Hepatitis B Virus Pre-S2 Mutant Surface Antigen Induces Degradation of Cyclin-Dependent Kinase Inhibitor P27kip1 Through C-Jun Activation Domain-Binding Protein 1

Hepatitis B Virus Pre-S2 Mutant Surface Antigen Induces Degradation of Cyclin-Dependent Kinase Inhibitor p27Kip1 through c-Jun Activation Domain-Binding Protein 1

Yi-Hsuan Hsieh,1,4 Ih-Jen Su,5,6 Hui-Ching Wang,6 Jui-He Tsai,4 Yu-Jun Huang,4 Wen-Wei Chang,1,2 Ming-Derg Lai,1,3,5 Huan-Yaw Lei,1,2 and Wenya Huang1,4,5

Departments of 1Basic Medical Sciences, 2Microbiology and Immunology, 3Biochemistry, 4Medical Laboratory Science and Biotechnology, 5Center for Gene Regulation and Signal Transduction Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan and 6Division of Clinical Research, National Health Research Institute, Taipei, Taiwan

Abstract and HCV, which together are responsible for the majority of The hepatitis B virus (HBV) large surface antigen (LHBS) hepatocellular carcinomas in humans. These viruses cause mutant with deletion at the pre-S2 region accumulates necroinflammatory liver disease of variable duration and in endoplasmic reticulum (ER) and is associated with severity. A major portion of the viral hepatitis progresses into HBV-induced hepatocellular carcinogenesis. In this liver cirrhosis and dysplasia and, ultimately, hepatocellular study, we found that the pre-S2 LHBS mutant directly carcinoma. In this process, the viral proteins are believed to be interacts with the Jun activation domain–binding important players, which cross-talk with various host proteins, protein 1 (JAB1). Association of pre-S2 LHBS with affecting the host signaling pathways. A number of HBV gene JAB1 dissociated JAB1 from the JAB1/IRE1 complex products have been identified as viral tumor proteins. The X in ER. The free (active) JAB1 then translocated into protein (pX) is oncogenic because it activates the Ras/Raf-1 cell nuclei and rendered the Cdk inhibitor p27Kip1 to signal transduction pathway and inhibits DNA repair (1, 2). cytosolic proteasome for degradation. The pre-S2 HBV surface protein (HBsAg) expression in the chronic phase LHBS mutant induced hyperphosphorylation of tumor of HBV infection is also associated with hepatocellular suppressor retinoblastoma (RB) via cyclin-dependent carcinoma incidence. HBsAg causes sustained hepatic inflam- kinase 2 (Cdk2), a downstream molecule regulated mation and injury, an important marker identifying chronic by p27Kip1. This effect is independent of the ER HBV carriers (3). stress signaling pathway. The transgenic mice In the chronic phase of HBV infection, the HBV genome carrying the pre-S2 mutant LHBS gene also exhibited often integrates into the host chromosome (4). In this status, the Cdk2 activation, p27Kip1 degradation, as well as large form of HBsAg (LHBS) becomes largely expressed (5). RB hyperphosphorylation. The mouse hepatocytes Compared with small-form HBsAg, the large form includes an exhibited morphologic abnormalities such as chromatin additional pre-S region, which is the upstream promoter region condensation, multinucleation, and dysplasia of for the small form (5). LHBS is pro-oncogenic: it induced hepatocytes. In summary, the pre-S2 LHBS mutant hepatocellular carcinoma in a transgenic mouse model (6). causes p27Kip1 degradation through direct interaction These findings indicate that LHBS interacts with host factors to with JAB1. The pre-S2 mutant LHBS is suggested to be regulate the mechanism of hepatocellular carcinogenesis. a potential oncoprotein for HBV-related hepatocellular Ground glass hepatocytes are the histologic hallmarks of carcinoma. (Mol Cancer Res 2007;5(10):1063–72) chronic HBV infection (7). The type II ground glass hepatocytes display marginal staining pattern of HBsAg and Introduction has been found to carry specific mutations in the pre-S2 region Chronic viral hepatitis is the major cause for hepatocellular (8). This mutant HBS gene is deleted in approximately carcinoma, the most frequent visceral neoplasm worldwide. The nucleotides 4 to 57 of the pre-S2 region and often contains a main causative agents for hepatocellular carcinoma are HBV point mutation in the start codon of the region, which leads to a dramatic decrease in the synthesis of small-sized and middle- sized surface antigens. Type II ground glass hepatocytes often Received 2/23/07; revised 6/6/07; accepted 6/21/07. appear in hepatic nodules and proliferate in clusters, strongly Grant support: Program for Promoting University Academic Excellence Project suggesting that they are involved in HBV-related hepatocarci- 91-B-FA09-1-4 (I-J. Su) and the National Health Research Institute Extramural nogenesis. This mutant form of the HBS gene, designated pre- Project NHRI-EX95-9520BI (W. Huang), Taiwan. The costs of publication of this article were defrayed in part by the payment of S2 HBS mutant, emerges only in the late or nonreplicative page charges. This article must therefore be hereby marked advertisement in phase of chronic HBV infection and eventually becomes a accordance with 18 U.S.C. Section 1734 solely to indicate this fact. dominant HBV gene product in hepatocytes (9, 10). Requests for reprints: Wenya Huang, Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung The pre-S2 type of LHBS mutant is predominant in University, Tainan 70101, Taiwan. Phone: 886-6-235-3535, ext. 5766; Fax: hepatocellular carcinoma patients with HBV infection (10-13). 886-6-236-3956. E-mail: [email protected] Copyright D 2007 American Association for Cancer Research. Based on epidemiologic studies, HBV carriers who presented doi:10.1158/1541-7786.MCR-07-0098 with the pre-S2 LHBS mutant in serum had worse disease

