Activation of NF-kappa B by hepatitis B virus X protein through an Ikappa B kinase-independent mechanism

Nicole H. Purcell*,1,2, Chenfei Yu*,1,2, Daoyao He*,2, Jialing Xiang1, Nir Paran3, Joseph A. DiDonato4, Shoji Yamaoka5, Yosef Shaul3, and Anning Lin1

1 Ben May Institute for Cancer Research, Committee on Cancer Biology, University of Chicago, Chicago, Illinois 60637; 2 Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294; 3 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel; 4 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195; and 5 Institute for Virus Research, Kyoto University, Kyoto 606, Japan


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pX, the hepatitis B virus-encoded transcription coactivator, is involved in viral infection in vivo. pX stimulates the activity of several transcription factors including nuclear factor-kappa B (NF-kappa B), but the mechanism of activation is poorly understood. The Ikappa B kinase complex (IKK) mediates activation of NF-kappa B in response to various extracellular stimuli, including inflammatory cytokines like tumor necrosis factor and interleukin 1, human T cell lymphoma virus 1 Tax protein, and tumor promoters like phorbol esters. It is not known whether IKK also mediates activation of NF-kappa B by pX. Here we report that IKK was not essential for activation of NF-kappa B by pX. Expression of pX resulted in the degradation of Ikappa Balpha in the absence of its phosphorylation at Ser32 and Ser36 residues. Although pX stimulated the activity of cotransfected IKK-beta when it was overexpressed, it failed to activate endogenous IKK. Furthermore, expression of pX stimulated NF-kappa B nuclear translocation and transcriptional activity in IKK-gamma -null fibroblast 5R cells. Our data indicate that pX stimulates NF-kappa B activity through a mechanism that is dependent on Ikappa Balpha degradation but not on IKK activation.

nuclear factor-kappa B; inhibitor of nuclear factor-kappa B; X protein of hepatitis B virus


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HEPATITIS B VIRUS (HBV) is a small, hepatotrophic DNA virus that is able to cause viral hepatitis and may be involved in the development of hepatocellular carcinoma (4, 29, 65). pX (also known as HBx) is a promiscuous viral protein encoded by HBV. The open reading frame of pX is conserved among all mammalian members of the Hepadnaviridae and is essential for woodchuck hepatitis virus infectivity (10, 72). pX functions as a coactivator that transactivates a variety of viral and cellular genes under the control of activator protein (AP)-1, nuclear factor (NF)-AT, and NF-kappa B (12, 23, 31, 56).

The mechanism(s) by which pX activates gene expression is not completely understood. One possible mechanism is that pX may interact directly with the transcription machinery. It is reported that pX interacts with several components of the basal transcription complex including the RPB5 unit of RNA Pol II enzyme, TATA-binding protein (TBP), transcription factor II (TFII)H, TFIIB (11, 24, 38, 51, 64), and some upstream transcription activators, such as cAMP response element binding protein (CREB)/activating transcription factor (ATF) (40) and p53 (60, 61). Another possibility is that pX may stimulate gene expression through the cellular signaling network, including Ras/Raf/mitogen-activated protein (MAP) kinase kinase (MEK)/extracellular signal-related kinase (ERK) MAP kinase pathway (6), protein kinase C (PKC) (28), c-Jun NH2-terminal kinase (JNK) (7), and the Janus kinase (Jak)-signal transducer and activator of transcription (STAT) pathway (32).

pX stimulates NF-kappa B activity (17, 43, 56); however, the mechanism of stimulation is not clear. The dimeric sequence-specific transcription factor NF-kappa B comprises a family of proteins that contain a common Rel homology domain, and the classic form NF-kappa B is a p65/Rel dimer (2). In most resting cells, NF-kappa B is bound to its cytoplasmic inhibitors, the Ikappa Bs, and remains in the cytoplasm as a latent-form transcription factor (1-3, 63). On stimulation by most NF-kappa B stimuli, the Ikappa B proteins become phosphorylated on specific serine residues (Ser32 and Ser36 in Ikappa Balpha ; Ser19 and Ser23 in Ikappa Bbeta ) (1-3, 8, 9, 63). Phosphorylation of the Ikappa B proteins triggers their ubiquitination, followed by degradation by the 26S proteasome (58, 59, 67). Proteolysis of the Ikappa B proteins results in the release of NF-kappa B, which translocates into the nucleus and stimulates transcription of its target genes (58).

