CARD11 mediates factor-specific activation of NF-κB by the T cell receptor complex
2002; Springer Nature; Volume: 21; Issue: 19 Linguagem: Inglês
10.1093/emboj/cdf505
ISSN1460-2075
AutoresJoel L. Pomerantz, Elissa M. Denny, David Baltimore,
Tópico(s)T-cell and B-cell Immunology
ResumoArticle1 October 2002free access CARD11 mediates factor-specific activation of NF-κB by the T cell receptor complex Joel L. Pomerantz Corresponding Author Joel L. Pomerantz Division of Biology, California Institute of Technology, Pasadena, CA, 91125 USA Search for more papers by this author Elissa M. Denny Elissa M. Denny Division of Biology, California Institute of Technology, Pasadena, CA, 91125 USA Search for more papers by this author David Baltimore Corresponding Author David Baltimore Division of Biology, California Institute of Technology, Pasadena, CA, 91125 USA Search for more papers by this author Joel L. Pomerantz Corresponding Author Joel L. Pomerantz Division of Biology, California Institute of Technology, Pasadena, CA, 91125 USA Search for more papers by this author Elissa M. Denny Elissa M. Denny Division of Biology, California Institute of Technology, Pasadena, CA, 91125 USA Search for more papers by this author David Baltimore Corresponding Author David Baltimore Division of Biology, California Institute of Technology, Pasadena, CA, 91125 USA Search for more papers by this author Author Information Joel L. Pomerantz 1, Elissa M. Denny1 and David Baltimore 1 1Division of Biology, California Institute of Technology, Pasadena, CA, 91125 USA *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2002)21:5184-5194https://doi.org/10.1093/emboj/cdf505 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info NF-κB is a critical target of signaling downstream of the T cell receptor (TCR) complex, but how TCR signaling activates NF-κB is poorly understood. We have developed an expression cloning strategy that can identify catalytic and noncatalytic molecules that participate in different pathways of NF-κB activation. Screening of a mouse thymus cDNA library yielded CARD11, a membrane-associated guanylate kinase (MAGUK) family member containing CARD, PDZ, SH3 and GUK domains. Using a CARD-deleted variant of CARD11 and RNA interference (RNAi), we demonstrate that CARD11 mediates NF-κB activation by αCD3/αCD28 cross-linking and PMA/ionomycin treatment, but not by TNFα or dsRNA. CARD11 is not required for TCR-mediated induction of NFAT or AP-1. CARD11 functions upstream of the IκB-kinase (IKK) complex and cooperates with Bcl10 in a CARD domain-dependent manner. RNAi-rescue experiments suggest that the CARD, coiled-coil, SH3 and GUK domains of CARD11 are critical for its signaling function. These results implicate CARD11 in factor- specific activation of NF-κB by the TCR complex and establish a role for a MAGUK family member in antigen receptor signaling. Introduction The NF-κB transcription factor is rapidly activated by diverse stimuli that alert a cell or organism to stressful or infectious conditions (Ghosh et al., 1998). These include UV and γ-irradiation, bacterial and viral products (e.g. lipopolysaccharide and dsRNA), pro-inflammatory cytokines (e.g. TNFα and IL-1), antigen recognition by the T and B cell receptor complexes, and apoptotic and necrotic stimuli. NF-κB regulates many genes involved in the development and function of the immune response, inflammation, cell growth control and anti-apoptotic responses. Many stimuli activate NF-κB by causing the phosphorylation and destruction of IκBs, inhibitory molecules that retain NF-κB in the cytoplasm. The signal-induced phosphorylation of IκBs is accomplished by the IκB-kinase (IKK) complex, which is composed of two kinase subunits, IKKα and IKKβ, and a noncatalytic subunit, NEMO/IKKγ (Karin and Ben-Neriah, 2000). Phos phorylated IκB is ubiquitylated and degraded by the 26S proteasome, allowing NF-κB to translocate to the nucleus to activate target genes. The IKK complex is activated by many stimuli, but the precise mechanism in most cases has not been firmly established. Mice deficient in NF-κB subunits, or in molecules that signal to NF-κB, have revealed that the proper regulation of NF-κB