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Originally published In Press as doi:10.1074/jbc.M110480200 on February 5, 2002

J. Biol. Chem., Vol. 277, Issue 16, 13745-13751, April 19, 2002
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The Human Herpes Virus 8-encoded Viral FLICE Inhibitory Protein Physically Associates with and Persistently Activates the Ikappa B Kinase Complex*

Li Liu, Michael T. Eby, Nisha Rathore, Suwan K. Sinha, Arvind Kumar, and Preet M. ChaudharyDagger

From the Hamon Center for Therapeutic Oncology Research and Division of Hematology-Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593

Received for publication, October 31, 2001, and in revised form, February 4, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human herpesvirus 8 (HHV8, also called Kaposi's sarcoma-associated herpesvirus) has been linked to Kaposi's sarcoma and primary effusion lymphoma (PEL) in immunocompromised individuals. We demonstrate that PEL cell lines have a constitutively active NF-kappa B pathway, which is associated with persistent phosphorylation of Ikappa Balpha . To elucidate the mechanism of NF-kappa B activation in PEL cell lines, we have investigated the role of viral FLICE inhibitory protein (vFLIP) in this process. We report that stable expression of HHV8 vFLIP in a variety of cell lines is associated with persistent NF-kappa B activation caused by constitutive phosphorylation of Ikappa Balpha . HHV8 vFLIP gets recruited to a ~700-kDa Ikappa B kinase (IKK) complex and physically associates with IKKalpha , IKKbeta , NEMO/IKKgamma , and RIP. HHV8 vFLIP is incapable of activating NF-kappa B in cells deficient in NEMO/IKKgamma , thereby suggesting an essential role of an intact IKK complex in this process. Our results suggest that HHV8 vFLIP might contribute to the persistent NF-kappa B activation observed in PEL cells by associating with and stimulating the activity of the cellular IKK complex.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear factor kappa B (NF-kappa B)1 is a heterodimeric transcription factor that is primarily composed of 50- and 65-kDa subunits of the Rel family and that is required for regulated expression of several genes involved in inflammation and immune response (1-3). NF-kappa B is present in the cytoplasm of cells in association with a family of inhibitory proteins, called Ikappa B (1, 4). Ikappa B proteins retain NF-kappa B in the cytoplasm by masking its nuclear localization signal. Stimulation by a number of cytokines, such as TNFalpha and interleukin-1, results in the activation of a large molecular mass (600-900 kDa), Ikappa B kinase complex that leads to inducible phosphorylation of the Ikappa B proteins at two N-terminal serine residues (5, 6). This complex consists of two catalytic subunits, IKKalpha (IKK1) and IKKbeta (IKK2), and a regulatory subunit, NEMO/IKKgamma (7-13). The phosphorylation of Ikappa B proteins results in their rapid ubiquitination and proteasome-mediated degradation, which releases NF-kappa B from their inhibitory influence. Once released, NF-kappa B is free to migrate to the nucleus and activate transcription of its target genes.

Some of the noteworthy genes activated by NF-kappa B include those for cytokines and growth factors, chemokines, cell adhesion molecules, acute phase proteins, anti-apoptotic proteins, and transcription factors p53 and c-Myc (2). The NF-kappa B pathway has been also shown to play a key role in the control of cell proliferation and oncogenesis. Several members of the NF-kappa B family have been associated with the development of tumors as a result of overexpression, gene amplification, or gene rearrangement (14). Activation of the NF-kappa B has been shown to be responsible for the transforming ability of human T-cell leukemia virus type I Tax and Epstein-Barr virus latent membrane protein 1 (15).

Caspase 8 (FLICE/MACH or Mch5) is one of the apical caspases of the caspase cascade, which is activated by signaling via the death receptors belonging to the TNF receptor family (16-18). Caspase 8 is recruited to the multimerized death-inducing signaling complex of these receptors via its N-terminal prodomain, which contains two homologous copies of a death effector domain. Death effector domain-containing prodomains are also found in two additional cellular proteins: caspase 10 (Mch4 and FLICE2) (18, 19), and MRIT (c-FLIP, Caspar, I-FLICE, FLAME, CASH, and CLARP), a caspase 8 homolog that is devoid of protease activity (20-26).

Several viruses also encode proteins containing two death effector domains (27-29). These virally encoded death effector domain-containing proteins (also called viral FLICE inhibitory proteins or vFLIPs) include the orf-K13 from the human herpesvirus 8 (HHV8)/KS-associated herpesvirus, MC159L and MC160L from the Molluscum contagiousum virus, and E8 from the equine herpesvirus 2. Recently, similar vFLIPs have been found in other Gammaherpesviridae of the genus Rhadinovirus, including rhesus rhadinovirus, herpesvirus saimiri, and bovine herpesvirus 4 (30-32).

