vCLAP, a Caspase-recruitment Domain-containing Protein of Equine Herpesvirus-2, Persistently Activates the Ikappa B Kinases through Oligomerization of IKKgamma *

Jean-Luc PoyetDagger, Srinivasa M. SrinivasulaDagger§, and Emad S. Alnemri

From the Center for Apoptosis Research and the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Received for publication, November 8, 2000, and in revised form, December 6, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

vCLAP, the E10 gene product of equine herpesvirus-2, is a caspase-recruitment domain (CARD)-containing protein that has been shown to induce both apoptosis and NF-kappa B activation in mammalian cells. vCLAP has a cellular counterpart, Bcl10/cCLAP, which is also an activator of apoptosis and NF-kappa B. Recent studies demonstrated that vCLAP activates NF-kappa B through an Ikappa B kinase (IKK)-dependent pathway, but the underlying mechanism remains unknown. In this report, we demonstrate that vCLAP associates stably with the IKK complex through direct binding to the C-terminal region of IKKgamma . Consistent with this finding, IKKgamma was found to be essential for vCLAP-induced NF-kappa B activation, and the association between vCLAP and the IKK complex induced persistent activation of the IKKs. Moreover, enforced oligomerization of the isolated C-terminal region of vCLAP, which interacts with IKKgamma , can trigger NF-kappa B activation. Finally, substitution of the C-terminal region of IKKgamma , which interacts with vCLAP, with the CARD of vCLAP or Bcl10 produced a molecule that was able to activate NF-kappa B when ectopically expressed in IKKgamma -deficient cells. These data suggest that vCLAP-induced oligomerization of IKKgamma , which is mediated by the CARD of vCLAP, could be the mechanism by which vCLAP induces activation of NF-kappa B.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Equine herpesvirus-2 (EHV-2)1 is a gammaherpesvirus related to other lymphotropic herpesviruses such as herpesvirus saimiri and Epstein-Barr virus. EHV-2 contains 79 reading frames that encode 77 distinct molecules, several of which show significant similarity to cellular genes. One of these molecules, vCLAP (also called vCIPER/E10/vCARMEN) (1-4), a CARD-containing apoptotic protein, was recently found to induce both apoptosis and activation of the transcription factor NF-kappa B in mammalian cells. vCLAP, like its cellular counterpart Bcl10, contains two domains, an N-terminal CARD that can oligomerize via homotypic interactions and a C-terminal domain that probably functions as the NF-kappa B activation domain. Because NF-kappa B activation is considered to be a survival signal, virally encoded proteins, such as vCLAP, may be utilized by viruses as a strategic tool to initiate self-replication or to suppress apoptosis in infected cells (5).

In most resting cells, NF-kappa B is sequestered in the cytoplasm through interaction with the Ikappa B inhibitory proteins. Ikappa Bs mask the NF-kappa B nuclear localization signal, thereby preventing its nuclear uptake. Exposure of cells to a wide variety of stimuli, such as viral or bacterial infection, inflammatory cytokines, or UV irradiation leads to the rapid phosphorylation, ubiquitination, and ultimately proteolitic degradation of the Ikappa Bs (6-9). This allows the activated NF-kappa B to translocate to the nucleus and activate the transcription of several NF-kappa B target genes.

The kinase activity responsible for phosphorylation of Ikappa Bs is present in a large (700-900 kDa) cytoplasmic complex composed of two catalytic subunits, IKKalpha and IKKbeta (10-14), and a noncatalytic subunit termed IKKgamma (also called NEMO, IKKAP1, or FIP-3) (15-18). We and others have recently demonstrated that activation of the IKK complex could be achieved through IKKgamma -mediated oligomerization of the IKK kinases, indicating that IKKgamma functions as an adaptor to link the IKKs with the upstream regulators of NF-kappa B (19, 20). Here we show that vCLAP associates directly and specifically with IKKgamma through its C-terminal glycine-rich domain and may regulate the activity of the IKK complex through CARD-mediated oligomerization of IKKgamma .