is critical for normal innate and adaptive immune responses (Gerondakis et al., 1999). In the adaptive immune response, NF-κB is a critical target of antigen receptor signaling in B and T cells. In T cells, engagement of the T cell receptor (TCR) complex in concert with costimulatory signals (CD28) leads to the activation of multiple signaling cascades which induce programs of gene expression through several transcription factors including NFAT, AP-1 and NF-κB. These signaling responses are required for normal T cell development, proliferation and activation (Alberola-Ila et al., 1997). Genetic studies have determined that NF-κB plays an important cell autonomous role in these processes. T cells lacking c-Rel, RelA or IKKβ develop, but fail to proliferate normally in response to TCR/CD28 costimulation (Kontgen et al., 1995; Doi et al., 1997; Senftleben et al., 2001). In addition, the properties of mice transgenic for non-degradable forms of IκB have suggested a role for NF-κB in TCR-mediated thymocyte selection and in pre-TCR survival signals (Hettmann and Leiden, 2000; Voll et al., 2000). The mechanism by which TCR signaling activates the IKK complex is poorly understood. Several molecules are required, including the ZAP70 tyrosine kinase, the SLP-76 adapter and the Vav GTP/GDP exchange factor (Costello et al., 1999; Herndon et al., 2001). These proteins participate in TCR-proximal events, signal to several targets and are not involved exclusively in NF-κB activation. Protein kinase Cθ (PKCθ) acts downstream of these molecules and is required in mature thymocytes for both NF-κB and AP-1 activation (Sun et al., 2000). A specific role in NF-κB induction by TCR triggering has been demonstrated for Bcl10, a caspase-recruitment domain (CARD)-containing protein first identified in the t(1;14)(p22;q32) translocation associated with B cell lymphomas of mucosa-associated lymphoid tissue (MALT; Willis et al., 1999; Zhang et al., 1999). This translocation places the bcl10 gene in the immunoglobulin heavy chain locus and results in its overexpression, which may provide an anti-apoptotic advantage via NF-κB activation. Mice deficient in Bcl10 are severely immunodeficient due to defects in antigen receptor-induced lymphocyte activation and NF-κB induction (Ruland et al., 2001). Although Bcl10 has been shown to act upstream of the IKK complex, its mechanism of action has not been elucidated. As an approach to increasing our understanding of the regulation and biological roles of NF-κB, we have been interested in developing new methods for identifying signaling molecules in NF-κB-inducing pathways. Here we describe an expression cloning strategy based upon the observation that several known signaling molecules in NF-κB-inducing pathways will activate NF-κB when overexpressed in tissue culture cells. In this method, a cDNA expression library is subdivided into pools, each of which is assayed for the ability to activate an NF-κB-responsive reporter after transfection into cells. Positive pools are assayed in secondary screens to confirm their specificity and the clone responsible for an interesting pool's activity is purified by sib selection. We demonstrate that this strategy identifies molecules of different biochemical types and can be used to isolate components of multiple signaling pathways. Using a mouse thymus expression library we have isolated the murine CARD11 cDNA. We present evidence that CARD11 cooperates with Bcl10 and functions between the TCR complex and the IKK complex in the activation of NF-κB in T cells. Results Establishment of the expression cloning screen For efficient and economical screening, we used a quantitative and highly sensitive reporter assay for NF-κB activation. In this assay, pool DNA cloned into an expression vector driven by the cytomegalovirus (CMV) promoter was transiently transfected into 293T cells with the Igκ2-IFN-LUC reporter, which contained two copies of the immunoglobulin κ light chain κB site (5′-GGGGACTTTCC-3′) upstream of the interferon-β minimal promoter (−55 to +19) (Fujita et al., 1987) driving luciferase expression. For normalization of transfection efficiency and extract recovery, the