We and others have previously demonstrated that overexpression of HHV8 vFLIP can protect against death receptor-induced apoptosis in vitro and to promote tumor growth in vivo (33-35). Furthermore, unlike MC159L and E8, HHV8 vFLIP was found to activate the NF-kappa B pathway when overexpressed in 293T and NIH3T3 cells by transient transfection (33). The present study was undertaken to better understand the mechanism of NF-kappa B activation by HHV8 vFLIP.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- pGEX-KG Ikappa Balpha (1-54) and pGEX-KG Ikappa Balpha (S32A/S36A) were generous gifts from Dr. Richard Gaynor. Retrovirus constructs containing C-terminal FLAG epitope tag vFLIP or MRIT/cFLIP were constructed in MSCVneo-based retroviral vectors, and the amphotropic retroviruses were generated as described previously (36).

Expression of Bacterially Produced GST-Ikappa B Proteins-- The wild-type and mutant Ikappa Balpha pGEX-KG constructs were transformed into Escherichia coli BL21 DE3. Cultures (400 ml) of E. coli were grown to an A600 nm of 0.6-0.8 and induced with 0.5 mM isopropyl-D-thiogalactopyranoside for 3 h. The cells were pelleted, resuspended in buffer A (20 mM HEPES, pH 7.9, 400 mM NaCl, 5 mM dithiothreitol (DTT), 50 mM manitol, 10 mM sodium ascorbate, 10% glycerol, 0.1 mM EDTA, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF)), mildly sonicated, and centrifuged. The supernatant was incubated with 0.5 ml of glutathione-agarose matrix (Sigma) for 1 h at 4 °C. The matrix was then washed four times with buffer A and two times with buffer B (50 mM Tris, pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, 5 mM DTT, 1 mM PMSF). These GST fusion proteins were eluted off the matrix by buffer B containing 10 mM glutathione, dialyzed with buffer containing 20 mM HEPES, pH 7.6, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 10% glycerol, and 0.5 mM PMSF, aliquoted, and stored at -80 °C.

Cell Culture and Protein Extraction-- Human non-small cell lung cancer cell line H460 (a kind gift of Dr. John Minna) was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Murine pre-B-cell line 70Z/3 and its NEMO-deficient mutant 1.3E2 (a kind gift of Dr. Carol Sibley) were cultured in RPMI 1640 medium supplemented with 7% fetal bovine serum and 50 µM beta -mercaptoethanol. Retrovirus-infected H460 cells were maintained in medium with 1000 µg/ml of G418, whereas clones of 70Z/3 and 1.3E2 cells were selected in 300 µg/ml of G418 (Invitrogen).

To obtain cytoplasmic proteins, the cells were washed with cold phosphate-buffered saline (pH 7.2), resuspended in buffer C (10 mM HEPES, pH 7.6, 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 50 mM NaF, 50 mM beta -glycophosphate, 5% glycerol, 1× protease inhibitor mixture (Roche Molecular Biochemicals)), and incubated on ice for 15 min. At the end of incubation, 1:20 volume of 10% Nonidet P-40 was added. The cells were vortexed for 30 s and then subjected to centrifugation for 30 s. The supernatants were collected as cytoplasmic extracts. The protein concentrations of the cytoplasmic extracts were determined by using Bio-Rad protein assay reagent.

Nuclei from parental and virus-infected H460, 70Z/3, or 1.3E2 were resuspended in buffer containing 20 mM HEPES, pH 7.6, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 10% glycerol, 1× protease inhibitor mixture and extracted at 4 °C on a rocker for 30 min, followed by Eppendorf centrifugation at 14,000 rpm for 10 min. The supernatants were collected as nuclear extracts.

To prepare whole cell extracts, the cells were washed with cold phosphate-buffered saline twice and lysed in lysis buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 10 mM beta -glycophosphate, 10 mM NaF, 1 mM DTT, 1× protease inhibitor mixture (Roche Molecular Biochemicals) at 4 °C for 30 min. After incubation, the mixture was pippetted five or six times to disperse the cells followed by centrifugation at 14,000 rpm at 4 °C for 10 min. The supernatants were collected as whole cell extracts, and protein concentration was determined as described above.

Electrophoretic Mobility Shift Assay-- 10 µg of each nuclear extract sample was incubated with 0.1 pmol of 32P-labeled double-stranded kappa B binding oligonucleotide (5'-GCTGGGGACTTTC-3') or SP1 binding oligonucleotide (5'-ATTCGATCGGGGCG GGGCGAGC-3') in buffer containing 1 µg of poly(dI-dC), 1 µg of bovine serum albumin, 10 mM HEPES, pH 7.6, 0.5 mM DTT, 0.1 mM EDTA, 60 mM KCl, 0.2 mM PMSF, 5 mM MgCl2, and 12% glycerol at room temperature for 30 min. The samples were analyzed by 5% native PAGE followed by autoradiography. For competition and antibody-mediated supershift experiments, kappa B-specific or -nonspecific oligonucleotides or specified antibodies were added to reaction for 15 min at room temperature before the addition of radio isotope-labeled kappa B probe.

