Rip2 Participates in Bcl10 Signaling and T-cell Receptor-mediated NF-κB Activation

Abstract

Engagement of the T-cell receptor (TCR) initiates a signaling cascade that ultimately results in activation of the transcription factor NF-κB, which regulates many T-cell functions including proliferation, differentiation and cytokine production. Herein we demonstrate that Rip2, a caspase recruitment domain (CARD)-containing serine/threonine kinase, plays an important role in this cascade and is required for optimal TCR signaling and NF-κB activation. Following TCR engagement, Rip2 associated with Bcl10, a CARD-containing signaling component of the TCR-induced NF-κB pathway, and induced its phosphorylation. Rip2-deficient mice were defective in TCR-induced NF-κB activation, interleukin-2 production, and proliferation in vitro and exhibited defective T-cell-dependent responses in vivo. The defect in Rip2-/- T-cells correlated with a lack of TCR-induced Bcl10 phosphorylation. Furthermore, deficiency in Bcl10-dependent NF-κB activation could be rescued in Rip2-/- embryonic fibroblasts by exogenous wild-type Rip2 but not a kinase-dead mutant. Together these data define an important role for Rip2 in TCR-induced NF-κB activation and T-cell function and highlight the significance of post-translational modification of Bcl10 by Rip2 in T-cell signaling. Engagement of the T-cell receptor (TCR) initiates a signaling cascade that ultimately results in activation of the transcription factor NF-κB, which regulates many T-cell functions including proliferation, differentiation and cytokine production. Herein we demonstrate that Rip2, a caspase recruitment domain (CARD)-containing serine/threonine kinase, plays an important role in this cascade and is required for optimal TCR signaling and NF-κB activation. Following TCR engagement, Rip2 associated with Bcl10, a CARD-containing signaling component of the TCR-induced NF-κB pathway, and induced its phosphorylation. Rip2-deficient mice were defective in TCR-induced NF-κB activation, interleukin-2 production, and proliferation in vitro and exhibited defective T-cell-dependent responses in vivo. The defect in Rip2-/- T-cells correlated with a lack of TCR-induced Bcl10 phosphorylation. Furthermore, deficiency in Bcl10-dependent NF-κB activation could be rescued in Rip2-/- embryonic fibroblasts by exogenous wild-type Rip2 but not a kinase-dead mutant. Together these data define an important role for Rip2 in TCR-induced NF-κB activation and T-cell function and highlight the significance of post-translational modification of Bcl10 by Rip2 in T-cell signaling. Many diverse stimuli activate NF-κB by inducing the phosphorylation and destruction of inhibitory molecules known as the IκBs that retain NF-κB in the cytoplasm (1.Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4607) Google Scholar). The IκB kinase (IKK) 1The abbreviations used are: IKK, IκB kinase; CARD, caspase recruitment domain; TCR, T-cell receptor; TLR, Toll-like receptor; LPS, lipopolysaccaride; HA, hemagglutinin; PMA, phorbol myristate acetate; IL-2, interleukin-2; MEF, mouse embryonic fibroblast; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase. complex, composed of two kinase subunits, IKKα and IKKβ, and a non-catalytic subunit, NEMO/IKKγ, is responsible for the phosphorylation of the IκBs. The association of Bcl10 and CARMA1 (CARD11), two caspase-recruitment domain (CARD)-containing proteins, has been shown to be essential to the transduction of the signal from the T-cell receptor (TCR) to the IKK complex (2.Thome M. Tschopp J. Trends Immunol. 