transfection included the pCSK-lacZ vector (Condie et al., 1990), which constitutively expresses β-galactosidase and is unaffected by NF-κB. To maximize the number of cDNAs that could be assayed, it was important to determine what complexity (number of cDNAs per pool) would allow reliable detection of a single active clone in a mixture of cDNAs. Pilot experiments using the cDNA for TRAF2, an adapter protein in the TNFα pathway (Rothe et al., 1995), suggested that a pool complexity of 100 cDNAs would allow detection of molecules possessing 3-fold lower specific activity than TRAF2 in this assay (data not shown). The sensitivity of detection of luciferase and β-galactosidase activities allowed us to scale down the size of the transfection and to minimize the amount of pool DNA required (see Materials and methods). To test this methodology, a portion of an arrayed human placenta cDNA expression library was subdivided into 561 pools of ∼100 cDNA complexity. Plasmid DNA from each pool was assayed and pools were considered positive if they activated the reporter 3-fold or more relative to the activity observed with the empty expression plasmid pcDNA3. In addition, some pools were considered positive if they were 3-fold or more active than the average activity of a cohort of pools assayed in parallel. Positive pools were reassayed and their ability to activate the Igκ2-IFN-LUC reporter confirmed. Of the 561 pools assayed in this way, 41 were positive by these criteria, ranging in fold activation from 2.3-fold to 256-fold. An example of primary screening is shown in Figure 1A, in which 67 pools were assayed and those considered positive were pools 10 (4.5-fold), 12 (4.4-fold), 24 (16.7-fold), and 52 (8.3-fold). Figure 1.The expression cloning strategy. (A) Primary screening of pools. Pool DNA was transfected into 293T cells with the Igκ2-IFN-LUC reporter and the pCSK-lacZ control vector and fold stimulation was determined as described in Materials and methods. (B) Secondary screening of positive pools. Positive pool DNA was assayed for the ability to stimulate the Igκ2-IFN-LUC reporter, the MUT-IFN-LUC reporter or the Igκ2-IFN-LUC reporter in the presence of IKKβ K44A or TBK1 K38A as indicated. The activities in the pools illustrated here were identified to be C/EBPδ (pool 73), TRAIL (pool 224), TNFR1 (pool 473), TRAF2 (pool 24), MyD88 (pool 72) and IKK-i/ϵ (pool 178). (C) Clone purification. The purification of the rhoB cDNA from pool 443 is illustrated. The activities of the original pool (complexity ∼100 cDNAs) and that of the derived positive subpool (complexity of 24 cDNAs) are shown, as well as the activities observed in the clone identification matrix and for the isolated clone. In this matrix, the coordinates of the positive well were D, 2, X. A schematic of the conceptual clone identification matrix is illustrated. Actual values reading from left to right are 3.6, 39.6, 0.9, 0.7, 0.9, 39.3, 1.4, 63.4, 0.6, 61.2, 0.7 and 114. Download figure Download PowerPoint We applied three secondary screens. First, we tested the NF-κB dependence of the activity of a pool by comparing its fold induction on the Igκ2-IFN-LUC reporter to that on the MUT-IFN-LUC reporter, which contains mutations in the Igκ κB motifs (5′-ATCCACTTTCC-3′). Secondly, we tested which activities might function upstream of the IKK complex by assessing their activity in the presence of the IKKβ K44A kinase-dead dominant negative. Thirdly, as a control, we tested each κB-specific positive pool in the presence of kinase-dead TANK-binding kinase 1 (TBK1 K38A), an IKK-related kinase (Pomerantz and Baltimore, 1999) which should not block most pathways leading to NF-κB. Examples of these secondary screens are shown in Figure 1B. Of the 41 positive pools, 34 were dependent on the κB sites for activity. Each of these specific pools was inhibited by co-expression with IKKβ K44A, and one pool (pool 178) was also inhibited by co-expression with TBK1 K38A (Figure 1B). To identify the cDNA responsible for a pool's activity, colonies derived from its glycerol stock were sib selected (see Materials and methods). For most pools, activity increased as the clone was purified, although the activity