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Hepatitis B Virus X Protein Activates a Survival Signaling by Linking Src to Phosphatidylinositol 3-Kinase*

  • Wen-Ling Shih
    Footnotes
    Affiliations
    Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 10063, Taiwan
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  • Min-Liang Kuo
    Affiliations
    Institute of Toxicology, College of Medicine, National Taiwan University, Taipei 10063, Taiwan
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  • Shuang-En Chuang
    Affiliations
    National Health Research Institute, Taipei 11564, Taiwan
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  • Ann-Lii Cheng
    Affiliations
    Cancer Research Center, College of Medicine, National Taiwan University, Taipei 10063, Taiwan

    Department of Oncology and Department of Internal Medicine, National Taiwan University Hospital, Taipei 10063, Taiwan
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  • Shin-Lian Doong
    Correspondence
    To whom correspondence should be addressed: Graduate Institute of Microbiology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Rd. Taipei, Taiwan 10063. Tel.: 886-2-23123456 (ext. 8288); Fax: 886-2-23915293
    Affiliations
    Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 10063, Taiwan

    Cancer Research Center, College of Medicine, National Taiwan University, Taipei 10063, Taiwan
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  • Author Footnotes
    * This work was supported in part by the National Health Research Institute, Department of Health (NHRI-Ex90-8829 SP and 91A1-CANT-1) and the National Science Council (NSC 89-2320-B-002-194) of Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Current address: Dept. of Life Sciences, Tzu Chi University, Hualien, Taiwan.
Open AccessPublished:June 12, 2003DOI:https://doi.org/10.1074/jbc.M302580200
      We have previously shown that transactivation-proficient hepatitis virus B X protein (HBx) protects Hep 3B cells from transforming growth factor-β (TGF-β)-induced apoptosis via activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway. This work further investigated how HBx activates PI 3-kinase. Src activity was elevated in Hep 3B cells following expression of transactivation-proficient HBx or HBx-GFP fusion proteins. The Src family kinase inhibitor PP2 and C-terminal Src kinase (Csk) both alleviated HBx-mediated PI 3-kinase activation and protection from TGF-β-induced apoptosis. Therefore, HBx activated a survival signal by linking Src to PI 3-kinase. Systemic subcellular fractionation and membrane flotation assays indicated that ∼1.5% of ectopically expressed HBxGFP was associated with periplasmic membrane where Src was located. However, neither nucleus-targeted nor periplasmic membrane-targeted HBxGFP was able to upregulate Src activity or to augment PI 3-kinase survival signaling pathway.
      Hepatitis B virus X protein (HBx),
      The abbreviations used are: HBx, hepatitis B virus X protein; PI, phosphatidylinositol; GFP, green fluorescent protein; Csk, C-terminal Src kinase.
      1The abbreviations used are: HBx, hepatitis B virus X protein; PI, phosphatidylinositol; GFP, green fluorescent protein; Csk, C-terminal Src kinase.
      a small regulatory protein that is required for the establishment of viral infection (
      • Chen H.S.
      • Kane K.O.
      • Girones R.
      • Anderson R.W.
      • Hornbuckle W.E.
      • Tennant B.C.
      • Cote P.J.
      • Gerin J.L.
      • Purcell R.H.
      • Miller R.H.
      ,
      • Zoulim F.
      • Saputelli J.
      • Seeger C.
      ,
      • Klein N.P.
      • Bouchard M.J.
      • Wang L.H.
      • Kobarg C.
      • Schneider R.J.
      ), has been claimed to contribute to the development of hepatocellular carcinoma (
      • Kim Y.C.
      • Song K.S.
      • Yoon G.
      • Nan M.J.
      • Ryu W.S.
      ,
      • Madden C.R.
      • Finegold M.J.
      • Slagle B.L.
      ). However, the molecular mechanisms involved in HBx-mediated hepatocarcinogenesis remain obscure.
      The first biological activity linked to HBx was transcriptional transactivation activity on a variety of viral and cellular promoters (
      • Murakami S.
      ). HBx does not directly bind to ds DNA but has been shown to interact with components of basal transcription machinery (
      • Qadri I.
      • Maguire H.F.
      • Siddiqui A.
      ,
      • Cheong J.H.
      • Yi M.K.
      • Lin Y.
      • Murakami S.
      ) and several transcription factors (
      • Maguire H.F.
      • Hoeffler J.P.
      • Siddiqui A.
      ,
      • Antunovic J.
      • Lemieux N.
      • Cromlish J.A.
      ,
      • Ohno H.
      • Kane K.O.
      • Lin Y.
      • Kobayashi K.
      • Murrakami S.
      ,
      • Yoo Y.B.
      • Ueda H.
      • Park K.
      • Flanders K.C.
      • Lee Y.I.
      • Jay G.
      • Kim S.J.
      ). These associations require the presence of HBx in the nucleus. However, HBx is predominantly localized to the cytoplasm, and very little, if any, is distributed to the nucleus of transfected cells (
      • Henkler F.
      • Hoare J.
      • Waseem N.
      • Goldin R.D.
      • McGarvey M.J.
      • Koshy R.
      • King I.A.
      ,
      • Hoare J.
      • Henkler F.
      • Dowling J.
      • Errington W.
      • Goldin R.D.
      • Fish D.
      • McGarvey M.J.
      ,
      • Doria M.
      • Klein N.
      • Lucito R.
      • Schneider R.J.
      ). Based upon its cytoplasmic localization, HBx has been shown to mediate the activation of signal transduction cascades, including Src/Ras/mitogen-activated protein kinase (MAPK) (
      • Klein N.P.