The Ikappa B kinase (IKK) was first identified as a high-molecular-weight protein complex (58). Two catalytic subunits of IKK, IKK-alpha and IKK-beta (also known as IKK-1 and IKK-2, respectively), that specifically phosphorylate Ikappa B proteins have been isolated (15, 42, 52, 59, 67, 68, 71). A regulatory subunit of IKK, IKK-gamma (also known as NEMO or IKKAP1) (41, 54, 69) has also been identified. Phosphorylation of IKK-beta on its two serine residues (Ser177 and Ser181) is essential for IKK activation (42, 68, 71). Two protein kinases in the MAP kinase kinase kinase (MEKK) family, NIK and MEKK, have been proposed as immediate upstream kinases of IKK (14, 19, 37, 41, 44, 45, 50, 54, 62, 69, 71, 72). Several other protein kinases including transforming growth factor-beta activated kinase-1 (TAK1) (46) and Akt (39, 48, 53) may also function as upstream activators. Activation of IKK is essential for mediating NF-kappa B activation by many extracellular stimuli including tumor necrosis factor (TNF)-alpha (15, 34, 35, 45, 57), interleukin (IL)-1 (15, 34-35, 45, 57), 12-O-tetradecanoylphorbol 13-acetate (TPA) (30), lipopolysaccharide (LPS) (18, 25, 47), gamma -irradiation (5, 33) and Tax, a viral protein of human T lymphocyte virus 1 (HTLV-1) (13, 19-20, 27, 62, 70).

Here we report that pX activates NF-kappa B through an IKK-independent mechanism. Expression of pX triggers the degradation of Ikappa Balpha without inducing its phosphorylation at Ser32 and Ser36 residues. Consistently, pX is unable to stimulate the enzymatic activity of endogenous IKK-beta in human embryonic kidney 293 cells. Furthermore, pX stimulates NF-kappa B transcriptional activity in IKK-gamma -null fibroblast 5R cells, in which IKK is refractory to activation by many NF-kappa B stimuli such as TNF-alpha . Thus our data indicate that pX stimulates NF-kappa B activity through a mechanism that does not depend on IKK activation and phosphorylation of Ikappa Balpha NH2-terminal serines.


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Cell culture and transfection. HeLa, COS-1, Rat-1, and 5R cells were grown in DMEM (Mediatech, Herndon, VA) supplemented with 10% FCS (Atlanta Biol., Atlanta, GA), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Human embryonic kidney 293 cells were grown in DMEM-F-12 (Mediatech) with the supplements. Transfections were described previously (36).

Plasmids, fusion proteins, and reagents. Hemagglutinin (HA)-tagged IKK-alpha , HA-IKK-beta , HA-Ikappa Balpha , HA-Ikappa Balpha (AA), in which Ser32 and Ser36 were replaced by alanines, M2-pX, or its transcription-inactive mutant M2-pXm7, were described previously (22, 45). Glutathione-S-transferase (GST)-Ikappa Balpha (1-54) and GST-c-Jun(1-79) were purified on glutathione-agarose as described previously (36, 45). Proteasome inhibitors AcLLnL (N-Ac-Leu-Leu-norleucinal) and lactacystin (clasto-lactacystin beta -lactone) were purchased from Calbiochem (San Diego, CA). Rabbit polyclonal anti-p65 was from Zymed (San Francisco, CA), and mouse monoclonal anti-p65 was from Boehringer Mannheim (Indianapolis, IN). Rabbit polyclonal anti-phospho-Ikappa Balpha (Ser32) antibody was from New England BioLabs (Beverly, MA). Anti-M2 and anti-actin monoclonal antibodies were from Sigma (St. Louis, MO).

Protein kinase assays and transcription assays. Transfected HA-IKK-alpha and HA-IKK-beta were immunoprecipitated from HeLa or COS-1 cell extracts with an anti-HA monoclonal antibody (12CA5, Santa Cruz, Santa Cruz, CA), and the activity of the immune complex was assayed at 30°C for 30-60 min in 30 µl of kinase buffer (45) in the presence of 10 µM ATP/10 µCi [gamma -32P]ATP (10 Ci/mmol) with GST-Ikappa Balpha (1-54) as a substrate. The reactions were terminated with 4× Laemmli sample buffer. The proteins were resolved by 13% SDS-PAGE followed by autoradiography. Radioactivity in the phosphorylated proteins was quantitated by a phospho/chemifluorescence imager (Molecular Dynamics, Sunnyvale, CA). To assay the activity of endogenous IKK or JNK, the kinase was immunoprecipitated from 293 cell extracts using a polyclonal anti-IKK-beta antibody (Zymed) or monoclonal anti-JNK antibody (PharMigen, San Diego, CA) and the kinase activity was measured by immunocomplex kinase assays with GST-Ikappa Balpha (1-54) or GST-c-Jun(1-79) as a substrate, respectively.

