A catalytic-independent role for the LUBAC in NF-κB activation upon antigen receptor engagement and in lymphoma cells

Abstract

Key Points• LUBAC elements HOIP and SHARPIN participate in T-cell receptor-mediated NF-kB activation independently of HOIP catalytic activity.• LUBAC silencing compromises constitutive NF-kB activation and cell survival in ABC DLBCL lines.Antigen receptor-mediated nuclear factor kB (NF-kB) activation relies on the formation of a large multi-protein complex that contains CARMA1, BCL10, and MALT1 (CBM complex). This signalosome is pirated in the activated B-cell-like subgroup of diffuse large B-cell lymphoma (ABC DLBCL) to drive aberrant NF-kB activation, thereby promoting cell survival and propagation. Using an unbiased proteomic approach, we screened for additional components of the CBM in lymphocytes. We found that the linear ubiquitin chain assembly complex (LUBAC), which was previously linked to cytokine-mediated NF-kB activation, dynamically integrates the CBM and marshals NF-kB optimal activation following antigen receptor ligation independently of its catalytic activity. The LUBAC also participates in preassembled CBM complex in cells derived from ABC DLBCL. Silencing the LUBAC reduced NF-kB activation and was toxic in ABC DLBCL cell lines. Thus, our findings reveal a role for the LUBAC during lymphocyte activation and in B-cell malignancy. (Blood. 2014;123(14):2199-2203) IntroductionThe activated B-cell like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) constitutes the most aggressive DLBCL entity. 1 In contrast to germinal center B-cell-like (GCB) subtype of DLBCL, ABC DLBCL survival and proliferation require the constitutive activation of nuclear factor kB (NF-kB) transcription factors, which often results from somatic mutations in CD79B, CARD11 (also called CARMA1), MYD88, and TNFAIP3 genes.2 New perspectives for treatments restricted to the lymphoid compartment came from genomic-scale RNA interference screens, which unveiled that ABC DLBCL exploited a multi-protein complex that contains CARMA1, BCL10, and MALT1 (CBM complex) normally engaged in conveying NF-kB following antigen receptor engagement. [3][4][5][6] Within the CBM, Lys-63 (K63)-linked ubiquitylation of BCL10 and MALT1 ensures the recruitment and activation of the inhibitor of NF-kB kinase (IKK, composed of IKKa, IKKb, and NEMO) through IKKb phosphorylation and NEMO poly-ubiquitylation.7-9 IKK subsequently authorizes NF-kB to shuttle in the nucleus and exert its transcriptional activity by phosphorylating its cognate inhibitors (IkBs), which further undergo proteasomal degradation. Here, we screened for additional CBM partners and identified the linear ubiquitin chain assembly complex (LUBAC), which comprises 2 E3 ligases, HOIL-1 and HOIP, and SHARPIN. 11Although this tripartite complex was previously linked to cytokine-, bacteria-, and genotoxic stress-mediated NF-kB signaling, 12-17 its involvement in adaptive immunity remains unknown. We now show that the LUBAC binds to the CBM and governs NF-kB activation upon antigen receptor engagement independently of HOIP catalytic activity. In ABC DLBCL cells, the LUBAC is integral to pre...

The paracaspase MALT1 cleaves the LUBAC subunit HOIL1 during antigen receptor signaling

“…Hence, MALT1 drives HOIL1 processing at R 165 when overexpressed. Because MALT1 catalytic activity is unleashed in the vicinity of the CBM complex (Pelzer et al, 2013;Rebeaud et al, 2008), where the LUBAC is dynamically recruited (Dubois et al, 2014), we next investigated the status of HOIL1 in Jurkat T lymphocytes. Stimulation with either antibodies to CD3 and CD28 or with phorbol 12-myristate 13-acetate (PMA) plus ionomycin, which both mimic TCR ligation, led to a robust cleavage of HOIL1 ( Fig.…”

“…This complex catalyzes linear ubiquitylation and participates in multiple signaling pathways converging on NF-κB (Iwai et al, 2014). Although the LUBAC is a central part of the CBM needed for IKK activation in lymphocytes and in ABC DLBCL cells, the exact role of HOIL1 remains elusive (Dubois et al, 2014;Yang et al, 2014). We now report that HOIL1 is a substrate of MALT1 that is cleaved in TCR-stimulated cells and in ABC DLBCL cells, and that this processing contributes to the optimal activation of NF-κB in lymphocytes.…”

“…S2). In stimulated Jurkat, HOIL1, but not HOIL1 Cter , bound to CK1α (also known as CSNK1A1), a kinase that dynamically interacts with the CBM and the LUBAC (Dubois et al, 2014), reinforcing the idea that HOIL1…”

“…This N-terminal fragment likely maintains the LUBAC activity and mediates aberrant NF-κB in ABC DLBCL (Dubois et al, 2014;Yang et al, 2014). HOIL1…”

“…HEK293T cells were transfected according to standard calcium phosphate protocol, and Jurkat cells were transfected by electroporation (BTX ECM 830, Harvard Apparatus) as previously described (Bidere et al, 2009). Luciferase gene reporter assays (Promega), ELISA for IL-2 secretion (R&D Systems), and cell transduction were performed as previously described (Bidere et al, 2009;Dubois et al, 2014). Antibodies against A20 (59A426, 1:1000), BCL10 (A-6, 1:1000), CK1α (C-19, 1:1000), CYLD (H-6, 1:1000), GAPDH (6C5, 1:20,000), HOIL1 (H-1, 1:1000), MALT1 (B-12, 1:1000), and to tubulin (TU-02, 1:1000) were from Santa Cruz Biotechnology.…”