      • Schneider R.J.
      ), protein kinase C (PKC) (
      • Kekule A.S.
      • Laner U.
      • Weiss L.
      • Luber B.
      • Hofschneider P.H.
      ,
      • Luber B.
      • Luber U.
      • Weiss L.
      • Hohne M.
      • Hofschneider P.H.
      • Kekule A.S.
      ), NF-κB (
      • Pan J.
      • Duan L.Y.
      • Sun B.S.
      • Feitelson M.A.
      ,
      • Su F.
      • Schneider R.J.
      ,
      • Weil R.
      • Sirma H.
      • Giannini C.
      • Kremsdorf D.
      • Bessia C.
      • Dargemont C.
      • Brechot C.
      • Israel A.
      ,
      • Chirillo P.
      • Falco M.
      • Puri P.L.
      • Artini M.
      • Balsano C.
      • Levrero M.
      • Natoli G.
      ), c-Jun N-terminal kinase (JNK) (
      • Diao J.
      • Khine A.A.
      • Sarangi F.
      • Hsu E.
      • Tibbles L.A.
      • Woodgett J.R.
      • Penninger J.
      • Richardson C.D.
      ), phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/Akt) (
      • Shih W.L.
      • Kuo M.L.
      • Chuang S.E.
      • Cheng A.L.
      • Doong S.L.
      ,
      • Lee Y.I.
      • Kang-Park S.
      • Do S.I.
      • Lee Y.I.
      ), Janus kinase/STAT pathways (
      • Lee Y.H.
      • Yun Y.
      ). Activation of these signaling pathways may contribute to HBx-mediated effects, including deregulation of cell cycle check points control (
      • Doria M.
      • Klein N.
      • Lucito R.
      • Schneider R.J.
      ,
      • Benn J.
      • Schneider R.J.
      ,
      • Bouchard M.
      • Giannakopoulos S.
      • Wang E.H.
      • Tanese N.
      • Schneider R.J.
      ), and protection of cells from apoptosis induced by Fas (
      • Pan J.
      • Duan L.Y.
      • Sun B.S.
      • Feitelson M.A.
      ), transforming growth factor-β (TGF-β) (
      • Shih W.L.
      • Kuo M.L.
      • Chuang S.E.
      • Cheng A.L.
      • Doong S.L.
      ), and etoposide (
      • Lee Y.I.
      • Kang-Park S.
      • Do S.I.
      • Lee Y.I.
      ). In contrast, apoptotic effects of HBx have been reported by several groups (
      • Wang X.W.
      • Gibson M.K.
      • Vermenlen W.
      • Forrester K.
      • Sturzbecher H.W.
      • Hoeijmaker J.H.J.
      • Harris C.C.
      ,
      • Chirillo P.
      • Pagano S.
      • Natoli G.
      • Puri P.L.
      • Burgio U.L.
      • Balsano C.
      • Levereo M.
      ,
      • Shintani Y.
      • Yotsuyanagi H.
      • Moriya K.
      • Fujie H.
      • Tsutsumi I.
      • Kanegae Y.
      • Kimura S.
      • Saito I.
      • Koike K.
      ,
      • Terradillos O.
      • Pollicino T.
      • LecoBver H.
      • Tripodi M.
      • Gougeon M.L.
      • Tiollais P.
      • Buendia M.A.
      ). In Chang liver cells, HBx failed to induce apoptosis; however, it did sensitize cells to apoptosis triggered by TNF-α through c-Myc activation and stress-related MAP kinase pathways (
      • Su F.
      • Schneider R.J.
      ). Taken together, these seemingly contradictory results suggest that HBx may modulate both preapoptotic and antiapoptotic pathways, dependent upon different cell settings.
      Previously, we demonstrated that HBx can effectively suppress TGF-β-induced apoptosis in Hep 3B hepatoma cell line by stimulating PI 3-kinase activity (
      • Shih W.L.
      • Kuo M.L.
      • Chuang S.E.
      • Cheng A.L.
      • Doong S.L.
      ). In the present study, we characterized the mechanism by which HBx activated PI 3-kinase. Src tyrosine kinase activity was activated by transactivation-proficient HBx. The Src-family kinase inhibitor PP2 and C-terminal Src kinase (Csk) alleviated HBx-mediated PI 3-kinase activation and protection from TGF-β-induced apoptosis. The link from Src to PI 3-kinase was demonstrated to provide a survival signal in HBx-expressing hepatoma cells. In addition to the reported compartmentalization of HBx in nucleus and cytoplasm (cytosol, mitochondria), the association of HBxGFP fusion proteins with periplasmic membrane was observed. Neither periplasmic membrane-targeted nor nucleus-targeted HBxGFP was able to activate Src activity or to augument p85 phosphorylation.

      MATERIALS AND METHODS

      Antibodies, Reagents, and Plasmids—Rabbit polyclonal antibodies against GFP expressed in Escherichia coli were employed for detection of HBx-GFP fusion protein. The anti-Csk antibody, anti-Src antibody and anti-phosphotyrosine(clone 4G10) were purchased from Upstate Biotechnology. Antibody for the p85 subunit of the PI 3-kinase was purchased from Santa Cruz Biotechnology. PP2, TGF-β, enolase, phosphatidylinositol were purchased from Sigma.
      pRT-GFP, pRT-HBxGFP, pRT-HBx7GFP, pRT-HBx61GFP, pRT-HBx69GFP, and pRT-HBx90–91GFP were generated in previous study (
      • Shih W.L.