Immunoprecipitation of vFLIP or MRIT/cFLIP-- Immobilized monoclonal antibody against FLAG (M2, Sigma) or mouse IgG-Sepharose beads as control was added to cellular extracts (4 mg) prepared from virus-infected H460 cells and incubated at 4 °C for 1 h. In the case of immunoprecipitation with alpha -NEMO antibody, 10 µl of soluble antibody were added to cellular extracts for 30 min on ice. Protein G-Sepharose Cl-4B were then added to the mix, and incubation was performed at 4 °C for 1 h. The beads were washed three times with buffer containing 40 mM HEPES, pH 7.9, 500 mM NaCl, 0.2 mM EDTA, 1 mM DTT, 10 mM NaF, 10 mM beta -glycophosphate, and 0.1% Nonidet P-40. In vitro kinase assay or Western blotting experiments were then performed.

In Vitro Kinase Assay-- Beads from the immunoprecipitation experiments were used to incubate with soluble GST-Ikappa Balpha proteins (10 µg/reaction) in buffer containing 20 mM HEPES, pH 7.6, 100 mM KCl, 10% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 8 µM MgCl2, 3.3 µg/µl bovine serum albumin, 4 µM ATP, 10 µCi of [gamma -32P]ATP, 5 mM NaF, 5 mM beta -glycophosphate, and protease inhibitor mixture (Roche Molecular Biochemicals) at 37 °C for 30 min. 30 µl of reaction solution was added to each sample. 10 µl of 4× SDS-PAGE sample buffer was added at the end of reaction, and the mixture was heated at 95 °C for 5 min and pelleted by centrifugation. The supernatants were then resolved on SDS-12% polyacrylamide gels followed by autoradiography. For in-solution kinase assay, protein samples (10 µl each of fractions from Superdex 200 column) were incubated with soluble GST-Ikappa Balpha protein in the same reaction conditions mentioned above.

Western Blot Analysis-- 100-200 µg of whole cell extracts, cytoplasmic extracts, or proteins immunoprecipitated on agarose beads were heated in the presence of SDS-PAGE sample buffer and loaded on 12% SDS-PAGE gel followed by transferring to nitrocellulose membranes. These membranes were incubated with antibodies against specified proteins in Tris-buffered-saline with Tween 20 followed by incubation with secondary antibody and development with enhanced chemiluminescence (Pierce). The primary antibodies used in these experiments were alpha -Ikappa Balpha (Santa Cruz Biotechnology, SC-371, 1:5000), p-Ikappa Balpha (New England Biolabs, 9241S, 1:1000), alpha -FLAG (Santa Cruz Biotechnology, SC-807, 1:5000), alpha -NEMO (Santa Cruz Biotechnology, SC-8330, 1:4000), alpha -RIP (Transduction Laboratories, R41220, 1:1000), alpha -IKKalpha (Santa Cruz Biotechnology, SC-7182, 1:4000), and alpha -IKKbeta (Santa Cruz Biotechnology, SC-7607, 1:5000).

Protein Fractionation-- S100 extracts were prepared from H460 cells infected with retroviruses encoding HHV8 vFLIP or MRIT/cFLIP as described previously (37). These extracts were fractionated on a Superdex 200 column (Amersham Biosciences) and eluted with buffer containing 20 mM HEPES, pH 7.6, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 10% glycerol, and 0.5 mM PMSF.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Constitutive Activation of NF-kappa B in HHV8-infected PEL Cell Lines Is Due to Persistent Activation of the IKK Complex-- NF-kappa B is normally sequestered in the cytoplasm of cells because of its association with a family of inhibitory proteins, called Ikappa B (1, 4). However, NF-kappa B is persistently present in the nuclei of human T-cell leukemia virus type I- and Epstein-Barr virus-transformed cells and has been shown to contribute to the transforming ability of these viruses (15). We were interested in checking whether infection by HHV8 virus might also lead to persistent NF-kappa B activation. To test this hypothesis we used electrophoretic mobility shift assay (EMSA) to examine the DNA binding activity of nuclear NF-kappa B in three HHV8-infected PEL cell lines, BC-1, BC-3, and BCBL, respectively. We also used two non-HHV8-infected lymphoid cell lines, CEM and Jurkat, respectively, as controls for the above experiment. Consistent with a recent report (38), persistent NF-kappa B activation was seen in all three HHV8-infected PEL cell lines, with the BC-1 cell line showing the highest and BCBL cell line showing the least NF-kappa B activation. In contrast, NF-kappa B binding activity was absent in the nuclear extracts of CEM and Jurkat cell lines (Fig. 1A).