2003; 24: 419-424Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Mice deficient for either Bcl10 or CARMA1 display profound defects in T-cell proliferation and cytokine production due to a lack of NF-κB activation (3.Ruland J. Duncan G.S. Elia A. del Barco Barrantes I. Nguyen L. Plyte S. Millar D.G. Bouchard D. Wakeham A. Ohashi P.S. Mak T.W. Cell. 2001; 104: 33-42Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 4.Newton K. Dixit V.M. Curr. Biol. 2003; 13: 1247-1251Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 5.Egawa T. Albrecht B. Favier B. Sunshine M.J. Mirchandani K. O'Brien W. Thome M. Littman D.R. Curr. Biol. 2003; 13: 1252-1258Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 6.Hara H. Wada T. Bakal C. Kozieradzki I. Suzuki S. Suzuki N. Nghiem M. Griffiths E.K. Krawczyk C. Bauer B. D'Acquisto F. Ghosh S. Yeh W.C. Baier G. Rottapel R. Penninger J.M. Immunity. 2003; 18: 763-775Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 7.Jun J.E. Wilson L.E. Vinuesa C.G. Lesage S. Blery M. Miosge L.A. Cook M.C. Kucharska E.M. Hara H. Penninger J.M. Domashenz H. Hong N.A. Glynne R.J. Nelms K.A. Goodnow C.C. Immunity. 2003; 18: 751-762Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar); however, the mechanism by which the CARMA1/Bcl10 complex activates IKK remains unclear. In vitro experiments have indicated that Bcl10 undergoes phosphorylation when over expressed with its viral homologue, E10 or CARMA1 (8.Thome M. Gaide O. Micheau O. Martinon F. Bonnet D. Gonzalez M. Tschopp J. J. Cell Biol. 2001; 152: 1115-1122Crossref PubMed Scopus (17) Google Scholar, 9.Gaide O. Martinon F. Micheau O. Bonnet D. Thome M. Tschopp J. FEBS Lett. 2001; 496: 121-127Crossref PubMed Scopus (169) Google Scholar, 10.Bertin J. Wang L. Guo Y. Jacobson M.D. Poyet J.L. Srinivasula S.M. Merriam S. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 276: 11877-11882Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). In these studies, Bcl10 phosphorylation correlated with its ability to activate NF-κB, suggesting that this modification was required for NF-κB activation. Indeed, the COOH-terminal domain of Bcl10 is rich in serine and threonine residues and has been proposed as the site of CARMA1-mediated phosphorylation (10.Bertin J. Wang L. Guo Y. Jacobson M.D. Poyet J.L. Srinivasula S.M. Merriam S. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 276: 11877-11882Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Since CARMA1 itself is not a kinase, the kinase responsible for Bcl10 phosphorylation has remained an open question. Rip2 is a serine/threonine kinase that contains a CARD domain at its carboxyl terminus and has been shown to induce NF-κB activation in over expression systems (11.McCarthy J.V. Ni J. Dixit V.M. J. Biol. Chem. 1998; 273: 16968-16975Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 12.Thome M. Hofmann K. Burns K. Martinon F. Bodmer J.L. Mattmann C. Tschopp J. Curr. Biol. 1998; 8: 885-888Abstract Full Text Full Text PDF PubMed Google Scholar, 13.Inohara N. del Peso L. Koseki T. Chen S. Nunez G. J. Biol. Chem. 1998; 273: 12296-12300Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Rip2 has also been shown to associate in vitro with members of the TRAF family, such as TRAF6, that plays an essential role in the innate immune response downstream of Toll-like receptors (TLRs) (14.Mak T.W. Yeh W.C. Nature. 2002; 418: 835-836Crossref PubMed Scopus (12) Google Scholar, 15.Lomaga M.A. Yeh W.C. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. van der Heiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z. Penninger J.M. Paige C.J. Lacey D.L. Dunstan C.R. Boyle W.J. Goeddel D.V. Mak T.W. Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1080) Google Scholar). In addition, Rip2 has been implicated in regulating both the innate and adaptive immune responses (16.Kobayashi K. Inohara N. Hernandez L.D. Galan J.E. Nunez G. Janeway C.A. Medzhitov R. Flavell R.A. Nature. 