of some reached saturation in the assay before complete purification. An example of clone purification is shown in Figure 1C. The identities of 25 specific clones are presented in Table I with the representative behavior of pools in primary and secondary screens, and their activities when purified. Six isolated molecules had been linked previously to pathways known to activate NF-κB. These included the ligand TRAIL, the TRAMP/DR3 and TNFR1 cell surface receptors, the TRAF2 and MyD88 adapter proteins and the IKK-i/ϵ kinase (see Table I for associated pathways). The other specific clones encoded the small GTPase rhoB, the MARCKS PKC substrate, the DLK cell surface protein and the Snk kinase. To our knowledge, NF-κB has not been linked to MARCKS, Snk or DLK previously, and rhoB has not been placed in a particular NF-κB-inducing pathway. Table 1. Summary of isolates from the human placenta library and representative pool characteristics cDNA No. of isolates Type Pathway Rep. poola Igκ2b MUTc IKKβ K44Ad TBK1 K38Ae Isolated clonef TRAIL 1 Ligand TRAIL 224 3.1 0.6 1.1 3.0 19.0 DLK 2 Ligand ??? 228 6.2 0.6 1.5 11.0 66.0 TNFR1 2 Receptor TNFα 473 87.1 1.8 0.7 67.7 108.0 TRAMP 4 Receptor Apo3L 110 74.5 1.0 0.8 75.4 109.0 TRAF2 2 Adapter TNFα 24 25.2 2.9 2.0 25.4 46.8 MyD88 1 Adapter IL-1/Toll 72 30.2 3.8 3.6 19.7 999.0 IKK-i/ϵ 1 Kinase PMA/TCR 178 7.6 1.3 0.9 1.4 53.5 rhoB 9 Small GTPase ??? 501 3.2 0.8 0.3 2.7 20.3 Snk 1 Kinase ??? 270 6.3 1.1 1.2 5.2 24.0 MARCKS 2 Kinase substrate ??? 525 40.9 4.4 2.9 47.0 69.0 See Supplementary data at The EMBO Journal Online for further discussion. a Pool identifier number. b Fold stimulation elicited by the pool on the Igκ2-IFN-LUC reporter. c Fold stimulation elicited by the pool on the MUT-IFN-LUC reporter. d Fold stimulation elicited by the pool on the Igκ2-IFN-LUC reporter in the presence of IKKβ K44A. e Fold stimulation elicited by the pool on the Igκ2-IFN-LUC reporter in the presence of TBK1 K38A. f Fold stimulation elicited by the isolated cDNA on the Igκ2-IFN-LUC reporter. ???, pathway not known. Isolation of murine CARD11 from a mouse thymus expression library The results from the human placenta library screen indicated that this methodology yields molecules of different biochemical types, both catalytic and non-catalytic, which mediate signaling in different pathways leading to NF-κB. With the aim of identifying molecules involved in lymphocyte signaling, we applied this screen using a mouse thymus cDNA expression library. Known molecules isolated from this library included: the TNFR1, LTβR, CD40 and TRAMP/DR3 receptors; the TRAF2, MyD88, TAB2, RIP3 and NOD1 adapters; the NIK kinase and the RelA subunit of NF-κB. In addition, we independently isolated four clones of a membrane-associated guanylate kinase (MAGUK) family member, the murine homolog of a human protein described while this work was in progress as CARD11 (Bertin et al., 2001) or CARMA1 (Gaide et al., 2001). The murine cDNA encoded 1159 amino acids (Figure 2B) and contained predicted CARD, PDZ, SH3 and GUK domains, in addition to a region predicted to form coiled coils (Figure 2A). The murine and human proteins are 88% identical. Figure 2.Murine CARD11. (A) Schematic of murine CARD11 showing domains predicted by sequence homology. (B) Amino acid sequence of murine CARD11 and comparison to the human homolog. Domains in (A) and (B) are color matched. Download figure Download PowerPoint The presence of PDZ, SH3 and GUK domains is characteristic of the MAGUK family of proteins, which function to cluster and localize multiprotein signaling complexes in several systems (Dimitratos et al., 1999). The CARD is found in caspases, regulators of apoptosis and in proteins that signal to NF-κB (Reed, 2000). The isolation of murine CARD11 in our screen suggested that it might function as an adapter to coordinate signaling in an NF-κB-inducing pathway. In 293T cells, overexpression of the CARD11 cDNA activated the Igκ2-IFN-LUC reporter, but not the MUT-IFN-LUC reporter (Figure 3A). Deletion of the first 115 amino acids containing the CARD abrogated this activity (Figure 3B), confirming the importance of this domain for NF-κB induction. Activation