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Analysis of potential phosphorylation sites in human T cell leukemia virus type 1 Tax


The human T cell leukemia virus type 1 (HTLV-1) Tax is a phosphoprotein, however, the contribution of phosphorylation to Tax activity is unknown. Previous studies have shown that phosphorylation of Tax occurs on serine residue(s), within one tryptic fragment, in response to 4β-phorbol-12β-myristate-13α-acetate, in both mouse and human cells. Studies were conducted in multiple cell lines to identify the specific phosphorylated serines as a prelude to functional analysis. The phosphorylation pattern of Tax was found to be different in 293T and COS-7 cells in comparison with MT-4 and Px-1 cells. However, one tryptic fragment remained consistent in comigration analyses among all cell lines. Using selected Tax serine mutants a tryptic fragment containing a serine at residue 113 believed to be the site of phosphorylation of Tax did not comigrate with the common phosphorylated tryptic fragment. Analysis of selected Tax mutants for ability totrans-activate the cytomegalovirus promoter demonstrated mutation of serine 77 to alanine reducedtrans-activation by 90% compared to wild-type Tax. However, examination of the phosphorylation pattern of the serine 77 mutant demonstrated that it is not the site of phosphorylation. These studies demonstrate the importance of using relevant cell lines to characterize the role of phosphorylation in protein function.

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  1. 1

    Ballard DW, Bohnlein E, Lowenthal JW, Wano Y, Franza BR, Greene WC. HTLV-1 Tax induces cellular proteins that activate the kappa B element in the IL-2 receptor alpha gene. Science 242:1652–1655;1988.

  2. 2

    Beimling P, Moelling K. Tax-independent binding of multiple cellular factors to Tax-response element DNA of HTLV-1. Oncogene 5:361–368;1990.

  3. 3

    Beraud C, Sun SC, Ganchi P, Ballard DW, Greene WC. Human T-cell leukemia virus type I Tax associates with and is negatively regulated by the NF-kappa B2 p 100 gene product: Implications for viral latency. Mol Cell Biol 14:1374–1382;1994.

  4. 4

    Bex F, Yin M-J, Burny A, Gaynor RB. Differential transcriptional activation by human T-cell leukemia virus type 1 Tax mutants is mediated by distinct interactions with CREB binding protein and p300. Mol Cell Biol 18:2392–2405.

  5. 5

    Bosilevac JM, Gilchrist CA, Jankowski PE, Paul S, Rees AR, Hinrichs SH. Inhibition of activating transcription factor 1- and cAMP-responsive element-binding protein-activated transcription by an intracellular single chain Fv fragment. J Biol Chem 273:16874–16879;1998.

  6. 6

    Boyle WJ, Ver Der Geer P, Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thinlayer cellulose plates. Methods Enzymol 201:110–149;1991.

  7. 7

    Brasier AR, Tate JE, Habener JF. Optimized use of firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7:1116–1122;1989.

  8. 8

    Brauweiler A, Garl P, Franklin AA, Giebler HA, Nyborg JK. A molecular mechanism for human T-cell leukemia virus latency and Tax transactivation. J Biol Chem 270:12814–12822;1995.

  9. 9

    Chen C, Okayama H. High efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752;1987.

  10. 10

    Fontes J, Strawhecker J, Bills ND, Lewis RE, Hinrichs S. Phorbol esters modulate phosphorylation of human T-cell leukemia virus type I tax. J Virol 67:4436–4441;1993.

  11. 11

    Fujii M, Tsuchiya H, Chuhjo T, Akizawa T, Seiki M. Interaction of HTLV-1 Tax1 with p67SRF causes the aberrant induction of cellular immediate early genes through CArG boxes. Genes Dev 6:2066–2076;1992.

  12. 12

    Gallo R, Kalyanaraman VS, Sarngaharan MG, Sliski A, Vonderheid EC, Maeda M, Nakao Y, Yamad K, Ito Y, Gutensohn N, Murphy S, Bunn PAJ, Catovsky D, Greaves MF, Blayney DW, Blattner W, Jarrett WFH, zur Hausen H, Seligmann M, Brouett JC, Haynes BF, Jegasothy BV, Jaffe E, Cossman J, Broder S, Fisher RI, Golde DW, Robert-Gournoff M. Association of the human type C retrovirus with a subset of adult T-cell cancers. Cancer Res 43:3892–3899;1983.

  13. 13

    Gessain A, Barin F, Vernant JC, Gout O, Maurs L, Calendar A, de The G. Antibodies to human T lymphotropic virus type I in patients with tropical spastic paraparesis. Lancet ii:407–409;1985.

  14. 14

    Gilchrist CA, Orten JD, Hinrichs SH. Evidence for the role of cyclic-AMP responsive elements in human virus replication and disease. J Biomed Sci 3:293–306;1996.

  15. 15

    Green JE. Trans-activation of nerve growth factor in transgenic mice containing the human T-cell lymphotropic virus type I Tax gene. Mol Cell Biol 11:4635–4641;1991.

