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Mol Cell Biol. 1997 November; 17(11): 6427–6436.
PMCID: PMC232495

Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras.

Abstract

The HBx protein of hepatitis B virus (HBV) is a small transcriptional transactivator that is essential for infection by the mammalian hepadnaviruses and is thought to be a cofactor in HBV-mediated liver cancer. HBx stimulates signal transduction pathways by acting in the cytoplasm, which accounts for many but not all of its transcriptional activities. Studies have shown that HBx protein activates Ras and downstream Ras signaling pathways including Raf, mitogen-activated protein (MAP) kinase kinase kinase (MEK), and MAP kinases. In this study, we investigated the mechanism of activation of Ras by HBx because it has been found to be central to the ability of HBx protein to stimulate transcription and to release growth arrest in quiescent cells. In contrast to the transient but strong stimulation of Ras typical of autocrine factors, activation of Ras by HBx protein was found to be constitutive but moderate. HBx induced the association of Ras upstream activating proteins Shc, Grb2, and Sos and stimulated GTP loading onto Ras, but without directly participating in complex formation. Instead, HBx is shown to stimulate Ras-activating proteins by functioning as an intracellular cytoplasmic activator of the Src family of tyrosine kinases, which can signal to Ras. HBx protein stimulated c-Src and Fyn kinases for a prolonged time. Activation of Src is shown to be indispensable for a number of HBx activities, including activation of Ras and the Ras-Raf-MAP kinase pathway and stimulation of transcription mediated by transcription factor AP-1. Importantly, HBx protein expressed in cultured cells during HBV replication is shown to activate the Ras signaling pathway. Mechanisms by which HBx protein might activate Src kinases are discussed.

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Selected References

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  • Aronheim A, Engelberg D, Li N, al-Alawi N, Schlessinger J, Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell. 1994 Sep 23;78(6):949–961. [PubMed]
  • Aufiero B, Schneider RJ. The hepatitis B virus X-gene product trans-activates both RNA polymerase II and III promoters. EMBO J. 1990 Feb;9(2):497–504. [PubMed]
  • Balsano C, Avantaggiati ML, Natoli G, De Marzio E, Will H, Perricaudet M, Levrero M. Full-length and truncated versions of the hepatitis B virus (HBV) X protein (pX) transactivate the cmyc protooncogene at the transcriptional level. Biochem Biophys Res Commun. 1991 May 15;176(3):985–992. [PubMed]
  • Beasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan. Lancet. 1981 Nov 21;2(8256):1129–1133. [PubMed]
  • Benn J, Schneider RJ. Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10350–10354. [PubMed]
  • Benn J, Schneider RJ. Hepatitis B virus HBx protein deregulates cell cycle checkpoint controls. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11215–11219. [PubMed]
  • Benn J, Su F, Doria M, Schneider RJ. Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases. J Virol. 1996 Aug;70(8):4978–4985. [PMC free article] [PubMed]
  • Blum HE, Zhang ZS, Galun E, von Weizsäcker F, Garner B, Liang TJ, Wands JR. Hepatitis B virus X protein is not central to the viral life cycle in vitro. J Virol. 1992 Feb;66(2):1223–1227. [PMC free article] [PubMed]
  • Boguski MS, McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. [PubMed]
  • Buday L, Downward J. Epidermal growth factor regulates the exchange rate of guanine nucleotides on p21ras in fibroblasts. Mol Cell Biol. 1993 Mar;13(3):1903–1910. [PMC free article] [PubMed]
  • Butel JS, Lee TH, Slagle BL. Is the DNA repair system involved in hepatitis-B-virus-mediated hepatocellular carcinogenesis? Trends Microbiol. 1996 Mar;4(3):119–124. [PubMed]
