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PNAS 104 (3): 908-913

Copyright © 2007 by the National Academy of Sciences.

Negative feedback loop in T cell activation through I{kappa}B kinase-induced phosphorylation and degradation of Bcl10

Camille Lobry, Tatiana Lopez, Alain Israël, and Robert Weil*

Unité de Signalisation Moléculaire et Activation Cellulaire, Unité de Recherche Associée 2582, Centre National de la Recherche Scientifique, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France


Figure 1
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Fig. 1. NEMO-, CARMA1-, and proteasome-dependent degradation of Bcl10 in T cells in response to PMA and CD3/CD28 costimulation. (A) Kinetics of Bcl10 degradation in response to CD3/CD28 costimulation. Jurkat parental cells (lanes 1–6) as well as NEMO-deficient (lanes 7–12) or CARMA1-deficient (lanes 13–18) cells were treated with CD3/CD28 antibodies for the indicated period, and the amount of Bcl10 was measured by immunoblotting. The level of expression of IKKbeta is shown as a loading control. (B) PMA/ionomycin but not TNF-{alpha} induces Bcl10 degradation. Jurkat cells (Left, lanes 1–4, and Right) or NEMO-deficient cells (lanes 5–8) were stimulated for the indicated period (hours) with PMA or TNF-{alpha}. Levels of Bcl10 and beta-tubulin as a loading control were determined by Western blotting. (C) Bcl10 is degraded by the proteasome. Jurkat cells were pretreated with ALLN for 30 min and treated with ALLN and CD3/CD28 antibodies for the indicated period, and the levels of Bcl10, IKK{alpha}, and IKKbeta were determined by Western blotting.

 

Figure 2
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Fig. 2. IKKbeta-dependent Bcl10 phosphorylation is required for its degradation. (A) Sequence alignment of proteins that are polyubiquitinated by the beta-TrCP E3 ligase. The ubiquitination of these proteins is caused by phosphorylation by IKK, except for VPU and beta-catenin. The phosphoacceptor sites are the S and T indicated in bold. In the consensus sequence, {psi} represents an hydrophobic amino acid, and X represents any amino acid. (B) Pharmacological inhibition of IKK inhibits Bcl10 degradation. Jurkat cells were pretreated with the IKK inhibitor Bay 11-7085 for 30 min. Time course analysis of Bcl10 expression (bottom gel) and I{kappa}B{alpha} phosphorylation [using a phosphospecific anti-I{kappa}B{alpha} antibody (middle gel)] reveals that Bay 11-7085 abrogates PMA/ionomycin-induced I{kappa}B{alpha} phosphorylation and Bcl10 degradation. IKK2 was used as a loading control. (C) siRNA-mediated depletion of IKKbeta, and to a lesser extent IKK{alpha}, inhibits Bcl10 degradation. Jurkat cells were either left untransfected or transfected with IKKbeta siRNA, IKKbeta siRNA, or both, as mentioned in the figure. Cells were then treated with PMA/ionomycin for the indicated time. The level of expression of IKK{alpha}, IKKbeta, actin (loading control), and Bcl10 were determined by Western blotting. (D) (Left) Ex vivo phosphorylation of Bcl10 by IKKbeta. (Left) Flag-tagged Bcl10 was expressed in HEK-293T cells in the absence (–) or presence (+) of increasing amounts of VSV-tagged IKKbeta. Bcl10 was analyzed by Western blotting with anti-Flag antibody and reveals the IKK-dependent accumulation of higher-molecular-weight products (* and **). Expression of transfected IKKbeta was also monitored by immunoblotting with anti-VSV. (Right) Lysates of cells cotransfected with Flag-Bcl10 and VSV-IKKbeta (lane 2) were treated with {lambda}-phosphatase alone (lane 4) or with {lambda}-phosphatase and phosphatase inhibitors (lane 3), and analyzed by Western blotting with anti-Bcl10 anti-serum. (E) In vitro phosphorylation of Bcl10 by IKKbeta. (Left) VSV-Rip2 and VSV-IKKbeta were transiently transfected in HEK293T cells, and kinase activity was assayed (KA) by immune complex reaction in the presence of increasing amounts of GST-Bcl10 (1, 2.5, 5, and 10 µg). (Right) VSV-IKKbeta WT, Flag-IKKbeta DA (a constitutively active mutant), and VSV-IKKbeta DN (a kinase-dead mutant) were transiently transfected into HEK-293T cells and immunoprecipitated. In vitro kinase assays (KA) were performed by using 2.5 µg of GST-Bcl10, GST-I{kappa}B{alpha} N-ter (as a positive control), or GST-I{kappa}Bbeta C-ter (as a negative control).

 

Figure 3
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Fig. 3. Mapping of IKKbeta-induced Bcl10 phosphorylation sites. (A) In vitro phosphorylation of Bcl10 fragments (F1–F6) by IKKbeta. VSV-tagged IKKbeta, either WT or dominant negative (DN), were expressed in HEK-293T cells, and immunoprecipitates were used for in vitro kinase assays (KA) with fragments of Bcl10 fused to GST, as indicated above the lanes (the relevant bands are indicated by asterisks). (B–D) Mutation analysis of IKK-mediated Bcl10 phosphorylation. GST fused to fragment F1 [amino acids 11–38 (B)], F3 [74–112 (C)], or F5 [155–196 (D)] of Bcl10 and mutants of the indicated Ser (S) or Thr (T) residues were subjected to in vitro kinase assays as described in A.

 

Figure 4
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Fig. 4. The presence of active IKKbeta is necessary for the association between Bcl10 and beta-TrCP. HEK-293T cells were transfected with the indicated constructs. Cell extracts were immunoprecipitated with anti-Flag and immunoblotted with anti-VSV (Upper), and total lysates (TL) were blotted with the indicated antibodies (Lower).

 

Figure 5
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Fig. 5. The Bcl10 T81A/S85A mutation prevents degradation and increases IL-2 production. (A) Bcl10 degradation in response to TCR stimulation is prevented by mutation of the T81/S85 phosphorylation site. E29.1 murine T cells transduced with WT human Bcl10 (lanes 1–4) or the T81A/S85A mutant (lanes 5–8) were treated with anti-TCR mAb for the indicated period in the presence of cycloheximide, and the expression of Bcl10 was determined by Western blotting. (B) Increased IL-2 production in cells expressing Bcl10 T81A/S85A. (B Lower) Levels of Bcl10 were measured in E29.1 cells expressing the empty vector MSCV (lane 1), in three individual clones expressing WT Bcl10 (lanes 2–4) and in three individual clones expressing Bcl10 T81A/S85A (lanes 5–7). (B Upper) IL-2 production after 14 h of PMA/ionomycin treatment was assayed in the E29.1 clones.

 

Figure 6
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Fig. 6. Subcellular localization of WT and mutant (T81A/S85A and S7A/T81A/S85A/S167A/T168A) Bcl10 in untreated (A) or TCR-activated (B and C) E29.1 cells. Mutation of the T81/S85 phosphorylation site is sufficient to induce nuclear localization of Bcl10 upon TCR stimulation. A similar localization can be observed for the WT molecule in the presence of proteasome inhibitors [MG132 (C)]. In each panel, E29.1 cells were stained with Hoechst to reveal DNA (Left) and with anti-Bcl10 antiserum (Center).

 


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