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture, Transfection, and Treatment-- Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) (HeLa, Rat-1, or 5R cells) or DMEM/F12 (293T cells; Life Technologies, Inc.), supplemented with 10% fetal bovine serum, 200 µg/ml penicillin, and 100 µg/ml streptomycin sulfate. Transfections were carried out using LipofectAMINE (Life Technologies, Inc.). Cells were stimulated with either 20 ng/ml recombinant human TNF-alpha (Sigma) or 0.1 µg/ml AP1510 (Arriad) for the indicated times. NEMO/IKKgamma -deficient Rat-1 cells (5R) are a gift from S. Yamaoka.

Expression Vectors and Antibodies-- Constructs encoding full-length IKKgamma , IKKalpha , IKKbeta , or vCLAP or truncated mutants have been described previously (1, 20, 21). The FKBP12 fusion of vCLAP C-terminal domain (CTD) was constructed in a modified pcDNA3-T7 vector, which contains a T7 tag sequence, by fusing three tandem repeats of FKBP12 cDNA in frame with the cDNA of vCLAP-CTD (residues 108-311) as described previously (1). The plasmids expressing the green fluorescent protein (GFP) (pEGFP-C1) and the red fluorescent protein (RFP) (pDsRed1-N1) were from CLONTECH. FLAG-M5 antibody was from Sigma. T7-horseradish peroxidase conjugate antibody was from Novagen. IKKalpha and IKKgamma polyclonal antibodies were from Santa Cruz.

Biochemical Analysis-- Immunoprecipitations were performed as described previously (20), and the precipitated proteins were analyzed by SDS polyacrylamide gel electrophoresis followed by immunoblotting. GST pull-down assays, luciferase reporter gene assays, and IKK kinase assays were performed as described (20, 22).

Confocal Microscopy-- 293T cells were grown on coverslips and then transfected with the GFP-tagged IKKgamma or RFP-tagged vCLAP separately or together, with the indicated vectors. 24 h after transfection, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 min. The coverslips were mounted on a glass slide, and the fluorescence was detected by confocal microscopy using an excitation wavelength of 488 nm and a detection wavelength of 522 nm (GFP) or an excitation wavelength of 568 nm and a detection wavelength of 585 nm (RFP). Images were Kalman-averaged with a Kalman filter to increase the signal/noise ratio.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

vCLAP Interacts with the IKK Complex and Persistently Activates the IKK Kinases-- Whereas in resting cells the IKK kinases are inactive, potent activators, such as TNF-alpha , interleukin-1, or lipopolysaccharide, induce a very rapid IKK activation, detectable within minutes. However, numerous studies have shown that this activation is only transient and after ~30 min decreases to about 25% of its peak value (12, 14, 23). Using a luciferase reporter assay, we and others demonstrated that expression of vCLAP results in a robust activation of NF-kappa B (1-4). Because of the very high level of this activation, we asked whether vCLAP could persistently activate the IKKs, resulting in a sustained rather than transient activation of NF-kappa B. To answer this question, endogenous IKKalpha was isolated by immunoprecipitation from extracts prepared from vCLAP-transfected or TNF-alpha -treated HeLa cells and assayed for IKK catalytic activity using a GST-Ikappa Balpha fusion protein as a substrate. Consistent with previous observations, TNF-alpha stimulation of HeLa cells induced high but transient IKKalpha kinase activity (Fig. 1A); the activity, which was maximum after 10 min of stimulation, declined sharply with time and was barely detectable after 90 min of stimulation. However, compared with TNF-alpha stimulation, overexpression of vCLAP in HeLa cells induced a robust and sustained IKKalpha kinase activity in the absence of any external stimulation (Fig. 1B). IKKalpha protein expression was comparable in vCLAP-transfected and nontransfected cells, indicating that vCLAP activates endogenous IKKalpha by a post-translational mechanism. To determine whether vCLAP associates stably with the IKK complex, immunoprecipitates obtained using anti-IKKalpha or anti-FLAG antibodies were assayed for the presence of vCLAP or the IKK components, respectively. As shown in Fig. 1B, vCLAP was readily detected after precipitation of endogenous IKKalpha . Moreover, IKKalpha and IKKgamma were also detected in immunocomplexes obtained after precipitation of vCLAP (Fig. 1C). The vCLAP immunoprecipitates also possessed IKK kinase activity (Fig. 1C). These results provide direct biochemical evidence that vCLAP associates stably with the IKK complex and is able to persistently activate the IKK kinases.