Rat-1, 5R, or 293 cells were cotransfected with a 2× NF-kappa B luciferase reporter plasmid (45) and various expression vectors, as indicated in Figures 1-5. Luciferase activity was determined as described previously (36).


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Fig. 1.   Activation of nuclear factor (NF)-kappa B by pX. A: pX, but not its transcription-inactive mutant pXm7, stimulates nuclear translocation of p65. Human embryonic kidney 293 cells grown on coverslips were transfected with M2-pX (c) or M2-pXm7 (d) (2 µg each). After 40 h, cells were treated with tumor necrosis factor (TNF)-alpha (20 ng/ml, 15 min; b) or left untreated (a) and fixed. Nuclear localization of p65 was detected by indirect immunofluorescence analysis using a monoclonal anti-p65 antibody as its primary antibody and FITC-conjugated rabbit anti-mouse antibody as secondary antibody. *Cells with nuclear p65 staining. B: 293 cells were cotransfected with a 2× NF-kappa B-luciferase (LUC) reporter plasmid (0.2 µg) and expression vectors encoding M2-pX or M2-pXm7 (50 and 100 ng each) as indicated. After 30 h, cells were treated with TNF-alpha (20 ng/ml) for 10 h or left untreated. Cells were then harvested, and relative LUC activity was determined as described previously (36, 45). Results are means ± SE and represent 4 individual experiments. LUC activity expressed by cells transfected with empty vector (C) was given an arbitrary value of 1.



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Fig. 2.   pX induces Ikappa Balpha degradation in the absence of Ser32 and Ser36 phosphorylation. A: time course of Ikappa Balpha degradation induced by pX. HeLa cells were transfected with the expression vector encoding M2-pX (2 µg; +) or empty vector (-) and treated with TNF-alpha (20 ng/ml, 15 min) or left untreated. Cells were harvested at various time points after transfection as indicated. Degradation of Ikappa Balpha was monitored by immunoblotting using a polyclonal anti-Ikappa Balpha antibody. In parallel, aliquots of each sample were analyzed for the level of actin, using an anti-actin antibody. B: pX-induced Ikappa Balpha degradation was catalyzed by ubiquitination-dependent proteasome but in the absence of phosphorylation of Ser32 and Ser36. HeLa cells were cotransfected with expression vectors encoding hemagglutinin (HA)-Ikappa Balpha or HA-Ikappa Balpha (32/36 AA) mutant (2 µg each) with or without M2-pX (2 µg) or empty vector. After 12 h, cells were treated with or without the proteasome inhibitor AcLLnL (40 µM) for 1 h before being treated with TNF-alpha (20 ng/ml) for 15 min or left untreated. Cells were harvested, and degradation of transfected or endogenous Ikappa Balpha was examined by immunoblotting with anti-Ikappa Balpha (alpha -Ikappa Balpha ) or anti-phospho-Ikappa Balpha (Ser32) (alpha -p-Ikappa Balpha ) antibody, respectively, as indicated. These experiments were repeated 3 times with similar results. WB, Western blot; ns, nonspecific; T, TNF-alpha .



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Fig. 3.   The biphasic effect of pX on Ikappa B kinase complex (IKK)-beta activity. A: HeLa and COS-1 cells were cotransfected with expression vectors encoding HA-IKK-beta (1 µg), with or without M2-pX, or M2-pXm7 (M7) or empty vector (3 µg each), as indicated. After 40 h, cells were harvested. HA-IKK-beta was immunoprecipitated, and its activity was measured by immunocomplex kinase assays with GST-Ikappa Balpha (1-54) as a substrate. Aliquots of each sample were analyzed for expression of HA-IKK-beta and M2-pX by immunoblotting using anti-HA (alpha -HA) or anti-M2 (alpha -M2) antibody, respectively. B: COS-1 cells were cotransfected with expression vector encoding HA-IKK-beta (0.1, 0.3, 0.5, and 1.0 µg) with or without M2-pX (2 µg). The activity of HA-IKK-beta was measured by immunocomplex kinase assays with GST-Ikappa Balpha (1-54) as a substrate. Aliquots of each sample were analyzed for expression of HA-IKK-beta by immunoblotting using anti-HA antibody. Fold stimulation and expression of HA-IKK-beta (bottom) were quantitated by a PhosphoImager. These experiments were repeated 3 times with similar results. KA, kinase assay.