      • Kuo M.L.
      • Chuang S.E.
      • Cheng A.L.
      • Doong S.L.
      ). Briefly, point mutations were introduced to alter the following amino acids HBx7 (amino acid 7; Cys changed to Ser), HBx61 (amino acid 61; Cys changed to Leu), HBx69 (amino acid 69; Cys changed to Leu), and HBx9091 (amino acid 90; Pro changed to Val, and amino acid 91; Lys changed to Leu). While HBx7GFP retained its transactivation activity, HBx61GFP, HBx69GFP, and HBx90–91GFP lost their transactivation ability (
      • Shih W.L.
      • Kuo M.L.
      • Chuang S.E.
      • Cheng A.L.
      • Doong S.L.
      ,
      • Becker S.A.
      • Lee T.H.
      • Butel J.S.
      • Slagle B.L.
      ).
      pSRα-Csk (kindly provided from Dr. M. Okada) was digested with MluI. The 1.4-kbp Csk cDNA fragment was isolated, treated with Klenow fragment, and ligated into BamHI/Klenow treated pREV-TRE retroviral vector, generating pRT-Csk.
      For construction of periplasmic membrane-targeted HBx-GFP fusion protein expression vector, pRT-HBxGFPRas4B, Ras4B prenylation signal was fused into the C-terminal domain of the HBx-GFP fusion protein. Briefly, two oligonucleotides (Ras4B-U: 5′-GAG CAA AGA TGG TAA AAA GAA GAA AAA GAA ATC AAA GAC AAA GTG TGT AAT TAT GTG A-3′, Ras4B-L: 5′-TCA CAT AAT TAC ACA CTT TGT CTT TGA CTT CTT TTT CTT CTT TTT ACC ATC TTT GCT C-3′) were synthesized, annealed, treated with T4 polynucleotide kinase, and ligated into SmaI-cut pGFPemd-bm, generating pGFPRas4B. An 800-bp DNA fragment, isolated from HindIII-digested pGFPRas4B, was ligated with the 7029-bp DNA fragment isolated from HindIII-digested pRT-HBxGFP, generating pRT-HBxGFPRas4B.
      For construction of nucleus-targeted HBx-GFP fusion protein expression vector, pRT-HBxGFPNLSMYC, nucleus localization signal of SV40 large T antigen was fused into the C-terminal of HBx-GFP fusion protein. A 130-bp DNA fragment, isolated from NotI- and XbaI-digested pCMV/GFP/NLS/myc (Invitrogen) was ligated with SmaI-cut pGFPemd-bm, generating pGFPNLSMYC. A 840-bp DNA fragment, isolated from HindIII-digested pGFPNLSMYC, was ligated with the 7029-bp DNA fragment isolated from HindIII-digested pRT-HBxGFP, generating pRT-HBxGFPNLSMYC.
      Cell Culture—Hep 3B cells were cultured in a minimum essential medium (MEM) supplemented with Earle's salt, 10% fetal calf serum, penicillin G (50 units/ml), streptomycin (50 μg/ml), fungizone 1.25 μg/ml at 37 °C in a 5% CO2 incubator. PT67, retrovirus packaging cell line, was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin G (50 units/ml), streptomycin (50 μg/ml), fungizone 1.25 μg/ml at 37 °C in a 5% CO2 incubator. Hep 3B stable transfectants, with doxycycline-inducible expression of GFP, HBx, HBx-GFP, HBx7-GFP, HBx61-GFP, HBx69-GFP, HBx90–91-GFP, HBxGFPNLSMYC, or HBxGFPRas4B were generated as described previously (
      • Shih W.L.
      • Kuo M.L.
      • Chuang S.E.
      • Cheng A.L.
      • Doong S.L.
      ) and cultured in complete MEM medium supplemented with 100 μg/ml G418 and 50 μg/ml hygromycin.
      Transfection and Retrovirus Infection—DNAs were introduced into cells through transfection or retrovirus infection. Transfection was performed by the calcium phosphate-DNA precipitation method according to the procedure described by Chen et al. (
      • Chen C.
      • Okayama H.
      ).
      For retrovirus infection, media was collected from virus-producing PT67 cell line, filtered through a 0.45-μm membrane. After addition of polybrene to final 0.4 μg/ml, the whole mixture was poured into target cells. After incubation for 16 h, the virus-containing media were aspirated. Cells were washed, incubated for two additional days before they were ready for selection or analysis.
      Src Kinase Assay—Cells were lysed with Src lysis buffer (150 mm NaCl, 20 mm Tris, pH 8.0, 50 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1% Nonidet-40, 2 mm orthovanadate, 10 mg/ml pepstatin, 2.5 mm EDTA), immunoprecipitated with anti-Src antibody followed by protein-A Sepharose. The immunocomplexes were washed four times with lysis buffer, once with 20 mm Hepes pH 7.4, once with kinase assay buffer (20 mm Hepes pH 7.4, 10 mm MnCl2), then resuspended in kinase assay buffer containing 0.2 μg of acid-denatured enolase, 10 μCi of [γ-32P]ATP, 10 μm ATP and incubated at 30 °C for 30 min. Kinase reaction was stopped with 2× sample loading buffer, boiled for 10 min, and resolved by 10% SDS-PAGE. Src kinase activity assay kit using peptide KVEKIGEGTYGVVYK was purchased from Upstate Biotechnology. The assay was performed according to the manufacturer's instructions.