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Fig. 1.   vCLAP associates stably with the IKK complex and persistently activates the IKK kinases. A, HeLa cells were either left untreated or were incubated with TNF-alpha for the indicated times and then lysed. The lysates were immunoprecipitated with anti-IKKalpha antibody, and the IKK activity associated with IKKalpha was determined by immune complex kinase assay (KA). Expression of endogenous IKKalpha was determined by immunoblotting (IB). B and C, HeLa cells were transfected with expression construct for FLAG-vCLAP. At the indicated times after transfection, cells were lysed, and equal amount of proteins were immunoprecipitated with anti-IKKalpha (B) or anti-FLAG (C) antibody. The immunoprecipitates were assayed for IKK activity by immune complex kinase assay and analyzed by SDS-PAGE and immunoblotted with anti-FLAG antibody (B) or anti-IKKalpha and anti-IKKgamma antibodies (C). The cellular extracts were also immunoblotted with anti-IKKalpha and anti-FLAG (B) or anti-FLAG (C) antibodies.

vCLAP Interacts Directly with and Requires IKKgamma for Activation of NF-kappa B-- To determine which component of the IKK complex interacts with vCLAP, 293T cells were transfected with expression vectors for FLAG-tagged vCLAP and T7-IKKbeta with or without T7-IKKgamma . As shown in Fig. 2A, a small amount of IKKbeta was coimmunoprecipitated with vCLAP in the absence of ectopic IKKgamma . However, a remarkably higher amount of IKKbeta was coimmunoprecipitated with vCLAP in the presence of coexpressed IKKgamma (Fig. 2A). The ectopic T7-IKKgamma was also detected in these complexes (Fig. 2A). No IKKbeta or IKKgamma were precipitated with the FLAG antibody in the absence of FLAG-vCLAP (Fig. 2A). This result shows that IKKgamma mediates the interaction of vCLAP with the IKK complex.



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Fig. 2.   IKKgamma mediates the assembly of the vCLAP-IKK complexes and is required for vCLAP-induced NF-kappa B activation. A, 293T cells were transfected with expression constructs for T7-IKKbeta , T7-IKKgamma , and FLAG-vCLAP as indicated. 24 h after transfection, cells were lysed, and the lysates immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were immunoblotted (IB) with anti-T7 antibody. Expression of T7-IKKbeta , T7-IKKgamma , or FLAG-vCLAP was determined by immunoblotting with anti-T7 or anti-FLAG antibodies, respectively. B, in vitro interaction of vCLAP with GST-IKKgamma . 35S-Labeled vCLAP (lane 1, 10% input) was incubated with an equal amount of GST (lane 2) or GST-IKKgamma (lane 3) proteins bound to glutathione-Sepharose beads. Bound proteins were then eluted and analyzed by SDS-PAGE and autoradiography. C, Rat-1 cells or the IKKgamma -deficient Rat-1 derivative (5R) cells were transfected with 5× kappa B-luciferase reporter together with either empty vector or vCLAP or Bcl10 expression construct. 24 h after transfection, cells were either left untreated or incubated with TNF-alpha for 5 h. Cells were then collected and lysed, and the luciferase activity in the cell lysates was determined. pRSC-LacZ was included in all transfection reactions to normalize the transfection efficiency. Mean values ± S.E. are shown from three independent experiments performed in duplicate.

To rule out the possibility that other proteins were necessary for the vCLAP-IKKgamma interaction, we analyzed the ability of a GST-IKKgamma fusion protein to associate with an in vitro-translated 35S-labeled vCLAP. In agreement with a direct interaction between vCLAP and IKKgamma , 35S-labeled vCLAP bound to the GST-IKKgamma fusion protein but not the GST control (Fig. 2B).

To address the physiological relevance of this finding, we transiently expressed vCLAP in wild type or IKKgamma -deficient Rat-1 cells (15). In contrast to wild type Rat-1 cells, no NF-kappa B activation was elicited in the IKKgamma -deficient 5R cells after transfection with the vCLAP construct or treatment with TNF-alpha (Fig. 2C). This result provides genetic proof for the requirement of IKKgamma in vCLAP-induced activation of NF-kappa B, confirming its role as a molecular adaptor in the assembly of the vCLAP-IKK complexes. The inability of vCLAP to induce NF-kappa B activation in 5R cells cannot be attributed to defects in the NF-kappa B pathway downstream of IKKgamma , because transfection of these cells with IKKgamma can restore NF-kappa B activation by Tax, which is expressed stably in this cell line (Ref. 15 and data not shown).