2002; 416: 194-199Crossref PubMed Scopus (744) Google Scholar, 17.Chin A.I. Dempsey P.W. Bruhn K. Miller J.F. Xu Y. Cheng G. Nature. 2002; 416: 190-194Crossref PubMed Scopus (330) Google Scholar). Mice deficient in Rip2 mounted only an attenuated immune response against Toll-like receptor agonists such as lipopolysaccaride (LPS) (16.Kobayashi K. Inohara N. Hernandez L.D. Galan J.E. Nunez G. Janeway C.A. Medzhitov R. Flavell R.A. Nature. 2002; 416: 194-199Crossref PubMed Scopus (744) Google Scholar, 17.Chin A.I. Dempsey P.W. Bruhn K. Miller J.F. Xu Y. Cheng G. Nature. 2002; 416: 190-194Crossref PubMed Scopus (330) Google Scholar). Interestingly, CD4+ T-cells from Rip2-deficient mice were unable to proliferate efficiently in response to antigen-induced T-cell activation, but no mechanism was provided for this striking observation (16.Kobayashi K. Inohara N. Hernandez L.D. Galan J.E. Nunez G. Janeway C.A. Medzhitov R. Flavell R.A. Nature. 2002; 416: 194-199Crossref PubMed Scopus (744) Google Scholar, 17.Chin A.I. Dempsey P.W. Bruhn K. Miller J.F. Xu Y. Cheng G. Nature. 2002; 416: 190-194Crossref PubMed Scopus (330) Google Scholar). We sought to define the role of Rip2 in antigen-induced NF-κB activation and T-cell proliferation. Generation of Rip2-/- Mice—A targeting vector that removed exon I of Rip2 was electroporated into ES cells. Homologous recombinants were used to generate chimeric founder mice by microinjection into C57BL/6J blastocysts. Germ line transmission was confirmed by Southern blot analysis of genomic tail DNA. Two independent ES clone lines resulted in mice with identical phenotypes. All mice used in experiments were backcrossed onto C57BL/6 five to seven generations and were confirmed to be >95% C57BL/6 by PCR analysis of genomic tail DNA. Immunoprecipitations—293 T-cells were transfected with expression vectors for NH2-terminal HA- or Myc/His-tagged Rip2, Rip2 311-541(ΔKinase) and Rip2 1-454 (ΔCARD) (11.McCarthy J.V. Ni J. Dixit V.M. J. Biol. Chem. 1998; 273: 16968-16975Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar), and V5-tagged Bcl10 (18.Yan M. Lee J. Schilbach S. Goddard A. Dixit V. J. Biol. Chem. 1999; 274: 10287-10292Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Tagged proteins were immunoprecipitated using monoclonal antibodies to Rip2 (Genentech, Inc.), V5 (Invitrogen), HA and Myc (Zymed Laboratories Inc.), and actin (ICN). Jurkat cells (4 × 107) were stimulated with cross-linking antibodies to the Jurkat TCR clone C305 (a gift from A. Chan, Genentech, Inc.). Cell lysates were prepared as described previously (10.Bertin J. Wang L. Guo Y. Jacobson M.D. Poyet J.L. Srinivasula S.M. Merriam S. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 276: 11877-11882Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Endogenous proteins were immunoprecipitated using anti-Bcl10 clone 331.3 (Santa Cruz) or anti-Rip2 and protein A/G beads (Pierce) and analyzed by Western blots using antibodies to Bcl10 (H-197) or Bcl10 (331.3) (Santa Cruz) and phospho-Zap-70 (Cell Signaling Technology). In some cases, immunoprecipitates were treated with λ phosphatase (New England Biolabs). Proliferation Assays—Splenic B and T-cells and CD4+ T were purified by negative selection using magnetic beads (Miltenyi Biotech) to >95% purity. Purified T-cells were activated with plate bound anti-CD3 (0-10 μg/ml) (BD Biosciences) with or without irradiated CD4-depleted APCs or plate-bound anti-CD28 (0-10 μg/ml) (BD Biosciences), phorbol myristate acetate (PMA) (2 ng/ml) plus ionomycin (0.1 μg/ml) (Sigma) in the presence or absence of IL-2 (50 ng/ml) (R & D Systems). B-cells were stimulated with anti-IgM (20 μg/ml) (Jackson Laboratories), LPS (20 μg/ml) (Sigma), or PMA (2 ng/ml) plus ionomycin (0.1 μg/ml). Cells were harvested at 24, 48, 72, and 96 h after an 8-h pulse with [3H]thymidine (1 