of NF-κB by CARD11 overexpression was inhibited by IKKβ K44A co-expression, but not by TBK1 K38A co-expression (Figure 3C), suggesting that overexpressed CARD11 activates NF-κB in 293T cells via IKK activation. Previous reports (Bertin et al., 2001; Gaide et al., 2001) also showed that overexpressed CARD11 can activate NF-κB but they did not place CARD11 in a physiological pathway. Figure 3.CARD11 activates the Igκ2-IFN-LUC reporter when overexpressed in 293T cells, in a CARD-dependent manner, upstream of the IKK complex. (A) Titration of the murine CARD11 expression vector (ng) in the presence of 2 ng pCSK-LacZ and 20 ng Igκ2-IFN-LUC or MUT-IFN-LUC reporters. (B) Titration of CARD11 or ΔCARD11 expression vectors in the presence of 20 ng Igκ2-IFN-LUC and 2 ng pCSK-LacZ. Western blotting confirmed comparable expression of wild-type and ΔCARD proteins. (C) Two hundred nanograms of CARD11 expression vector were transfected with 20 ng Igκ2-IFN-LUC and 2 ng pCSK-LacZ in the absence or presence of 100 ng of constructs expressing IKKβ K44A or TBK1 K38A. Download figure Download PowerPoint Involvement of CARD11 in TCR signaling to NF-κB The presence of CARD11 in the mouse thymus cDNA library suggested that it might function to signal to NF-κB in T cells. Since the CARD-deleted mutant of CARD11 (ΔCARD) could not activate NF-κB in 293T cells, but retained other domains of the protein, we tested its potential inhibitory effect in Jurkat T cells on various stimuli. As shown in Figure 4, ΔCARD inhibited the activation of the Igκ2-IFN-LUC reporter by αCD3/αCD28 cross-linking, while the wild-type protein enhanced the stimulation achieved with no effect on basal reporter activity (Figure 4A and B). The inhibitory effect of ΔCARD was specific; it had no effect on NF-κB activation by TNFα (Figure 4C) or dsRNA (Figure 4D), and did not inhibit the activation of an NFAT-responsive reporter, NFAT-LUC, by αCD3/αCD28 cross-linking (Figure 4E). Finally, ΔCARD also inhibited the activation of the Igκ2-IFN-LUC reporter by PMA/ionomycin treatment but not that of the NFAT-LUC reporter (Figure 4F and G). These specific effects of the ΔCARD variant suggested that CARD11 mediates pathway-specific, factor-specific activation of NF-κB by TCR/CD28 cross-linking, and that the CARD was necessary for this signaling. Figure 4.ΔCARD specifically blocks NF-κB activation by αCD3/αCD28 cross-linking in Jurkat T cells. The indicated amounts (ng) of expression vectors for CARD11 or ΔCARD were co-transfected with 200 ng pCSK-LacZ and 1500 ng of either Igκ2-IFN-LUC or NFAT-LUC into Jurkat T cells. Cells were stimulated with αCD3/αCD28 cross-linking, TNFα, dsRNA or PMA/ionomycin co-treatment as indicated. Download figure Download PowerPoint CARD11 functions upstream of the IKK complex In order to investigate how ΔCARD inhibited NF-κB reporter activation by αCD3/αCD28 cross-linking, we generated pools of Jurkat cells stably expressing myc-tagged wild-type and mutant CARD11 proteins by infection with Moloney murine leukemia virus-based retroviruses expressing these cDNAs linked to puromycin resistance (Figure 5A). A control, puro-resistant pool of Jurkat cells (puroR pool) was generated by infection with a parental virus containing no cDNA insert. Treatment of the ΔCARD Jurkat pool with αCD3/αCD28 cross-linking activated the NFAT-responsive reporter, but failed to activate the Igκ2-IFN-LUC reporter, while the wild-type CARD11 pool and control puroR pool activated both reporters (Figure 5B and D). Similar results were obtained with PMA/ionomycin treatment (Figure 5C and E). Thus, the stable pools recapitulated the phenotypes observed in transient transfections. Importantly, the levels of stably expressed myc-tagged proteins in these pools did not affect basal NF-κB activity in the absence of signaling (Figures 5 and 6). Figure 5.Generation of puro-resistant Jurkat pools expressing wild-type murine CARD11, the ΔCARD mutant or no murine CARD11 variant. (A) Schematic of viral constructs. Following integration, the CMV enhancer/chicken β-actin promoter fusion drives expression of an mRNA containing the inserted cDNA, an internal ribosomal entry site (IRES) and the puromycin resistance gene (PURO). (B–E) Jurkat pools were transfected with 200 ng pCSK-LacZ and 2800 ng of either Igκ2-IFN-LUC or NFAT-LUC and stimulated with either αCD3/αCD28 cross-linking or PMA/ionomycin co-treatment as indicated. Download figure Download PowerPoint Figure 6.