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MALT1 auto-proteolysis is essential for NF-κB-dependent gene transcription in activated lymphocytes.

Baens M, Bonsignore L, Somers R, Vanderheydt C, Weeks SD, Gunnarsson J, Nilsson E, Roth RG, Thome M, Marynen P - PLoS ONE (2014)

Bottom Line: MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity.Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits.Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells.

View Article: PubMed Central - PubMed

Affiliation: Human Genome Laboratory, VIB Center for the Biology of Disease, Leuven, Belgium; Human Genome Laboratory, Center for Human Genetics, KU Leuven, Leuven, Belgium.

ABSTRACT
Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.

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MALT1 p76 activates NF-κB signalling in 293T cells.A) Features of Flag-tagged MALT1, p76, MALT1-C and MALT1-p19. Numbers indicate the start and stop AA position for p76, MALT1-C and MALT1-p19 relative to the MALT1 protein sequence (Refseq NP_006776.1, 824 AA). B-C) NF-κB-reporter assays of 293T cells transiently expressing MALT1, mp-MALT1, MALT1-p19, p76 and mutants or MALT1-C. NF-κB-dependent luciferase activity is shown as fold induction of vector-transfected cells and represents the mean +/- S.D. (n = 3). Immunoblot of cell lysates with a-Flag – a-β-actin shows equal expression/loading of the different MALT1 constructs (B) and a-MALT1-C (C) shows equal expression of the different MALT1 constructs. Bottom (C): streptavidin pull-down (bio-IP) of MALT1, p76 or MALT1-C, transiently expressed in 293T cells, and immunoblotted with a-MALT1C, a-TRAF6 and a-BCL10 antibodies. e-MALT1, e-TRAF6, e-BCL10: endogenous MALT1, TRAF6 and BCL10. D) immunoblot of bio-IPs of Avi-tagged Ub-p76 expressed together with Flag-p76 or Flag MALT1 with a-MALT1-C, a-Flag and a-TRAF6. * indicate non-specific bands.
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pone-0103774-g004: MALT1 p76 activates NF-κB signalling in 293T cells.A) Features of Flag-tagged MALT1, p76, MALT1-C and MALT1-p19. Numbers indicate the start and stop AA position for p76, MALT1-C and MALT1-p19 relative to the MALT1 protein sequence (Refseq NP_006776.1, 824 AA). B-C) NF-κB-reporter assays of 293T cells transiently expressing MALT1, mp-MALT1, MALT1-p19, p76 and mutants or MALT1-C. NF-κB-dependent luciferase activity is shown as fold induction of vector-transfected cells and represents the mean +/- S.D. (n = 3). Immunoblot of cell lysates with a-Flag – a-β-actin shows equal expression/loading of the different MALT1 constructs (B) and a-MALT1-C (C) shows equal expression of the different MALT1 constructs. Bottom (C): streptavidin pull-down (bio-IP) of MALT1, p76 or MALT1-C, transiently expressed in 293T cells, and immunoblotted with a-MALT1C, a-TRAF6 and a-BCL10 antibodies. e-MALT1, e-TRAF6, e-BCL10: endogenous MALT1, TRAF6 and BCL10. D) immunoblot of bio-IPs of Avi-tagged Ub-p76 expressed together with Flag-p76 or Flag MALT1 with a-MALT1-C, a-Flag and a-TRAF6. * indicate non-specific bands.

Mentions: To investigate whether auto-processing of MALT1 has a role in NF-κB signalling, we first tested the capacity of the p19 and p76 fragments of MALT1 to promote NF-κB activation. Like full length MALT1 alone, expression of the p19 fragment (MALT1-p19, Figure 4A) did not activate a NF-κB reporter in 293T cells (Figure 4B). In contrast, the p76 fragment potently activated the NF-κB reporter despite the fact that it has lost the ability to bind BCL10 (Figure 4C). This suggests that removal of the N-terminal part of MALT1 might promote its capacity to activate NF-κB in a BCL10-independent manner. NF-κB activation by MALT1 involves TRAF6 binding via two distinct binding sites [27], [45] located within the Ig2 domain (T6-Ig) and at the MALT1 C-terminus (T6-C), respectively, and both are present in the p76 cleavage fragment (Figure 4A). Mutation of either one of the two TRAF6 binding sites, E313A/E316A (T6Ig-m) or E806A (T6C-m), strongly impaired the potential of p76 to activate NF-κB signalling in 293T cells (Figure 4C). Steptavidin pull-down experiments with Avi-tagged p76 constructs (bio-IP) confirmed that each of these individual mutations severely weakened the p76/TRAF6 interaction, while a complete inhibition of TRAF6 binding required mutation of both sites (T6Ig/C-m) (Figure 4C, bottom). Thus, p76-mediated NF-κB activation was clearly TRAF6-dependent. A shorter MALT1-C construct comprising only AA 334 to 824, which retained efficient TRAF6 binding via the T6C binding site, was unable to activate NF-κB signalling, suggesting an additional requirement for the intact Ig1 and Ig2 domains (Figure 4, A and C). Collectively, these data suggest that the MALT1 p76 fragment promotes NF-κB activation in a TRAF6-dependent but BCL10-independent manner.