      TGF-β-induced Cytotoxicity Assay—2 × 105 Hep 3B cells were seeded onto a 35-mm tissue culture plate. 24 h after inoculation, cells were washed with phosphate-buffered saline and cultured in serum free MEM for 48 h. TGF-β were then added to a culture medium at various concentrations. 48 h after treatment, cells were collected by trypsinization and suspended in MEM supplemented with 10% fetal calf serum. Viable and non-viable cells were then determined by direct counting using hemocytometer in the presence of trypan blue.
      Phosphatidylinositol 3-Kinase Assay—PI 3-kinase activities were assayed according to a procedure described previously (
      • Whitman M.
      • Kaplan D.R.
      • Schaffhausen B.
      • Cantley L.
      • Roberts T.M.
      ). Briefly, 107 cells were washed twice with ice-cold PBS and lysed with 1 ml of lysis buffer (137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2,1mm CaCl2, 1% Nonidet P-40, 10% glycerol, 1 mg/ml bovine serum albumin, 20 mm Tris, pH 8.0, 2 mm orthovanadate). Cell extracts were incubated with 1 μg of anti-phosphotyrosine antibody (Clone 4G10) overnight at 4 °C. The immunocomplex was precipitated with 50 μl of protein A-Sepharose for1hat 4 °C, washed three times with lysis buffer, twice with LiCl buffer (0.5 m LiCl, 100 mm Tris, pH 7.6) and twice with TNE buffer (10 mm Tris, pH 7.6, 100 mm NaCl, 1 mm EDTA). The immunocomplex was preincubated on ice for 10 min with 10 μlof20mm Hepes (pH 7.4), containing 2 mg/ml phosphatidylinositol (PI) (Sigma). Kinase reaction was performed by the addition of 40 μl of the reaction buffer (10 μCi of [γ-32P]ATP, 20 mm Hepes, pH 7.4, 20 μm ATP, 5 mm MgCl2) at room temperature for 15 min. The reaction was stopped by the addition of 100 μl of 1 m HCl and extracted with 200 μl of a 1:1 mixture of chloroform and methanol. The radiolabeled lipids were separated by thin-layer chromatography and visualized using a phosphorimager.
      DNA Fragmentation Analysis—Cells were collected, washed with phosphate-buffered saline, and lysed with lysis buffer (50 mm Tris-HCl, pH 7.5, 20 mm EDTA, 1% Nonidet P-40). The supernatant was collected and incubated with RNase at a final concentration of 500 μg/ml for 1 h at 37 °C. Subsequently, proteinase K was added to a final concentration of 500 μg/ml. The mixtures were then incubated overnight at 55 °C. The DNA was extracted with phenol/chloroform, precipitated with ethanol, dissolved in TE8.0 and subjected to 1.7% agarose gel electrophoresis.
      Subcellular Fractionation—This experiment was performed as described by Krajewski et al. (
      • Krajewski S.
      • Tanaka S.
      • Takayama S.
      • Schibler M.J.
      • Fenton W.
      • Reed J.C.
      ). Briefly, cells were lysed in hypotonic buffer (5 mm Tris, pH 7.4, 5 mm KCl, 1.5 mm MgCl2, 0.1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride) and incubated on ice for 15 min before disruption of cells by passing through 26-gauge needle 15 times. Unbroken cells and nuclei were obtained by centrifugation at 1,000 × g for 5 min. The resulting supernatant was subjected to centrifugation at 10,000 × g for 15 min at 4 °C. The pellets (containing mitochondria, lysosome, Golgi, and rough endoplasmic reticulum) were collected and designated as heavy membrane fraction. The supernatant was subjected to centrifugation again at 150,000 × g in a Beckman SW50.1 rotor for 60 min. The pellets were collected and designated as light membrane fraction. The resulting supernatant was designated as the cytosolic fraction. All fractions were resuspended in Laemmli sample buffer, subjected to SDS-PAGE and Western blot analysis using various antibodies.
      Membrane Flotation—The analysis was performed as described by Matsumato et al. (
      • Matsumato M.
      • Hsieh J.Y.
      • Zhu N.
      • Vanarscdle T.
      • Hwang S.B.
      • Jeng K.S.
      • Gorbalenya A.E.
      • Lo S.Y.
      • Ou J.H.
      • Ware C.F.
      • Lai M.M.C.
      ). Cells were lysed in hypotonic buffer (5 mm Tris pH 7.4, 5 mm KCl, 1.5 mm MgCl2, 0.1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride) and incubated on ice for 15 min before disruption of cells by passing through 26-gauge needle 15 times. Unbroken cells and nuclei were obtained by centrifugation at 1,000 × g for 5 min. 0.5 ml of the resulting supernatant was dispersed into 2 ml 72%(wt/wt) sucrose in low salt buffer (LSB) (50 mm Tris pH 7.5, 25 mm KCl, 5 mm MgCl2) and overlaid with 2.5 ml of 55%(w/w) sucrose in LSB and 0.6 ml of 10% (w/w) sucrose in LSB. Centrifugation was done in a Beckman SW50.1 rotor at 4 °C for 12 h at 38,000 rpm. After centrifugation, fractions (0.8 ml per fraction) were collected from the top of the gradient. The pelleted fraction contained cytoplasmic organelles. The first fraction from the top contained plasma membrane. The plasma membrane fraction was diluted with 4 ml of LSB and subjected to centrifugation in Beckman SW50.1 rotor at 4 °C for 1.5 h at 46,000 rpm. Proteins from nuclei, organelles and plasma membrane pellets were resuspended in Laemmli sample buffer, boiled for 10 min and loaded onto SDS-polyacrylamide gel, transferred onto polyvinylidene difluoride membrane then detected by Western blot using various antibody.

