Address correspondence to Josef M. Penninger, IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, c/o Dr. Bohr Gasse 3-5, A-1030 Vienna, Austria. Phone: 43-1-79730-454; Fax: 43-1-79730-459; email: Josef.penninger{at}imba.oeaw.ac.at
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Abstract |
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Key Words: Carma1/CARD11/Bimp3 MAGUK T cell IKK immune synapse
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Introduction |
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Membrane-associated guanylate kinase (MAGUK) family proteins control the polarity of membrane domains at epithelial cell junctions and play critical roles in synaptic development and plasticity (1114). Because immune and neuronal synapses share structural similarities (3, 4), it has been speculated that MAGUK family proteins have a function in the formation and/or organization of the immune synapse in lymphocytes. Carma1/caspase recruitment domain (CARD)11/Bimp3 is a CARD-containing MAGUK family protein that is abundantly expressed in lymphoid tissues. Using gene targeting, we and others have recently demonstrated that Carma1 is essential for TCR-induced activation of T cells (1518). However, it is not known whether Carma1 is essential to form the immune synapse.
The recognition of pathogens by innate or adaptive immune receptors leads to activation of the NF-B family of transcription factors (19). NF-
B proteins are present in the cytoplasm in association with inhibitory proteins known as inhibitor of NF-
Bs (I
Bs). The signals generated by activated receptors result in phosphorylation of I
B proteins. Phosphorylated I
B proteins become targets for ubiquitination and proteasome-mediated degradation, allowing NF-
B to translocate to the nucleus. Phosphorylation of I
B is mediated by the I
B kinase (IKK) complex, consisting of two catalytic subunits, IKK1/
and IKK2/ß, and the NEMO/IKK
regulatory subunit (20,21). Therefore, the understanding of NF-
B regulation and function is tightly linked to the understanding of IKK regulation and function (22).
Activation of NF-B mediated by TCR and CD28 costimulation is essential for activation, expansion, and effector functions of T cells (19, 23). PKC
integrates the TCR and CD28 signals that lead to NF-
B activation, and after T cell activation, PKC
translocates to lipid rafts at the immune synapse (2426). Importantly, among the several isoenzymes expressed in T cells, it appears that PKC
is the only PKC isoform that translocates to the immune synapse (1). Genetic approaches have confirmed that PKC
is a component in the TCR-induced NF-
B activation pathway because T cells from PKC
/ mice cannot be activated in response to antigen and display impaired NF-
B activation (27). Other essential components in this pathway are Bcl10, MALT1, and Carma1 (1518, 28, 29). These three molecules interact with each other and seem to form a signalosome to activate NF-
B (3032). Recent reports have shown that the IKK complex translocates to lipid rafts at the immune synapse after TCR/CD28 or PMA/ionomycin stimulation and this localization is sufficient to induce NF-
B activation (33, 34). These findings suggested that the recruitment of IKK into the lipid rafts is a key event to connect IKK to upstream components. However, the exact molecular mechanisms that control the recruitment of IKK into lipid rafts are not yet defined. Moreover, despite the similarity in function and phenotype of knockout mice, the mechanistic connections between PKC
, Carma1, Bcl10, and IKK remain unclear.
Because data on Carma1-regulated NF-B activation were based on antibody cross-linking studies, we analyzed the role of Carma1 in peptideAPC-induced T cell activation and assembly of the immune synapse. We show that Carma1/ T cells can still proliferate but display markedly abrogated cytokine production when activated by specific peptides. This is not due to defects in the immune synapse formation between T cells and APCs, nor due to defective recruitment of PKC
to the synapse. Moreover, peptide-induced cell adhesion and LFA-1 accumulation at the immune synapse appear normal. Intriguingly, Carma1 was found to be essential for recruitment of IKK into the central region of the immune synapse. Moreover, genetic inactivation of Carma1 completely abolishes PKC-dependent IKK recruitment to aggregated lipid rafts and IKK activation. We also show that Bcl10 and IKK recruitment to lipid rafts is independently regulated. Thus, the MAGUK family protein Carma1 is the first molecular adaptor identified in vivo that couples antigen receptor signaling to the assembly of supramolecular signaling complexes downstream of immune receptor and lipid raft clustering.
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Materials and Methods |
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T Cell Responses.