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Fig. 4.   pX activates endogenous c-Jun NH2-terminal kinase (JNK) but not IKK. A: human embryonic kidney 293 cells were transfected with the expression vector encoding M2-pX (0.5 and 2 µg). After 40 h, cells were treated with TNF-alpha (20 ng/ml) for 15 min or left untreated. Endogenous IKK (A) or JNK (B) was immunoprecipitated from cell extracts, and the kinase activity was measured by immunocomplex kinase assays with GST-Ikappa Balpha (1-54) (for IKK assays) or GST-c-Jun-(1-79) (for JNK assays) as a substrate, respectively. Aliquots of each sample were analyzed by immunoblotting using an anti-IKK-beta (alpha -IKK-beta ) antibody or an anti-JNK (alpha -JNK) antibody, respectively. This experiment was repeated 3 times with similar results. C, control.



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Fig. 5.   pX is able to stimulate NF-kappa B activity in IKK-gamma -null fibroblast 5R cells. A: pX-induced nuclear translocation of p65 in IKK-gamma -null 5R cells. Rat-1 or IKK-gamma -null variant 5R cells grown on coverslips were transfected with the expression vector M2-pX or empty vector (2 µg each). After 40 h, cells were treated with TNF-alpha (20 ng/ml) for 15 min or left untreated. Cells were fixed and incubated with either monoclonal anti-p65 (a, b, e, and f) or a mixture of polyclonal anti-p65 and monoclonal anti-M2 (c, d, g, and h). After washing, cells were stained with rabbit anti-mouse FITC (a, b, e, and f) or a mixture of donkey anti-mouse FITC and donkey anti-rabbit TRITC (c, d, g, and h), respectively. p65 nuclear localization was visualized by a Leitz fluorescence microscope. *Cells that express M2-pX or p65 nuclear staining. B: pX stimulated NF-kappa B transcriptional activity in IKK-gamma -null 5R cells. Rat-1 (filled bars) or 5R (open bars) cells were cotransfected with a 2× NF-kappa B-LUC reporter plasmid (0.2 µg) and expression vectors encoding M2-pX (100 ng) as indicated. After 30 h, cells were treated with TNF-alpha (20 ng/ml) for 10 h or left untreated. Cells were then harvested, and relative luciferase activity was determined as described previously (36, 45). The results are means ± SE and represent 4 individual experiments. LUC activity expressed by cells transfected with empty vector was given an arbitrary value of 1.

Immunoblotting and immunofluorescence. Immunoblotting was performed as described previously (45), and the expression of proteins was quantitated using a phospho/chemifluorescence imager (Molecular Dynamics). Detection of subcellular localization of p65 and M2-pX was performed as described previously (54, 73).


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pX stimulates NF-kappa B nuclear translocation and transcription activity. To study activation of NF-kappa B by pX, we determined the effect of pX on NF-kappa B nuclear translocation. 293 Cells grown on coverslips were transiently transfected with expression vectors encoding M2-pX or its inactive mutant M2-pXm7 or empty vector. After 40 h, cells were treated with TNF-alpha for 15 min or left untreated. Cells were fixed, and the nuclear translocation of NF-kappa B was examined by indirect immunofluorescence analysis using a monoclonal anti-p65 antibody. Treatment with TNF-alpha induced the accumulation of p65 in the nucleus (Fig. 1A, b) as reported previously (69). Expression of M2-pX also induced the accumulation of p65 in the nucleus as evident by the enhanced nuclear staining (Fig. 1A, c). Under the same conditions, pXm7 had no detectable effect (Fig. 1A, d). The pX effect appeared to be specific, because it was blocked by treatment with the inhibitor AcLLnL, which inhibits the proteasome-mediated degradation of Ikappa B proteins and prevents p65 nuclear translocation (data not shown).

