Triggering of antigen receptors on lymphocytes is critical for initiating adaptive immune response against pathogens. T‐cell receptor (TCR) engagement induces the formation of the Carma1–Bcl10–Malt1 (CBM) complex that is essential for activation of the IκB kinase (IKK)/NF‐κB pathway. However, the molecular mechanisms that link CBM complex formation to IKK activation remain unclear. Here we report that Malt1 is polyubiquitinated upon T‐cell activation. Ubiquitin chains on Malt1 provide a docking surface for the recruitment of the IKK regulatory subunit NEMO/IKKγ. TRAF6 associates with Malt1 in response to T‐cell activation and can function as an E3 ligase for Malt1 in vitro and in vivo, mediating lysine 63‐linked ubiquitination of Malt1. Multiple lysine residues in the C‐terminus of Malt1 serve as acceptor sites for the assembly of polyubiquitin chains. Malt1 mutants that lack C‐terminal ubiquitin acceptor lysines are impaired in rescuing NF‐κB signaling and IL‐2 production in Malt1−/− T cells. Thus, our data demonstrate that induced Malt1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing TCR signals to the canonical NF‐κB pathway.

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Bcl10 is a critical regulator of NF-B activity in T and

T cell receptor (TCR) signaling to IkappaB kinase (IKK)/NF-kappaB is controlled by PKCtheta-dependent activation of the Carma1, Bcl10, and Malt1 (CBM) complex. Antigen-induced phosphorylation of Bcl10 has been reported, but its physiological function is unknown. Here we show that the putative downstream kinase IKKbeta is required for initial CBM complex formation. Further, upon engagement of IKKbeta/Malt1/Bcl10 with Carma1, IKKbeta phosphorylates Bcl10 in the C terminus and thereby interferes with Bcl10/Malt1 association and Bcl10-mediated IKKgamma ubiquitination. Mutation of the IKKbeta phosphorylation sites on Bcl10 enhances expression of NF-kappaB target genes IL-2 and TNFalpha after activation of primary T cells. Thus, our data provide evidence that IKKbeta serves a dual role upstream of its classical substrates, the IkappaB proteins. While being essential for triggering initial CBM complex formation, IKKbeta-dependent phosphorylation of Bcl10 exhibits a negative regulatory role in T cell activation.

The 'classical' NF-κB activation pathway proceeds via IκB kinase (IKK)-β/γ-mediated phosphorylation, induced ubiquitination and the degradation of small IκBs. An alternative, NF-κB-inducing kinase and IKK-α-dependent pathway, which stimulates the processing of NF-κB2/p100, has recently been suggested. However, no physiological stimulus has been shown to trigger the activation of this pathway. Here we demonstrate that persistent stimulation with lymphotoxin β (LT-β) receptor agonists or lipopolysaccharide (LPS), but not with interleukin-1β, tumour necrosis factor-α or 12-Otetradecanoylphorbol-13-acetate, induces the generation of p52 DNA-binding complexes by activating the processing of the p100 precursor. Induction of p52 DNA-binding activity is delayed in comparison with p50/p65 complexes and depends on de novo protein synthesis. p100 is constitutively and inducibly polyubiquitinated, and both ubiquitination and p52 generation are coupled to continuing p100 translation. Thus, both LT-β receptor agonists and LPS induce NF-κB/p100 processing to p52 at the level of the ribosome.