Lymph node CD4+ T cells were purified from DO11.10-Tg mice using magnetic beads (Dynal) to remove CD8+, B220+, Mac-1+, and Gr-1+ cells. 5 x 104 purified CD4+ T cells (>95% CD4+ by FACS) were stimulated for 48 h with 10 µg/ml mitomycin c (Sigma-Aldrich) treated A20 B lymphoma cells (3 x 104; derived from BALB/c mice) as APCs in the presence or absence of various doses of OVA323339 peptide (ISQAVHAAHAEINEAGR; reference 35) in 96-well plates in RPMI 1640 media supplemented with 10% FCS and 105 M ß-mercaptoethanol. Cells were stimulated in triplicate followed by an 8-h pulse with 1 µCi per well of [3H]thymidine (Amersham Biosciences) to determine proliferation. Culture supernatants were assayed in triplicate for the production of IL-2, IL-4, and IFN- by ELISA (R&D Systems).
Conjugate Formation and Cell Adhesion.
Unless otherwise indicated, A20 B lymphoma cells were loaded with 106 M of OVA323339 peptide for 4 h at 37°C. 106 lymph node CD4+ T cells and peptide-loaded or nonloaded APCs (A20 B cells) were then mixed at a 1:1 ratio, centrifuged briefly to promote conjugate formation, and incubated at 37°C for different times in serum-free medium. The reactions were stopped and cells were fixed by adding 4% paraformaldehyde. Cell conjugates were stained with anti-DO11.10 TCR PE (BD Biosciences) and anti-B220 APC (BD Biosciences) to detect T cells and APCs, respectively. The percentages of TAPC conjugates (DO11.10 TCR and B220 double positive cells) were analyzed by flow cytometry.
Immunofluorescence Confocal Microscopy.
Primary T lymphoblasts were made by culturing CD4+ T cells with 106 M peptide-pulsed A20 APCs for 2 d followed by an additional 4-d culture in the presence of 50 U/ml of rmIL-2 (R&D Systems) without antigen stimulation. For peptide-specific stimulation, CD4+ T cells or the primary T lymphoblasts from DO11.10-Tg/Carma1+/+, Carma1+/, and Carma1/ mice were stained with 5 µg/ml Alexa Fluor 488 cholera toxin (CTx; Molecular Probes), and then incubated with peptide-pulsed APCs (A20 B cells) as described above. CTx specifically binds to the lipid raft enriched in glycosphingolipid GM-1 (5, 36). For raft cross-linking experiments, Alexa Fluor 488 CTxstained T cells were incubated with 4 µg/ml goat anti-CTx (Calbiochem) and 50 ng/ml PMA plus 100 ng/ml calcium ionophore for 10 min at 37°C. Cells were fixed with 4% paraformaldehyde, transferred onto slides using cytospin, and permeabilized with 0.1% Triton X-100 in PBS. Cells were then stained with rabbit anti-PKC (C-18; Santa Cruz Biotechnology, Inc.), anti-Bcl10 mAb (331.3; Santa Cruz Biotechnology, Inc.), rabbit anti-IKK
/ß (H-470; Santa Cruz Biotechnology, Inc.), anti-IKK
(C73-764; BD Biosciences), antiLFA-1 (M17/4; BD Biosciences), biotinylated anti-DO11.10 TCR mAb (Caltag), and Texas redconjugated phalloidin (Molecular Probes). Goat antirabbit, antirat, or antimouse IgG secondary antibodies conjugated to Alexa Fluor 594 or Alexa Fluor 633 (Molecular Probes) were used to visualize the primary antibodies. For biotinylated antibodies, Alexa Fluor 594conjugated streptavidin (Molecular Probes) was used. Confocal images were obtained using an Olympus 1X-70 inverted microscope equipped with fluorescence optics and Deltavision Deconvolution Microscopy software (Applied Precision). For the detection of cSMACs and pSMACs, fixed cells were stained with rat antiLFA-1 (M17/4; BD Biosciences) and rabbit anti-PKC
or rabbit anti-IKK
/ß, followed by staining with Alexa Fluor 568conjugated goat antirabbit IgG and Alexa Fluor 633conjugated goat antirat IgG, respectively. Confocal images of TAPC interfaces were obtained and analyzed using confocal scanning microscopy.
Kinase Assays.
