Negative feedback loop in T cell activation through IkappaB kinase-induced phosphorylation and degradation of Bcl10

Abstract

Activation of the transcription factor NF-kappaB after stimulation through antigen receptors is important for lymphocyte differentiation, activation, proliferation, and protection against apoptosis. Much progress has been made in understanding the molecular events leading to NF-kappaB activation, but how this activation is eventually down-regulated is less well understood. Recent studies have indicated that Bcl10 functions downstream of lymphocyte antigen receptors to promote the activation of the IkappaB kinase complex leading to the phosphorylation and degradation of the IkappaB inhibitors of NF-kappaB. Bcl10 has also been implicated in the pathogenesis of mucosa-associated lymphoid tissue lymphoma, possibly in association with its nuclear localization. Here, we provide evidence that the IkappaB kinase complex phosphorylates Bcl10 after T cell antigen receptor stimulation and causes its proteolysis via the beta-TrCP ubiquitin ligase/proteasome pathway. These findings document a negative regulatory activity of the IKK complex and suggest that Bcl10 degradation is part of the regulatory mechanisms that precisely control the response to antigens. Mutants of Bcl10 in the IKK phosphorylation site are resistant to degradation, accumulate in the nucleus, and lead to an increase in IL-2 production after T cell antigen receptor stimulation.

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 IKKβ is shown as a loading control. (B) PMA/ionomycin but not TNF-α 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-α. Levels of Bcl10 and β-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, IKKα, and IKKβ were determined by Western blotting.

Fig. 2.

IKKβ-dependent Bcl10 phosphorylation is required for its degradation. (A) Sequence alignment of proteins that are polyubiquitinated by the β-TrCP E3 ligase. The ubiquitination of these proteins is caused by phosphorylation by IKK, except for VPU and β-catenin. The phosphoacceptor sites are the S and T indicated in bold. In the consensus sequence, ψ 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 IκBα phosphorylation [using a phosphospecific anti-IκBα antibody (middle gel)] reveals that Bay 11-7085 abrogates PMA/ionomycin-induced IκBα phosphorylation and Bcl10 degradation. IKK2 was used as a loading control. (C) siRNA-mediated depletion of IKKβ, and to a lesser extent IKKα, inhibits Bcl10 degradation. Jurkat cells were either left untransfected or transfected with IKKβ siRNA, IKKβ siRNA, or both, as mentioned in the figure. Cells were then treated with PMA/ionomycin for the indicated time. The level of expression of IKKα, IKKβ, actin (loading control), and Bcl10 were determined by Western blotting. (D) (Left) Ex vivo phosphorylation of Bcl10 by IKKβ. (Left) Flag-tagged Bcl10 was expressed in HEK-293T cells in the absence (−) or presence (+) of increasing amounts of VSV-tagged IKKβ. 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 IKKβ was also monitored by immunoblotting with anti-VSV. (Right) Lysates of cells cotransfected with Flag-Bcl10 and VSV-IKKβ (lane 2) were treated with λ-phosphatase alone (lane 4) or with λ-phosphatase and phosphatase inhibitors (lane 3), and analyzed by Western blotting with anti-Bcl10 anti-serum. (E) In vitro phosphorylation of Bcl10 by IKKβ. (Left) VSV-Rip2 and VSV-IKKβ 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-IKKβ WT, Flag-IKKβ DA (a constitutively active mutant), and VSV-IKKβ 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-IκBα N-ter (as a positive control), or GST-IκBβ C-ter (as a negative control).

Fig. 3.

Mapping of IKKβ-induced Bcl10 phosphorylation sites. (A) In vitro phosphorylation of Bcl10 fragments (F1–F6) by IKKβ. VSV-tagged IKKβ, 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.

Fig. 4.

The presence of active IKKβ is necessary for the association between Bcl10 and β-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).

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.

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).