FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1993648889> ?p ?o ?g. }

• W1993648889 endingPage "3187" @default.
• W1993648889 startingPage "3183" @default.
• W1993648889 abstract "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-κB activation in mammalian cells. vCLAP has a cellular counterpart, Bcl10/cCLAP, which is also an activator of apoptosis and NF-κB. Recent studies demonstrated that vCLAP activates NF-κB through an IκB 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 IKKγ. Consistent with this finding, IKKγ was found to be essential for vCLAP-induced NF-κB 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 IKKγ, can trigger NF-κB activation. Finally, substitution of the C-terminal region of IKKγ, which interacts with vCLAP, with the CARD of vCLAP or Bcl10 produced a molecule that was able to activate NF-κB when ectopically expressed in IKKγ-deficient cells. These data suggest that vCLAP-induced oligomerization of IKKγ, which is mediated by the CARD of vCLAP, could be the mechanism by which vCLAP induces activation of NF-κB. 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-κB activation in mammalian cells. vCLAP has a cellular counterpart, Bcl10/cCLAP, which is also an activator of apoptosis and NF-κB. Recent studies demonstrated that vCLAP activates NF-κB through an IκB 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 IKKγ. Consistent with this finding, IKKγ was found to be essential for vCLAP-induced NF-κB 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 IKKγ, can trigger NF-κB activation. Finally, substitution of the C-terminal region of IKKγ, which interacts with vCLAP, with the CARD of vCLAP or Bcl10 produced a molecule that was able to activate NF-κB when ectopically expressed in IKKγ-deficient cells. These data suggest that vCLAP-induced oligomerization of IKKγ, which is mediated by the CARD of vCLAP, could be the mechanism by which vCLAP induces activation of NF-κB. equine herpesvirus tumor necrosis factor caspase-recruitment domain IκB kinase receptor-interacting protein glutathione S-transferase green fluorescent protein red fluorescent protein C-terminal domain polyacrylamide gel electrophoresis Dulbecco's modified Eagle's medium 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) (1Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 2Koseki T. Inohara N. Chen S. Carrio R. Merino J. Hottiger M.O. Nabel G.J. Nunez G. J. Biol. Chem. 1999; 274: 9955-9961Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 3Costanzo A. Guiet C. Vito P. J. Biol. Chem. 1999; 274: 20127-20132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 4Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), a CARD-containing apoptotic protein, was recently found to induce both apoptosis and activation of the transcription factor NF-κB 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-κB activation domain. Because NF-κB 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 (5Meinl E. Fickenscher H. Thome M. Tschopp J. Fleckenstein B. Immunol. Today. 1998; 19: 474-479Abstract Full Text PDF PubMed Scopus (76) Google Scholar). In most resting cells, NF-κB is sequestered in the cytoplasm through interaction with the IκB inhibitory proteins. IκBs mask the NF-κB 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 IκBs (6Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1307) Google Scholar, 7Burke J.R. Miller K.R. Wood M.K. Meyers C.A. J. Biol. Chem. 1998; 273: 12041-12046Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 8Scherer D.C. Brockman J.A. Chen Z. Maniatis T. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11259-11263Crossref PubMed Scopus (500) Google Scholar, 9Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1159) Google Scholar). This allows the activated NF-κB to translocate to the nucleus and activate the transcription of several NF-κB target genes. The kinase activity responsible for phosphorylation of IκBs is present in a large (700–900 kDa) cytoplasmic complex composed of two catalytic subunits, IKKα and IKKβ (10Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar, 11Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1831) Google Scholar, 12DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1890) Google Scholar, 13Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1060) Google Scholar, 14Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1562) Google Scholar), and a noncatalytic subunit termed IKKγ (also called NEMO, IKKAP1, or FIP-3) (15Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar, 16Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (836) Google Scholar, 17Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, 18Li Y. Kang J. Friedman J. Tarassishin L. Ye J. Kovalenko A. Wallach D. Horwitz M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1042-1047Crossref PubMed Scopus (155) Google Scholar). We and others have recently demonstrated that activation of the IKK complex could be achieved through IKKγ-mediated oligomerization of the IKK kinases, indicating that IKKγ functions as an adaptor to link the IKKs with the upstream regulators of NF-κB (19Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nunez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 20Poyet J.L. Srinivasula S.M. Lin J. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Here we show that vCLAP associates directly and specifically with IKKγ through its C-terminal glycine-rich domain and may regulate the activity of the IKK complex through CARD-mediated oligomerization of IKKγ. 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-α (Sigma) or 0.1 μg/ml AP1510 (Arriad) for the indicated times. NEMO/IKKγ-deficient Rat-1 cells (5R) are a gift from S. Yamaoka. Constructs encoding full-length IKKγ, IKKα, IKKβ, or vCLAP or truncated mutants have been described previously (1Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 20Poyet J.L. Srinivasula S.M. Lin J. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 21Lin X. Mu Y. Cunningham Jr., E.T. Marcu K.B. Geleziunas R. Greene W.C. Mol. Cell. Biol. 1998; 18: 5899-5907Crossref PubMed Google Scholar). 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 (1Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). 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. IKKα and IKKγ polyclonal antibodies were from Santa Cruz. Immunoprecipitations were performed as described previously (20Poyet J.L. Srinivasula S.M. Lin J. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), 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 (20Poyet J.L. Srinivasula S.M. Lin J. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 22Ahmad M. Srinivasula S.M. Wang L. Talanian R.V. Litwack G. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1997; 57: 615-619PubMed Google Scholar). 293T cells were grown on coverslips and then transfected with the GFP-tagged IKKγ 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. Whereas in resting cells the IKK kinases are inactive, potent activators, such as TNF-α, 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 (12DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1890) Google Scholar, 14Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1562) Google Scholar, 23Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (741) Google Scholar). Using a luciferase reporter assay, we and others demonstrated that expression of vCLAP results in a robust activation of NF-κB (1Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 2Koseki T. Inohara N. Chen S. Carrio R. Merino J. Hottiger M.O. Nabel G.J. Nunez G. J. Biol. Chem. 1999; 274: 9955-9961Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 3Costanzo A. Guiet C. Vito P. J. Biol. Chem. 1999; 274: 20127-20132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 4Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). 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-κB. To answer this question, endogenous IKKα was isolated by immunoprecipitation from extracts prepared from vCLAP-transfected or TNF-α-treated HeLa cells and assayed for IKK catalytic activity using a GST-IκBα fusion protein as a substrate. Consistent with previous observations, TNF-α stimulation of HeLa cells induced high but transient IKKα kinase activity (Fig.1 A); 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-α stimulation, overexpression of vCLAP in HeLa cells induced a robust and sustained IKKα kinase activity in the absence of any external stimulation (Fig. 1 B). IKKα protein expression was comparable in vCLAP-transfected and nontransfected cells, indicating that vCLAP activates endogenous IKKα by a post-translational mechanism. To determine whether vCLAP associates stably with the IKK complex, immunoprecipitates obtained using anti-IKKα or anti-FLAG antibodies were assayed for the presence of vCLAP or the IKK components, respectively. As shown in Fig.1 B, vCLAP was readily detected after precipitation of endogenous IKKα. Moreover, IKKα and IKKγ were also detected in immunocomplexes obtained after precipitation of vCLAP (Fig.1 C). The vCLAP immunoprecipitates also possessed IKK kinase activity (Fig. 1 C). These results