outcomes than those who did not (13). Although the correlation inhibitor lactacystin, the loss in p27Kip1 protein level was between the prevalence of pre-S2 LHBS mutant and hepato- abrogated (Fig. 3A). By coimmunoprecipitation studies, it cellular carcinoma has been clearly shown, the molecular was found that the amount of p27Kip1 associated with JAB1 was mechanism of hepatocellular carcinogenesis induced by the pre- diminished in cells carrying the pre-S2 mutant LHBS but was S2 LHBS mutant is not yet clear. In a recent study (14), we fully recovered after lactacystin treatment (Fig. 3B). This found that pre-S2 mutant LHBS accumulated in ER and induced revealed that pre-S2 LHBS mutant activated JAB1 and caused Kip1 strong ER stress, which induced oxidative stress, DNA damage, p27 to be degraded via proteolysis. Kip1 and mutagenesis, all of which resulted in genomic instability in p27 is an inhibitor for the kinase activity of the cyclin- hepatocytes. The pre-S2 LHBS mutant also induced over- Cdk2 complex (20). To detect whether the Cdk2 was indeed expression of cyclin A, which is associated with the G1-S activated by the pre-S2 mutant LHBS, the activated Cdk2, cyclin-dependent kinases (Cdk), leading to cell cycle progres- indicated by its phosphorylation on the threonine residue at sion in the presence of DNA lesions (15). In the present study, amino acid 160, was detected in the HuH-7 cells carrying the WT or the pre-S LHBS mutant (21). It was found that the we searched for the molecule directly targeted by the pre-S2 2 phosphorylation on Thr160 of Cdk2 was significantly greater in LHBS mutant and the mechanism for pre-S2 LHBS mutant– induced hepatocellular carcinogenesis. cells carrying the pre-S2 LHBS mutant than in cells carrying the WT LHBS (Fig. 3C). On the contrary, the levels of cyclin D1, associated with Cdk4 in the G1-Cdk complex, were not changed Results in various transfected cells (Fig. 3C; ref. 22). These findings Pre-S2 LHBS Mutant Is Associated with Jun Activation indicate that the pre-S2 LHBS mutant causes activation of the Domain–Binding Protein 1 cyclin-Cdk2 complex through p27Kip1 degradataion. In addi- We used yeast two-hybrid assays to identify the human tion, these effects were dependent on JAB1 because the p27Kip1 protein directly targeted by the pre-S2 LHBS mutant. We found degradation and CdK2 activation were nearly abolished when that Jun activation domain–binding protein 1 (JAB1) was cells were transfected with siRNA knockdown construct of associated with the pre-S2 mutant LHBS (Fig. 1A; ref. 16). In JAB1 (Fig. 3D). the human hepatoma HuH-7 cells transfected with the wild-

