, western blotting and cell-based assays show that HAUSP is overexpressed in glioma and contributes towards glioma progression. However, HAUSP activity on Rb is abrogated in glioma (cancer), where these two proteins show an inverse relationship. MDM2 (a known substrate of HAUSP) serves as a better target for HAUSP mediated deubiquitination in cancer cells, facilitating degradation of Rb and oncogenic progression. This novel regulatory axis is proteasome mediated, p53 independent and the level of MDM2 is critical. The shift in equilibrium by differential deubiquitination in regulation of Rb explains a subtle difference existing between normal and cancer cells. This is speculative of a new possibility to distinguish cancer cells from normal cells at the molecular level, which may be investigated for therapeutic intervention in future. This article is protected by copyright. All rights reserved.
    FEBS Journal 05/2014; DOI:10.1111/febs.12843 · 3.99 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The adenovirus E1A gene is the first gene expressed upon viral infection. E1A re-models the cellular environment to maximize permissivity for viral replication. E1A is also the major transactivator of viral early gene expression and a co-regulator of a large number of cellular genes. E1A carries out its functions predominantly by binding to cellular regulatory proteins and altering their activities. The unstructured nature of E1A enables it to bind to a large variety of cellular proteins and form new molecular complexes with novel functions. The C-terminus of E1A is the least characterized region of the protein with few known binding partners. Here we report the identification of a cellular factor DREF (ZBED1) as a novel, and direct binding partner of E1A. Our studies identify a dual role for DREF in the viral life cycle. DREF contributes to activation of gene expression from all viral promoters early in infection. Unexpectedly, it also functions as a growth restriction factor for adenovirus as knockdown of DREF enhances virus growth and increases viral genome copy number late in the infection. We also identify DREF as a component of viral replication centers. E1A affects the sub-cellular distribution of DREF within PML bodies and enhances DREF SUMOylation. Our findings identify DREF as a novel E1A C-terminus binding partner and provide evidence supporting a role for DREF in viral replication.
    Journal of Virology 09/2014; 88(22). DOI:10.1128/JVI.02538-14 · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cervical cancer is the second most common cancer among women worldwide and remains a clinical problem despite improvements in early detection and therapy. The human papillomavirus (HPV) type 16 (HPV16) E7 oncoprotein expressed in cervical carcinoma cells are considered as attractive tumor-specific antigen targets for immunotherapy. Since the transformation potential of the oncogenes, vaccination based of these oncogenes is not safe. In present study, DNA vaccine expressing the modified variant with mutation in pRb-binding motif of the HPV-16 E7 oncoprotein was generated. A novel modified E7 gene with mutation in LYCYE motif was designed and constructed and the immunogenicity and antitumor effect of therapeutic DNA vaccines encoding the mutant and wild type of E7 gene were investigated. The L-Y-C-Y-E pRb-binding motif of E7 proteins has been involved in the immortalization and transformation of the host cell. The results showed that the mutant and wild type HPV-16 E7 vectors expressed the desired protein. Furthermore, the immunological mechanism behind mutant E7 DNA vaccine can be attributed at least partially to increased cytotoxic T lymphocyte, accompanied by the up-regulation of Th1-cytokine IFN- γ and TNF- β and down-regulation of Th3-cytokine TGF-β. Immunized mice with mutant plasmid demonstrated significantly stronger cell immune responses and higher levels of tumor protection than wild-type E7 DNA vaccine. The results exhibit that modified E7 DNA vaccine may be a promising candidate for development of therapeutic vaccine against HPV-16 cancers.
    Journal of Virological Methods 05/2014; DOI:10.1016/j.jviromet.2014.05.013 · 1.88 Impact Factor

Preview

Download
0 Downloads
Available from