Purified lymph node CD4+ T cells were stimulated with 50 ng/ml PMA plus 100 ng/ml calcium ionophore as described above. After immunoprecipitation with anti-IKK (M-280; Santa Cruz Biotechnology, Inc.), IKK activity was assayed using 3 µg of recombinant GST-IB
(amino acids 154) as a substrate as described previously (37).
Flow Cytometry.
Cells were stained with FITC-, PE-, and APC-conjugated antibodies. For analysis of surface antigen expression, the following antibodies were used: anti-CD3 APC, anti-DO11.10 TCR PE, anti-CD4 FITC, anti-CD4 PE, anti-CD28 FITC, and antiLFA-1 PE. All antibodies were purchased from BD Biosciences. Samples were analyzed by flow cytometry using a FACScan (Becton Dickinson).
Cellular Fractionation.
Cellular fractionations were performed as described previously (33). In brief, 107 purified lymph node T cells were left untreated or stimulated with 50 ng/ml PMA plus 100 ng/ml calcium ionophore. Cells were lysed on ice for 30 min in 1 ml of NP-40 lysis buffer (1% NP-40, 10 mM Tris, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM Na3VO4, and protein inhibitors), and then homogenized with a Dounce homogenizer (20 strokes) and spun at 500 g for 7 min at 4°C. The postnuclear supernatant was centrifuged at 100,000 g for 1 h at 4°C and the supernatant, referred to as the detergent-soluble membrane and cytosolic fraction (S), was collected. The pellet was rinsed once in the NP-40 lysis buffer and solubilized in 100 µl of RIPA buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 10 mM DTT, 10 mM MgCl2, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and protein inhibitors), and the insoluble material was removed by centrifugation for 10 min at 10,000 g. This preparation was designated as detergent insoluble material (DIM). 25-µl aliquots of each fraction were resolved by SDS-PAGE and analyzed by Western blotting using antibodies specific for anti-IKK mAb (14A231; Upstate Biotechnology) and anti-PKC
(C-18; Santa Cruz Biotechnology, Inc.). For quantification of expression levels, Western blot images were scanned and the intensity of bands was quantified using IPLab Gel (Signal Analytics).
Online Supplemental Material.
Fig. S1 shows defective recruitment of IKK into lipid rafts at the immune synapse in naive Carma1/ CD4+ T cells. Fig. S2 shows defective subcellular translocation of IKK from detergent-soluble fractions to detergent-insoluble membrane fractions in Carma1/ T cells after PMA plus calcium ionophore stimulation. Fig. S3 shows a schematic model of the role for Carma1 in the TCR-mediated NF-B pathway. Figs. S1S3 are available at http://www.jem.org/cgi/content/full/jem.20032246/DC1.
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Results |
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Carma1 Is Not Involved in the Formation of the Immune Synapse.
It has been reported that Carma1 is recruited to the immune synapse and becomes associated with the antigen receptor complex after TCR stimulation (38). Antigen receptor stimulation in T cells results in TCR clustering at the immune synapse, reorganization of the actin cytoskeleton at the site of assembled TCRs, and clustering of integrins (39). In addition, lipid rafts are also assembled at the immune synapse and membrane receptors including TCR and many intracellular signaling molecules are recruited to lipid rafts during T cell activation (36, 40). Thus, similar to the function of other MAGUK family proteins in neuronal synapse, we speculated that lack of Carma1 could affect immune synapse formation in T cells. Therefore, we examined whether loss of Carma1 in T cells results in defective formation of antigen clustering, clustering of cell adhesion receptors, alterations in the cytoskeleton, and/or defective recruitment of downstream effector molecules using confocal imaging of peptide-specific immune synapses between DO11.10-Tg T cells and peptide-loaded APCs. Loss of Carma1 expression had no apparent effect on conjugation of DO11.10-Tg T cells with peptide-pulsed APCs (Fig. 2 A). Lipid raft clustering as well as TCR clustering and recruitment of TCRs to lipid rafts at the T cellAPC interfaces were also comparable between DO11.10-Tg/Carma1/ and control DO11.10-Tg/Carma1+/+ T cells (Fig. 2, A and D). Moreover, F-actin (Fig. 2, B and D) as well as LFA-1 clustering (Fig. 2, C and D) at the immune synapses were comparable between Carma1+/+ and Carma1/ T cells.
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Carma1 Is Required for Recruitment of IKK into the cSMAC.
