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PMID: 11333893 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Identification of major phosphorylation sites of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP): ability of EBNA-LP to induce latent membrane protein 1 cooperatively with EBNA-2 is regulated by phosphorylation.

Journal of virology ·Vol. 75 ·No. 11 ·2001-06-00 ·Pages 5119-28

Yokoyama A, Tanaka M, Matsuda G, Kato K, Kanamori M, Kawasaki H, Hirano H, Kitabayashi I, Ohki M, Hirai K, Kawaguchi Y

Abstract

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a phosphoprotein suggested to play important roles in EBV-induced immortalization of B cells. One of the potential functions of EBNA-LP is a cooperative induction with EBNA-2 of viral and cellular gene expression, including that of the genes for viral latent membrane protein 1 (LMP-1) and cellular cyclin D2. We report here that the phosphorylation of EBNA-LP by cellular kinase(s) is critical to its ability to cooperate with EBNA-2 in up-regulating the expression of LMP-1 in a B-lymphoma cell line. Our conclusion is based on the following observations. (i) Mass-spectrometric analysis of purified EBNA-LP and mutational analyses of EBNA-LP revealed that the serine residue at position 35 in the W2 repeat domain is the major phosphorylation site of EBNA-LP in vivo. (ii) Substitutions of this site in each W2 repeat domain with alanine markedly reduced the ability of the protein to induce LMP-1 expression in combination with EBNA-2 in Akata cells. (iii) Replacement at the major phosphorylation sites with glutamic acids restored the wild-type phenotype. It is well established that this substitution mimics constitutive phosphorylation. These results indicated that the coactivator function of EBNA-LP is regulated by phosphorylation.