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  		Fig. 1.   PEL cell lines have constitutive NF-kappa B activation. A, EMSA demonstrating constitutive NF-kappa B DNA binding activity in the PEL cell lines. The position of the NF-kappa B complex is marked with an arrow. Nuclear extracts from the following cell lines were used. Lane 1, Jurkat; lane 2, CEM; lane 3, BC-1; lane 4, BC-3; lane 5, BCBL. B, EMSAs demonstrating the nature and specificity of NF-kappa B complex in BC-1 cell line. The following nuclear extracts were used. Lane 1, BC-1 + cold nonspecific competitor oligonucleotide; lane 2, BC-1 + cold kappa B competitor oligonucleotide; lane 3, BC-1 + p65 antiserum; lane 4, BC-1 + p50 antiserum; lane 5, BC-1 + c-Rel antiserum. The position of the NF-kappa B complex is marked with arrow, whereas an asterisk marks the position of a nonspecific band. The arrowheads mark the position of the supershifted complexes. C, status of phosphorylated and total Ikappa Balpha in PEL cell lines. Cellular extracts containing equal amount of protein were resolved by SDS-PAGE, and phosphorylation of Ikappa Balpha was analyzed by Western blot using phospho-Ikappa Balpha antibody (top panel). The blot was stripped and reprobed with an antibodies directed against Ikappa Balpha to demonstrate degradation of Ikappa Balpha . Western blotting with antibodies against IKKalpha , IKKbeta , and actin is used to demonstrate equal loading of proteins in different lanes. Treatment of Jurkat cells with TNFalpha (10 ng/ml) was carried out for 15 min and served as a positive control.

We next sought to analyze the nature and composition of the observed complex in the BC-1 cell line. As shown in Fig. 1B, the observed complex in the BC-1 cell line could be effectively competed with an excess cold probe containing kappa B binding sites but was unaffected by competition with a nonspecific DNA duplex. Finally, a supershift assay utilizing subunit-specific antibodies demonstrated that the observed complex contained the p65 and p50 subunits of NF-kappa B (Fig. 1B).

To determine the mechanism of persistent NF-kappa B activation in PEL cell lines, we examined the phosphorylation status of Ikappa Balpha protein by Western blot analysis. Consistent with the EMSA results, phosphorylation of Ikappa Balpha was totally absent in CEM and Jurkat cells (Fig. 1C). In contrast, phosphorylation of Ikappa Balpha was readily detected in all three PEL cell lines and was present in the following order of magnitude: BC-1 > BC-3 > BCBL (Fig. 1C). Of interest, persistent phosphorylation of Ikappa Balpha observed in BC-1 and BC-3 cell lines was significantly stronger than the TNF-induced Ikappa Balpha -phosphorylation observed in Jurkat cells. Reprobing of the above blot with an Ikappa Balpha antibody revealed a decrease in the level of total Ikappa Balpha protein in the PEL cell lines (Fig. 1C). The above results suggest that the constitutive NF-kappa B activation in the PEL cell lines is probably due to persistent phosphorylation and subsequent degradation of the Ikappa Balpha protein.

Retroviral-mediated Expression of HHV8 vFLIP Leads to Persistent NF-kappa B Activation-- We have previously demonstrated that transient transfection-based overexpression of HHV8 vFLIP can lead to NF-kappa B activation (33). We were interested in determining whether the constitutive NF-kappa B activation observed in PEL cell lines might be mediated by HHV8 vFLIP. To test this hypothesis, we began by checking whether stable expression of HHV8 vFLIP can lead to constitutive NF-kappa B activation. For this purpose, we used retroviral-mediated gene transfer to generate mass culture of H460 cells with stable expression of FLAG epitope-tagged HHV8 vFLIP or MRIT/cFLIP. As shown in Fig. 2A, stable expression of HHV8 vFLIP in H460 cells lead to strong NF-kappa B binding activity as measured by gel shift assay. In contrast, cells expressing an empty vector or MRIT/cFLIP demonstrated low level basal NF-kappa B binding activity. The specificity of the NF-kappa B complex seen in the vFLIP-expressing cells was confirmed by competition with a cold NF-kappa B probe or a nonspecific probe (Fig. 2A). A supershift assay utilizing subunit specific antibodies demonstrated that the complex was a heterodimer of p65 and p50 subunits of NF-kappa B (Fig. 2A). Essentially similar results were obtained upon stable expression of HHV8 vFLIP in 293T and TF-1 cells (Fig. 2B and data not shown). Finally, expression of vFLIP in 293T and H460 cells was associated with an increase in NF-kappa B transcriptional activity as measured by a luciferase-based reporter assay (Fig. 2C and data not shown).