provide direct biochemical evidence that vCLAP associates stably with the IKK complex and is able to persistently activate the IKK kinases. To determine which component of the IKK complex interacts with vCLAP, 293T cells were transfected with expression vectors for FLAG-tagged vCLAP and T7-IKKβ with or without T7-IKKγ. As shown in Fig. 2 A, a small amount of IKKβ was coimmunoprecipitated with vCLAP in the absence of ectopic IKKγ. However, a remarkably higher amount of IKKβ was coimmunoprecipitated with vCLAP in the presence of coexpressed IKKγ (Fig. 2 A). The ectopic T7-IKKγ was also detected in these complexes (Fig. 2 A). No IKKβ or IKKγ were precipitated with the FLAG antibody in the absence of FLAG-vCLAP (Fig.2 A). This result shows that IKKγ mediates the interaction of vCLAP with the IKK complex. To rule out the possibility that other proteins were necessary for the vCLAP-IKKγ interaction, we analyzed the ability of a GST-IKKγ fusion protein to associate with an in vitro-translated35S-labeled vCLAP. In agreement with a direct interaction between vCLAP and IKKγ, 35S-labeled vCLAP bound to the GST-IKKγ fusion protein but not the GST control (Fig. 2 B). To address the physiological relevance of this finding, we transiently expressed vCLAP in wild type or IKKγ-deficient Rat-1 cells (15Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar). In contrast to wild type Rat-1 cells, no NF-κB activation was elicited in the IKKγ-deficient 5R cells after transfection with the vCLAP construct or treatment with TNF-α (Fig. 2 C). This result provides genetic proof for the requirement of IKKγ in vCLAP-induced activation of NF-κB, confirming its role as a molecular adaptor in the assembly of the vCLAP-IKK complexes. The inability of vCLAP to induce NF-κB activation in 5R cells cannot be attributed to defects in the NF-κB pathway downstream of IKKγ, because transfection of these cells with IKKγ can restore NF-κB activation by Tax, which is expressed stably in this cell line (Ref. 15Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar and data not shown). In contrast to vCLAP, Bcl10 was unable to interact with IKKγ in vitro (data not shown). However, like vCLAP, Bcl10 was able to induce NF-κB activation in Rat-1, but not in 5R cells (Fig.2 C), suggesting that Bcl10 could relay its signal to IKKγ indirectly. We next mapped the regions of vCLAP and IKKγ that are required for their interaction. FLAG-tagged IKKγ 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 IKKγ (Fig.3 A). In contrast, the CARD did not interact with IKKγ (Fig. 3 A). The same results were obtained using a GST-IKKγ pull-down assay, which showed that the recombinant GST-IKKγ 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 IKKγ. To extend the characterization of the vCLAP-IKKγ interaction, we expressed T7-tagged vCLAP in 293T cells together with several FLAG-tagged full-length or truncated IKKγ. vCLAP was found to specifically associate with full-length IKKγ but not with the C-terminally truncated IKKγ () or IKKγ () (Fig.3 B). Removal of the last 119 amino acids of IKKγ strongly reduced its interaction with the vCLAP (Fig. 3 B). Taken together, these data indicate that the interaction between vCLAP and IKKγ involves sequences in the C-terminal region of IKKγ. To confirm that vCLAP associates intercellularly with IKKγ when the two proteins are coexpressed in the same cell, we cotransfected 293T cells with constructs encoding GFP-IKKγ and RFP-vCLAP fusion proteins and then monitored the subcellular localization of these proteins by confocal microscopy. As shown in Fig. 3 C, the two proteins exhibited different patterns of cellular localization when expressed alone. Whereas vCLAP exhibited a clear pattern of discrete and interconnecting cytoplasmic filaments, IKKγ displayed a somewhat punctuate cytoplasmic or whole-cell distribution. However, coexpression of the two proteins resulted in redistribution of IKKγ to the vCLAP filaments. This observation is consistent with a direct interaction between vCLAP and IKKγ. Our data demonstrate that IKKγ mediates the association of vCLAP with the IKK kinases and is required for vCLAP-induced activation of NF-κB. This suggest that IKKγ is an adaptor molecule with a C-terminal half that binds to NF-κB activators like vCLAP (see above), RIP (18Li Y. Kang J. Friedman J. Tarassishin L. Ye J. Kovalenko A. Wallach D. Horwitz M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1042-1047Crossref PubMed Scopus (155) Google Scholar, 19Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nunez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 20Poyet J.L. Srinivasula S.M. Lin J. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 24Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar), or Tax (25Chu Z.L. Shin Y.A. Yang J.M. DiDonato J.A. Ballard D.W. J. Biol. Chem. 1999; 274: 15297-15300Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 26Harhaj E.W. Sun S.C. J. Biol. Chem. 1999; 274: 22911-22914Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 27Jin D.Y. Giordano V. Kibler K.V. Nakano H. Jeang K.T. J. Biol. Chem. 1999; 274: 17402-17405Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) and an N-terminal half that is required for interaction with the IKK kinases (20Poyet J.L. Srinivasula S.M. Lin J. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 28Ye J. Xie X. Tarassishin L. Horwitz M.S. J. Biol. Chem. 2000; 275: 9882-9889Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). vCLAP has a bipartite structure consisting of an N-terminal CARD and a C-terminal domain that interacts with IKKγ (see above). Interestingly, we and others have shown that the CARDs of cellular and viral CLAP proteins are important for homo- and heterotypic interactions (1Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 2Koseki T. Inohara N. Chen S. Carrio R. Merino J. Hottiger M.O. Nabel G.J. Nunez G. J. Biol. Chem. 1999; 274: 9955-9961Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 3Costanzo A. Guiet C. Vito P. J. Biol. Chem. 1999; 274: 20127-20132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 4Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). It is therefore likely that CARD-mediated self-association of vCLAP could induce oligomerization of IKKγ resulting in activation of the IKK complex. To test this hypothesis, we generated a fusion protein (CARD-IKKγ-ΔC) composed of the CARD of vCLAP linked to the N-terminal part (residues 1–200) of IKKγ (IKKγ-ΔC) and determined its ability to induce NF-κB activation. To rule out the possibility that the CARD-IKKγ-ΔC chimera functions through interaction with the endogenous IKKγ protein, we examined its ability to activate NF-κB in the IKKγ-deficient 5R cells. As shown in Fig.4 A, transient transfection of the CARD-IKKγ-ΔC chimera resulted in a large increase of NF-κB activity in a dose-dependent manner. In contrast, neither the separate CARD of vCLAP nor IKKγ-ΔC were able to activate the NF-κB when transfected at either low or high doses (Fig.4 A). Moreover, a single point mutation of a conserved residue in the CARD of vCLAP that abrogates homodimerization (L49R) (1Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) prevented NF-κB activation by the chimeric protein (Fig.4 A). Similar results were obtained when the CARD of Bcl10 was used instead of vCLAP-CARD in the above experiments (data not shown). We then tested whether enforced oligomerization of the CTD of vCLAP could induce NF-κB 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 (29MacCorkle R.A. Freeman K.W. Spencer D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3655-3660Crossref PubMed Scopus (173) Google Scholar). As shown in Fig. 4 B, transient tranfection of this construct into Rat-1 cells, but not in the IKKγ-deficient 5R cells, induced a large NF-κB activation in a ligand-dependent manner. No NF-κB activation was detected when the FKBP12-CTD construct was cotransfected with kinase-inactive IKKβ (not shown) or after treatment of empty vector-transfected 293T cells with AP1510 (Fig. 4 B). Taken together, these results demonstrate that vCLAP-induced oligomerization of IKKγ is the triggering event leading to activation of the IKK complex. Although previous studies have shown that the EHV-2-encoded vCLAP protein activates NF-κB in mammalian cells (1Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 4Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), the mechanism by which vCLAP interfaces with the cell's NF-κB-activating machinery remains unclear. In this paper, we report several observations that, when combined, provide a potential mechanism for vCLAP-induced NF-κB activation. First, we show that the IKK kinases are persistently activated in vCLAP-expressing cells, which might explain the robust NF-κB activity observed in vCLAP-transfected cells (1Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 4Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). 