  • Chen HS, Kaneko S, Girones R, Anderson RW, Hornbuckle WE, Tennant BC, Cote PJ, Gerin JL, Purcell RH, Miller RH. The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks. J Virol. 1993 Mar;67(3):1218–1226. [PMC free article] [PubMed]
  • Cheong JH, Yi M, Lin Y, Murakami S. Human RPB5, a subunit shared by eukaryotic nuclear RNA polymerases, binds human hepatitis B virus X protein and may play a role in X transactivation. EMBO J. 1995 Jan 3;14(1):143–150. [PubMed]
  • Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol. 1995;13:29–60. [PubMed]
  • Courtneidge SA. Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. EMBO J. 1985 Jun;4(6):1471–1477. [PubMed]
  • Courtneidge SA, Dhand R, Pilat D, Twamley GM, Waterfield MD, Roussel MF. Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor. EMBO J. 1993 Mar;12(3):943–950. [PubMed]
  • Courtneidge SA, Smith AE. Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature. 1983 Jun 2;303(5916):435–439. [PubMed]
  • Cross JC, Wen P, Rutter WJ. Transactivation by hepatitis B virus X protein is promiscuous and dependent on mitogen-activated cellular serine/threonine kinases. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8078–8082. [PubMed]
  • Dandri M, Schirmacher P, Rogler CE. Woodchuck hepatitis virus X protein is present in chronically infected woodchuck liver and woodchuck hepatocellular carcinomas which are permissive for viral replication. J Virol. 1996 Aug;70(8):5246–5254. [PMC free article] [PubMed]
  • Dikic I, Tokiwa G, Lev S, Courtneidge SA, Schlessinger J. A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation. Nature. 1996 Oct 10;383(6600):547–550. [PubMed]
  • Dilworth SM, Brewster CE, Jones MD, Lanfrancone L, Pelicci G, Pelicci PG. Transformation by polyoma virus middle T-antigen involves the binding and tyrosine phosphorylation of Shc. Nature. 1994 Jan 6;367(6458):87–90. [PubMed]
  • Doria M, Klein N, Lucito R, Schneider RJ. The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors. EMBO J. 1995 Oct 2;14(19):4747–4757. [PubMed]
  • Downward J, Graves JD, Warne PH, Rayter S, Cantrell DA. Stimulation of p21ras upon T-cell activation. Nature. 1990 Aug 23;346(6286):719–723. [PubMed]
  • Erpel T, Courtneidge SA. Src family protein tyrosine kinases and cellular signal transduction pathways. Curr Opin Cell Biol. 1995 Apr;7(2):176–182. [PubMed]
  • Ganem D, Varmus HE. The molecular biology of the hepatitis B viruses. Annu Rev Biochem. 1987;56:651–693. [PubMed]
  • Haviv I, Vaizel D, Shaul Y. pX, the HBV-encoded coactivator, interacts with components of the transcription machinery and stimulates transcription in a TAF-independent manner. EMBO J. 1996 Jul 1;15(13):3413–3420. [PubMed]
  • Haviv I, Vaizel D, Shaul Y. The X protein of hepatitis B virus coactivates potent activation domains. Mol Cell Biol. 1995 Feb;15(2):1079–1085. [PMC free article] [PubMed]
  • Huang J, Kwong J, Sun EC, Liang TJ. Proteasome complex as a potential cellular target of hepatitis B virus X protein. J Virol. 1996 Aug;70(8):5582–5591. [PMC free article] [PubMed]
  • Kaplan KB, Swedlow JR, Morgan DO, Varmus HE. c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism. Genes Dev. 1995 Jun 15;9(12):1505–1517. [PubMed]
  • Kekulé AS, Lauer U, Weiss L, Luber B, Hofschneider PH. Hepatitis B virus transactivator HBx uses a tumour promoter signalling pathway. Nature. 1993 Feb 25;361(6414):742–745. [PubMed]
  • Kim CM, Koike K, Saito I, Miyamura T, Jay G. HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature. 1991 May 23;351(6324):317–320. [PubMed]
  • Koike K, Moriya K, Iino S, Yotsuyanagi H, Endo Y, Miyamura T, Kurokawa K. High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice. Hepatology. 1994 Apr;19(4):810–819. [PubMed]
  • Koike K, Moriya K, Yotsuyanagi H, Iino S, Kurokawa K. Induction of cell cycle progression by hepatitis B virus HBx gene expression in quiescent mouse fibroblasts. J Clin Invest. 1994 Jul;94(1):44–49. [PMC free article] [PubMed]