In contrast to vCLAP, Bcl10 was unable to interact with IKKgamma in vitro (data not shown). However, like vCLAP, Bcl10 was able to induce NF-kappa B activation in Rat-1, but not in 5R cells (Fig. 2C), suggesting that Bcl10 could relay its signal to IKKgamma indirectly.

Mapping of the Interaction Domains of vCLAP and IKKgamma -- We next mapped the regions of vCLAP and IKKgamma that are required for their interaction. FLAG-tagged IKKgamma was expressed in 293T cells with T7-tagged full-length domain, CARD (residues 1-107), or CTD (residues 108-311) of vCLAP. Extracts prepared from the transfected cells were immunoprecipitated with an anti-FLAG antibody, and the resulting immune complexes were analyzed by Western blotting with an anti-T7 antibody that recognizes the T7-vCLAP variants. Both the full-length domain and the CTD of vCLAP were able to bind to IKKgamma (Fig. 3A). In contrast, the CARD did not interact with IKKgamma (Fig. 3A). The same results were obtained using a GST-IKKgamma pull-down assay, which showed that the recombinant GST-IKKgamma fusion protein is able to bind the in vitro-translated 35S-labeled full-length domain or the CTD of vCLAP, but not the CARD of vCLAP (not shown). Combined, these results show that the CTD of vCLAP mediates its interaction with IKKgamma .



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Fig. 3.   Interaction domains of IKKgamma and vCLAP. The CTD of vCLAP mediates its interaction with IKKgamma . vCLAP, IKKgamma , and their deletion mutants are represented on the top. A, 293T cells were transfected with FLAG-IKKgamma together with either empty vector or T7-tagged full-length (FL) vCLAP, vCLAP-CARD, or vCLAP-CTD expression constructs. 24 h after transfection, cells were lysed, and the lysates were immunoprecipitated (IP) with anti-FLAG antibody. The immunoprecipitates were immunoblotted (IB) with anti-T7 antibody. The expression of FLAG-IKKgamma and the different T7-CLAP chimeras was determined by immunoblotting with anti-FLAG or anti-T7 antibodies, respectively. B, IKKgamma interacts with vCLAP via its C terminus. 293T cells were transfected with T7-vCLAP and different FLAG-tagged full-length (FL) or C-terminally truncated IKKgamma expression constructs. 24 h after transfection, cells were lysed, and the lysates were immunoprecipitated (IP) with anti-FLAG antibody. The immunoprecipitates were immunoblotted (IB) with anti-T7 antibody. The expression of T7-vCLAP and the different FLAG-IKKgamma truncated mutants was determined by immunoblotting with anti-T7 or anti-FLAG antibodies, respectively. LZ, leucine zipper. C, recruitment of IKKgamma into vCLAP filaments. RFP-tagged vCLAP and GFP-tagged IKKgamma were expressed either alone or together in 293T cells. 24 h after transfection, cells were then examined and photographed under a fluorescent microscope.

To extend the characterization of the vCLAP-IKKgamma interaction, we expressed T7-tagged vCLAP in 293T cells together with several FLAG-tagged full-length or truncated IKKgamma . vCLAP was found to specifically associate with full-length IKKgamma but not with the C-terminally truncated IKKgamma () or IKKgamma () (Fig. 3B). Removal of the last 119 amino acids of IKKgamma strongly reduced its interaction with the vCLAP (Fig. 3B). Taken together, these data indicate that the interaction between vCLAP and IKKgamma involves sequences in the C-terminal region of IKKgamma .