Translocation into the nucleus allows NF-kappa B to stimulate expression of its target genes. Thus we determined whether pX can stimulate NF-kappa B transcriptional activity with a NF-kappa B reporter gene assay. 293 Cells were transiently transfected with a 2× NF-kappa B luciferase reporter gene (NF-kappa B-LUC) with or without expression vectors encoding M2-pX or M2-pXm7. After 30 h, cells were treated with TNF-alpha for another 10 h or left untreated. As reported previously (45), treatment with TNF-alpha resulted in stimulation of NF-kappa B-LUC activity (Fig. 1B). Expression of M2-pX also stimulated the activity of NF-kappa B-LUC in a dose-dependent manner (Fig. 1B). Under the same conditions, M2-pXm7 failed to stimulate NF-kappa B-LUC activity (Fig. 1B), although its expression was similar to that of the wild-type M2-pX (data not shown). Together, these findings show that expression of pX was able to stimulate NF-kappa B activity, consistent with previous reports (17, 43, 56).

pX-induced Ikappa Balpha degradation requires ubiquitination-dependent proteasome but not Ser32 and Ser36 phosphorylation. A key step that leads to NF-kappa B activation in response to many extracellular stimuli is the degradation of Ikappa Balpha by the ubiquitination-dependent proteasome, which is generally triggered by Ikappa Balpha phosphorylation at Ser32 and Ser36 (1-3, 8, 9, 63). To understand how pX activates NF-kappa B, we determined whether pX induces degradation of Ikappa Balpha , and, if so, whether it depends on Ser32 and Ser36 phosphorylation.

HeLa cells were transfected with or without the expression vector encoding M2-pX or empty vector and harvested at various time points. Cells treated with TNF-alpha were used as a positive control. The degradation of Ikappa Balpha was analyzed by immunoblotting using an anti-Ikappa Balpha antibody. Degradation of Ikappa Balpha by M2-pX was noticeable during the period from 11 to 13 h after transfection (Fig. 2A, top). The degradation was specific because the expression level of actin was not affected (Fig. 2A, bottom). pX-induced Ikappa Balpha degradation was partial, as reported previously (56). The pX effect is probably underestimated, however, because the efficiency of transient transfection was low (~5-10%).

Degradation of Ikappa Balpha can be a phosphorylation-dependent (1-3, 8, 9, 63) or -independent (5, 33) process. To determine the mechanism by which pX induces Ikappa Balpha degradation, HeLa cells were cotransfected with expression vectors encoding M2-pX, HA-Ikappa Balpha , HA-Ikappa Balpha (32/36AA) mutant, in which Ser32 and Ser36 residues were replaced with nonphosphorylatable alanines, or empty vector. Cells were treated with or without the proteasome inhibitors AcLLnL or lactacystin. Before harvesting, cells were treated with TNF-alpha for 15 min or left untreated. As shown in Fig. 2A, treatment with TNF-alpha resulted in a rapid and complete degradation of endogenous Ikappa Balpha . This degradation can be blocked by pretreatment of the cells with the proteasome inhibitor AcLLnL (16), resulting in the accumulation of a slower-migrating form of Ikappa Balpha (Fig. 2B, top), which corresponds to its phosphorylation form (see Fig. 2B, bottom). Expression of M2-pX induced a partial degradation of endogenous Ikappa Balpha (Fig. 2A, top; Fig. 2B, top). In the presence of AcLLnL, M2-pX induced a slight accumulation of the slower-migrating form of Ikappa Balpha (Fig. 2B, top). This accumulation, however, was not pX specific, because there was a similar level of accumulation of the slower-migrating form of Ikappa Balpha in the control, which was only treated with AcLLnL (Fig. 2B, top). Similar results were obtained when lactacystin was used (data not shown). Consistently, the immunoblotting analysis with anti-phospho-Ikappa Balpha antibody showed that expression of M2-pX was unable to significantly induce endogenous Ikappa Balpha phosphorylation over the control (Fig. 2B, bottom).

The degradation profile of the transfected HA-Ikappa Balpha was similar to that of endogenous Ikappa Balpha under the same conditions (Fig. 2B). Interestingly, expression of M2-pX also induced a partial degradation of the HA-Ikappa Balpha (32/36AA) mutant (Fig. 2B, top). As previously reported (1-3, 8, 9, 61), the HA-Ikappa Balpha (32/36AA) mutant was resistant to TNF-alpha -induced phosphorylation (Fig. 2B, bottom) and degradation (Fig. 2B, top). These results indicate that, unlike TNF-alpha , M2-pX may induce Ikappa Balpha degradation in the absence of Ser32 and Ser36 phosphorylation.

The biphasic effect of pX on the activity of HA-IKK-beta . The finding that pX can induce Ikappa Balpha degradation in the absence of its phosphorylation at Ser32 and Ser36 prompts us to examine the effect of pX on IKK, the key enzyme that controls NF-kappa B activation in response to various extracellular stimuli (14, 15, 41, 42, 52, 54, 68, 69, 71).