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 IKKγ. Consistent with this observation, IKKγ was found to be essential for vCLAP-induced activation of NF-κB. Third, we demonstrate that vCLAP activates the IKK complex through oligomerization of IKKγ. Indeed, CARD-dependent clustering of the N-terminal part of IKKγ, which we have previously shown to interact with the IKK kinases (20Poyet J.L. Srinivasula S.M. Lin J. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), was able to activate NF-κB. Moreover, enforced oligomerization of the CTD of vCLAP was able to induce a large increase in NF-κB activity in Rat-1 cells but not in the IKKγ-deficient 5R cells. This mechanism of NF-κB activation by vCLAP, namely oligomerization-induced activation of the IKK kinases via IKKγ, is reminiscent of the model we proposed for RIP-induced activation of NF-κB after ligation of TNF-R1 (20Poyet J.L. Srinivasula S.M. Lin J. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Based on this model, TNF-α stimulation induces binding of RIP to, and concomitant oligomerization of IKKγ, which in turn passes the oligomerization signal to the effector kinases, resulting in their activation through autophosphorylation of their T-loop serines (20Poyet J.L. Srinivasula S.M. Lin J. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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-κB 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-κB activation. One well documented example of this is the human T-cell leukemia virus-Tax protein (30Yin M.J. Christerson L.B. Yamamoto Y. Kwak Y.T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 31Uhlik M. Good L. Xiao G. Harhaj E.W. Zandi E. Karin M. Sun S.C. J. Biol. Chem. 1998; 273: 21132-21136Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 32Geleziunas R. Ferrell S. Lin X. Mu Y. Cunningham Jr., E.T. Grant M. Connelly M.A. Hambor J.E. Marcu K.B. Greene W.C. Mol. Cell. Biol. 1998; 18: 5157-5165Crossref PubMed Google Scholar, 33Chu Z.L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Tax has been shown to interact with the IKK complex through direct interaction with IKKγ (25Chu Z.L. Shin Y.A. Yang J.M. DiDonato J.A. Ballard D.W. J. Biol. Chem. 1999; 274: 15297-15300Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 26Harhaj E.W. Sun S.C. J. Biol. Chem. 1999; 274: 22911-22914Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 27Jin D.Y. Giordano V. Kibler K.V. Nakano H. Jeang K.T. J. Biol. Chem. 1999; 274: 17402-17405Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Moreover, Tax mutants defective in IKKγ binding failed to activate NF-κB (34Xiao G. Harhaj E.W. Sun S.C. J. Biol. Chem. 2000; 275: 34060-34067Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Interestingly, Tax has been shown to function as a dimerizer that stabilizes dimer formation in other proteins (35Bex F. Gaynor R.B. Methods. 1998; 16: 83-94Crossref PubMed Scopus (101) Google Scholar). It is therefore possible that Tax-induced oligomerization of IKKγ 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-κB when expressed in cells, although at a lesser degree than vCLAP. Bcl10 requires IKKγ for activation of NF-κB (Fig. 2 C). 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-IKKγ or GST-IKKα (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. We thank W. C. Green for the IKKβ kinase inactive construct and S. Yamaoka and A. Israel for the NEMO/IKKγ-deficient Rat-1." @default.
• W1993648889 created "2016-06-24" @default.
• W1993648889 creator A5055976857 @default.
• W1993648889 creator A5077708146 @default.
• W1993648889 creator A5079412406 @default.
• W1993648889 date "2001-02-01" @default.
• W1993648889 modified "2023-10-14" @default.
• W1993648889 title "vCLAP, a Caspase-recruitment Domain-containing Protein of Equine Herpesvirus-2, Persistently Activates the IκB Kinases through Oligomerization of IKKγ" @default.
• W1993648889 cites W1607664081 @default.
• W1993648889 cites W1608832731 @default.
• W1993648889 cites W1614707550 @default.
• W1993648889 cites W1661180389 @default.
• W1993648889 cites W1978434703 @default.
• W1993648889 cites W1979520552 @default.
• W1993648889 cites W1980768491 @default.
• W1993648889 cites W1987172924 @default.
• W1993648889 cites W1989844987 @default.
• W1993648889 cites W1997509253 @default.
• W1993648889 cites W2000594485 @default.
• W1993648889 cites W2013202536 @default.
• W1993648889 cites W2028494050 @default.
• W1993648889 cites W2032901533 @default.
• W1993648889 cites W2033098442 @default.
• W1993648889 cites W2036770241 @default.
• W1993648889 cites W2036789689 @default.
• W1993648889 cites W2042919664 @default.
• W1993648889 cites W2049634835 @default.
• W1993648889 cites W2056831005 @default.
• W1993648889 cites W2070853191 @default.
