CD3/CD28 costimulation-induced NF-kappaB activation is mediated by recruitment of protein kinase C-theta, Bcl10, and IkappaB kinase beta to the immunological synapse through CARMA1

Abstract

CARMA1 (also known as CARD11) is a scaffold molecule and contains a caspase-recruitment domain (CARD) and a membrane-associated guanylate kinase-like (MAGUK) domain. It plays an essential role in mediating CD3/CD28 costimulation-induced NF-kappaB activation. However, the molecular mechanism by which CARMA1 mediates costimulatory signals remains to be determined. Here, we show that CARMA1 is constitutively associated with the cytoplasmic membrane. This membrane association is essential for the function of CARMA1, since a mutant of CARMA1, CARMA1(L808P), that is defective in the membrane association cannot rescue CD3/CD28 costimulation-induced NF-kappaB activation in JPM50.6 CARMA1-deficient T cells. Although CD3/CD28 costimulation effectively induces the formation of the immunological synapse in CARMA1-deficient T cells, the recruitment of protein kinase C-theta (PKC-theta), Bcl10, and IkappaB kinase beta (IKKbeta) into lipid rafts of the immunological synapse is defective. Moreover, expression of wild-type CARMA1, but not CARMA1(L808P), restores the recruitment of PKC-theta, Bcl10, and IKKbeta into lipid rafts in CARMA1-deficient T cells. Consistently, expression of a mutant CARMA1, CARMA1(DeltaCD), that cannot associate with Bcl10 failed to restore CD3/CD28 costimulation-induced NF-kappaB activation in JPM50.6 cells, whereas expression of Bcl10-CARMA(DeltaCD) fusion protein effectively restored this NF-kappaB activation. Together, these results indicate that CARMA1 mediates CD3/CD28 costimulation-induced NF-kappaB activation by recruiting downstream signaling components into the immunological synapse.

FIG. 1.

CARMA1(L808P) is functionally defective. (A) JPM50.6 cells were transfected with plasmids encoding an NF-κB-dependent luciferase gene in the presence of expression plasmids encoding the wild-type CARMA1 [CARMA1(WT)] or L808P mutant [CARMA1(L808P)]. Twenty-four hours after transfection, the transfected cells were stimulated with (i) media, (ii) 1 μg of anti-CD3 and 1 μg of anti-CD28 per ml, or (iii) 10 ng of PMA and 1 μg of anti-CD28 antibodies per ml for 6 h. The cells were lysed, and luciferase activity was determined. All of the samples were also transfected with an EF1α promoter-dependent Renilla luciferase. The constitutively expressed Renilla luciferase activity was used to normalize the transfection efficiency. All data are presented as fold induction. The expression levels of the transfected CARMA1 were detected by Western blotting with anti-Myc antibodies (inset). Unstim, unstimulated. (B) JPM50.6 cells stably expressing Myc-tagged wild-type CARMA1 or CARMA1(L808P) were unstimulated (a and c) or stimulated with PMA (10 ng/ml) plus anti-CD28 antibodies (1 μg/ml) (b and d) for 6 h. The cells were collected and examined for the expression of NF-κB-dependent GFP in these cells by fluorescence-activated cell sorting (FACS). The y axis indicates the cell numbers (10⁴), and the x axis indicates the fluorescence intensity.

FIG. 2.

CARMA1(L808P) is defective in its subcellular localization. JPM50.6 cells stably expressing Myc-tagged wild-type CARMA1 (A) or CARMA1(L808P) (B) were labeled with anti-Myc antibodies, followed by staining with secondary Alexa-labeled antibodies. Subcellular localization of wild-type CARMA1 or CARMA1(L808P) was visualized by immunofluorescence with a confocal fluorescent microscope.

FIG. 3.

CARMA(L808P) is defective in its recruitment to the immunological synapse. (A) JPM50.6 cells expressing Myc-tagged wild-type CARMA1 (JPM50.6/WT) or CARMA1(L808P) (JPM50.6/L808P) were stimulated with SEE-primed or nonprimed Raji cells (blue) at 37°C for 15 min. The cells were labeled with anti-Myc monoclonal antibodies and Alexa-labeled rabbit anti-mouse antibodies (red). The subcellular localization of CARMA1 (red) was visualized by a confocal fluorescent microscope. (B) The percentage of CARMA1 recruitment to the immunological synapse was calculated by counting the number of the recruitment-like cells among 25 to 30 JMP50.6-Raji conjugates (blue-plus-red cells) in random fields.