HeLa or COS-1 cells were cotransfected with expression vectors encoding HA-IKK-beta with or without M2-pX or M2-pXm7, respectively. After 40 h, cells were harvested and the IKK activity was measured by immunocomplex kinase assays with GST-Ikappa Balpha as a substrate (45). In both cell lines, the activity of HA-IKK-beta was enhanced by cotransfected M2-pX but not by M2-pXm7 (Fig. 3A, top). However, the expression level of HA-IKK-beta was also increased by M2-pX (Fig. 3A, middle). The inability of M2-pXm7 to enhance IKK-beta activity and expression was not a result of differences in expression between M2-pXm7 and M2-pX, as demonstrated by immunoblotting analysis (Fig. 3A, bottom). Under the same conditions, activation of HA-IKK-alpha by pX was undetectable (data not shown).

To further analyze IKK-beta activation by pX, COS-1 cells were transfected with various amounts of HA-IKK-beta in the presence or absence of M2-pX. Coexpression of M2-pX enhanced both activity and expression of HA-IKK-beta (Fig. 3B). Interestingly, the effect of M2-pX on HA-IKK-beta activity showed a biphasic profile. At lower concentrations of HA-IKK-beta the increased HA-IKK-beta activity was entirely caused by the increase in its protein expression (Fig. 3B). At higher concentrations, however, there was a net increase in the activity of HA-IKK-beta (Fig. 3B). Thus M2-pX was able to stimulate the enzymatic activity of cotransfected HA-IKK-beta when the kinase was overexpressed. Consistently, in HeLa cells, where the expression level of HA-IKK-beta was much lower than that in COS-1 cells (Fig. 3A, middle), coexpression of M2-pX led to a net increase in HA-IKK-beta activity only at higher concentrations of cotransfected HA-IKK-beta (data not shown). The net increase in HA-IKK-beta induced by M2-pX was minimal (~3-fold in COS-1 and 1.5-fold in HeLa) but repeatable.

pX does not activate endogenous IKK. To determine whether pX can stimulate the activity of endogenous IKK, 293 cells were transfected with expression vectors encoding M2-pX or HA-MEKK1. After 40 h, cells were treated with TNF-alpha for 15 min or left untreated. Endogenous IKK was immunoprecipitated, and its activity was measured by immunocomplex kinase assays with GST-Ikappa Balpha as a substrate. As reported previously, treatment with TNF-alpha strongly activated endogenous IKK (Fig. 4A). In contrast, expression of M2-pX failed to do so (Fig. 4A). The inability of M2-pX to activate endogenous IKK was not the result of the expression of M2-pX or the transfection efficiency (data not shown). Under the same conditions, M2-pX was able to stimulate NF-kappa B transcription activity as measured by NF-kappa B reporter assays (data not shown; see Fig. 1B). Furthermore, expression of M2-pX was able to stimulate the activity of endogenous JNK as measured by immunocomplex kinase assays with GST-c-Jun as a substrate (Fig. 4B). These results suggest that pX has no detectable stimulatory effect on endogenous IKK activity.

pX induces NF-kappa B activation in IKK-gamma -null fibroblast 5R cells. IKK-gamma is the regulatory subunit of the IKK complex and is essential for IKK-mediated NF-kappa B activation in response to many stimuli (41, 54, 69). For instance, no NF-kappa B activity can be induced by TNF-alpha , IL-1, or Tax in IKK-gamma -null fibroblast 5R cells (69). Because pX was able to stimulate NF-kappa B activity in the absence of IKK activation, we examined the effect of pX on NF-kappa B activity in IKK-gamma -null 5R cells.

Rat fibroblast Rat-1 cells or IKK-gamma -null 5R variant cells grown on coverslips were transfected with the expression vector encoding M2-pX or empty vector, respectively. After 40 h, the cells were treated with TNF-alpha for 15 min or left untreated. The effect of pX on NF-kappa B activation was determined by indirect immunofluorescence. The subcellular distribution of p65 was detected by staining with monoclonal anti-p65 antibody, whereas M2-pX-transfected cells were identified by double staining with polyclonal anti-p65 and monoclonal anti-M2. Treatment with TNF-alpha only induced p65 nuclear accumulation in Rat-1 cells but not in IKK-gamma -null 5R cells (Fig. 5A, b and f). In contrast, expression of M2-pX induced nuclear staining of p65 in both Rat-1 and 5R cells (Fig. 5A, c and g).