• W1993648889 cites W2071877809 @default.
• W1993648889 cites W2088102742 @default.
• W1993648889 cites W2089152744 @default.
• W1993648889 cites W2091161875 @default.
• W1993648889 cites W2093293884 @default.
• W1993648889 cites W2095340633 @default.
• W1993648889 cites W2122275228 @default.
• W1993648889 cites W2152044329 @default.
• W1993648889 cites W2152530163 @default.
• W1993648889 cites W2155933617 @default.
• W1993648889 cites W2158708535 @default.
• W1993648889 cites W2159979956 @default.
• W1993648889 cites W2728081960 @default.
• W1993648889 doi "https://doi.org/10.1074/jbc.c000792200" @default.
• W1993648889 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11113112" @default.
• W1993648889 hasPublicationYear "2001" @default.
• W1993648889 type Work @default.
• W1993648889 sameAs 1993648889 @default.
• W1993648889 citedByCount "28" @default.
• W1993648889 countsByYear W19936488892012 @default.
• W1993648889 countsByYear W19936488892015 @default.
• W1993648889 countsByYear W19936488892018 @default.
• W1993648889 countsByYear W19936488892020 @default.
• W1993648889 countsByYear W19936488892023 @default.
• W1993648889 crossrefType "journal-article" @default.
• W1993648889 hasAuthorship W1993648889A5055976857 @default.
• W1993648889 hasAuthorship W1993648889A5077708146 @default.
• W1993648889 hasAuthorship W1993648889A5079412406 @default.
• W1993648889 hasConcept C100175707 @default.
• W1993648889 hasConcept C159047783 @default.
• W1993648889 hasConcept C184235292 @default.
• W1993648889 hasConcept C185592680 @default.
• W1993648889 hasConcept C2522874641 @default.
• W1993648889 hasConcept C2775976905 @default.
• W1993648889 hasConcept C2776409557 @default.
• W1993648889 hasConcept C2777730290 @default.
• W1993648889 hasConcept C2780727368 @default.
• W1993648889 hasConcept C2910050888 @default.
• W1993648889 hasConcept C62478195 @default.
• W1993648889 hasConcept C86803240 @default.
• W1993648889 hasConcept C95444343 @default.
• W1993648889 hasConcept C97029542 @default.
• W1993648889 hasConceptScore W1993648889C100175707 @default.
• W1993648889 hasConceptScore W1993648889C159047783 @default.
• W1993648889 hasConceptScore W1993648889C184235292 @default.
• W1993648889 hasConceptScore W1993648889C185592680 @default.
• W1993648889 hasConceptScore W1993648889C2522874641 @default.
• W1993648889 hasConceptScore W1993648889C2775976905 @default.
• W1993648889 hasConceptScore W1993648889C2776409557 @default.
• W1993648889 hasConceptScore W1993648889C2777730290 @default.
• W1993648889 hasConceptScore W1993648889C2780727368 @default.
• W1993648889 hasConceptScore W1993648889C2910050888 @default.
• W1993648889 hasConceptScore W1993648889C62478195 @default.
• W1993648889 hasConceptScore W1993648889C86803240 @default.
• W1993648889 hasConceptScore W1993648889C95444343 @default.
• W1993648889 hasConceptScore W1993648889C97029542 @default.
• W1993648889 hasIssue "5" @default.
• W1993648889 hasLocation W19936488891 @default.
• W1993648889 hasOpenAccess W1993648889 @default.
• W1993648889 hasPrimaryLocation W19936488891 @default.
• W1993648889 hasRelatedWork W1511369931 @default.
• W1993648889 hasRelatedWork W1698057790 @default.
• W1993648889 hasRelatedWork W2036331617 @default.
• W1993648889 hasRelatedWork W2091800772 @default.
• W1993648889 hasRelatedWork W2096837432 @default.
• W1993648889 hasRelatedWork W2097798460 @default.
• W1993648889 hasRelatedWork W2170617996 @default.
• W1993648889 hasRelatedWork W3026238348 @default.
• W1993648889 hasRelatedWork W3119138534 @default.

• next