      RESULTS

      Enhancement of Src Tyrosine Kinase Activity by Transactivation-proficient HBx—The synthetic peptide KVEKIGEGTYGVVYK or enolase was utilized as a substrate to measure Src kinase activity in Hep 3B cells with doxycycline-regulatable expression of GFP, HBx, and various HBx-GFP fusion proteins. The activity assay was performed 24h after induction of the indicated proteins. As shown in Fig. 1, A and B, transactivation-proficient HBx, HBx-GFP, HBx7-GFP resulted in a substantial increase of Src kinase activity but transactivation-deficient HBx61-GFP, HBx69-GFP, HBx90–91-GFP did not. The same results were observed 48 h after induction, indicating that activation of Src kinase activity by HBx was constitutive. The ability of HBx to activate Src correlated well with its ability to activate PI 3-kinase and to protect cells from TGF-β-induced apoptosis (
      • Shih W.L.
      • Kuo M.L.
      • Chuang S.E.
      • Cheng A.L.
      • Doong S.L.
      ).
      Figure thumbnail gr1
      Fig. 1Up-regulation of Src kinase by HBx. Twenty-four hours after incubation with or without doxycycline, lysates of cells overexpressing the indicated products were immunoprecipitated with anti-Src antibody. The immunocomplexes were incubated with the peptide KVEKIGEGTYGVVYK (A) or enolase (B) in the presence of [γ-32P]ATP to examine Src activity. In panel A, the phosphorylated peptide was precipitated by TCA and quantitated using scintillation counting. The amount of Src in the cell lysates was verified by immunoprecipitation followed by Western blot analysis using anti-Src antibody; these results are shown in the bottom of panel A. Src activity was expressed as fold activation relative to activity in cells expressing GFP only. Data are presented as mean ± S.D. of three independent experiments. In panel B, the phosphorylated enolase was separated by SDS-PAGE and visualized by autoradiography.
      Src Family Kinase Inhibitor, PP2, Alleviated the HBx Anti-apoptotic Response—To examine the involvement of Src tyrosine kinase in HBx-mediated anti-apoptotic effects in Hep 3B cells, the Src family tyrosine kinase inhibitor, PP2, was utilized. As shown in Fig. 2, cells overexpressing the transactivation-proficient HBx (including HBx, HBx-GFP, HBx7-GFP) lost their ability to resist TGF-β-induced apoptosis in the presence of PP2. This result indicated that Src plays an important role in mediating the HBx anti-apoptotic signaling against TGF-β.
      Figure thumbnail gr2
      Fig. 2PP2 alleviated HBx-mediated anti-TGF-β induced apoptosis. Hep 3B cells with doxycycline-regulatable expression of the indicated products were treated with 5 ng/ml TGF-β, or 5 ng/ml TGF-β combined with 10 μm PP2 for 48 h. Viable cells were visualized by trypan blue staining and counted by hemocytometer. Data are presented as mean ± S.D. of two independent experiments, each performed in duplicate.
      Csk Diminished HBx-induced PI 3-Kinase Activation and HBx-mediated Anti-apoptotic Effect Against TGF-βHaving identified both Src and PI 3-kinase as important signaling molecules in mediating the HBx anti-apoptotic effect, we next attempted to determine the relationship between the two signaling molecules. Csk is a tyrosine kinase that specifically phosphorylates the C-terminal tyrosine of Src kinases and returns them to an inactive state (
      • Okada M.
      • Nakagawa H.
      ). Overexpression of Csk can be used to block the activity of Src family kinases and test whether Src is functioning upstream of PI 3-kinase. Vectors with doxycycline-inducible expression of Csk were introduced into Hep 3B cells with doxycycline-regulatable expression of GFP, HBx, and HBx-GFP. As shown in Fig. 3, A and B, expression of Csk diminished Src activation mediated by HBx or HBx-GFP. Csk alleviated HBx-mediated tyrosine phosphorylation (Fig. 4A) of p85, the regulatory subunit of PI 3-kinase, and consequentially PI 3-kinase activity (Fig. 4B). HBx-mediated protection against TGF-β-induced DNA fragmentation was not evident in cells expressing Csk as shown in Fig. 4C. Thus, HBx activated Src and subsequently PI 3-kinase, thereby transmitting a crucial survival signaling against TGF-β-induced apoptosis.
      Figure thumbnail gr3
      Fig. 3Csk alleviated HBx-mediated elevation of Src activity. Hep 3B cells with doxycycline-regulatable expression of GFP, HBx, or HBx-GFP were infected with (filled box) or without (empty box) recombinant retrovirus containing a doxycycline-regulatable expression cassette of Csk. Forty-eight hours after infection, cells were collected and treated with doxycycline for 24 h or were not treated. Subsequently, lysates from cells treated with (+) or without (–) doxycycline were analyzed for Src activity using synthetic peptide (A) or enolase (B) as substrate.