μCi/well), and incorporation of [3H]thymidine was measured using a Matrix 96 direct β counter system (Hewlett-Packard). Data represent triplicate samples and are representative of at least three separate experiments. Neonatal Heart Allograft—Neonatal hearts from BALB/c (H-2d) mice were surgically implanted behind the dorsum of the ear pinna of 12-week-old male Rip2-/- and wild-type mice (Both H-2b). Heart grafts were examined with a stereomicroscope at 10-20-fold magnification every other day until rejection. Western Blots—Purified T-cells (2 × 106) were stimulated with 10 μg/ml plate-bound anti-CD3 for 0-30 min. Western blots were performed using phospho-specific antibodies for IκBα, ERK1/2, and JNK (Cell Signaling Technology). Blots were stripped and re-probed with antibodies to IκBα and p44 (Cell Signaling Technology). NF-κB Luciferase Assays—Mouse embryonic fibroblasts (MEFs) were transfected using Superfect (Qiagen) with 2-μg expression vectors (V5-Bcl10, HA-Rip2, Myc/His-K47ARip2 (11.McCarthy J.V. Ni J. Dixit V.M. J. Biol. Chem. 1998; 273: 16968-16975Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar), or HA-pcDNA3), 0.25 μg pLam3 (NF-κB-responsive luciferase), and 0.025 μg of pR-TK (control luciferase). Luciferase activity was measured 48 h post-transfection using the dual luciferase reporter assay system (Promega). Data represent triplicate samples and are representative of at least three separate experiments. Isolation of Phosphorylated Proteins—Splenic T-cells from Rip2-/- and wild-type mice (4 × 107) cells/ml) were stimulated with 10 μg/ml plate-bound anti-CD3 for 0, 15, and 30 min. Cells were lysed under denaturing conditions to disrupt protein-protein interactions and diluted to 0.1 mg/ml in phospho-lysis buffer (Qiagen). Phosphorylated proteins were separated using the phospho-protein purification kit (Qiagen) according to the manufacturer's instructions. Rip2 Associates with Bcl10 and Induces Its Phosphorylation—We investigated whether Rip2 could associate with molecules known to play essential roles in the TCR-induced signaling cascade. Initially, we tested whether Rip2 and Bcl10 could associate by overexpressing tagged versions of both proteins in 293 T-cells. V5-tagged Bcl10 could be co-immunoprecipitated with HA-tagged Rip2 (Fig. 1A). Interestingly, two bands representing Bcl10 were observed. The upper band was determined to be a hyperphosphorylated form of Bcl10, since it could be collapsed to the lower band by λ phosphatase treatment (Fig. 1A). Hyperphosphorylation of Bcl10 was also apparent by mobility shift in whole cell lysates from 293T-cells co-transfected with Rip2 and Bcl10, compared with the very low levels of phosphorylation seen with Bcl10 alone (Fig. 1B). To establish the domains of Rip2 responsible for hyperphosphorylation of Bcl10, mutants with deletions of either the kinase domain or the CARD domain were used in co-expression studies. Bcl10 hyperphosphorylation required both a functional kinase domain and CARD domain of Rip2 as neither mutant induced phosphorylation of Bcl10 (Fig. 1B). Moreover, phosphorylation of Bcl10 was specific for Rip2, as overexpression of RIP or Rip3 did not induce Bcl10 phosphorylation (Fig. 1C). To determine whether Rip2 was involved in Bcl10-dependent signaling pathways, we studied the interaction of endogenous proteins in Jurkat cells stimulated with cross-linking antibodies to CD3-TCR. Rip2 and Bcl10 consistently associated in a transient and time-dependent manner after TCR engagement (Fig. 1D, bottom panel). Induction of phosphorylated Zap-70 confirmed TCR activation (Fig. 1E). We next examined the phosphorylation status of Bcl10 using phosphoserine-specific antibodies. Lysates from anti-CD3 treated and untreated Jurkats were immunoprecipitated using antibodies to Bcl10 and