To confirm that vCLAP associates intercellularly with IKKgamma when the two proteins are coexpressed in the same cell, we cotransfected 293T cells with constructs encoding GFP-IKKgamma and RFP-vCLAP fusion proteins and then monitored the subcellular localization of these proteins by confocal microscopy. As shown in Fig. 3C, the two proteins exhibited different patterns of cellular localization when expressed alone. Whereas vCLAP exhibited a clear pattern of discrete and interconnecting cytoplasmic filaments, IKKgamma displayed a somewhat punctuate cytoplasmic or whole-cell distribution. However, coexpression of the two proteins resulted in redistribution of IKKgamma to the vCLAP filaments. This observation is consistent with a direct interaction between vCLAP and IKKgamma .

vCLAP Mediates Activation of the IKKs through Oligomerization of IKKgamma -- Our data demonstrate that IKKgamma mediates the association of vCLAP with the IKK kinases and is required for vCLAP-induced activation of NF-kappa B. This suggest that IKKgamma is an adaptor molecule with a C-terminal half that binds to NF-kappa B activators like vCLAP (see above), RIP (18-20, 24), or Tax (25-27) and an N-terminal half that is required for interaction with the IKK kinases (20, 28). vCLAP has a bipartite structure consisting of an N-terminal CARD and a C-terminal domain that interacts with IKKgamma (see above). Interestingly, we and others have shown that the CARDs of cellular and viral CLAP proteins are important for homo- and heterotypic interactions (1-4). It is therefore likely that CARD-mediated self-association of vCLAP could induce oligomerization of IKKgamma resulting in activation of the IKK complex. To test this hypothesis, we generated a fusion protein (CARD-IKKgamma -Delta C) composed of the CARD of vCLAP linked to the N-terminal part (residues 1-200) of IKKgamma (IKKgamma -Delta C) and determined its ability to induce NF-kappa B activation. To rule out the possibility that the CARD-IKKgamma -Delta C chimera functions through interaction with the endogenous IKKgamma protein, we examined its ability to activate NF-kappa B in the IKKgamma -deficient 5R cells. As shown in Fig. 4A, transient transfection of the CARD-IKKgamma -Delta C chimera resulted in a large increase of NF-kappa B activity in a dose-dependent manner. In contrast, neither the separate CARD of vCLAP nor IKKgamma -Delta C were able to activate the NF-kappa B when transfected at either low or high doses (Fig. 4A). Moreover, a single point mutation of a conserved residue in the CARD of vCLAP that abrogates homodimerization (L49R) (1) prevented NF-kappa B activation by the chimeric protein (Fig. 4A). Similar results were obtained when the CARD of Bcl10 was used instead of vCLAP-CARD in the above experiments (data not shown).



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Fig. 4.   Activation of NF-kappa B through vCLAP-induced oligomerization of IKKgamma . A, IKKgamma -deficient (5R) cells were transfected with 5× kappa B-luciferase reporter together with different amounts (0.1 and 0.3 µg) of the indicated expression constructs, with or without kinase-inactive mutant of IKKbeta (DN IKKbeta ). 24 h after transfection, cells were lysed, and the luciferase activity was determined as in the legend of Fig. 2. The CARD-IKKgamma -Delta C chimera is represented by a bar diagram. B, enforced oligomerization of the CTD of vCLAP induces activation of NF-kappa B. Rat-1 cells or the IKKgamma -deficient Rat-1 derivative (5R) cells were transfected with 5× kappa B-luciferase reporter together with either empty vector or the indicated expression constructs. 24 h after transfection, cells were either left untreated or incubated with AP1510 for 6 h. The luciferase activity in the transfected cell lysates was assayed and normalized as in the legend of Fig. 2.