      Figure thumbnail gr4
      Fig. 4Csk alleviated HBx-mediated anti-apoptotic effect. Hep 3B cells with doxycycline-regulatable expression of GFP, HBx or HBx-GFP were infected with recombinant retrovirus containing a doxycycline-regulatable expression cassette of Csk. Forty-eight hours after infection, cells were collected and treated with doxycycline for 24 h or were not treated. Subsequently, lysates from cells treated with (+) or without (–) doxycycline were subjected to immunoprecipitation and Western blot analysis (panel A) or subjected to PI 3-kinase activity assay (panel B). A, elevation of p85 phosphorylation level by HBx or HBx-GFP was abolished by the expression of Csk. Cell lysates were immunoprecipitated with anti-p85 antibody. The immunocomplex was separated by SDS-PAGE and subjected to immunoblotting with anti-phosphotyrosine (clone 4G10) or anti-p85 antibody. Cell lysates were separated on polyacrylamide gels and subjected to direct immunoblotting with anti-Csk or anti-HBx antibody. B, the elevated PI 3-kinase activity induced by HBx or HBx-GFP was diminished by the expression of Csk. Cell lysates were immunoprecipitated with an anti-phosphotyrosine antibody. The immunocomplex was subjected to in vitro kinase assay using phosphatidylinositol as the substrate. The production of 32P-labeled phosphatidylinositol 3-phosphate was analyzed by thinlayer chromatography. C, HBx-mediated protection against TGF-β-induced DNA fragmentation was not evident in cells with Csk expression. Cells treated with (+) or without (–) doxycycline were cultured in serum-free media for 48 h, treated with (upper panel) or without (lower panel) TGF-β (5 ng/ml) for 16 h. DNAs were then extracted and analyzed by agarose gel electrophoresis.
      HBx Was Able to Associate with Periplasmic Membrane— Analogous to its promiscuous biological functions, the subcellular localization of HBx also remains controversial. HBx has been reported to be located in the nucleus, the cytoplasm, or both (
      • Murakami S.
      ,
      • Henkler F.
      • Hoare J.
      • Waseem N.
      • Goldin R.D.
      • McGarvey M.J.
      • Koshy R.
      • King I.A.
      ,
      • Hoare J.
      • Henkler F.
      • Dowling J.
      • Errington W.
      • Goldin R.D.
      • Fish D.
      • McGarvey M.J.
      ,
      • Doria M.
      • Klein N.
      • Lucito R.
      • Schneider R.J.
      ). Several reports have demonstrated that HBx forms intracellular aggregates associated with mitochondria around the nuclear periphery (
      • Rahmani Z.
      • Huh K.W.
      • Lasher R.
      • Siddiqui A.
      ,
      • Takada S.
      • Shirakata Y.
      • Kaneniwa N.
      • Koike K.
      ) or proteasome (
      • Sirma H.
      • Weil R.
      • Rosmorduc U.
      • Urban S.
      • Israel A.
      • Kremsforf D.
      • Brechot C.
      ). Based on the observation that HBx stimulates the activity of several periplasmic membrane-associated signaling molecules, such as Src, Ras, and PI 3-kinase, HBx might work in the vicinity of the plasma membrane. We examined the possible distribution of HBx in the periplasmic cellular membrane and elucidated its role in activation of Src. Systemic subcellular fractionation was utilized to examine the distribution of ectopically expressed HBx-GFP in different cellular compartment. Antibodies against specific molecules in each compartment were utilized to verify the fractionation efficiency. As shown in Fig. 5A, 51.4% of HBx-GFP was found to be associated with the nucleus, 14.5% in the cytosol, 32.6% in the heavy membrane fraction, and 1.5% in the light membrane fraction. Membrane flotation assay revealed that a small proportion of HBx-GFP was indeed associated with the plasma membrane (Fig. 5B). GFP alone was never detected in the plasma membrane fraction. As expected, the majority of Src was found to be associated with the plasma membrane, as revealed by the presence of Src in the light membrane fraction.
      Figure thumbnail gr5
      Fig. 5Subcellular distribution of HBx-GFP was examined by systemic subcellular fraction and membrane flotation methods. Cells were transfected with the expression plasmids pRT-GFP or pRT-HBxGFP in combination with pRevTet-On vector containing the rtTA regulatory element. Cells were treated with 1 μg/ml doxycycline 48 h after transfection. Cells were harvested and resuspended in hypotonic lysis buffer 24 h after addition of doxycycline. After disruption of cells, nuclei and unbroken cells were pelleted by low speed centrifugation. The resulting supernatants were subjected to systemic subcellular fractionation (A) or membrane flotation analysis (B) as described under “Materials and Methods.” Equal proportion of each fraction was subjected to Western blot analysis using anti-GFP, anti-Src, anti-sc35 (nuclear protein), anti-cytochrome c (mitochondria marker), anti-E-cadherin (periplasmic membrane marker), or anti-CPP32 (cytosolic marker). The core/enhancer elements for HBV core gene lie within the HBx open reading frame region and drive an independent transcription of the GFP gene downstream of the HBx gene. The appearance of GFP protein in cells expressing HBx-GFP fusion protein was due to the presence of this GFP transcript (data not shown).
      Neither Membrane-bound nor Nucleus-targeted HBx-GFP Fusion Protein Was Able to Activate Src and Augument Tyrosine Phosphorylation of p85—To examine the biological role played by HBx-GFP in the nucleus or periplasmic membrane, vectors with periplasmic membrane-targeted (HBxGFPRas4B) or nucleus-targeted (HBxGFPNLSMYC) fusion proteins were constructed. When introduced into cells, HBxGFPNLSMYC was primarily detected in the nucleus and HBxGFPRas4B was mainly localized in the plasma membrane fraction (Fig. 6). However, in contrast with HBx or HBx-GFP, induced expression of HBxGFPRas4B or HBxGFPNLSMYC (Fig. 7A) failed to activate Src (Fig. 7B). The level of tyrosine phosphorylation of p85 was not altered by either nucleus-targeted or periplasmic membrane-targeted forms of HBx-GFP (Fig. 7C).