type (WT) or pre-S2 mutant LHBS, we found that the JAB1 Pre-S2 LHBS Mutant–Induced Retinoblastoma Hyper- protein was associated with the pre-S2 LHBS mutant, but not phosphorylation with the WT LHBS, shown by coimmunoprecipitation experi- The cyclin A-Cdk2 complex plays a key role in the entry ments (Fig. 1B and C). One study (17) reported that JAB1 is into S phase of the cell cycle by phosphorylating the tumor associated with the ER transmembrane kinase/RNase IRE1 in suppressor retinoblastoma protein (RB; refs. 23). We found ER lumen and dissociated from it on ER stress. We found that that human hepatoma HuH-7 cells with the pre-S2 LHBS the association was nearly completely abolished in cells mutant, but not those carrying the WT LHBS, showed RB expressing pre-S2 LHBS mutant (Fig. 1D-F). In addition, such hyperphosphorylation (Fig. 4A). Such RB hyperphosphoryla- association between JAB1 and IRE1 could not be recovered tion was indeed caused primarily by Cdk2, which phosphor- even in the presence of ER stress inhibitor vomitoxin, ylates RB at the threonine residue at amino acid 821 (Fig. 4B; indicating that the pre-S2 LHBS mutant disrupted JAB1-IRE1 ref. 24). To show that such an effect induced by pre-S2 LHBS binding by specifically interacting with JAB1 (Fig. 1F). mutant was not cell type specific, the T24 human bladder Macrophage migration inhibitory factor (MIF) protein has also cancer cell line was also used for the present study, and it been associated with JAB1 in cytosol (18). Coimmunopreci- showed RB hyperphosphorylation by Cdk2 when transfected pitation studies showed that pre-S2 LHBS mutant also with the pre-S2 LHBS mutant gene (Fig. 4C and D), disrupted the association between MIF and JAB1 (Fig. 1G). indicating that RB hyperphosphorylation induced by the Therefore, the pre-S2 LHBS mutant seems to competitively pre-S2 LHBS mutant is solely dependent on expression of bind with JAB1 and dissociate JAB1/IRE1 and JAB1/MIF the pre-S2 LHBS mutant gene. It was also found that the complexes. pre-S2 mutant LHBS rendered cells to progress through cell cycle after serum starvation, whereas the WT LHBS did not

Pre-S2 LHBS Mutant–Induced Degradation of Cdk (Fig. 4E). Inhibitor p27Kip1 The nuclear JAB1 has been shown to target the Cdk RB Hyperphosphorylation in Transgenic Mice with the Kip1 inhibitor p27 and bring it to proteasome 26S for degradation Pre-S2 LHBS Mutant (19). To confirm that the pre-S2 LHBS mutant triggered nuclear We established an FVB/N mouse model containing the pre- localization of JAB1, the nuclear and cytosolic fractions were S2 HBS mutant transgene (Fig. 5A; ref. 14). Liver tissue analyzed for JAB1 protein levels. We found that, in the showed a large number of HBsAg+ hepatocytes (Fig. 5B) with presence of pre-S2 LHBS mutant, nuclear JAB1 levels were abnormal morphologies (i.e., centrosome condensation, aneu- consistently significantly higher than those induced by the WT ploidy, and dysplasia; Fig. 5C). After examining the non- (Fig. 2A and B). In addition, JAB1 also enhanced the activity of tumorous sections of liver in these mice, we found that the transcription activator protein 1 (AP-1), which was known to be majority of transgenic mice with the pre-S2 LHBS mutant gene regulated by JAB1 (Fig. 2C). showed RB hyperphosphorylation, Cdk2 activation, and Kip1 We also found that pre-S2 LHBS mutant down-regulated decrease in p27 protein levels, as compared with the mice p27Kip1; however, after treatment with the 26S proteasome carrying the WT LHBS gene (Fig. 5D). These findings were

