1887

Abstract

Using a yeast two-hybrid screen of a B-cell cDNA library with an Epstein–Barr nuclear antigen 5 (EBNA5) molecule containing seven repeats of the WW domain as bait, we have isolated the EBNA5-interacting protein HAX-1. HAX-1 has previously been shown to associate with HS1, a protein specifically expressed in cells of the haematopoietic lineage, and is thought to be involved in signal transduction in B-cells. Immunofluorescence experiments showed that HAX-1 co-localized with the hsp60 protein that is associated with the mitochondria in the cell cytoplasm. Pull down experiments with a fusion protein between glutathione -transferase and the seven copy repeat EBNA5 synthesized in bacteria and in yeast cells confirmed that HAX-1 can interact with EBNA5 . Conventionally, EBNA5 is regarded as a nuclear protein. However, we show here that the smallest EBNA5 species, composed of the unique Y domain and only one copy of the WW repeat domain, like HAX-1, co-localizes with the mitochondrial hsp60 protein in the B-cell cytoplasm. Furthermore, immunoprecipitation experiments demonstrate that the single repeat EBNA5 associates with HAX-1 in transfected B-lymphoblastoid cells.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-82-7-1581
2001-07-01
2019-12-14
Loading full text...

Full text loading...

/deliver/fulltext/jgv/82/7/0821581a.html?itemId=/content/journal/jgv/10.1099/0022-1317-82-7-1581&mimeType=html&fmt=ahah