Western blots were performed using antibodies for phosphoserine and Bcl10. Serine phosphorylated Bcl10 was detected after 15-min treatment with anti-CD3 (Fig. 1F, top panel) and treatment of immunoprecipitates with λ phosphatase (λPPase) significantly diminished levels of serine-phosphorylated Bcl10. Phosphorylation of endogenous Bcl10 was also apparent after treatment with anti-CD3, as evidenced by a slower migrating band that could be collapsed by treatment with λ phosphatase (Fig. 1F, bottom panel). Taken together, these results were consistent with Rip2 binding Bcl10 upon TCR engagement and inducing its phosphorylation. Defective T-cell Proliferation and Function in Rip2-/- the of Rip2 T cell activation in an in we Rip2-deficient mice by Rip2-/- T-cells were deficient in anti-CD3 induced proliferation (Fig. and defect could not be rescued by with anti-CD28 or activation using PMA in with (Fig. The levels of IL-2 after treatment with anti-CD3 anti-CD3 with or with PMA and ionomycin were compared with wild-type T-cells not Moreover, of exogenous IL-2 was not to the defect in proliferation in Rip2-/- T-cells after (Fig. with proliferation in response to and LPS was Rip2-deficient and wild-type B-cells not (16.Kobayashi K. Inohara N. Hernandez L.D. Galan J.E. Nunez G. Janeway C.A. Medzhitov R. Flavell R.A. Nature. 2002; 416: 194-199Crossref PubMed Scopus (744) Google Scholar). Taken together, these results that the defect in proliferation in Rip2-/- mice was to T-cells and due to of IL-2 transcription and NF-κB activation. in experiments Rip2-/- mice tested T-cell using such as and T-cell-dependent responses (16.Kobayashi K. Inohara N. Hernandez L.D. Galan J.E. Nunez G. Janeway C.A. Medzhitov R. Flavell R.A. Nature. 2002; 416: 194-199Crossref PubMed Scopus (744) Google Scholar, 17.Chin A.I. Dempsey P.W. Bruhn K. Miller J.F. Xu Y. Cheng G. Nature. 2002; 416: 190-194Crossref PubMed Scopus (330) Google Scholar), which also of and other innate signaling and to T-cell in we a functional the ability to in a which not from of the innate immune from BALB/c (H-2d) mice were into the ear pinna of wild-type and Rip2-deficient mice and hearts were by wild-type mice by day 15, over of the hearts were in Rip2-deficient mice and to function for an (Fig. Rip2-deficient mice wild-type consistent with in vitro data and a defect in T-cell activation and Defective NF-κB in Rip2-deficient determine the of the in T-cell receptor signaling in the absence of Rip2, we analyzed activated by TCR engagement in wild-type and Rip2-/- T-cells. T-cells from wild-type and Rip2-/- mice were treated with plate-bound anti-CD3 or and lysates were by Western blot using phospho-specific antibodies to IκBα was phosphorylated and in wild-type T-cells but not in Rip2-/- cells (Fig. In treatment of both wild-type and Rip2-/- T-cells with phosphorylation and of IκBα (Fig. NF-κB signaling downstream of other receptors remained in Rip2-/- TCR engagement also activation of the protein Western using phospho-specific antibodies that and were phosphorylated with in wild-type and Rip2-/- T-cells after TCR engagement (Fig. upper panel). activation of the JNK signaling engagement was in both wild-type and Rip2-deficient T-cells (Fig. lower panel). results confirmed that the defect was specific for NF-κB signaling downstream of the TCR and that activated by TCR engagement remained To the role of Rip2 kinase activity in Bcl10-dependent NF-κB activation, we transfected from wild-type and Rip2-/- mice with Bcl10 and a luciferase reporter for Bcl10 could induce NF-κB activation in wild-type NF-κB activation by Bcl10 was significantly in Rip2-/- (Fig. of exogenous wild-type Rip2, but not a kinase-dead could NF-κB reporter activity in Rip2-/- (Fig. the kinase activity of Rip2 is required for optimal NF-κB