To determine the effect of pX on NF-kappa B transcriptional activity in 5R cells, Rat-1 and 5R cells were also transfected with the NF-kappa B-LUC reporter gene with or without the expression vector encoding M2-pX. Before harvesting, cells were treated with TNF-alpha for 10 h or left untreated. Consistent with the results of indirect immunofluorescence shown in Fig. 5A, TNF-alpha was only able to stimulate the activity of NF-kappa B-LUC in the parental Rat-1 cells but not in the IKK-gamma -null 5R cells (Fig. 5B). In contrast, expression of M2-pX stimulated the activity of NF-kappa B-LUC in both Rat-1 and 5R cells (Fig. 5B). These results indicate that pX may stimulate NF-kappa B activity in the absence of a functional IKK complex.


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pX, the transcription cofactor encoded by HBV, is a promiscuous viral protein that is able to transactivate numerous viral and cellular genes controlled by transcription factors such as AP-1, NF-AT, and NF-kappa B (12, 23, 31, 56). However, the mechanism of action of pX is not completely understood. In this study, we examined whether pX-induced NF-kappa B activation is mediated by IKK, which is a key enzyme that controls NF-kappa B activation in response to various stimuli. Our results indicate that pX may stimulate NF-kappa B activity but bypass the IKK signaling pathway. This conclusion is based on several lines of evidence. First, pX-induced Ikappa Balpha degradation is mediated by the ubiquitination-dependent proteasome. However, the degradation occurs in the absence of Ser32 and Ser36 phosphorylation (Fig. 2B). Second, pX is unable to stimulate the enzymatic activity of IKK unless IKK is overexpressed (Figs. 3 and 4). Third, pX induces NF-kappa B activation in IKK-gamma -null fibroblast 5R cells (Fig. 5).

Phosphorylation of Ser32 and Ser36 residues is a critical step that triggers Ikappa Balpha degradation by ubiquitination-dependent proteasome in response to many NF-kappa B activators, including TNF-alpha (15, 34, 35, 45, 57), IL-1 (15, 34, 35, 45, 57), TPA (30), LPS (18, 25, 47), gamma -irradiation (5, 33), and Tax (13, 19, 20, 27, 62, 70). Surprisingly, pX induces Ikappa Balpha degradation in the absence of phosphorylation at these serines, as determined by immunoblotting with the anti-phospho-Ikappa Balpha antibody that specifically recognizes phosphorylated Ser32 (Fig. 2B). Consistently, pX also induces degradation of the nonphosphorylatable HA-Ikappa Balpha (32/36AA) mutant, although to a lesser extent (Fig. 2B). It is plausible that the basal IKK-beta might facilitate the degradation of wild-type Ikappa Balpha by pX but not the Ikappa Balpha (32/36AA) mutant. We also do not detect tyrosine phosphorylation of Ikappa Balpha (unpublished results), which is a novel mechanism by which intact Ikappa Balpha disassociates from NF-kappa B in hypoxic cells on reoxidation (26). Although the known phosphorylation events are apparently not involved, degradation of Ikappa Balpha by pX was still mediated by ubiquitination-dependent proteasome (Fig. 2B), consistent with previous reports (56). Thus pX, along with ultraviolet radiation, may form a unique class of NF-kappa B activators, which induce degradation of Ikappa Balpha in a proteasome-dependent manner but bypass IKK activation and Ikappa Balpha phosphorylation. The mechanism by which pX induces degradation of Ikappa Balpha is currently under investigation.

These findings contradict an earlier report that expression of pX induces Ikappa Balpha phosphorylation (56). This discrepancy may have resulted from different methods used to analyze the status of Ikappa Balpha phosphorylation. In the earlier report, phosphorylation of Ikappa Balpha was concluded on the basis of the observation that a small portion of Ikappa Balpha had a slightly slower migration on a SDS gel (56). However, this slightly slower-migrating form of Ikappa Balpha may not have resulted from pX-induced phosphorylation on Ser32 and Ser36. As shown in Fig. 2B, treatment of cells with the proteasome inhibitor AcLLnL alone results in a similar slightly slower-migrating form of Ikappa Balpha . This slower-migrating form of Ikappa Balpha can be recognized by the anti-phospho-Ikappa Balpha antibody, suggesting that at least low-level phosphorylation of Ser32 may be involved in the basal turnover of Ikappa Balpha proteins. Nevertheless, expression of pX does not enhance phosphorylation of Ikappa Balpha at these serines. We cannot, however, formally exclude the possibility that pX may stimulate other protein kinases that phosphorylate Ikappa Balpha on other unknown region(s), leading to its ubiquitination and degradation.