      Figure thumbnail gr6
      Fig. 6Subcellular distribution of GFP, HBx-GFP, HBxGFPNLSMYC, and HBxGFPRas4B fusion proteins. Cells were transfected with expression plasmids pRT-GFP, pRT-HBxGFP, pRT-HBxGFPNLSMYC, or pRT-HBxGFPRas4B along with pRevTet-On vector containing the rtTA regulatory element. Cells were treated with 1 μg/ml doxycycline 48 h after transfection. Cells were harvested and resuspended in hypotonic lysis buffer 24h after addition of doxycycline. After disruption of cells, nuclei and unbroken cells were pelleted by low speed centrifugation. The resulting supernatant was fractionated by equilibrium sucrose gradient centrifugation (see “Materials and Methods”). Fractions were collected from the top of the gradient. The pelleted fraction contained organelles. The first fraction from the top contained plasma membrane. Proteins from plasma membrane, organelle, and nuclei fractions were separated on polyacrylamide gels and detected by immunoblotting using polyclonal antibody against GFP, PCNA (nucleus marker), cytochrome c (mitochondria marker), or E-cadherin (periplasmic membrane marker).
      Figure thumbnail gr7
      Fig. 7Src activity and tyrosine phosphorylation level of p85 were not altered by HBxGFPNLSMYC or HBxGFPRas4B. After incubation with doxycycline for 24 h or not, Hep 3B cells with expression of indicated gene products were harvested and cell lysates were subjected to Western blot analysis or immunoprecipitation. A, Western blot analysis showing inducible expression of indicated gene products using anti-HBx polyclonal antibody. Actin was included as an internal control. B, lysates of cells were immunoprecipitated with anti-Src antibody. The immunocomplex was analyzed for Src activity (upper panel) using synthetic peptide as a substrate or immunoblotted with anti-Src antibody (lower panel) to monitor the expression level of Src. C, cell lysates were immunoprecipitated with anti-p85 antibody. The immunocomplex was separated by SDS-PAGE and subjected to Western blot analysis using anti-phosphotyrosine antibody (clone 4G10) (upper panel) or anti-p85 antibody (lower panel).

      DISCUSSION

      Previously, we have shown that HBx activates PI 3-kinase and protect Hep 3B cells from TGF-β-induced apoptosis (
      • Shih W.L.
      • Kuo M.L.
      • Chuang S.E.
      • Cheng A.L.
      • Doong S.L.
      ). In this study, we tried to elucidate mechanism(s) by which HBx activates PI 3-kinase. Src activity was elevated in Hep 3B cells following expression of transactivation-proficient HBx and HBx-GFP. Both Csk and PP2 alleviated the effect of HBx on activation of PI 3-kinase and protection against TGF-β-induced apoptosis. Taken together, these results indicate that HBx up-regulated a survival signal by linking Src to PI 3-kinase. HBx activation of Src promotes reverse transcription in the HBV DNA replication (
      • Klein N.P.
      • Bouchard M.J.
      • Wang L.H.
      • Kobarg C.
      • Schneider R.J.
      ), cell cycle transition from G1 to S (
      • Bouchard M.
      • Giannakopoulos S.
      • Wang E.H.
      • Tanese N.
      • Schneider R.J.
      ), as well as stimulation of several transcription factors (
      • Klein N.P.
      • Schneider R.J.
      ). Our study provided an additional biological function of HBx via the activation of Src.
      Biochemical studies of Src-dependent signaling in vitro and genetic studies of knockout mice have revealed an extraordinary range of functions for Src family kinases, including control of development, regulation of apoptosis, and control of cell cycle progression (
      • Martin G.S.
      ). It is conceivable that many of the other phenotypic effects of HBx expression that have been described may be attributable to the signaling pathway that is dependent on Src. However, HBx protein has been shown to stimulate many additional signaling cascades, such as PKC (
      • Kekule A.S.
      • Laner U.
      • Weiss L.
      • Luber B.
      • Hofschneider P.H.
      ,
      • Luber B.
      • Luber U.
      • Weiss L.
      • Hohne M.
      • Hofschneider P.H.
      • Kekule A.S.
      ), Jak1 (
      • Lee Y.H.
      • Yun Y.
      ), NF-κB (
      • Pan J.
      • Duan L.Y.
      • Sun B.S.
      • Feitelson M.A.
      ,
      • Su F.
      • Schneider R.J.
      ,
      • Weil R.
      • Sirma H.
      • Giannini C.
      • Kremsdorf D.
      • Bessia C.
      • Dargemont C.
      • Brechot C.
      • Israel A.
      ,
      • Chirillo P.
      • Falco M.
      • Puri P.L.
      • Artini M.
      • Balsano C.
      • Levrero M.
      • Natoli G.
      ), PI 3-kinase (
      • Shih W.L.
      • Kuo M.L.
      • Chuang S.E.
      • Cheng A.L.
      • Doong S.L.
      ,
      • Lee Y.I.
      • Kang-Park S.
      • Do S.I.
      • Lee Y.I.
      ), SAPK (
      • Diao J.
      • Khine A.A.
      • Sarangi F.
      • Hsu E.
      • Tibbles L.A.
      • Woodgett J.R.
      • Penninger J.
      • Richardson C.D.
      ), and Akt/PKB (
      • Shih W.L.
      • Kuo M.L.
      • Chuang S.E.
      • Cheng A.L.
      • Doong S.L.