In neurons, MAGUK family proteins are required for synapse formation and function as scaffolds for the assembly of signaling complexes and cytoskeletal components at cellcell contact sites (12). It has been shown previously that Carma1 is an essential component in the TCR-mediated NF-B activation pathway (15, 16, 38, 41). Because immune synapse formation appeared normal in DO11.10-Tg/Carma1/ T cells, we determined whether the loss of Carma1 expression affects the recruitment of defined downstream signaling molecules involved in the NF-
B activation pathway to the immune synapse. Moreover, we wanted to determine whether the segregation of the immune synapse into defined cSMAC and pSMAC subregions occurred in the absence of Carma1 expression (1, 2).
Because it has been suggested that NF-B activation by PKC
and IKK is regulated by their recruitment into lipid rafts at the immune synapse (25, 33, 34), we first examined the recruitment of these molecules after stimulation with peptide-pulsed APCs (Figs. 3 and 4). When T cells were incubated with control APCs, PKC
and IKK were localized in the cytoplasm and did not colocalize with GM-1containing lipid rafts in both Carma1+/ and Carma1/ T cells (Figs. 3 A and 4 A). After conjugation of DO11.10-Tg/Carma1+/ T cells with OVA peptidepulsed APCs, PKC
and IKK translocated to the T cellAPC contact sites (Figs. 3 A and 4 A). Localization experiments with GM-1 rafts to visualize cSMACs (5) and LFA-1 to mark pSMAC (1, 2) regions showed that PKC
was confined to the cSMAC area and colocalized with the clustered GM-1 lipid rafts. Although IKK can be detected in both cSMAC and pSMAC subregions, IKK preferentially segregated into the cSMAC area and colocalized with the clustered GM-1 lipid rafts (Figs. 3 B and 4, B and C). Similar to control T cells, peptide-mediated stimulation of DO11.10-Tg/Carma1/ T cells resulted in the recruitment of PKC
to the immune synapse (Fig. 3 A). Moreover, in DO11.10-Tg/Carma1/ T cells, PKC
colocalized with GM-1 lipid rafts at the cSMAC of the immune synapse and formed typical cSMAC and pSMAC structure characterized by central PKC
surrounded by a peripheral LFA-1 ring (Fig. 3 B and Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20032246/DC1). Intriguingly, although IKK translocated to the focal contact sites, colocalization of IKK with lipid rafts at the cSMAC of the immune synapse was markedly decreased in DO11.10-Tg/Carma1/ T cells (Fig. S1). Z axis analysis of the T cellAPC interface revealed that IKK and lipid rafts were indeed spatially segregated at the contact area of DO11.10-Tg/Carma1/ T cells (Fig. 4 B). Moreover, although PKC
and IKK colocalize at the cSMAC region in Carma1-expressing T cells, IKK was retained in the pSMAC area and did not colocalize with PKC
at the cSMAC in DO11.10-Tg/Carma1/ T cells (Fig. 4, BD). This segregation of lipid rafts and IKK at immune synapses between Carma1/ T cells and APCs followed a typical pattern: clustered lipid rafts and PKC
at the cSMAC are surrounded by LFA-1 and IKK in the pSMAC areas (Fig. 4, B and C). Together, these results indicate that Carma1 is dispensable for translocation of PKC
and IKK to the focal contact sites between DO11.10-Tg T cells and peptide-loaded APCs, but Carma1 is essential to recruit the IKK complex to the cSMAC of the immune synapse where PKC
localizes.
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Because it has been reported that Carma1/ T cells are defective in recruitment of Bcl10 to lipid rafts after TCR stimulation and CTx cross-linking (15), we tested whether Bcl10 recruitment is also impaired after PMA/ionophore stimulation. Bcl10 was recruited to the plasma membrane and colocalized with lipid rafts in Carma1+/+ T cells. In contrast, recruitment and redistribution of Bcl10 to clustered lipid rafts did not occur in Carma1/ T cells (Fig. 5 C). However, although PMA/ionophore stimulation of Carma1/ T cells results in the formation of IKK clusters, albeit clustered IKK molecules are excluded from the lipid rafts (Fig. 5 B), Bcl10 did not translocate to the plasma membrane and remained localized throughout the cytoplasm (Fig. 5 C). These results indicate that Carma1 controls recruitment of both IKK and Bcl10 into lipid rafts, but their recruitment is likely to be regulated differently.