How pX stimulates NF-kappa B activity has yet to be determined. It is reported that pX may activate NF-kappa B through inducing the degradation of Ikappa Balpha and the NF-kappa B precursor/inhibitor p105 (56). Our results support this model, although it is not clear how pX induces Ikappa Balpha degradation. Alternatively, pX may directly associate with Ikappa Balpha and prevent its association with or induce its disassociation from NF-kappa B. Recently, Israel and colleagues (66) have elegantly shown that cotransfected Ikappa Balpha interacted with pX and transported it into the nucleus. Interestingly, they found that pX interacts with the newly synthesized endogenous Ikappa Balpha induced by TNF-alpha but not with endogenous Ikappa Balpha in nonstimulated cells and that it does not disassociate the preformed NF-kappa B-Ikappa Balpha complex (66). It is proposed that the nuclear localization of the Ikappa Balpha -pX complex prevented Ikappa Balpha from disrupting the NF-kappa B-DNA complex, resulting in sustained NF-kappa B activation (66). However, we found that transfected pX mainly locates in the nucleus (Fig. 5A), consistent with previous reports (24). It is also not clear how pX activates NF-kappa B in the first place, i.e., in the absence of cotransfected Ikappa Balpha or other stimuli. We propose here that degradation of Ikappa Balpha induced by pX may set NF-kappa B activation in motion. This effect of pX is then reinforced by retention of newly synthesized Ikappa Balpha in the nucleus by pX. It is also plausible that pX may stimulate the activity of the proteasome, which in turn degrades Ikappa Balpha . This is not likely, however, because pX is reported to inhibit, rather than activate, proteasome activity (21). It is more likely, however, that the interaction with pX may bring the proteasome near Ikappa Balpha proteins, resulting in its degradation. Future studies must test this hypothesis.

The effect of pX on IKK activity is intriguing. In both COS-1 and HeLa cells expression of pX only stimulates IKK activity when IKK is overexpressed (Fig. 3B). More importantly, expression of pX is unable to activate endogenous IKK (Fig. 4A), although it does activate endogenous JNK (Fig. 4B). IKK must be incorporated into the IKK complex for its proper regulation (54). Overexpression of HA-IKK-beta may result in a portion of the transfected HA-IKK-beta staying as a nonincorporated form and being activated by pX through a yet to be identified mechanism. It would be interesting to determine whether expression of IKK is upregulated in HBV-infected hepatocytes so that pX could activate IKK. How pX induces expression of the cotransfected IKK-beta is not clear. It may be plausible that pX stimulates yet to be identified transcription factors that bind to the promoter of the cotransfected IKK-beta and induce its expression. Nevertheless, our data indicate that in the absence of a functional IKK, pX is still able to induce nuclear translocation and activation of NF-kappa B, as demonstrated by the use of IKK-gamma -null fibroblast 5R cells (Fig. 5). Thus IKK activation may not be essential for pX activation of NF-kappa B. This conclusion is quite unexpected, considering the fact that the properties of pX are very similar to those of Tax, a viral transforming protein of HTLV-1 that activates NF-kappa B through activation of IKK (13, 19, 20, 27, 62, 70). We cannot, however, rule out the possibility that activation of NF-kappa B by pX might be mediated by the atypical IKK-i/IKK-epsilon (49, 55) or some unknown IKKs. In fact, we found that activation of NF-kappa B by pX was blocked by the dominant-negative mutants of MEKK1, NIK, and Akt, the putative IKK upstream kinases (unpublished results). This possibility is currently under investigation in our laboratory. It is possible that activation of NF-kappa B by pX may be utilized by HBV for its infection and may also contribute to the development of hepatic carcinogenesis.


    ACKNOWLEDGEMENTS

We thank Tony Hunter, Michael Karin, and David A. Brenner for critical comments and helpful discussions.


    FOOTNOTES

* N. H. Purcell, C. Yu, and D. He contributed equally to this work.

This work was supported by National Cancer Institute Grant CA-73740 and American Cancer Society Grant RPG-99-171-01-CCC.

Address for reprint requests and other correspondence: A. Lin, Ben May Institute for Cancer Research, Univ. of Chicago, 5841 S. Maryland, MC 6027, Chicago, IL 60637 (E-mail:alin{at}huggins.bsd.uchicago.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 July 2000; accepted in final form 10 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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