The recurrent translocation t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue (MALT) lymphoma results in the expression of an API2⅐MALT1 fusion protein that constitutively activates NF-B. The first baculovirus IAP repeat (BIR) domain of API2 and the C terminus of MALT1, which contains its caspase-like domain, are present in all reported fusion variants and interact with TRAF2 and TRAF6, respectively, suggesting their contribution to NF-B signaling by API2⅐MALT1. Also, the involvement of BCL10 has been suggested via binding to BIR1 of API2 and via its interaction with the immunoglobulin domains of MALT1, present in half of the fusion variants. However, conflicting reports exist concerning their roles in API2⅐MALT1-induced NF-B signaling. In this report, streptavidin pulldowns of biotinylated API2⅐MALT1 fusion variants showed that none of the fusion variants interacted with endogenous BCL10; its role in NF-B signaling by API2⅐MALT1 was further questioned by RNA interference experiments. In contrast, TRAF6 was essential for NF-B activation by all fusion variants, and we identified a novel TRAF6 binding site in the second immunoglobulin domain of MALT1, which enhanced NF-B activation when present in the fusion protein. Furthermore, inclusion of both immunoglobulin domains in API2⅐MALT1 further enhanced NF-B signaling via intramolecular TRAF6 activation. Finally, binding of TRAF2 to BIR1 contributed to NF-B activation by API2⅐MALT1, although additional mechanisms involving BIR1-mediated raft association are also important. Taken together, these data reveal distinct mechanisms of NF-B activation by the different API2⅐MALT1 fusion variants with an essential role for TRAF6.Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) 4 represents with 8% one of the most common types of non-Hodgkin B-cell lymphoma. Two translocations specific for MALT lymphoma are t(1,14)(p22; q32) and t(14,18)(q32;q21), which up-regulate the expression of the BCL10 and MALT1 gene, respectively, via their fusion to the enhancer of IgH (1-4). The most common genetic aberration is the translocation t(11;18)(q21;q21), where fusion of API2 to MALT1 results in the expression of an API2⅐MALT1 fusion protein (5).BCL10 (6) and MALT1 (7, 8) play a crucial role in the signaling cascade from the antigen receptors to the transcription factor NF-B. Upon T-cell receptor stimulation, CARMA1 (CARD-containing MAGUK protein-1) recruits the BCL10⅐MALT1⅐TRAF6 (9, 10) and the IKK (11) (IKK␣⅐IKK⅐IKK␥/NEMO) complexes to the lipid rafts (12-14) surrounding the receptor. Here it is thought that oligomerization of TRAF6 elicits its ubiquitin ligase activity, resulting in polyubiquitination of IKK␥, the regulatory subunit of the IKK complex (15,16). This together with phosphorylation of IKK by the TAK1⅐TAB complex (16) fully activates the IKK complex, which then triggers phosphorylation and proteasome-mediated degradation of IB-␣, the inhibitor of NF-B. In this way NF-B is released as a free complex allowing it to translo...
Enhanced Green Fluorescent Protein (EGFP) is the most commonly used live cell reporter despite a number of conflicting reports that it can affect cell physiology. Thus far, the precise mechanism of GFP-associated defects remained unclear. Here we demonstrate that EGFP and EGFP fusion proteins inhibit polyubiquitination, a posttranslational modification that controls a wide variety of cellular processes, like activation of kinase signalling or protein degradation by the proteasome. As a consequence, the NF-κB and JNK signalling pathways are less responsive to activation, and the stability of the p53 tumour suppressor is enhanced in cell lines and in vivo. In view of the emerging role of polyubiquitination in the regulation of numerous cellular processes, the use of EGFP as a live cell reporter should be carefully considered.
The translocation t(11;18)(q21;q21) that generates an API2-MALT1 fusion protein is the most common structural abnormality among the genetic defects reported in mucosaassociated lymphoid tissue (MALT)-type lymphomas, and its presence correlates with the apparent lack of further genetic instability or chromosomal imbalances. Hence, constitutive nuclear factor-KB (NF-KB) activation induced by the API2-MALT1 fusion protein is considered essential for B-cell transformation. To examine its role in B-cell development and lymphomagenesis, EM-API2-MALT1 transgenic mice were produced. Our data show that expression of the API2-MALT1 fusion protein alone is not sufficient for the development of lymphoma masses within 50 weeks. Nevertheless, API2-MALT1 expression affected B-cell maturation in the bone marrow and triggered the specific expansion of splenic marginal zone B cells. Polyubiquitination of IKB kinase ; (IKK;), indicative for enhanced NF-KB activation, was increased in splenic lymphocytes and promoted the survival of B cells ex vivo. In addition, we show that the API2-MALT1 fusion resided in the cholesterol-and sphingolipid-enriched membrane microdomains, termed lipid rafts. We provide evidence that association of the MALT1 COOH terminal with the lipid rafts, which is mediated by the API2 portion, is sufficient to trigger NF-KB activation via enhanced polyubiquitination of IKK;. Taken together, these data support the hypothesis that the API2-MALT1 fusion protein can contribute to MALT lymphoma formation via increased NF-KB activation. (Cancer Res 2006; 66(10): 5270-7)
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