Defective IKK Activation in Carma1/ T Cells.
To further confirm impaired raft recruitment of IKK in Carma1/ T cells, we isolated detergent-soluble membrane and cytosolic (S) and lipid raftcontaining DIM fractions (33). Consistent with our confocal microscopy studies, in both Carma1+/+ and Carma1/ T cells, PKC translocated from the S fraction into DIM fractions after PMA/ionophore stimulation, and the ratio of DIM/S fractions was increased by three to four times (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20032246/DC1). IKK was detected in S fractions as well as DIM fractions at similar levels in unstimulated Carma1+/+ and Carma1/ T cells. After stimulation of Carma1+/+ T cells, the amount of IKK in the S fractions was decreased and consequently the DIM/S ratio was markedly increased, indicating that IKK translocates from S into DIM fractions. In Carma1/ T cells, however, the amount of IKK in S fractions did not change after stimulation (Fig. S2). Because it has been shown that IKK recruitment to lipid rafts is important for activation (33, 34) and our results indicated that IKK lipid raft recruitment is defective in Carma1/ T cells, we evaluated IKK function by in vitro kinase activity. In parallel with defective recruitment of IKK to the lipid rafts, IKK activation after PMA/ionophore stimulation was significantly impaired in Carma1/ T cells (Fig. 5 D). Thus, genetic inactivation of Carma1 abolishes stimulation-dependent IKK recruitment to lipid rafts and IKK activation.
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Discussion |
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Our data using a peptideAPC system show that Carma1 does not simply determine an all or nothing response as previously reported, but under physiological stimulation conditions Carma1 sets the threshold for T cell proliferation. Importantly, on the other hand, signals required for cytokine production definitely depend on Carma1 expression. This differential requirement of Carma1 may explain the in vivo T cell phenotype in Carma1/ mice. Carma1-deficient mice show normal numbers and development of thymocytes and normal populations of peripheral T cells in lymph nodes and spleen (1517), indicating that loss of Carma1 does not affect thymocyte expansion during development, thymocyte selection, or homeostatic expansion of peripheral T cells in vivo. In contrast, Carma1/ mice show impaired T-dependent immunity in vivo, which is consistent with the abrogated cytokine production of Carma1/ T cells observed in our study. Thus, our findings provide a novel aspect of Carma1-mediated TCR signaling in T cell homeostasis and T cell activation. Whether compensatory cytokine production in the absence of Carma1 could drive proliferation of these cells needs to be determined. Moreover, it would be interesting to elucidate whether Carma1 might control localized cytokine secretion at the focal contact site.
The nervous system and immune system use specialized cell surface contacts to transduce signals between their constituent cell populations (4). These two synaptic junctions in neurons and lymphocytes share common structural features. In neurons and epithelial cells, MAGUK family proteins such as CASK, DLG, or PDS-95 have been shown to assemble receptors, cytoskeletal components, and signaling molecules at sites of cellcell contact, including synapses, cellular junctions, and polarized membrane domains (13, 46). Carma1 is a lymphocyte-specific member of the MAGUK family of proteins and is an essential component required for TCR- or B cell receptorinduced lymphocyte activation. Thus, Carma1 was predicted to be a candidate molecule for the assembly and/or organization of supramolecular signaling complexes in lymphocytes.
Using the DO11.10 immune synapse formation system, our data show that loss of Carma1 does not typically affect features of immune synapses at the T cellAPC interface. DO11.10-Tg/Carma1/ T cells display normal antigen receptor clustering, lipid raft clustering, integrin clustering, as well as reorganization of filamentous actin at the focal contact sites. Importantly, inactivation of Carma1 also did not affect the normal segregation of the immune synapse into cSMACs and pSMACs. Consistent with normal surface receptor clustering and cytoskeletal reorganization, conjugate formation and peptide-induced adhesion between T cells and APCs were also found to be comparable between DO11.10-Tg/Carma1/ and DO11.10-Tg/Carma1+/+ T cells. Our experiments refute the hypothesis that Carma1 functions as a scaffold that assembles multiple surface receptors and the actin cytoskeleton at the synapse. Another MAGUK family protein, the human lymphocyte homologue of the Drosophila discs large tumor suppressor protein, has also been implicated in T cell activation and is recruited to SMACs upon CD2 cross-linking (47). Whether MAGUK family proteins other than Carma1 are indeed essential for immune synapse formation needs to be determined. Importantly, our data provide the first genetic evidence on a new class of molecular scaffold essential for the selective recruitment of IKK into lipid rafts and spatial segregation of IKK into the cSMAC at immune synapses.