ΔCARD blocks αCD3/αCD28 cross-linking-induced IKK activation, IκBα degradation and nuclear translocation of NF-κB. Jurkat pools were treated with αCD3/αCD28 cross-linking or TNFα for the indicated times in minutes, and nuclear and cytoplasmic extracts were prepared. (A) EMSA assays were performed with nuclear extracts using probes for NF-κB, NFAT and AP-1. The arrows indicate the specific inducible complexes as verified by their lack of binding to control probes containing binding site mutations (data not shown). Unbound probes are not shown. (B) Western blot assays were performed on cytoplasmic extracts using antibodies for IκBα, IκBβ or myc-tagged proteins. The asterisk indicates an ∼77 kDa truncation of wild-type CARD11 that contains the myc-tagged N-terminus. No truncation was observed in the ΔCARD-expressing pool. (C) Jurkat pools were treated with αCD3/αCD28 cross-linking for the indicated times in minutes, and IKK IP-kinase assays were performed. The amount of radioactivity incorporated into substrate (top panel) was quantitated and fold activations were, from left to right, 1.0, 3.9, 4.8, 1.0, 2.9, 3.1, 1.0, 1.3 and 1.2. The lower panel shows a western blot with αIKKα to indicate the relative amount of IKKα in each sample. Download figure Download PowerPoint We treated these Jurkat pools with a time course of αCD3/αCD28 cross-linking, and made nuclear and cytoplasmic extracts to assess the induction of NF-κB, AP-1 and NFAT DNA-binding activity. As shown in Figure 6A, the puroR control and wild-type CARD11-expressing pools activated NF-κB DNA-binding activity in response to αCD3/αCD28 cross-linking, while the ΔCARD pool failed to induce NF-κB. In contrast, all three pools induced NF-κB in response to TNFα treatment. The failure of the ΔCARD pool to induce NF-κB in response to αCD3/αCD28 cross-linking was specific since AP-1 and NFAT activities were induced in a manner comparable with that observed with the control and wild-type CARD11 pools (Figure 6A, lower two panels). Western blot analysis of cytoplasmic extracts revealed degradation of IκBα in the puroR control and wild-type CARD11 pools in response to αCD3/αCD28 cross-linking and TNFα treatment, while degradation of IκBα was only observed in the ΔCARD pool in response to TNFα (Figure 6B). Degradation of IκBβ was not observed in response to these stimuli. Western blotting with anti-c-myc antibodies revealed slightly lower expression of the myc-tagged ΔCARD protein than the myc-tagged wild-type CARD11 protein in their respective pools (Figure 6B, lower panel). IKK IP-kinase assays confirmed a failure to activate the IKK complex in the ΔCARD pool in response to αCD3/αCD28 cross-linking, as compared with that observed in the control and wild-type CARD11 pools (Figure 6C). These results indicated that the ΔCARD mutant blocks NF-κB induction upstream of IKK complex activation, IκBα degradation and nuclear localization of NF-κB. Functional cooperation and association of CARD11 with Bcl10 Bcl10-deficient T cells display a factor-specific defect in NF-κB activation in response to TCR triggering or PMA/ionomycin treatment (Ruland et al., 2001). These cells fail to activate the IKK complex in response to TCR triggering, while other signaling events are unperturbed. To assess a functional relationship between Bcl10 and CARD11, we transfected a Bcl10 expression vector into the puroR control, CARD11- and ΔCARD11-expressing Jurkat pools and assayed Igκ2-IFN-LUC activity in the absence and presence of αCD3/αCD28 cross-linking. As shown in Figure 7A, transfection of Bcl10 into the control puroR Jurkat pool had no significant effect on reporter activity either in the absence or presence of stimulation by αCD3/αCD28 cross-linking. However, in the CARD11-expressing pool, transfection of Bcl10 resulted in a robust induction in the absence of TCR triggering, which was not significantly enhanced by αCD3/αCD28 cross-linking. In