  • Lee TH, Elledge SJ, Butel JS. Hepatitis B virus X protein interacts with a probable cellular DNA repair protein. J Virol. 1995 Feb;69(2):1107–1114. [PMC free article] [PubMed]
  • Lee TH, Finegold MJ, Shen RF, DeMayo JL, Woo SL, Butel JS. Hepatitis B virus transactivator X protein is not tumorigenic in transgenic mice. J Virol. 1990 Dec;64(12):5939–5947. [PMC free article] [PubMed]
  • Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B, Schlessinger J. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature. 1995 Aug 31;376(6543):737–745. [PubMed]
  • Levrero M, Balsano C, Natoli G, Avantaggiati ML, Elfassi E. Hepatitis B virus X protein transactivates the long terminal repeats of human immunodeficiency virus types 1 and 2. J Virol. 1990 Jun;64(6):3082–3086. [PMC free article] [PubMed]
  • Levrero M, Jean-Jean O, Balsano C, Will H, Perricaudet M. Hepatitis B virus (HBV) X gene expression in human cells and anti-HBx antibodies detection in chronic HBV infection. Virology. 1990 Jan;174(1):299–304. [PubMed]
  • Li BQ, Kaplan D, Kung HF, Kamata T. Nerve growth factor stimulation of the Ras-guanine nucleotide exchange factor and GAP activities. Science. 1992 Jun 5;256(5062):1456–1459. [PubMed]
  • Li N, Batzer A, Daly R, Yajnik V, Skolnik E, Chardin P, Bar-Sagi D, Margolis B, Schlessinger J. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature. 1993 May 6;363(6424):85–88. [PubMed]
  • Lowell CA, Soriano P. Knockouts of Src-family kinases: stiff bones, wimpy T cells, and bad memories. Genes Dev. 1996 Aug 1;10(15):1845–1857. [PubMed]
  • Lowenstein EJ, Daly RJ, Batzer AG, Li W, Margolis B, Lammers R, Ullrich A, Skolnik EY, Bar-Sagi D, Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. [PubMed]
  • Lucito R, Schneider RJ. Hepatitis B virus X protein activates transcription factor NF-kappa B without a requirement for protein kinase C. J Virol. 1992 Feb;66(2):983–991. [PMC free article] [PubMed]
  • Luttrell LM, Hawes BE, van Biesen T, Luttrell DK, Lansing TJ, Lefkowitz RJ. Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases. J Biol Chem. 1996 Aug 9;271(32):19443–19450. [PubMed]
  • Maguire HF, Hoeffler JP, Siddiqui A. HBV X protein alters the DNA binding specificity of CREB and ATF-2 by protein-protein interactions. Science. 1991 May 10;252(5007):842–844. [PubMed]
  • Messerschmitt AS, Dunant N, Ballmer-Hofer K. DNA tumor viruses and Src family tyrosine kinases, an intimate relationship. Virology. 1997 Jan 20;227(2):271–280. [PubMed]
  • Moodie SA, Paris MJ, Kolch W, Wolfman A. Association of MEK1 with p21ras.GMPPNP is dependent on B-Raf. Mol Cell Biol. 1994 Nov;14(11):7153–7162. [PMC free article] [PubMed]
  • Natoli G, Avantaggiati ML, Chirillo P, Costanzo A, Artini M, Balsano C, Levrero M. Induction of the DNA-binding activity of c-jun/c-fos heterodimers by the hepatitis B virus transactivator pX. Mol Cell Biol. 1994 Feb;14(2):989–998. [PMC free article] [PubMed]
  • Natoli G, Avantaggiati ML, Chirillo P, Puri PL, Ianni A, Balsano C, Levrero M. Ras- and Raf-dependent activation of c-jun transcriptional activity by the hepatitis B virus transactivator pX. Oncogene. 1994 Oct;9(10):2837–2843. [PubMed]
  • Pelicci G, Lanfrancone L, Grignani F, McGlade J, Cavallo F, Forni G, Nicoletti I, Grignani F, Pawson T, Pelicci PG. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell. 1992 Jul 10;70(1):93–104. [PubMed]
  • Pruett W, Yuan Y, Rose E, Batzer AG, Harada N, Skolnik EY. Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin. Mol Cell Biol. 1995 Mar;15(3):1778–1785. [PMC free article] [PubMed]
  • Qadri I, Maguire HF, Siddiqui A. Hepatitis B virus transactivator protein X interacts with the TATA-binding protein. Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1003–1007. [PubMed]
