CARD11 mediates factor-specific activation of NF-kappaB by the T cell receptor complex - PubMed (original) (raw)
CARD11 mediates factor-specific activation of NF-kappaB by the T cell receptor complex
Joel L Pomerantz et al. EMBO J. 2002.
Abstract
NF-kappaB is a critical target of signaling downstream of the T cell receptor (TCR) complex, but how TCR signaling activates NF-kappaB is poorly understood. We have developed an expression cloning strategy that can identify catalytic and noncatalytic molecules that participate in different pathways of NF-kappaB 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-kappaB activation by alphaCD3/alphaCD28 cross-linking and PMA/ionomycin treatment, but not by TNFalpha or dsRNA. CARD11 is not required for TCR-mediated induction of NFAT or AP-1. CARD11 functions upstream of the IkappaB-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-kappaB by the TCR complex and establish a role for a MAGUK family member in antigen receptor signaling.
Figures
Fig. 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.
Fig. 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.
Fig. 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.
Fig. 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.
Fig. 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.
Fig. 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.
Fig. 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.
Fig. 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.
Fig. 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 ng Igκ2-IFN-LUC in the absence or presence of 100 ng of the sihCARD11-1 expression construct and stimulated with TNFα as indicated.
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