FIGURE 1. Association of the pre-S2 mutant LHBS with JAB1. A. Yeast two-hybrid assays. The yeast colonies on growth plates and filter papers after the 5-bromo-4-chloro-3-indolyl-h-D-galactopyranoside reactions were shown as mirror images. B. Association of JAB1 with LHBS in the cells transfected with WT or pre-S2 mutant (D2) LHBS genes in HuH-7 cells detected by coimmunoprecipitation assays with the antibody against hemagglutinin (HA) epitope fused to various types of LHBS. gp42, the 42-kDa glycosylated formof LHBS (39 kDa). C, control cells transfected with the plasmidvector pIRES-hrGFP-2a onl y. C. Reciprocal immunoprecipitation analysis to detect the association of pre-S2 mutant LHBS with JAB1. D. Induction of ER stress by WT or pre-S2 mutant LHBS in HuH-7 cells, detected by the alternative splicing patterns of XBP1 mRNA by mutiplex reverse transcription-PCR analysis. Form I, total XBP1 (unspliced and spliced); formII, spliced XBP1. TG, cells treated with thapsigargin (1 Amol/L); TM, cells treated with tunicamycin (10 Ag/mL). Levels of the form II and form I products were quantified and indicated below the gel electrophoresis. Columns, mean fold value (form II/I) from three independent experiments; bars, SD. E. Dissociation of IRE1 fromJAB1, induced by the ER stress inducers tunicamycinand thapsigargin. F. Association of IRE1 with JAB1 in the cells transfected with WT or pre-S2 mutant LHBS, shown by coimmunoprecipitation assays. VT, vomitoxin (150 ng/mL). G. Association of MIF with JAB1 in the cells transfected with WT or pre-S2 mutant LHBS, shown by coimmunoprecipitation assays.

FIGURE 2. JAB1 subcellular localization and AP-1 activation by the pre-S2 mutant LHBS. A. JAB1 levels in the nuclear and cytosolic fractions in the HuH-7 cells transfected with WT or pre-S2 (D2) mutant LHBS, shown by Western blots. Cdk4 and a-actin were used as markers for proteins in nuclei and cytosol, respectively. V, cells transfected with pIRES-hrGFP-2a vector only. The relative levels of nuclear/cytosolic JAB1 were quantified and indicated below the Western blots. B. Subcellular localization of JAB1 detected by immunofluorescence analysis. T24 cells transfected with WT, pre-S2 (D2) mutant LHBS, tagged with hemagglutinin epitope, or mock treated (C) were stained with mouse anti-JAB1 and rabbit antihemagglutinin antibodies. The antimouse antibody conjugated with Alexa 488 (green) and antirabbit antibody conjugated with Alexa 594 (red) were then simultaneously incubated with the cells. Cell nuclei were stained with Hoechst 3324 (blue) fluorescent dye. Merge, the merged images of JAB1 and nuclear stainings. The relative levels of nuclear JAB1 in cells were quantified and indicated below the photographs. Col- umns, mean from 10 examined cells of each type; bars, SD. C. AP-1 transcriptional activities in cells carrying the WT or pre-S2 mutant LHBS or mitogen-activated protein kinase kinase kinase (MEKK). A, and C, columns, mean from three independent experiments; bars, SD.

Kip1 Kip1 FIGURE 3. p27 degradation affected by the pre-S2 mutant LHBS. A. Degradation of p27 in the HuH-7 cells carrying the WT or pre-S2 LHBS shown by Western blots. HA, Western blot with antihemagglutinin antibody to detect WT and pre-S2 mutant LHBS. LACTA, lactacystin (10 Amol/L). EtOH, the solvent for lactacystin. The levels of p27Kip1 in each experiment were quantified and indicated below the Western blots. Columns, mean from three independent experiments; bars, SD. B. Levels of p27Kip1 associated with JAB1 shown by coimmunoprecipitations. C. Activation of Cdk2 in the HuH-7 cells carrying the Kip1 pre-S2 mutant LHBS by Western blot. D. Inhibition of p27 degradation and CdK2 activation by siRNA against JAB1. V, HuH-7 cells transfected with pSUPER vector only. consistent with ours in human HuH-7 in vitro cell cultures. This Therefore, JAB1 also plays an active role in the ER stress means that the pre-S2 LHBS mutant induces RB hyper- signaling pathway, which activates the NFnB proto-oncogene phosphorylation through p27Kip1 degradation in in vivo mouse (26). It has been documented that JAB1 is highly expressed in models. hepatocellular carcinoma and other common types of cancer (27, 28). In addition, the ER stress signaling pathways likely contribute to the JAB1-related carcinogenesis process. Discussion Previous studies have shown that the pre-S2 LHBS mutant In this study, we found that the pre-S2 LHBS mutant directly induced strong ER stress (10). In the present study, we found interacts with the JAB1 protein and subsequently results in that association of pre-S2 LHBS mutant with JAB1 is hyperphosphorylation of the RB tumor suppressor protein independent of ER stress because the ER stress inhibitor did (summarized in Fig. 6). JAB1 is a multifunctional protein not block such association (data not shown), suggesting that the associated with the signaling pathway, cell cycle regulation, and ER stress signaling pathway is not an essential mechanism for development, and acts as a key subunit of the COP9 the pre-S2 LHBS mutant–induced JAB1 activation. signalosome (CSN; ref. 16). Overexpression of CSN5/JAB1 We previously showed that the pre-S2 mutant LHBS promotes cell proliferation, increases AP-1 transcription, and induced strong ER stress–dependent oxidative DNA lesions stimulates or inhibits turnover of a number of proteins (16). that activated the DNA repair mechanism and mutagenesis CSN5/JAB1 binding has also been shown to stimulate protein (14). The DNA damage potentially deactivates certain tumor degradation, whereas in other cases, such as hypoxia-inducible suppressors or proto-oncogenes, leading to cellular transfor- factor 1a, CSN5/JAB1 binding tends to promote stabilization mation and hepatocellular carcinogenesis. The results of the (25). Given the multiple functionalities of JAB1, there might be present study have added another important mechanism for other pathways of pre-S2 LHBS mutant–induced carcinogen- pre-S2 LHBS mutant–induced hepatocellular carcinogenesis: Kip1 esis in addition to p27 degradation. It was also reported that RB hyperphosphorylation and defects in G1-S cell cycle JAB1 participates in unfolded protein response through its arrest. Taking these findings together, we hypothesize that in association with and dissociation from ER factor IRE1 (17). the presence of DNA damage, the failure of cell cycle arrest