  • Rosen LB, Ginty DD, Weber MJ, Greenberg ME. Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras. Neuron. 1994 Jun;12(6):1207–1221. [PubMed]
  • Rozakis-Adcock M, McGlade J, Mbamalu G, Pelicci G, Daly R, Li W, Batzer A, Thomas S, Brugge J, Pelicci PG, et al. Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature. 1992 Dec 17;360(6405):689–692. [PubMed]
  • Rusanescu G, Qi H, Thomas SM, Brugge JS, Halegoua S. Calcium influx induces neurite growth through a Src-Ras signaling cassette. Neuron. 1995 Dec;15(6):1415–1425. [PubMed]
  • Schek N, Bartenschlager R, Kuhn C, Schaller H. Phosphorylation and rapid turnover of hepatitis B virus X-protein expressed in HepG2 cells from a recombinant vaccinia virus. Oncogene. 1991 Oct;6(10):1735–1744. [PubMed]
  • Seto E, Mitchell PJ, Yen TS. Transactivation by the hepatitis B virus X protein depends on AP-2 and other transcription factors. Nature. 1990 Mar 1;344(6261):72–74. [PubMed]
  • Slagle BL, Lee TH, Medina D, Finegold MJ, Butel JS. Increased sensitivity to the hepatocarcinogen diethylnitrosamine in transgenic mice carrying the hepatitis B virus X gene. Mol Carcinog. 1996 Apr;15(4):261–269. [PubMed]
  • Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, et al. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. [PubMed]
  • Spandau DF, Lee CH. trans-activation of viral enhancers by the hepatitis B virus X protein. J Virol. 1988 Feb;62(2):427–434. [PMC free article] [PubMed]
  • Su F, Schneider RJ. Hepatitis B virus HBx protein activates transcription factor NF-kappaB by acting on multiple cytoplasmic inhibitors of rel-related proteins. J Virol. 1996 Jul;70(7):4558–4566. [PMC free article] [PubMed]
  • Su F, Schneider RJ. Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor alpha. Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8744–8749. [PubMed]
  • Truant R, Antunovic J, Greenblatt J, Prives C, Cromlish JA. Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation. J Virol. 1995 Mar;69(3):1851–1859. [PMC free article] [PubMed]
  • Twu JS, Robinson WS. Hepatitis B virus X gene can transactivate heterologous viral sequences. Proc Natl Acad Sci U S A. 1989 Mar;86(6):2046–2050. [PubMed]
  • Wang HD, Yuh CH, Dang CV, Johnson DL. The hepatitis B virus X protein increases the cellular level of TATA-binding protein, which mediates transactivation of RNA polymerase III genes. Mol Cell Biol. 1995 Dec;15(12):6720–6728. [PMC free article] [PubMed]
  • Wang XW, Forrester K, Yeh H, Feitelson MA, Gu JR, Harris CC. Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2230–2234. [PubMed]
  • Williams JS, Andrisani OM. The hepatitis B virus X protein targets the basic region-leucine zipper domain of CREB. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3819–3823. [PubMed]
  • Yen TSB. Hepadnaviral X Protein:Review of Recent Progress. J Biomed Sci. 1996 Jan;3(1):20–30. [PubMed]
  • Yueh A, Schneider RJ. Selective translation initiation by ribosome jumping in adenovirus-infected and heat-shocked cells. Genes Dev. 1996 Jun 15;10(12):1557–1567. [PubMed]
  • Zentgraf H, Herrmann G, Klein R, Schranz P, Loncarevic I, Herrmann D, Hübner K, Schröder CH. Mouse monoclonal antibody directed against hepatitis B virus X protein synthesized in Escherichia coli: detection of reactive antigen in liver cell carcinoma and chronic hepatitis. Oncology. 1990;47(2):143–148. [PubMed]
  • Zhang K, Papageorge AG, Lowy DR. Mechanistic aspects of signaling through Ras in NIH 3T3 cells. Science. 1992 Jul 31;257(5070):671–674. [PubMed]
  • Zheng YW, Riegler J, Wu J, Yen TS. Novel short transcripts of hepatitis B virus X gene derived from intragenic promoter. J Biol Chem. 1994 Sep 9;269(36):22593–22598. [PubMed]
  • Zoulim F, Saputelli J, Seeger C. Woodchuck hepatitis virus X protein is required for viral infection in vivo. J Virol. 1994 Mar;68(3):2026–2030. [PMC free article] [PubMed]

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