activation. Bcl10 Phosphorylated after TCR Engagement in but Rip2-/- together, data that Rip2 functions to T-cell activation by Bcl10. we to establish whether the lack of NF-κB activation in Rip2-/- T-cells correlated with a lack of Bcl10 phosphorylation after TCR and Rip2-deficient T-cells were treated with and cell lysates were using a these protein-protein interactions are and only phosphorylated proteins the proteins Western of the phosphorylated protein using antibodies to and Bcl10 that phosphorylated could be detected in the purified phosphorylated of both wild-type and Rip2-/- T-cells (Fig. Bcl10 was only in the purified phosphorylated of treated wild-type T-cells (Fig. top panel). levels of Bcl10 were detected in the from wild-type and T-cells (Fig. bottom panel). To that no proteins the phosphorylated protein lysates from both were Western using antibodies for was in the but in the phosphorylated protein (Fig. data demonstrate that Bcl10 is phosphorylated in mouse T cells after TCR and deficiency of Rip2 phosphorylation of Bcl10. Herein we for the of Rip2 in NF-κB activation and Bcl10-dependent signaling. Phosphorylation of Bcl10 after TCR engagement, and lack of phosphorylation with a defect in NF-κB activation and T-cell proliferation. have shown that Bcl10 is phosphorylated upon over expression of however, the of phosphorylation in T-cell signaling was unclear. data that phosphorylation of Bcl10 by Rip2 plays a role in signaling the TCR and the IKK K. Dixit V.M. Curr. Biol. 2003; 13: 1247-1251Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 5.Egawa T. Albrecht B. Favier B. Sunshine M.J. Mirchandani K. O'Brien W. Thome M. Littman D.R. Curr. 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J. 2002; PubMed Scopus Google have that CARMA1 is involved in TCR-induced NF-κB activation. remains whether Bcl10 phosphorylation is required for its association with The of the association Rip2 and Bcl10 and Bcl10 phosphorylation in Jurkat cells with the of the interaction CARMA1 and Bcl10 in Jurkat T-cells O. Favier B. Bonnet D. B. S. C. Tschopp J. Thome M. Immunol. 2002; PubMed Scopus Google Scholar). Phosphorylation of Bcl10 either its recruitment to or to activate other molecules in downstream signaling that ultimately activate the IKK a has also been shown to associate with Bcl10 and NF-κB activation K. L.A. S. Dixit V.M. Cell. Full Text Full Text PDF PubMed Google Scholar, M. Inohara N. Chen S. M. Nunez G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google and is also required for TCR-induced proliferation, cytokine production, and NF-κB activation Dixit V.M. 2003; PubMed Scopus Google Scholar). data are consistent with that Rip2-deficient mice from defects in the adaptive immune response due to lack of antigen-induced T-cell proliferation and NF-κB activation (16.Kobayashi K. Inohara N. Hernandez L.D. Galan J.E. Nunez G. Janeway C.A. Medzhitov R. Flavell R.A. Nature. 2002; 416: 194-199Crossref PubMed Scopus (744) Google Scholar, 17.Chin A.I. Dempsey P.W. Bruhn K. Miller J.F. Xu Y. Cheng G. Nature. 2002; 416: 190-194Crossref PubMed Scopus (330) Google Scholar). to we also a defect in cytokine production in stimulated with LPS and other Toll-like an defect in innate not (16.Kobayashi K. Inohara N. Hernandez L.D. Galan J.E. Nunez G. Janeway C.A. Medzhitov R. Flavell R.A. Nature. 2002; 416: 194-199Crossref PubMed Scopus (744) Google Scholar, 17.Chin A.I. Dempsey P.W. Bruhn K. Miller J.F. Xu Y. Cheng G. Nature. 2002; 416: 190-194Crossref PubMed Scopus (330) Google Scholar). Since Rip2 with signaling molecules in both the adaptive and innate immune such as Bcl10 and it is that the absence of this kinase signaling and in in immune system We F. D. N. and the Dixit members for and of this and S. and the for the and of used in this