FIGURE 4. RB hyperphosphorylation by Cdk2 in the HuH-7 cells carrying the pre-S2 mutant LHBS. A and C. Western blot to detect total RB hyperphosphorylation in the HuH-7 (A) and T24 (C) cells carrying the WT or pre-S2 mutant (D2) LHBS. C, control cells transfected with pIRES-hrGFP-2a only. The relative levels of hyperphosphorylated/hypophosphorylated forms of RB were quantified and shown at the bottom of Western blots. B and D. 821 T821 Western blot to detect RB hyperphosphorylation at Thr in the HuH-7 (B) or T24 (D) cells carrying the WT or pre-S2 LHBS. pRB was detected with the monoclonal antibody specifically recognizing RB phosphorylated at Thr821. EGF, epidermal growth factor; positive control for Cdk2 activation. E. Cell cycle analyses of the T24 cells carrying the WT, pre-S2 mutant (D2) LHBS, or vector only (V). The cells were serumstarved for 48 h before harvest. C, control cells without serumstarvation. The relative numbersof cells in G 1-S + G2 phases were quantified and shown below the histograms. Columns, mean from three independent experiments; bars, SD. evidently increases gene mutation rates and genomic insta- hepatocellular carcinoma at significantly higher rates (odds bilities and further enhances the carcinogenesis process (29). ratio, 3.2; data not shown) than those without. The results Our recent epidemiologic studies found that patients who of the present study provide an explanation of the mecha- presented with pre-S2 LHBS mutant in serum developed nism that yields such a high association of pre-S2 LHBS

FIGURE 5. RB hyperphosphorylation in the transgenic mice carrying the pre-S2 mutant LHBS. A. Serum HBS gene levels were detected by PCR. M, DNA size marker. C, nega- tive control; PCR reaction without addition of DNA template. pHBV3.6, plasmid containing the HBV genome DNA, used as a control for HBS gene (658 bp) in PCR reaction. GAPDH (293 bp), internal control. B. Expres- sions of the WT and pre-S2 mutant (D2) LHBS in hepatocytes of the transgenic mice. The brown-colored cells on the arrows are the LHBS- expressing cells. C, control, naı¨ve mice. C. Morphologies of the liver sections in the transgenic mice, shown by H&E staining. Arrows, cells that display morphologic abnormalities. D. RB and Cdk2 phosphoryla- tions, as well as p27Kip1 levels, in the nontumor liver tissues of representa- tive mice carrying the WT or pre-S2 mutant HBS transgenes. For each detected mouse, the serum HBsAg level (ng/mL), detected by the ELISA assays, was shown at the bottomof its PCR products. The relative levels of Cdk2 phosphorylation, p27Kip1, and hyperphosphorylated/hypophosphorylated forms of RB were quantified and shown at the bottomof Western blots.