MALT1 auto-proteolysis is essential for NF-κB-dependent gene transcription in activated lymphocytes.

Baens M, Bonsignore L, Somers R, Vanderheydt C, Weeks SD, Gunnarsson J, Nilsson E, Roth RG, Thome M, Marynen P - PLoS ONE (2014)

MALT1 p76 activates NF-κB signalling in 293T cells.A) Features of Flag-tagged MALT1, p76, MALT1-C and MALT1-p19. Numbers indicate the start and stop AA position for p76, MALT1-C and MALT1-p19 relative to the MALT1 protein sequence (Refseq NP_006776.1, 824 AA). B-C) NF-κB-reporter assays of 293T cells transiently expressing MALT1, mp-MALT1, MALT1-p19, p76 and mutants or MALT1-C. NF-κB-dependent luciferase activity is shown as fold induction of vector-transfected cells and represents the mean +/- S.D. (n = 3). Immunoblot of cell lysates with a-Flag – a-β-actin shows equal expression/loading of the different MALT1 constructs (B) and a-MALT1-C (C) shows equal expression of the different MALT1 constructs. Bottom (C): streptavidin pull-down (bio-IP) of MALT1, p76 or MALT1-C, transiently expressed in 293T cells, and immunoblotted with a-MALT1C, a-TRAF6 and a-BCL10 antibodies. e-MALT1, e-TRAF6, e-BCL10: endogenous MALT1, TRAF6 and BCL10. D) immunoblot of bio-IPs of Avi-tagged Ub-p76 expressed together with Flag-p76 or Flag MALT1 with a-MALT1-C, a-Flag and a-TRAF6. * indicate non-specific bands.
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Related In: Results  -  Collection

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pone-0103774-g004: MALT1 p76 activates NF-κB signalling in 293T cells.A) Features of Flag-tagged MALT1, p76, MALT1-C and MALT1-p19. Numbers indicate the start and stop AA position for p76, MALT1-C and MALT1-p19 relative to the MALT1 protein sequence (Refseq NP_006776.1, 824 AA). B-C) NF-κB-reporter assays of 293T cells transiently expressing MALT1, mp-MALT1, MALT1-p19, p76 and mutants or MALT1-C. NF-κB-dependent luciferase activity is shown as fold induction of vector-transfected cells and represents the mean +/- S.D. (n = 3). Immunoblot of cell lysates with a-Flag – a-β-actin shows equal expression/loading of the different MALT1 constructs (B) and a-MALT1-C (C) shows equal expression of the different MALT1 constructs. Bottom (C): streptavidin pull-down (bio-IP) of MALT1, p76 or MALT1-C, transiently expressed in 293T cells, and immunoblotted with a-MALT1C, a-TRAF6 and a-BCL10 antibodies. e-MALT1, e-TRAF6, e-BCL10: endogenous MALT1, TRAF6 and BCL10. D) immunoblot of bio-IPs of Avi-tagged Ub-p76 expressed together with Flag-p76 or Flag MALT1 with a-MALT1-C, a-Flag and a-TRAF6. * indicate non-specific bands.
Mentions: To investigate whether auto-processing of MALT1 has a role in NF-κB signalling, we first tested the capacity of the p19 and p76 fragments of MALT1 to promote NF-κB activation. Like full length MALT1 alone, expression of the p19 fragment (MALT1-p19, Figure 4A) did not activate a NF-κB reporter in 293T cells (Figure 4B). In contrast, the p76 fragment potently activated the NF-κB reporter despite the fact that it has lost the ability to bind BCL10 (Figure 4C). This suggests that removal of the N-terminal part of MALT1 might promote its capacity to activate NF-κB in a BCL10-independent manner. NF-κB activation by MALT1 involves TRAF6 binding via two distinct binding sites [27], [45] located within the Ig2 domain (T6-Ig) and at the MALT1 C-terminus (T6-C), respectively, and both are present in the p76 cleavage fragment (Figure 4A). Mutation of either one of the two TRAF6 binding sites, E313A/E316A (T6Ig-m) or E806A (T6C-m), strongly impaired the potential of p76 to activate NF-κB signalling in 293T cells (Figure 4C). Steptavidin pull-down experiments with Avi-tagged p76 constructs (bio-IP) confirmed that each of these individual mutations severely weakened the p76/TRAF6 interaction, while a complete inhibition of TRAF6 binding required mutation of both sites (T6Ig/C-m) (Figure 4C, bottom). Thus, p76-mediated NF-κB activation was clearly TRAF6-dependent. A shorter MALT1-C construct comprising only AA 334 to 824, which retained efficient TRAF6 binding via the T6C binding site, was unable to activate NF-κB signalling, suggesting an additional requirement for the intact Ig1 and Ig2 domains (Figure 4, A and C). Collectively, these data suggest that the MALT1 p76 fragment promotes NF-κB activation in a TRAF6-dependent but BCL10-independent manner.

Bottom Line: MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity.Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits.Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells.

View Article: PubMed Central - PubMed

Affiliation: Human Genome Laboratory, VIB Center for the Biology of Disease, Leuven, Belgium; Human Genome Laboratory, Center for Human Genetics, KU Leuven, Leuven, Belgium.

ABSTRACT
Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.

Show MeSH
Related in: MedlinePlus