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  		Fig. 2.   Expression of HHV8 vFLIP leads to persistent NF-kappa B activation. A, electrophoretic mobility shift assays. Upper panel, nuclear extracts were prepared from parental H460 cells (lane 1) or those expressing empty vector (lane 2), vFLIP (lane 3), or MRIT/cFLIP (lane 4), and the assay was carried out as described under "Experimental Procedures." The position of the NF-kappa B complex is marked by an arrow. The specificity of the complex is demonstrated by competition with excess cold nonspecific probe (lane 5), wild-type NF-kappa B probe (lane 6), or mutant NF-kappa B probe (lane 7). Supershift assay was performed using antiserum against p65 (lane 8), p50 (lane 9), or c-Rel (lane 10) subunits of NF-kappa B or a control antiserum (lane 11). Lower panel, expression of vFLIP does not affect the SP1 binding activity. B, retroviral-mediated expression of vFLIP leads to persistent NF-kappa B activation in 293T cells as measured by EMSA. C, increased NF-kappa B transcriptional activity in 293T cells with stable expression of vFLIP as measured by a luciferase-based reporter assay. 293T cells were transfected with an NF-kappa B/luciferase reporter construct (75 ng/well) and a Rous sarcoma virus promoter-driven LacZ (beta -galactosidase) reporter construct (75 ng/well), and the experiment was performed as described previously (65). The values shown are the averages of one representative experiment of two in which each transfection was performed in duplicate. D, Western blot analysis demonstrating increased phosphorylation (top panel) and decrease in the total Ikappa Balpha protein (bottom panel) in H460 cells expressing vFLIP.

Next, we examined the status of total and phosphorylated Ikappa Balpha in H460 cells expressing empty vector, HHV8 vFLIP, or MRIT/cFLIP. Consistent with previous results with the PEL cell lines, expression of HHV8 vFLIP was associated with a decrease in the steady-state level of total Ikappa Balpha and an increase in its phosphorylated form (Fig. 2D). Taken together, the above results suggest that HHV8 vFLIP leads to persistent NF-kappa B activation by constitutive phosphorylation of Ikappa Balpha .

HHV8 vFLIP Complex Possesses IKK Activity-- To test the possibility that HHV8 vFLIP leads to persistent Ikappa Balpha phosphorylation by interacting with and activating the IKK complex, FLAG-tagged HHV8 vFLIP was immunoprecipitated from the cytosolic extracts of H460-vFLIP cells using FLAG antibody beads and assayed for the Ikappa Balpha kinase activity in an in vitro kinase reaction using GST-Ikappa Balpha and [gamma -32P]ATP. Parallel experiments utilizing a nonspecific antibody or an antibody against NEMO/IKKgamma served as negative and positive controls. As shown in Fig. 3A, immunoprecipitate of vFLIP with the FLAG antibody was able to phosphorylate GST-Ikappa Balpha , whereas an immunoprecipitate using a control antibody failed to do so. Similarly, immunoprecipitates of MRIT/cFLIP- or empty vector-expressing cells failed to phosphorylate GST-Ikappa Balpha (Fig. 3A, top and bottom panels). The vFLIP-associated IKK activity was specific for Ser-32 and Ser-36 of Ikappa Balpha , because it failed to phosphorylate a GST-Ikappa Balpha mutant substrate in which both the above residues were mutated to alanine (Fig. 3A, middle panel). Collectively, these results suggest that HHV8 vFLIP associates with a cytosolic complex that possesses IKK activity.


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  		Fig. 3.   HHV8 vFLIP has associated IKK activity. A, cellular extracts from H460 cells expressing empty vector, FLAG-vFLIP, or FLAG-MRIT/cFLIP were immunoprecipitated (IP) using a control antibody (Con), FLAG (M2) antibody, or NEMO antibody and subjected to immune complex kinase assay using wild-type (wt) or mutant (mt) GST-Ikappa Balpha (1-54) fusion proteins as substrates. The presence of vFLIP-associated IKK activity is demonstrated by the in vitro phosphorylation of wild-type but not mutant GST-Ikappa Balpha in the lanes containing FLAG immunoprecipitate. In addition, immune complex kinase assay with the NEMO antibody demonstrates an increase in the total IKK activity in the vFLIP-expressing cells. B, Coomassie blue-stained gel demonstrating that equal amounts of wild-type and mutant GST-Ikappa Balpha (1-54) substrate were used in each of the in vitro kinase reactions.

Components of HHV8 vFLIP-associated IKK Complex-- To determine the components of the HHV8 vFLIP-associated IKK activity, coimmunoprecipitation experiments were carried out. FLAG-tagged vFLIP and MRIT/cFLIP were immunoprecipitated using a FLAG monoclonal or a control mouse antibody, and the nature of the coimmunoprecipitated proteins was determined by Western analysis. As shown in Fig. 4A, IKKalpha , IKKbeta , and NEMO/IKKgamma readily coimmunoprecipitated with vFLIP but were not detected in the immunoprecipitate of MRIT/cFLIP. In contrast, RIP was detected in the immunoprecipitates of both vFLIP and MRIT/cFLIP.