contrast, in the ΔCARD-expressing pool, transfection of Bcl10 resulted in no stimulation in the absence of αCD3/αCD28 treatment and no rescue of reporter induction by αCD3/αCD28 cross-linking. These results indicate that Bcl10 and CARD11 can cooperate in T cells to signal to NF-κB in a manner dependent on the CARD11 CARD. In addition, the block in TCR signaling to NF-κB that is imposed by the ΔCARD variant cannot be bypassed by Bcl10 overexpression. These data suggest that CARD11 and Bcl10 function at the same level of the signaling pathway between the TCR and IKK complexes. Figure 7.CARD11 and Bcl10 functionally cooperate and associate in T cells. (A) Igκ2-IFN-LUC (1800 ng) was transfected into Jurkat pools expressing wild-type CARD11, ΔCARD or no cDNA insert in the presence of the indicated amounts (nanograms) of a Bcl10 expression vector (pc-FL-CIPER) (Koseki et al., 1999). Cells were treated with αCD3/αCD28 cross-linking as indicated. (B) Jurkat pools were treated with αCD3/αCD28 cross-linking for the indicated times in minutes, and immunoprecipitations using αBcl10 antibodies were performed. The immunoprecipitates (top panel) and lysates (6.5% of the IP input; bottom panel) were developed with α-myc primary antibody. Download figure Download PowerPoint To examine whether CARD11 and Bcl10 associate during TCR signaling, we assayed for myc-tagged proteins precipitating with anti-Bcl10 antibodies in lysates from puroR control, myc-tagged CARD11- and ΔCARD-expressing Jurkat pools. As shown in Figure 7B, we detected wild-type CARD11 co-precipitating with endogenous Bcl10 to an extent that was not reproducibly enhanced by αCD3/αCD28 cross-linking. In contrast, we failed to detect co-precipitation of the ΔCARD variant with endogenous Bcl10. These data suggest that CARD11 and Bcl10 associate in a manner that is dependent on the CARD of CARD11 and independent of TCR signaling. RNA interference of CARD11 inhibits TCR signaling to NF-κB To independently assess a role for CARD11 in TCR signaling to NF-κB, we used RNA interference (RNAi). Short hairpin RNAs can be expressed from RNA pol III promoters in vivo and processed to small interfering RNAs that silence genes in a sequence-specific manner (Brummelkamp et al., 2002). We designed two short hairpin RNAs to silence the endogenous human CARD11 mRNA (Figure 8A) in Jurkat T cells and expressed them downstream of the human H1 RNA promoter. The sihCARD11-1 hairpin targets a region 260 nucleotides 5′ to the termination codon while sihCARD11-2 targets a region 1400 nucleotides 3′ to the initiation codon. As shown in Figure 8B, transient transfection of Jurkat T cells with either sihCARD11-1 or sihCARD11-2 inhibited induction of the Igκ2-IFN-LUC reporter by TCR cross-linking by 70–75%. A control hairpin targeted to the luciferase mRNA of Renilla reniformis (siRenLuc) did not significantly inhibit. Co-transfection of sihCARD11-1 and sihCARD11-2 did not inhibit more than either transfected alone. Figure 8.siRNAs targeted to CARD11 inhibit NF-κB activation by αCD3/αCD28 cross-linking. (A) The sihCARD11-1 and sihCARD11-2 shRNAs are depicted with the murine sequence corresponding to the human target sequence of sihCARD11-1 (mismatches indicated in green). (B) The indicated amounts (nanograms) of Pol III expression constructs for shRNAs were co-transfected with 200 ng pCSK-LacZ and 2700 ng Igκ2-IFN-LUC into Jurkat T cells. Cells were stimulated with αCD3/αCD28 as indicated. (C) Jurkat T cells were transfected with 200 ng pCSK-LacZ and 2500 ng Igκ2-IFN-LUC in the absence or presence of 100 ng of the sihCARD11-1 expression construct and 200 ng of the indicated CARD11 variant expression constructs. Cells were stimulated with αCD3/αCD28 as indicated. Western blot analysis indicated that expression constructs for CARD11 deletion variants express comparable protein levels within a ∼2-fold range. (D) Jurkat T cells were transfected with 200 ng pCSK-LacZ and 2700 ng NFAT-LUC in the absence or presence of 100 ng of the sihCARD11-1 expression construct and stimulated with αCD3/αCD28 as indicated. (E) Jurkat T cells were transfected with 200 ng pCSK-LacZ and 2700
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