FIG. 4.

Formation of the immunological synapse is independent of CARMA1. Jurkat or JPM50.6 cells were stimulated with or without anti-CD3 and anti-CD28 antibody-coated polystyrene beads for 45 min. The mixtures of cells and beads were labeled with CTxB and visualized by confocal fluorescent microscopy.

FIG. 5.

Recruitment of PKC-θ to the immunological synapse is defective in JPM50.6 cells. (A) JPM50.6 cells or JPM50.6 cells expressing wild-type CARMA1 (JPM50.6/WT) were stimulated with SEE-primed or nonprimed Raji cells (blue) at 37°C for 15 min. The cells were labeled with mouse anti-PKC-θ monoclonal antibodies and Alexa-labeled rabbit anti-mouse antibodies (red). The subcellular localization of PKC-θ (red) was visualized by confocal fluorescent microscopy. (B) The percentage of PKC-θ recruitment to the immunological synapse was calculated by counting the number of the synapse-associated cells among 25 to 30 JPM50.6-Raji conjugates (blue-plus-red cells) in random fields.

FIG. 6.

PKC-θ forms a complex with CARMA1. (A) HEK293 cells were transfected with plasmids encoding Myc-tagged PKC-θ together with Flag-tagged CARMA1, Flag-tagged RIP, Flag-tagged Vav, and the vector control (A) or Flag-tagged CARMA1 and its deletion mutants (B). The transfected cells were lysed and immunoprecipitated (IP) with anti-Flag antibody-conjugated agarose. The resulting immunoprecipitates were subjected to SDS-PAGE and analyzed by Western blotting (immunoblotting [IB]) with antibodies specific for Myc tag (top panel) or Flag tag (bottom panel).

FIG. 7.

The recruitment of PKC-θ, Bcl10, and IKKβ to lipid rafts is defective in JPM50.6 cells. Jurkat (A) or JPM50.6 (B) cells (∼3 × 10⁶ to 5 × 10⁶) were stimulated with or without anti-CD3 antibodies (1 μg/ml) and anti-CD28 antibodies (1 μg/ml). The cells were then lysed with 1% Triton X-100 and subjected to sucrose density gradient centrifugation to isolate lipid rafts. Proteins from equal volumes of representative collected fractions were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for PKC-θ, LAT, Bcl10, or IKKβ.

FIG. 8.

Expression of CARMA1 rescued the defect of the localization of signaling components to lipid rafts in JPM50.6 cells. CARMA1-reconstituted JPM50.6 cells (3 × 10⁶ to ∼5 × 10⁶) were stimulated with or without anti-CD3 antibodies (1 μg/ml) and anti-CD28 antibodies (1 μg/ml). The lipid rafts were prepared as described in the legend to Fig. 7. Proteins from equal volumes of representative collected fractions were separated by SDS-PAGE and analyzed by Western blotting with antibodies specific for PKC-θ, LAT, Bcl10, or IKKβ.

FIG. 9.

Expression of CARMA1(L808P) failed to rescue the defect of the localization of signaling components to lipid rafts in JPM50.6 cells. CARMA1(L808P)-reconstituted JPM50.6 cells (JPM50.6/L808P [3 × 10⁶ to ∼5 × 10⁶]) were stimulated with or without anti-CD3 antibodies (1 μg/ml) and anti-CD28 antibodies (1 μg/ml). The lipid rafts were prepared as described in the legend to Fig. 7. Proteins from equal volumes of representative collected fractions were separated by SDS-PAGE and analyzed by Western blotting with antibodies specific for PKC-θ, Lck, Bcl10, or IKKβ.

FIG. 10.

Bcl10-CARMA1(ΔCD) fusion protein rescues the functional defect in JPM50.6 cells. (A) JPM50.6 cells were transfected with plasmids encoding a NF-κB-dependent luciferase gene in the presence of expression constructs encoding Bcl10, CARMA1(ΔCD), Bcl10-CARMA1(ΔCD), CARMA1, or the vector control. The transfected cells were stimulated with (i) medium, (ii) 1 μg (each) of anti-CD3 and anti-CD28 antibodies per ml, or (iii) 10 ng of PMA per ml and 1 μg of anti-CD28 antibodies per ml for 6 h. The cells were lysed, and luciferase activity was determined as described in the legend to Fig. 1. Unstim, unstimulated. (B) The expression of transfected constructs was examined by Western blotting (WB) with anti-Myc or anti-Bcl10 antibodies.