      ,
      • Lee Y.I.
      • Kang-Park S.
      • Do S.I.
      • Lee Y.I.
      ). Dependent upon different cell settings, the possibility that HBx could result in different ultimate functions within a given cell line cannot be excluded. Genistein, a general tyrosine kinase inhibitor, was also able to alleviate HBx-mediated anti-apoptotic effect in our model system (data not shown). In addition to the Src kinase, the involvement of other tyrosine kinase signaling molecules cannot be excluded. The exact contribution and significance of various signaling transduction pathways on HBx-mediated biological functions remains to be defined.
      Benn and Schneider (
      • Benn J.
      • Schneider R.J.
      ) indicated that HBx leads to the formation of the active Ras-GTP complex in NIH 3T3 and Chang liver cells. Activation of Src family kinases was demonstrated to be indispensable for HBx-mediated activation of Ras (
      • Klein N.P.
      • Schneider R.J.
      ). The activated Ras-GTP complex was reported to bind to p110, the catalytic subunit of PI 3-kinase, thereby resulting in the activation of PI 3-kinase (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). Is Ras activation required by HBx in the signaling pathway from Src to PI 3-kinase? Ras activity was not altered in the presence of HBx in Hep 3B cells (data not shown). Therefore, HBx activated Src and subsequently PI 3-kinase in the absence of Ras activation in our model system. The observation that the binding of the SH3 domain of Src family kinases to a proline-rich region within the p85 subunit of PI 3-kinase could result in the activation of PI 3-kinase (
      • Pleiman C.M.
      • Hertz M.W.
      • Cambier J.C.
      ) implies that Src does not require Ras or another intermediate molecule for activation of PI 3-kinase, and therefore substantiates our finding.
      Polyoma Middle T Antigen (PMT) is identified as the tumorigenic component of the polyoma virus. PMT forms a complex with Src and PI 3-kinase, subsequently activating PI 3-kinase (
      • Courtneidge S.A.
      • Heber A.
      ). Unlike PMT, HBx does not directly bind to Src (data not shown, Refs.
      • Klein N.P.
      • Bouchard M.J.
      • Wang L.H.
      • Kobarg C.
      • Schneider R.J.
      and
      • Klein N.P.
      • Schneider R.J.
      ). HBx does not act on Src α-phosphatase or Csk either (
      • Klein N.P.
      • Bouchard M.J.
      • Wang L.H.
      • Kobarg C.
      • Schneider R.J.
      ,
      • Klein N.P.
      • Schneider R.J.
      ). These data indicate that some other upstream regulators of the Src family are involved in HBx-mediated effects. Recently, Bouchard et al. (
      • Bouchard M.J.
      • Wang L.H.
      • Schneider R.J.
      ) demonstrated that HBx increased cytosolic calcium concentrations leading to the activation of Pyk2, a Src kinase activator, and consequentially activation of Src kinase. It remains to be elucidated whether Pyk2 is involved in the HBx-mediated survival signaling pathway from Src to PI 3-kinase.
      The cellular localization of HBx has been controversial, but the general consensus is that most HBx is located in the cytoplasm, with only a small fraction in the nucleus. Henkler et al. (
      • Henkler F.
      • Hoare J.
      • Waseem N.
      • Goldin R.D.
      • McGarvey M.J.
      • Koshy R.
      • King I.A.
      ) reported that HBx was predominantly localized in nuclei in weakly expressing cells. However, at elevated levels, HBx accumulated in the cytoplasm leading to a proportional decrease of its nuclear fraction. These data indicate that compartmentalization of HBx is complex and may be influenced by its level of expression. In our study, the fusion of GFP did not affect HBx-mediated effects on transcriptional transactivation or anti-TGF-β-induced apoptosis, but greatly increased the half-life of HBx (data not shown). Systemic subcellular fractionation indicated that HBx-GFP distributed not only in the nucleus, cytosol, and organelles such as mitochondria but also in the periplasmic membrane, albeit at a very low level (Fig. 5). Since HBx did not possess a membrane-targeting signal, the nature of this association might be through the interaction with membrane-bound proteins. However, we cannot exclude the possibility that these phenomena might occur only when HBx-GFP is overexpressed. The biological significance of the association remains to be investigated.
      The intracellular distribution of HBx has not been considered adequately in explorations of its biological role, and it is still difficult to relate its expression patterns with proposed functions. We applied artificial methods to recruit HBx to the nucleus by nuclear localization signal (NLS) tagging or to the periplasmic membrane by prenylation signal tagging to investigate the functional sites of HBx involved in mediating Src activation. Neither nucleus-targeted nor periplasmic membrane-targeted HBx-GFP fusion proteins were able to activate Src (Fig. 7). Cyclosporin A (CsA), which could disrupt mitochondrial calcium signaling (
      • Calpham D.E.
      ), prevents HBx activation of Pyk2 (
      • Bouchard M.J.
      • Wang L.H.
      • Schneider R.J.
      ). CGP37157, an inhibitor of the mitochondrial sodium-calcium pump (
      • Baron K.T.
      • Thayer S.A.
      ), abolishes HBx-mediated Src activation of HBV DNA replication (
      • Bouchard M.J.
      • Wang L.H.
      • Schneider R.J.
      ). These data suggest that HBx acts on mitochondria calcium control. Since a large proportion (about 1/3) of HBx-GFP fusion proteins were found to be associated with heavy membrane fraction (Fig. 5), it is tempting to speculate that heavy membrane associated HBx might influence the activation of Src through the regulation of Ca+2 release from these organelles.

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