We then tested whether enforced oligomerization of the CTD of vCLAP could induce NF-kappa B activation. For this purpose, the CTD of vCLAP was fused to a 3-fold repeat of the FKBP12 polypeptide, which oligomerizes when it binds to the cell-permeable synthetic organic ligand AP1510 (29). As shown in Fig. 4B, transient tranfection of this construct into Rat-1 cells, but not in the IKKgamma -deficient 5R cells, induced a large NF-kappa B activation in a ligand-dependent manner. No NF-kappa B activation was detected when the FKBP12-CTD construct was cotransfected with kinase-inactive IKKbeta (not shown) or after treatment of empty vector-transfected 293T cells with AP1510 (Fig. 4B). Taken together, these results demonstrate that vCLAP-induced oligomerization of IKKgamma is the triggering event leading to activation of the IKK complex.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although previous studies have shown that the EHV-2-encoded vCLAP protein activates NF-kappa B in mammalian cells (1, 4), the mechanism by which vCLAP interfaces with the cell's NF-kappa B-activating machinery remains unclear. In this paper, we report several observations that, when combined, provide a potential mechanism for vCLAP-induced NF-kappa B activation. First, we show that the IKK kinases are persistently activated in vCLAP-expressing cells, which might explain the robust NF-kappa B activity observed in vCLAP-transfected cells (1, 4). Second, we demonstrate that vCLAP, via its C-terminal domain, interacts physically with the IKK complex through direct binding to the C-terminal part of IKKgamma . Consistent with this observation, IKKgamma was found to be essential for vCLAP-induced activation of NF-kappa B. Third, we demonstrate that vCLAP activates the IKK complex through oligomerization of IKKgamma . Indeed, CARD-dependent clustering of the N-terminal part of IKKgamma , which we have previously shown to interact with the IKK kinases (20), was able to activate NF-kappa B. Moreover, enforced oligomerization of the CTD of vCLAP was able to induce a large increase in NF-kappa B activity in Rat-1 cells but not in the IKKgamma -deficient 5R cells. This mechanism of NF-kappa B activation by vCLAP, namely oligomerization-induced activation of the IKK kinases via IKKgamma , is reminiscent of the model we proposed for RIP-induced activation of NF-kappa B after ligation of TNF-R1 (20). Based on this model, TNF-alpha stimulation induces binding of RIP to, and concomitant oligomerization of IKKgamma , which in turn passes the oligomerization signal to the effector kinases, resulting in their activation through autophosphorylation of their T-loop serines (20). However, in contrast to vCLAP, which binds stably to the IKK complex, RIP releases the activated IKK complex after its oligomerization, resulting in transient rather than persistent activation. Therefore, our results illustrate the ubiquitous role of oligomerization in IKK activation.

Activation of NF-kappa B by expression of a single viral protein has been described in several studies, indicating that infection with an intact virus is not always required for NF-kappa B activation. One well documented example of this is the human T-cell leukemia virus-Tax protein (30-33). Tax has been shown to interact with the IKK complex through direct interaction with IKKgamma (25-27). Moreover, Tax mutants defective in IKKgamma binding failed to activate NF-kappa B (34). Interestingly, Tax has been shown to function as a dimerizer that stabilizes dimer formation in other proteins (35). It is therefore possible that Tax-induced oligomerization of IKKgamma is, as for vCLAP, the triggering event in the activation of the IKK kinases by Tax.

Recently, several independent groups have demonstrated that the cellular homologue of vCLAP, Bcl10 (also called cCLAP/CIPER/hE10/CARMEN), was also able to activate NF-kappa B when expressed in cells, although at a lesser degree than vCLAP. Bcl10 requires IKKgamma for activation of NF-kappa B (Fig. 2C). Our preliminary results suggest that cCLAP interacts with the IKK complex, as immunoprecipitation of the endogenous cCLAP results in isolation of the IKK components. However, in a GST-IKK pull-down assay, in vitro-translated 35S-labeled cCLAP was not able to bind to recombinant GST-IKKgamma or GST-IKKalpha (data not shown), indicating that the cCLAP-IKK complex interaction could be indirect or regulated by a post-translational modification of cCLAP, such as phosphorylation. Future studies will reveal the precise physiological function of cCLAP and how it activates the IKK complex.


    ACKNOWLEDGEMENT

We thank W. C. Green for the IKKbeta kinase inactive construct and S. Yamaoka and A. Israel for the NEMO/IKKgamma -deficient Rat-1.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA85421 (to E. S. A.).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 Contributed equally to this work.

Special fellow of the Leukemia and Lymphoma Society.

§ To whom correspondence should be addressed: Thomas Jefferson University, Kimmel Cancer Inst., Bluemle Life Sciences Bldg., Rm. 904, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4632; Fax: 215-923-1098; E-mail: E_Alnemri@lac.jci.tju.edu.

Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.C000792200


    ABBREVIATIONS

The abbreviations used are: EHV, equine herpesvirus; TNF, tumor necrosis factor; CARD, caspase-recruitment domain; IKK, Ikappa B kinase; RIP, receptor-interacting protein; GST, glutathione S-transferase; GFP, green fluorescent protein; RFP, red fluorescent protein; CTD, C-terminal domain; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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