References

  1. Alfieri, C., Birkenbach, M. & Kieff, E. ( 1991; ). Early events in Epstein–Barr virus infection of human B lymphocytes. Virology 181, 595-608.[CrossRef]
    [Google Scholar]
  2. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A. & Struhl, K. (1996). Current Protocols in Molecular Biology, unit 20.2. New York: John Wiley & Sons.
  3. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A. & Struhl, K. (1997). Current Protocols in Molecular Biology, unit 13.6.2. New York: John Wiley & Sons.
  4. Ben Bassat, H., Goldblum, N., Mitrani, S., Goldblum, T., Yoffey, J. M., Cohen, M. M., Bentwich, Z., Ramot, B., Klein, E. & Klein, G. ( 1977; ). Establishment in continuous culture of a new type of lymphocyte from a ‘Burkitt like’ malignant lymphoma (line D.G.-75). International Journal of Cancer 19, 27-33.[CrossRef]
    [Google Scholar]
  5. Boyd, J. M., Malstrom, S., Subramanian, T., Venkatesh, L. K., Schaeper, U., Elangovan, B., D’Sa-Eipper, C. & Chinnadurai, G. ( 1994; ). Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell 79, 341–351: erratum 1120.[CrossRef]
    [Google Scholar]
  6. Chen, G., Ray, R., Dubik, D., Shi, L., Cizeau, J., Bleackley, R. C., Saxena, S., Gietz, R. D. & Greenberg, A. H. ( 1997; ). The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis. Journal of Experimental Medicine 186, 1975-1983.[CrossRef]
    [Google Scholar]
  7. Cludts, I. & Farrell, P. J. ( 1998; ). Multiple functions within the Epstein–Barr virus EBNA-3A protein. Journal of Virology 72, 1862-1869.
    [Google Scholar]
  8. Dillner, J. & Kallin, B. ( 1988; ). The Epstein–Barr virus proteins. Advances in Cancer Research 50, 95-158.
    [Google Scholar]
  9. Dillner, J., Kallin, B., Alexander, H., Ernberg, I., Uno, M., Ono, Y., Klein, G. & Lerner, R. A. ( 1986; ). An Epstein–Barr virus (EBV)-determined nuclear antigen (EBNA5) partly encoded by the transformation-associated Bam WYH region of EBV DNA: preferential expression in lymphoblastoid cell lines. Proceedings of the National Academy of Sciences, USA 83, 6641-6645.[CrossRef]
    [Google Scholar]
  10. Finke, J., Rowe, M., Kallin, B., Ernberg, I., Rosén, A., Dillner, J. & Klein, G. ( 1987; ). Monoclonal and polyclonal antibodies against Epstein–Barr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitt’s lymphoblastoid cell lines. Journal of Virology 61, 3870-3878.
    [Google Scholar]
  11. Fukuda, T., Kitamura, D., Taniuchi, I., Maekawa, Y., Benhamou, L. E., Sarthou, P. & Watanabe, T. ( 1995; ). Restoration of surface IgM-mediated apoptosis in an anti-IgM-resistant variant of WEHI-231 lymphoma cells by HS1, a protein-tyrosine kinase substrate. Proceedings of the National Academy of Sciences, USA 92, 7302-7306.[CrossRef]
    [Google Scholar]
  12. Gallagher, A. R., Cedzich, A., Gretz, N., Somlo, S. & Witzgall, R. ( 2000; ). The polycystic kidney disease protein PKD2 interacts with Hax-1, a protein associated with the actin cytoskeleton. Proceedings of the National Academy of Sciences, USA 97, 4017-4022.[CrossRef]
    [Google Scholar]
  13. Harada, S. & Kieff, E. ( 1997; ). Epstein–Barr nuclear protein LP stimulates EBNA-2 acidic domain mediated transcriptional activation. Journal of Virology 71, 6611-6618.
    [Google Scholar]
  14. Hess, J. L. & Korsmeyer, S. J. ( 1998; ). Life, death and nuclear spots. Nature Genetics 20, 220-222.[CrossRef]
    [Google Scholar]
  15. Inman, G. J. & Farrell, P. J. ( 1995; ). Epstein–Barr virus EBNA-LP and transcription regulation of pRB, p107 and p53 in transfection assays. Journal of General Virology 76, 2141-2149.[CrossRef]
    [Google Scholar]
  16. Kawaguchi, Y., Nakajima, K., Igarashi, M., Morita, T., Tanaka, M., Suzuki, M., Yokoyama, A., Matsuda, G., Kato, K., Kanamori, M. & Hirai, K. ( 2000; ). Interaction of Epstein–Barr virus nuclear antigen leader protein (EBNA-LP) with HS1-associated protein X-1: implication of cytoplasmic function of EBNA-LP. Journal of Virology 74, 10104-10111.[CrossRef]
    [Google Scholar]
  17. Kieff, E. ( 1996; ). Epstein–Barr virus and its replication. In Fields Virology , pp. 2343-2396. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia:Lippincott–Raven.
  18. Kitay, M. K. & Rowe, D. T. ( 1996a; ). Cell cycle stage-specific phosphorylation of the Epstein–Barr virus immortalization protein EBNA-LP. Journal of Virology 70, 7885-7893.
    [Google Scholar]
  19. Kitay, M. K. & Rowe, D. T. ( 1996b; ). Protein–protein interactions between Epstein–Barr nuclear antigen-LP and cellular gene products: binding of 70-kilodalton heat shock proteins. Virology 220, 91-99.[CrossRef]
    [Google Scholar]
  20. Mannick, J. B., Tong, X., Hemnes, A. & Kieff, E. ( 1995; ). The Epstein–Barr nuclear antigen leader protein associates with hsp72/hsc73. Journal of Virology 69, 8169-8172.
    [Google Scholar]
  21. Matera, A. G. ( 1999; ). Nuclear bodies: multifaceted subdomains of the interchromatin space. Trends in Cell Biology 9, 302-309.[CrossRef]
    [Google Scholar]
  22. Nitsche, F., Bell, A. & Rickinson, A. ( 1997; ). Epstein–Barr leader protein enhances EBNA-2 mediated transactivation of latent membrane protein 1 expression: a role for the W1W2 repeat domain. Journal of Virology 71, 6619-6627.
    [Google Scholar]
  23. Peng, R., Tan, J. & Ling, P. D. ( 2000; ). Conserved regions in the Epstein–Barr virus leader protein define distinct domains required for nuclear localization and transcriptional cooperation with EBNA2. Journal of Virology 74, 9953-9963.[CrossRef]
    [Google Scholar]
  24. Sinclair, A. J., Ignacio, P., Peters, G. & Farrell, P. ( 1994; ). EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalization of resting human B lymphocytes by Epstein–Barr virus. EMBO Journal 13, 3321-3328.
    [Google Scholar]
  25. Soltys, B. J. & Gupta, R. S. ( 1996; ). Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (Hsp60) in mammalian cells. Experimental Cell Research 222, 16-27.[CrossRef]
    [Google Scholar]
  26. Suzuki, Y., Demoliere, C., Kitamura, D., Takeshita, H., Deuschle, U. & Watanabe, T. ( 1997; ). HAX-1, a novel intracellular protein, localized on mitochondria, directly associates with HS1, a substrate of Src family tyrosine kinases. Journal of Immunology 158, 2736-2744.
    [Google Scholar]
  27. Szekely, L., Selivanova, G., Magnusson, K. P., Klein, G. & Wiman, K. G. ( 1993; ). EBNA-5, an Epstein–Barr virus encoded nuclear antigen, binds to the retinoblastoma and p53 proteins. Proceedings of the National Academy of Sciences, USA 90, 5455-5459.[CrossRef]
    [Google Scholar]
  28. Szekely, L., Jiang, W.-Q., Pokrovskaja, K., Wiman, K. G., Klein, G. & Ringertz, N. ( 1995a; ). Reversible nucleolar translocation of Epstein–Barr virus-encoded EBNA-5 and hsp70 proteins after exposure to heat shock or cell density congestion. Journal of General Virology 76, 2423-2432.[CrossRef]
    [Google Scholar]
  29. Szekely, L., Pokrovskaja, K., Jiang, W.-Q., Selivanova, G., Löwbeer, M., Ringertz, N., Wiman, K. G. & Klein, G. ( 1995b; ). Resting B-cells, EBV-infected B-blasts and established lymphoblastoid cell lines differ in their Rb, p53 and EBNA-5 expression patterns. Oncogene 10, 1869-1874.
    [Google Scholar]
  30. Szekely, L., Pokrovskaja, K., Jiang, W.-Q., de The, H., Ringertz, N. & Klein, G. ( 1996; ). The Epstein–Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies. Journal of Virology 70, 2562-2568.
    [Google Scholar]
  31. Taniuchi, I., Kitamura, D., Maekawa, Y., Fukuda, T., Kishi, H. & Watanabe, T. ( 1995; ). Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired in mice lacking HS1 protein, a substrate of the antigen-receptor-coupled tyrosine kinases. EMBO Journal 14, 3664-3678.
    [Google Scholar]
  32. Yamanashi, Y., Fukuda, T., Nishizumi, H., Inazu, T., Higashi, K., Kitamura, D., Ishida, T., Yamamura, H., Watanabe, T. & Yamamoto, T. ( 1997; ). Role of tyrosine phosphorylation of HS1 in B cell antigen receptor-mediated apoptosis. Journal of Experimental Medicine 185, 1387-1392.[CrossRef]
    [Google Scholar]
  33. Yasuda, M., Theodorakis, P., Subramanian, T. & Chinnadurai, G. ( 1998; ). Adenovirus E1B-19K/BCL-2 interacting protein BNIP3 contains a BH3 domain and a mitochondrial targeting sequence. Journal of Biological Chemistry 273, 12415-12421.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-82-7-1581
Loading
/content/journal/jgv/10.1099/0022-1317-82-7-1581
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error