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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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BCL10 mediates cleavage of MALT1 at R149 in 293T cells.A) Immunoblot of lysates of 293T cells transiently expressing MALT1 alone or in combination with BCL10 with antibodies against MALT1 [31], BCL10 and tubulin. B) Immunoblot of streptavidin pull-downs (bio-IPs) of Avi-tagged MALT1 and its mutants co-expressed with BCL10 in 293T cells as specified. AS: a-specific band. Arrows indicate the N-terminal p19 cleavage fragment. All molecular mass standards are in kDa.
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pone-0103774-g002: BCL10 mediates cleavage of MALT1 at R149 in 293T cells.A) Immunoblot of lysates of 293T cells transiently expressing MALT1 alone or in combination with BCL10 with antibodies against MALT1 [31], BCL10 and tubulin. B) Immunoblot of streptavidin pull-downs (bio-IPs) of Avi-tagged MALT1 and its mutants co-expressed with BCL10 in 293T cells as specified. AS: a-specific band. Arrows indicate the N-terminal p19 cleavage fragment. All molecular mass standards are in kDa.

Mentions: Lucas et al.[2] reported that co-expression of BCL10 resulted in strong MALT1 oligomerization and activation of an NF-κB reporter in 293T cells [2]. We and others had demonstrated that co-expression of BCL10 with MALT1 in 293T cells triggers MALT1 protease activity and cleavage of its substrates A20 and BCL10 [30], [31]. When co-expressing BCL10 and MALT1 in 293T cells, we again observed the appearance of the p19 N-terminal cleavage fragment of MALT1 (Figure 2A, lane 3), in addition to a previously described hyper-phosphorylation of BCL10 [30], [31]. Processing did not occur when BCL10 was co-expressed with the C464A or the R149A mutant of MALT1 separately (Figure 2B). However, formation of the p19 fragment was restored when the R149A and C464A mutants were co-expressed together with BCL10, indicating that in this experimental setting, BCL10 triggers the protease activity of the MALT1-R149A mutant that induces – directly or indirectly - the cleavage of the MALT1-C464A mutant at R149 (Figure 2B).


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)

BCL10 mediates cleavage of MALT1 at R149 in 293T cells.A) Immunoblot of lysates of 293T cells transiently expressing MALT1 alone or in combination with BCL10 with antibodies against MALT1 [31], BCL10 and tubulin. B) Immunoblot of streptavidin pull-downs (bio-IPs) of Avi-tagged MALT1 and its mutants co-expressed with BCL10 in 293T cells as specified. AS: a-specific band. Arrows indicate the N-terminal p19 cleavage fragment. All molecular mass standards are in kDa.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126661&req=5

pone-0103774-g002: BCL10 mediates cleavage of MALT1 at R149 in 293T cells.A) Immunoblot of lysates of 293T cells transiently expressing MALT1 alone or in combination with BCL10 with antibodies against MALT1 [31], BCL10 and tubulin. B) Immunoblot of streptavidin pull-downs (bio-IPs) of Avi-tagged MALT1 and its mutants co-expressed with BCL10 in 293T cells as specified. AS: a-specific band. Arrows indicate the N-terminal p19 cleavage fragment. All molecular mass standards are in kDa.
Mentions: Lucas et al.[2] reported that co-expression of BCL10 resulted in strong MALT1 oligomerization and activation of an NF-κB reporter in 293T cells [2]. We and others had demonstrated that co-expression of BCL10 with MALT1 in 293T cells triggers MALT1 protease activity and cleavage of its substrates A20 and BCL10 [30], [31]. When co-expressing BCL10 and MALT1 in 293T cells, we again observed the appearance of the p19 N-terminal cleavage fragment of MALT1 (Figure 2A, lane 3), in addition to a previously described hyper-phosphorylation of BCL10 [30], [31]. Processing did not occur when BCL10 was co-expressed with the C464A or the R149A mutant of MALT1 separately (Figure 2B). However, formation of the p19 fragment was restored when the R149A and C464A mutants were co-expressed together with BCL10, indicating that in this experimental setting, BCL10 triggers the protease activity of the MALT1-R149A mutant that induces – directly or indirectly - the cleavage of the MALT1-C464A mutant at R149 (Figure 2B).

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