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  		Fig. 4.   HHV8 vFLIP physically associates with the components of IKK complex. Cellular extracts from H460 cells expressing empty vector, FLAG-vFLIP, or FLAG-MRIT/cFLIP were immunoprecipitated using a control antibody (C) or FLAG monoclonal antibody (F) and the presence of different proteins in the immunoprecipitates detected by Western blot analysis. A, Western blot with a rabbit polyclonal antibody against the FLAG tag confirms the expression of vFLIP and MRIT in the immunoprecipitates. B, IKKalpha , IKKbeta , and NEMO/IKKgamma coimmunoprecipitate with vFLIP but fail to coimmunoprecipitate with MRIT/cFLIP, whereas RIP coimmunoprecipitates with both vFLIP and MRIT/cFLIP. IB, immunoblot.

HHV8 vFLIP Physically Interacts with an ~700-kDa IKK Signalsome Complex-- Previous studies have demonstrated that cytokine-induced IKK activity is present in a multiprotein signalsome complex of ~700 kDa (5, 6). To determine whether HHV8 vFLIP stimulates IKK activation by interacting with this large molecular mass complex, we compared the chromatographic distribution of vFLIP in extracts prepared from H460-vFLIP cells. A parallel experiment with cellular extracts prepared from H460-MRIT/cFLIP cells served as a negative control. Following Superdex-200 fractionation of the above extracts, the column fractions were immunoprecipitated with FLAG (M2) monoclonal antibody. The immunoprecipitate was subsequently used for Western analysis with a rabbit polyclonal antibody against the FLAG tag to detect the presence of FLAG-tagged vFLIP or MRIT/cFLIP as well as in an in vitro kinase assay using GST-Ikappa Balpha as a substrate. As shown in Fig. 5, A and B, the majority of vFLIP was found migrating between 600 and 700 kDa, which also correlated with the fraction containing the IKK activity. In contrast, MRIT/cFLIP was found migrating between 443 and 200 kDa (Fig. 5A).


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  		Fig. 5.   Chromatographic distribution of vFLIP and MRIT/cFLIP isolated from H460 cell extracts. A, cytosolic extracts from H460 cells expressing FLAG-vFLIP (top panel) or FLAG-MRIT/cFLIP (bottom panel) were fractionated by Superdex-200 gel filtration chromatography, and the elution fractions were immunoprecipitated with a FLAG monoclonal antibody (M2). The immunoprecipitated proteins were resolved on a SDS-polyacrylamide gel and analyzed by Western blot using a rabbit polyclonal antibody against the FLAG tag. Lane C, control antibody; lane F, FLAG antibody. B, kinase assay was performed on the indicated column fractions of vFLIP-expressing cells using GST-Ikappa Balpha (1-54) as a substrate. Molecular mass markers for the column are shown at the top of the figure. In, input.

We also examined the distribution of NEMO/IKKgamma in the cell extracts prepared from vFLIP- and MRIT/cFLIP-expressing cells. Although the majority of NEMO/IKKgamma was found migrating between 600 and 700 kDa, a smaller peak migrating near 450 kDa was detected in both cell lines (Fig. 6, A and B). As compared with MRIT-expressing cells, a relatively larger amount of NEMO/IKKgamma in vFLIP-expressing cells was found in the ~700-kDa fraction. Because this fraction has been previously shown to contain the IKK activity (5, 6), these results suggest that expression of vFLIP leads to incorporation of NEMO/IKKgamma into a constitutively active high molecular mass IKK complex. Finally, we examined the elution profile of IKKalpha and IKKbeta in the cell extracts prepared from vFLIP-expressing cells using Western blot analysis. As shown in Fig. 6C, both of the above kinases were found to coelute with vFLIP in the column fractions 8-10, thereby demonstrating that vFLIP coelutes with both the catalytic and regulatory subunits of the IKK complex.


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  		Fig. 6.   Chromatographic distribution of the IKKs in vFLIP- and MRIT/cFLIP-expressing H460 cellular extracts. Column fractions obtained following Superdex-200 gel filtration were fractionated on a SDS-polyacrylamide gel and analyzed by Western blot using a NEMO/IKKgamma antibody (A and B) or antibodies against IKKalpha and IKKbeta (C).

Because the majority of vFLIP and the IKKs were detected in the same elution fractions, we next examined whether they are physically associated with each other. For this purpose, FLAG-tagged vFLIP or MRIT/cFLIP present in the various column fractions obtained following Superdex-200 fractionation were immunoprecipitated using the M2 FLAG antibody, and the presence of any associated NEMO/IKKgamma was detected using Western blot analysis. As shown in Fig. 7A, NEMO/IKKgamma was found to coimmunoprecipitate with vFLIP in the column fractions 8-10, suggesting that the two proteins not only comigrate but are the components of the same IKK signalsome complex. Similarly, both IKKalpha and IKKbeta were found to coimmunoprecipitate with vFLIP (see below). Consistent with previous results, no NEMO/IKKgamma was found to coimmunoprecipitate with MRIT/cFLIP (Fig. 7B).