FIGURE 6. A model of hepatocellular carcinogenesis caused by the HBV pre-S2 mutant LHBS. In the absence of ER stress or the pre-S2 mutant LHBS (top), JAB1 is associated with IRE1 in ER lumen and with MIF in cytosol. When the pre-S2 mutant LHBS accumulates in ER, it induces strong ER stress (bottom). It also directly associates with IRE1 in ER lumen and with MIF in cytosol, freeing JAB1 from the JAB1/IRE1 and JAB1/MIF complexes. The free JAB1 translocates into cell nuclei in which it associates with Cdk inhibitor p27Kip1, bringing p27 to cytosolic 26S proteasome for degradation. Degradation of Kip1 p27 then activates G1-to-S cyclin/Cdk2, which phosphorylates RB leading to S-phase progression.

mutant with hepatocellular carcinoma. Based on our studies, Materials and Methods we propose that the pre-S2 LHBS mutant is a potential onco- Cell Lines and Mice protein and may represent an important predictive marker Human hepatoma HuH-7 and bladder cancer T24 cell lines for the hepatocellular carcinoma caused by chronic HBV were used for in vitro cell culture studies. These cells were infection. maintained and grown as previously described (14). The

In summary, we found that the pre-S2 LHBS mutant directly transgenic mice carrying WT or pre-S2 LHBS mutant genes interacts with the ER factor JAB1 and triggers p27Kip1 were constructed in our previous study (14). degradation and RB hyperphosphorylation. This is a novel mechanism for hepatocellular carcinogenesis caused by HBV. Yeast Two-Hybrid Assays Based on the results of the present study, we conclude that the The protocols for yeast two-hybrid assays followed those pre-S2 LHBS mutant is an important prognostic marker for described elsewhere (30). Briefly, the pre-S2 LHBS/pGBKT7 chronic HBV infection and should be widely applied in clinical (BD Clontech), which contains the Gal4 DNA-binding domain, medicine. was transformed into AH109 yeast strain, and then screened for

the pre-S2 mutant LHBS–interacting proteins by using the slide (Nalgene Nunc). After 48 h of transfection, the cells were human yeast two-hybrid testis cDNA library (BD Clontech). fixed with 10% formalin and then washed with PBS (pH 7.4). The positive genes were sequenced and identified by National The slides were incubated with mouse anti-JAB1 and rabbit Center for Biotechnology Information Blast search. antihemagglutinin antibodies for 2 h at room temperature. The secondary monoclonal antimouse antibody conjugated with Western Blotting Alexa 488 and antirabbit antibody conjugated with Alexa 594 The protocols for Western blotting basically followed those fluorescent dye (Molecular Probes) were then simultaneously described elsewhere (14). The protein products of the large incubated with cells for 30 min at room temperature. Cell nuclei HBsAg were detected with monoclonal antibody raised against were stained with Hoechst 3324 fluorescent dye. Following the reactions, the slides were mounted and then observed by Leica the sequence in the pre-S1 region (IgMedica Biotechnology) or TCS SP2 spectral confocal microscopy. The relative level of with the antihemagglutinin antibody because the WT or pre-S2 mutant LHBS genes were cloned into the pIRES-hrGFP-2a nuclear JAB1 in each cell was quantified by using the plasmid (Stratagene), which is tagged with hemagglutinin Metamorph 7 software (Molecular Devices). epitope. RB protein was detected with mouse monoclonal antibodies 11D7 and G3-245 (generously supplied by Dr. W-H. AP-1 Transactivation Assay Lee, University of California, Irvine, CA), which recognize The AP-1 activity was measured by using the PathDetect total RB protein (31, 32). To detect phosphorylated RBT821 and in vivo signal transduction pathway cis-reporting systems Cdk2T160, antibodies that specifically recognized the phosphor- (Stratagene). Briefly, the cells were cotransfected with pAP-1- ylated residues were used (AbCam plc and Novus Biologicals, Luc reporter plasmid and WT or pre-S2 mutant LHBS in Inc.). The other primary antibodies used in this study were pIRES-hrGFP-2a vector. The plasmid pFC-MEKK, which mouse monoclonal antibodies against human JAB1 (Becton contains mitogen-activated protein kinase kinase kinase Dickinson Biosciences), MIF (Novus), cyclin D1, Cdk2, cyclin (MEKK), was used as the positive control for AP-1 activity. A, Cdk4, IRE1, p27Kip1, and hemagglutinin epitope (Santa The cells were assayed for luciferase activities by using the Cruz Biotechnology). luciferase assay kit (Applied Biosystems).