PKC, Carma1, Bcl10, and IKK are all essential components in the TCR-induced NF-
B activation pathway (1518, 2729). Although understanding of this pathway has progressed rapidly, the molecular and structural relationship between these molecules still remains unsolved. It has been suggested that PKC
, Bcl10, and IKK are located in the cytoplasm in resting cells and these molecules translocate to lipid rafts at the immune synapse after TCR activation (33, 34, 38). Carma1 is located in both the cytoplasm and lipid rafts in resting T cells and the amount of Carma1 in lipid rafts is increased after antigen receptor activation. Moreover, kinase activation of IKK is regulated by the recruitment into lipid rafts, but the molecule(s) that mediates the recruitment was not clear. Because previous reports showed that PKC
physically associates with the IKK complex in lipid rafts after TCR stimulation (33), the same molecular mechanism that regulates PKC
recruitment was a potential candidate pathway to also mediate the recruitment of IKK. However, our data clearly show that Carma1 acts downstream of PKCs in the recruitment of IKK into aggregated lipid rafts. In addition, Carma1 is essential for the recruitment of IKK, but not PKC
, into lipid rafts at the immune synapse. Consistent with this notion, a recent report has shown that Carma1 can physically interact with IKK
(48). Thus, Carma1 is an essential molecular adaptor that controls IKK recruitment into lipid rafts and cSMACs downstream of immune synapse formation and PKC activation. Our results also demonstrate that activation-induced recruitment of PKC
and IKK into lipid rafts are separately regulated.
It has been proposed that Bcl10 is the molecule that acts upstream of IKK and may recruit IKK to membranes (38). Our data here and a recent report by Egawa et al. (15) have shown that Carma1 is indeed essential for the recruitment of Bcl10 into lipid rafts. Importantly, although Bcl10 does not translocate to plasma membrane at all and remains distributed in the cytoplasm after TCR activation in Carma1/ T cells, IKKs translocate to the membrane region after stimulation and form aggregates at the immune synapse or PMA/ionophore-induced receptor caps, but does not enter into the lipid rafts. Thus, although the same molecule, Carma1, regulates the recruitment of both IKK and Bcl10 to lipid rafts, our data suggest that the underlying mechanisms of recruitment are different. Carma1 not only regulates translocation of Bcl10 to the membrane, but also lipid raft recruitment of Bcl10. In contrast, translocation of IKK from the cytoplasm to the focal contact sites at the immune synapse does occur in Carma1/ T cells, but Carma1 is essential for the recruitment of the IKK complex into aggregated GM-1 lipid rafts and the cSMAC. Based on our results, we propose that Bcl10 is required for IKK activation in the lipid rafts. Alternatively, Bcl10 recruitment into lipid rafts through Carma1 might be the prerequisite for the translocation of the IKK complex into GM-1containing rafts and cSMAC. For a model of Carma1-regulated IKK and Bcl10 recruitment and activation, see Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20032246/DC1.
In conclusion, we have found that Carma1 has no apparent role in the formation of the immune synapse, PKC recruitment, lipid raft aggregations, cSMAC and pSMAC segregation, or activation-dependent cell adhesion. Carma1 acts downstream of immune synapse formation and PKC activation and controls stimulation-dependent IKK recruitment into the cSMAC of the immune synapse, thereby regulating IKK activation. Moreover, Carma1 was found to be essential for cytokine production, but set the activation threshold for proliferation in peptide-activated T cells. Our data establish the molecular hierarchies by which different components of the NF-
B activation pathway are recruited into defined areas of the immune synapse. Moreover, these results elucidate the structural complexities by which the adaptor Carma1 couples antigen receptor signaling to IKK and NF-
B activation in T cells.
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Acknowledgments |
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J.M. Penninger is supported by grants from IMBA, The Austrian Academy of Sciences, The Austrian Ministry of Science and Education, and grants from the 6th EU Science framework program. This work is also supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
The authors have no conflicting financial interests.
Submitted: 29 December 2003
Accepted: 20 August 2004
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References |
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