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  		Fig. 7.   HHV8 vFLIP physically interacts with components of the ~700-kDa IKK signalsome complex. A and B, column fractions containing extracts prepared from vFLIP- and MRIT/cFLIP-expressing H460 cells were immunoprecipitated using FLAG antibody and resolved on a SDS-polyacrylamide gel, and the presence of NEMO/IKKgamma in the immunoprecipitates was detected by Western blot analysis. Lane C, control antibody; lane F, FLAG antibody. C, column fractions containing extracts prepared from vFLIP-expressing H460 cells were immunoprecipitated using FLAG antibody beads. The supernatant (S) and the pellet (P) fractions were resolved by a SDS-polyacrylamide gel followed by Western blot analysis using antibodies directed against NEMO/IKKgamma , IKKalpha , and IKKbeta , respectively. IN, input.

We next examined the proportion of the IKKs that associate with vFLIP in various column fractions. For this purpose, various column fractions were immunoprecipitated with M2 FLAG antibody beads, and the amount of IKKs found to coimmunoprecipitate with the beads and the unbound fraction remaining in the supernatant was examined by Western blot analysis. As shown in Fig. 7C, significant proportions of IKKalpha , IKKbeta , and NEMO/IKKgamma were found to coimmunoprecipitate with vFLIP in the column fractions 8-10. A densitometry analysis revealed that 27 and 38% of IKKalpha , 36 and 42% of IKKbeta , and 34 and 51% of NEMO/IKKgamma were associated with vFLIP in the column fractions 8 and 9, respectively.

NEMO/IKKgamma Is Essential for HHV8 vFLIP-induced NF-kappa B-- We were next interested in demonstrating that the IKK complex not only associates with HHV8 vFLIP but is also essential for its ability to activate NF-kappa B. For this purpose we took advantage of the murine pre-B-cell lines 70Z/3 and 1.3E2, respectively. The 1.3E2 cell line is a NEMO-deficient mutant of 70Z/3 cells and has been previously shown to be incapable of activating NF-kappa B in response to multiple stimuli (39). We used retroviral-mediated gene transfer to generate stable clones of the above cell lines expressing empty vector or FLAG-tagged vFLIP or MRIT/cFLIP. As shown in Fig. 8A, expression of vFLIP in 70Z/3 cells led to constitutive activation of NF-kappa B as measured by EMSA, whereas no NF-kappa B activation was seen in control vector- or MRIT-expressing cells. More importantly, expression of vFLIP failed to activate NF-kappa B in the 1.3E2 cells, thereby indicating the essential role of NEMO/IKKgamma and the IKK complex in vFLIP-mediated NF-kappa B activation. Control experiments confirmed the expression of equivalent amounts of vFLIP in both 70Z/3-vFLIP and 1.3E2-vFLIP cell (Fig. 8D) and the lack of NEMO expression in 1.3E2 cells (Fig. 8C).


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  		Fig. 8.   NEMO/IKKgamma is essential for vFLIP-induced NF-kappa B activation. A, EMSA demonstrating increased NF-kappa B DNA binding activity in nuclear extracts prepared from vFLIP-expressing 70Z/3 cells but not in the corresponding 1.3E2 cells or those expressing empty vector or MRIT. The specificity of the NF-kappa B complex present in the vFLIP-expressing 70Z/3 cells is demonstrated by competition with excess cold kappa B or nonspecific (NS) probes. B, control EMSA demonstrating equivalent SP1 DNA binding activity in the nuclear extracts prepared from 70Z/3 and 1.3E2 cells. C, Western blot analysis confirming the lack of NEMO expression in 1.3E2 cells. D, control experiment demonstrating equivalent expression of vFLIP in 70Z/3 and 1.3E2 clones. Cellular lysates (L) from the indicated cells were immunoprecipitated using control mouse IgG beads (C) or FLAG beads (F), and the presence of vFLIP was detected using a rabbit polyclonal antibody against the FLAG tag.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

KS is the most common malignancy found in the patients with HIV infection. The isolation of a novel gamma herpesvirus, designated HHV8, as a potential etiological agent for KS was a major step in understanding the pathogenesis of KS (40). HHV8 genomes have also been consistently found in patients with PEL, also known as body cavity-associated lymphoma, a rare form of B-cell lymphoma characterized by malignant pleural, pericardial, or peritoneal effusion in the absence of a tumor mass (41). In addition to KS and PEL, HHV8 genome has been detected in multicentric Castleman's disease, angioimmunoblastic lymphadenopathy, and some cases of reactive lymphadenopathies (42-44).