Coimmunoprecipitation RNA Interference Protocols for coimmunoprecipitation experiments basically JAB1 coding region, nucleotides 509 to 527 (5¶-ttgatccaa- followed those previously described (14). To pull down HBV caagaacaat-3¶), was cloned into pSUPER (33) to establish the large HBsAg, mouse antihemagglutinin epitope antibody and RNAi construct. HuH-7 cells were transfected with JAB1- protein A/G agarose beads (Santa Cruz Biotechnology) were RNAi plasmid for 96 h. After transfections, the levels of mixed with cell-free extracts of HuH-7 cells transiently JAB1, p27Kip1, and phosphorylated Cdk2T168 were assessed by transfected with WT or pre-S2 LHBS mutant genes cloned in Western blot. pIRES-hrGFP-2a plasmid. Immunoprecipitants were repeatedly washed with radioimmunoprecipitation assay buffer [150 ER Stress mmol/L NaCl, 10 mmol/L Tris (pH 7.2), 0.1% SDS, 1.0% Human HuH-7 cells were treated with an ER stress inducer, Triton X-100, 1% deoxycholate, and 5 mmol/L EDTA] tunicamycin (10 Ag/mL) or thapsigargin (1 Amol/L), for 3 h supplemented with protease inhibitor cocktail (Sigma-Aldrich), and then returned to cell medium for 3 h for further incubation and then analyzed by Western blotting. (34, 35). The cells were washed with PBS (pH 7.4), then lysed in radioimmunoprecipitation assay buffer supplemented with Isolation of Nuclear and Cytosolic Fractions protease inhibitors. ER stress was detected by examining the Human HuH-7 cells were washed with ice-cold PBS, alternative splicing patterns of the ER stress marker XBP1 collected with a cell scraper, and harvested by centrifugation. using multiplex reverse transcription-PCR. The amounts of The cell pellets were lysed in solution A [50 mmol/L HEPES functionally active XBP1, which was unconventionally spliced (pH 7.9), 10 mmol/L KCl, 1 mmol/L EDTA (pH 8.0), 1 mmol/L by IRE1 on ER stress, were measured by using PCR primers for DTT, 1 mmol/L phenylmethylsulfonyl fluoride, and protease total (unspliced and spliced) XBP1 transcripts and those inhibitors]. After cell lysis, 10% Triton X was gradually specifically for spliced XBP1 transcripts (36). The PCR primers added to a final concentration of 0.5% to extract the cell nuclei. used for XBP-1 were forward (total), 5¶-agcactcagactacgtgcac- After brief centrifugation, the supernatant (cytosol) was 3¶; reverse (spliced form), 5¶-acagagaaagggaggctggt-3¶; and collected. The precipitated cell nuclei were lysed in solution reverse (total), 5¶-accacattagcttggctctc-3¶. C [50 mmol/L HEPES (pH 7.9), 0.4 mol/L NaCl, 1 mmol/L Western blot analysis for RB was conducted with anti- EDTA (pH 8.0), 1 mmol/L DTT, 1 mmol/L phenylmethyl- T821 pRB antibody. To detect whether pre-S2 LHBS mutant– sulfonyl fluoride, and protease inhibitors] with vigorous vorte- induced RB hyperphosphorylation is dependent on ER stress, xing for 15 min at 4jC, and then centrifuged. The supernatant, the cells transiently transfected with WT or pre-S2 mutant genes which contained the nuclear extracts, was collected. were treated with ER stress inhibitor vomitoxin (150 ng/mL; Sigma-Aldrich) or mock treated for 24 h (37). After the cells Immunofluorescence Studies had been washed with ice-cold PBS (pH 7.4), they were lysed Â 5 T24 cells (5 10 ) transfected with WT or pre-S2 mutant in radioimmunoprecipitation assay buffer and then analyzed LHBS genes were seeded in a four-well Lab-Tek II chamber by Western blotting.

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Mol Cancer Res 2007;5(10). October 2007 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Hepatitis B Virus Pre-S2 Mutant Surface Antigen Induces Degradation of Cyclin-Dependent Kinase Inhibitor p27 Kip1 through c-Jun Activation Domain-Binding Protein 1

Yi-Hsuan Hsieh, Ih-Jen Su, Hui-Ching Wang, et al.

Mol Cancer Res 2007;5:1063-1072.

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