MeSH Terms
Alanine/metabolism Amino Acid Substitution Animals B-Lymphocytes Binding Sites Burkitt Lymphoma COS Cells Cell Line Cell Line, Transformed Epstein-Barr Virus Nuclear Antigens/metabolism Gene Deletion Glutamic Acid/metabolism Herpesvirus 4, Human/metabolism Humans Mass Spectrometry Phosphorylation Phosphotransferases/metabolism Transfection Up-Regulation Viral Matrix Proteins/biosynthesis Viral Proteins/genetics,isolation & purification,metabolism
Chemicals
EBNA-2 protein, Human herpesvirus 4 EBNA-LP protein, Human herpesvirus 4 EBV-associated membrane antigen, Epstein-Barr virus Epstein-Barr Virus Nuclear Antigens Viral Matrix Proteins Viral Proteins Glutamic Acid Phosphotransferases Alanine
Authors & Affiliations
11 authors, click to expand affiliations / ORCID
Yokoyama A
Department of Tumor Virology, Division of Virology and Immunology, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan.
Tanaka M
Matsuda G
Kato K
Kanamori M
Kawasaki H
Hirano H
Kitabayashi I
Ohki M
Hirai K
Kawaguchi Y
References (40)
40 references, click to expand
  1. Characterization of a latent protein encoded by the large internal repeats and the BamHI Y fragment of the Epstein-Barr virus (EBV) genome.
    Virology. 1988 Oct;166(2):586-90 PMID: 2845663
  2. Interaction of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) with HS1-associated protein X-1: implication of cytoplasmic function of EBNA-LP.
    J Virol. 2000 Nov;74(21):10104-11 PMID: 11024139
  3. Genetic analysis of immortalizing functions of Epstein-Barr virus in human B lymphocytes.
    Nature. 1989 Aug 3;340(6232):393-7 PMID: 2547164
  4. Subnuclear localization and phosphorylation of Epstein-Barr virus latent infection nuclear proteins.
    Virology. 1990 Jun;176(2):563-74 PMID: 2161150
  5. The Epstein-Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation.
    J Virol. 1991 Dec;65(12):6826-37 PMID: 1658376
  6. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations.
    Methods Enzymol. 1991;200:62-81 PMID: 1956339
  7. Co-localization of the retinoblastoma protein and the Epstein-Barr virus-encoded nuclear antigen EBNA-5.
    Exp Cell Res. 1991 Dec;197(2):314-8 PMID: 1659990
  8. The consensus sequences for cdc2 kinase and for casein kinase-2 are mutually incompatible. A study with peptides derived from the beta-subunit of casein kinase-2.
    FEBS Lett. 1992 Apr 13;301(1):111-4 PMID: 1451779
  9. EBNA-5, an Epstein-Barr virus-encoded nuclear antigen, binds to the retinoblastoma and p53 proteins.
    Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5455-9 PMID: 8390666
  10. Production of high-titer helper-free retroviruses by transient transfection.
    Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392-6 PMID: 7690960
  11. The conserved domain CR2 of Epstein-Barr virus nuclear antigen leader protein is responsible not only for nuclear matrix association but also for nuclear localization.
    Virology. 2001 Jan 20;279(2):401-13 PMID: 11162796
  12. Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.
    J Cell Biol. 1984 Jun;98(6):1973-84 PMID: 6202700
  13. DNA sequence and expression of the B95-8 Epstein-Barr virus genome.
    Nature. 1984 Jul 19-25;310(5974):207-11 PMID: 6087149
  14. Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization.
    Proc Natl Acad Sci U S A. 1984 Oct;81(19):5951-5 PMID: 6091123
  15. Protein serine/threonine kinases.
    Annu Rev Biochem. 1987;56:567-613 PMID: 2956925
  16. Monoclonal and polyclonal antibodies against Epstein-Barr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitt's lymphoma and lymphoblastoid cell lines.
    J Virol. 1987 Dec;61(12):3870-8 PMID: 2824821
  17. EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalization of resting human B lymphocytes by Epstein-Barr virus.
    EMBO J. 1994 Jul 15;13(14):3321-8 PMID: 8045261
  18. Epstein-Barr virus EBNA-LP and transcription regulation properties of pRB, p107 and p53 in transfection assays.
    J Gen Virol. 1995 Sep;76 ( Pt 9):2141-9 PMID: 7561751
  19. Reversible nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 and hsp70 proteins after exposure to heat shock or cell density congestion.
    J Gen Virol. 1995 Oct;76 ( Pt 10):2423-32 PMID: 7595346
  20. The Epstein-Barr virus nuclear antigen leader protein associates with hsp72/hsc73.
    J Virol. 1995 Dec;69(12):8169-72 PMID: 7494344
  21. The Epstein-Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies.
    J Virol. 1996 Apr;70(4):2562-8 PMID: 8642686
  22. Protein-protein interactions between Epstein-Barr virus nuclear antigen-LP and cellular gene products: binding of 70-kilodalton heat shock proteins.
    Virology. 1996 Jun 1;220(1):91-9 PMID: 8659132
  23. Cell cycle stage-specific phosphorylation of the Epstein-Barr virus immortalization protein EBNA-LP.
    J Virol. 1996 Nov;70(11):7885-93 PMID: 8892911
  24. Interaction of herpes simplex virus 1 alpha regulatory protein ICP0 with elongation factor 1delta: ICP0 affects translational machinery.
    J Virol. 1997 Feb;71(2):1019-24 PMID: 8995621
  25. HAX-1, a novel intracellular protein, localized on mitochondria, directly associates with HS1, a substrate of Src family tyrosine kinases.
    J Immunol. 1997 Mar 15;158(6):2736-44 PMID: 9058808
  26. Inducible expression and phosphorylation of coactivator BOB.1/OBF.1 in T cells.
    Science. 1997 Jul 11;277(5323):221-5 PMID: 9211847
  27. Epstein-Barr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation.
    J Virol. 1997 Sep;71(9):6611-8 PMID: 9261383
  28. Epstein-Barr virus leader protein enhances EBNA-2-mediated transactivation of latent membrane protein 1 expression: a role for the W1W2 repeat domain.
    J Virol. 1997 Sep;71(9):6619-28 PMID: 9261384
  29. Histone acetylation in chromatin structure and transcription.
    Nature. 1997 Sep 25;389(6649):349-52 PMID: 9311776
  30. Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3.
    J Virol. 1997 Oct;71(10):7328-36 PMID: 9311810
  31. Eukaryotic elongation factor 1delta is hyperphosphorylated by the protein kinase encoded by the U(L)13 gene of herpes simplex virus 1.
    J Virol. 1998 Mar;72(3):1731-6 PMID: 9499021
  32. Histone acetylation and transcriptional regulatory mechanisms.
    Genes Dev. 1998 Mar 1;12(5):599-606 PMID: 9499396
  33. Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A.
    Nature. 1998 Nov 12;396(6707):184-6 PMID: 9823900
  34. Phosphorylation by p44 MAP Kinase/ERK1 stimulates CBP histone acetyl transferase activity in vitro.
    Biochem Biophys Res Commun. 1999 Aug 19;262(1):157-62 PMID: 10448085
  35. Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses.
    J Virol. 2000 Jan;74(1):379-89 PMID: 10590127
  36. A protein kinase activity associated with Epstein-Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro.
    J Virol. 2000 Apr;74(7):3093-104 PMID: 10708424
  37. hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response.
    Nature. 2000 Mar 9;404(6774):201-4 PMID: 10724175
  38. Regulation of transcription factor function by phosphorylation.
    Cell Mol Life Sci. 2000 Aug;57(8-9):1172-83 PMID: 11028910
  39. Conserved regions in the Epstein-Barr virus leader protein define distinct domains required for nuclear localization and transcriptional cooperation with EBNA2.
    J Virol. 2000 Nov;74(21):9953-63 PMID: 11024123
  40. Synchronous and sequential activation of latently infected Epstein-Barr virus genomes.
    J Virol. 1989 Jan;63(1):445-9 PMID: 2462063
Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.75.11.5119-5128.2001
Published
2001-06-00
Pages
5119-28
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC114917
Subset
IM
External Links
PubMed source DOI: 10.1128/JVI.75.11.5119-5128.2001 PMC Full Text View journal details
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