  16. 16

    Harrod R, Tang Y, Nicot C, Lu H, Vassilev A, Nakatani Y, Giam C-Z. An exposed KID-like domain in human T-cell lymphotropic virus type 1 Tax is responsible for the recruitment of coactivators CBP/p300. Mol Cell Biol 18:5052–5061;1998.

  17. 17

    Hinrichs SH, Nerenberg M, Reynolds RK, Khoury G, Jay G. A transgenic mouse model for human neurofibromatosis. Science 237:1340–1343;1987.

  18. 18

    Jeang K-T, Brady J, Radonovich M, Duvall J, Khoury G. p40x Trans-activation of the HTLV-1 LTR promoter. Proceedings of the UCLA Symposium on mechanisms of Control of Gene Expression 67:181–189;1988.

  19. 19

    Jeang K-T, Boros I, Brady J, Radonovich M, Khoury G. Characterization of cellular factors that interact with the human T-cell leukemia virus type I p40x-responsive 21-base-pair sequence. J Virol 62:4499–4509;1988.

  20. 20

    Jensen FC, Girardi AJ, Gilden RV, Koprowski H. Infection of human and simian tissue cultures with rous sarcoma virus. Proc Natl Acad Sci USA 52:53–59;1964.

  21. 21

    Jin DY, Spencer F, Jeang KT. Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell 93:81–91;1998.

  22. 22

    Lewis RE, Volle DJ, Sanderson SD. Phorbol ester stimulates phosphorylation on serine 1327 of the human insulin receptor. J Biol Chem 269:262549–26266;1994.

  23. 23

    Low KG, Dorner LF, Dinali BF, Grossman J, Jean K-T. HTLV-1 Tax releases cell-cycle arrest induced by p16INK4a. J Virol 71:1956–1962;1997.

  24. 24

    Miyoshi I, Kubonishi I, Yaoshimoto S, Akagi T, Ohtsuki Y, Shiraishi Y, Nagata K, Hinuma Y. Detection of type C particles in a cord T-cell line derived by co-cultivation of normal human cord leukocytes and human leukemic T-cells. Nature 296:770–771;1981.

  25. 25

    Moch H, Lang D, Stamminger T. Strong transactivation of the human cytomegalovirus major immediate-early enhancer by p40tax of human T-cell leukemia virus type I via two repetitive tax-responsive sequence elements. J Virol 66:7346–7354;1992.

  26. 26

    Osame M, Igata A, Matsumoto M, Izumo S, Usuku K, Rosales RL, Tara M. HTLV-1 associated myelopathy, a new clinical entity. Lancet i:1031–1032;1986.

  27. 27

    Radonovich M, Jeang K-T. Activation of the human T-cell leukemia virus type I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate by Tax (p40x) occurs through similar but functionally distinct target sequences. J Virol 63:2987–2994;1989.

  28. 28

    Ressler S, Morris GF, Marriott SJ. Human T-cell leukemia virus type I Tax transactivates the human proliferating cell nuclear antigen promoter. J Virol 71:1181–1190;1997.

  29. 29

    Rotondaro L, Mele A, Rovera G. Efficiency of different viral promoters in directing gene expression in mammalian cells: Effect of 3′-untranslated sequences. Gene 168:195–198;1996.

  30. 30

    Semmes OJ, Jeang K-T. Mutational analysis of human T-cell leukemia virus type 1 Tax: Regions necessary for function determined with 47 mutant proteins. J Virol 66:7183–7192;1992.

  31. 31

    Smith M, Greene W. Identification of HTLV-1 tax transactivator mutants exhibiting novel transcriptional phenotypes. Genes Dev 4:1875–1885;1990.

  32. 32

    Sodroski J, Rosen C, Goh WC, Haseltime W. A transcriptional activator protein encoded by the x-lor region of the human T-cell leukemia virus. Science 228:1430–1434;1985.

  33. 33

    Suzuki T, Kitao S, Matsushime H, Yoshida M. HTLV-1 Tax protein interacts with cyclin-dependent kinase inhibitor p161NK4A and counteracts its inhibitory activity towards CDK4. EMBO J 15:1607–1614;1996.

  34. 34

    Yin MJ, Paulssen EJ, Seeler JS, Gaynor RB. Protein domains involved in both in vivo and in vitro interactions between human T-cell leukemia virus type I tax and CREB. J Virol 69:3420–3432;1995.

  35. 35

    Yin MJ, Christerson LB, Yamamoto Y, Kwak YT, Xu S, Mercurio F, Barbosa M, Cobb MH, Gaynor RB. HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation. Cell 93:875–884;1988.

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Boehm, A.K., Stawhecker, J.A., John Semmes, O. et al. Analysis of potential phosphorylation sites in human T cell leukemia virus type 1 Tax. J Biomed Sci 6, 206–212 (1999).

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Key Words

  • Tax
  • HTLV-1
  • Trans-activation
  • Phosphorylation
  • Mutagenesis
  • Transcription
  • Genetics