Despite the increasing evidence linking the presence of KS-associated herpesvirus/HHV8 with KS and lymphoproliferative disorders, the mechanism by which this virus leads to a transformed phenotype is still unknown. In the present study, we have demonstrated that Ikappa Balpha is persistently phosphorylated in the PEL cell lines and is associated with constitutive NF-kappa B activation in these cells. Because constitutive NF-kappa B activation has been previously implicated in cellular transformation seen in association with infection by Epstein-Barr virus and human T-cell leukemia virus type I (15), it may play a causative role in the pathogenesis of KS and HHV8-associated lymphoproliferative disorders as well.

We have discovered that stable expression HHV8 vFLIP in both hematopoietic and nonhematopoietic cell lines can lead to constitutive NF-kappa B activation. We would like to point out that although we have used a retroviral vector to express vFLIP, it did not result in the expression of abnormally high levels of this protein. On the contrary, vFLIP protein was undetectable in the cellular lysates using a highly sensitive Western blot analysis and could be detected only after severalfold concentration of the protein by immunoprecipitation. Furthermore, PEL cell lines are known to harbor multiple copies (50-100) of HHV8 genome (41, 45). Therefore, taken together, it is highly unlikely that the NF-kappa B activation observed in the present study is due to expression of abnormally high and supraphysiological levels of vFLIP protein. In addition to vFLIP, two other HHV8-encoded proteins have been shown to lead to NF-kappa B activation, i.e. K1 and viral G-protein-coupled receptor (vGPCR), respectively (46-50). However, among these proteins, only vFLIP is expressed in latently infected KS spindle and PEL cells (51-54), making it a prime candidate for the constitutive NF-kappa B activation observed in the PEL cell lines.

Our study suggests that persistent NF-kappa B activation seen in the PEL cell lines is due to constitutive phosphorylation of Ikappa Balpha , a feature also seen in vFLIP-expressing cells. Inducible phosphorylation of Ikappa Balpha at Ser-32 and Ser-37 followed by its destruction by the ubiquitin-proteasome-dependent pathway is a known mechanism for NF-kappa B activation by cytokines, such as TNF and interleukin-1. This signal-dependent phosphorylation of Ikappa Balpha has been shown to be mediated by the activation of a ~700-kDa signalsome complex comprising IKKalpha , IKKbeta , and IKKgamma /NEMO (7-13). Our results suggest that HHV8 vFLIP leads to constitutive NF-kappa B activation by associating with this high molecular mass complex. In this regard, HHV8 vFLIP may resemble human T-cell leukemia virus type I Tax protein, which has been also shown to lead to NF-kappa B activation by associating with and persistently activating the IKK complex (55-60).

The mechanism by which interaction of HHV8 vFLIP with the IKK complex results in persistent increase in IKK activity remains to be determined. It is conceivable that HHV8 vFLIP recruits an upstream kinase to the IKK complex. For example, we have demonstrated that in addition to the various IKKs, HHV8 vFLIP also interacts with RIP, a protein kinase known to be crucial for TNFalpha -mediated NF-kappa B activation. RIP in turn may recruit and activate NF-kappa B-inducing kinase or mitogene-activated protein kinase kinase kinase, which are known to activate the IKK complex (61-64). Studies to address the role of RIP, NF-kappa B-inducing kinase, and mitogene-activated protein kinase kinase kinase in vFLIP-induced NF-kappa B activation are currently in progress.

    ACKNOWLEDGEMENTS

We thank Dr. Richard Gaynor's laboratory for assistance with the gel filtration experiment, Dr. John Minna and Carol Sibley for cell lines, and the National Cell Culture Center (Minneapolis, MN) for large scale culture of H460 cells expressing vFLIP and MRIT/cFLIP.

    FOOTNOTES

* This work was supported in part by a grant from the Leukemia Research Foundation, National Institutes of Health Grants CA85177-01 and AI47230-01), and a grant from the Howard Hughes Medical Institute.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.

Dagger To whom correspondence should be addressed: Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8593. Tel.: 214-648-1837; Fax: 214-648-4940; E-mail: preet.chaudhary@utsouthwestern.edu.

Published, JBC Papers in Press, February 5, 2002, DOI 10.1074/jbc.M110480200

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; FLIP, FLICE inhibitory protein(s); GST, glutathione S-transferase; HHV8, human herpesvirus 8; KS, Kaposi's sarcoma; MRIT, Mach-related inducer of toxicity; NEMO, NF-kappa B essential modulator; PMSF, phenylmethylsufonyl fluoride; PEL, primary effusion lymphoma; Ikappa B, inhibitor of NF-kappa B; IKK, Ikappa B kinase; RIP, receptor-interacting protein; vFLIP, viral FLICE inhibitory protein; TNF, tumor necrosis factor; HIV, human immunodeficiency virus.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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