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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 auto-proteolysis is required for IL-2 production by Jurkat T cells.A) Jurkat T cells were left untreated or stimulated with P/I for 30 min, with or without pre min, with or without pre-treatment with 50 µM z-VRPR-fmk for 30 min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p-ERK (activation control) and tubulin (loading control). B) IL-2 production (ELISA) of Jurkat T cells stably expressing MALT1, MALT1-R149A, MALT1-C464A or MALT1-RACA, either untreated (-) or stimulated for 18 hrs with PMA hrs with PMA/ionomycin (P/I). Data shown as mean +/- S.D. (n = 3). Inset: Immunoblot with a-MALT1-C and a-Flag showing expression of ectopic MALT1 and mutants relative to endogenous MALT1 (lane 1). Numbers indicate fold overexpression relative to endogenous MALT1. AS: a-specific band obtained with a-Flag that serves as loading control. C) Immunoblot of cell lysates (top) and bio-IPs (bottom) from Jurkat T cells and Jurkat T cells with stable expression of Avi-tagged MALT1 or MALT1 mutants R149A, C464A and RACA with indicated antibodies.
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pone-0103774-g006: MALT1 auto-proteolysis is required for IL-2 production by Jurkat T cells.A) Jurkat T cells were left untreated or stimulated with P/I for 30 min, with or without pre min, with or without pre-treatment with 50 µM z-VRPR-fmk for 30 min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p-ERK (activation control) and tubulin (loading control). B) IL-2 production (ELISA) of Jurkat T cells stably expressing MALT1, MALT1-R149A, MALT1-C464A or MALT1-RACA, either untreated (-) or stimulated for 18 hrs with PMA hrs with PMA/ionomycin (P/I). Data shown as mean +/- S.D. (n = 3). Inset: Immunoblot with a-MALT1-C and a-Flag showing expression of ectopic MALT1 and mutants relative to endogenous MALT1 (lane 1). Numbers indicate fold overexpression relative to endogenous MALT1. AS: a-specific band obtained with a-Flag that serves as loading control. C) Immunoblot of cell lysates (top) and bio-IPs (bottom) from Jurkat T cells and Jurkat T cells with stable expression of Avi-tagged MALT1 or MALT1 mutants R149A, C464A and RACA with indicated antibodies.

Mentions: Next, we investigated whether MALT1 auto-processing affects T-cell activation. Stimulation of Jurkat T cells induced MALT1 protease activity, as demonstrated by the appearance of cleaved BCL10 after 30 minutes of P/I stimulation (Figure 6A). Simultaneously, the MALT1 p19 fragment was detected in lysates of these cells, and both cleavage events could be prevented by treatment of the cells with the MALT1 protease inhibitor z-VRPR-fmk (Figure 6A). To assess the relevance of MALT1 cleavage in T-cell activation, we generated Jurkat T cells that overexpress wild-type MALT1, the cleavage insensitive R149A mutant, the catalytically inactive C464A mutant or the double R149A/C464A mutant (RACA). Stimulation of these cells with P/I showed that none of the mutants affected the levels of inducible phosphorylation of IκBα or JNK or the nuclear accumulation of NF-κB subunits (Figure S4), events that are known to depend on the scaffold function of MALT1. Moreover, and in contrast to the C464A and RACA mutants, the R149A mutant did not affect cleavage of the MALT1 substrates A20, CYLD or RELB (Figure S4). Similar observations were made in the C464A and R149A cells in which endogenous MALT1 was inactivated using TALENs that target exon 2 of MALT1 (Figure S5 and S6).


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 auto-proteolysis is required for IL-2 production by Jurkat T cells.A) Jurkat T cells were left untreated or stimulated with P/I for 30 min, with or without pre min, with or without pre-treatment with 50 µM z-VRPR-fmk for 30 min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p-ERK (activation control) and tubulin (loading control). B) IL-2 production (ELISA) of Jurkat T cells stably expressing MALT1, MALT1-R149A, MALT1-C464A or MALT1-RACA, either untreated (-) or stimulated for 18 hrs with PMA hrs with PMA/ionomycin (P/I). Data shown as mean +/- S.D. (n = 3). Inset: Immunoblot with a-MALT1-C and a-Flag showing expression of ectopic MALT1 and mutants relative to endogenous MALT1 (lane 1). Numbers indicate fold overexpression relative to endogenous MALT1. AS: a-specific band obtained with a-Flag that serves as loading control. C) Immunoblot of cell lysates (top) and bio-IPs (bottom) from Jurkat T cells and Jurkat T cells with stable expression of Avi-tagged MALT1 or MALT1 mutants R149A, C464A and RACA with indicated antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126661&req=5

pone-0103774-g006: MALT1 auto-proteolysis is required for IL-2 production by Jurkat T cells.A) Jurkat T cells were left untreated or stimulated with P/I for 30 min, with or without pre min, with or without pre-treatment with 50 µM z-VRPR-fmk for 30 min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p-ERK (activation control) and tubulin (loading control). B) IL-2 production (ELISA) of Jurkat T cells stably expressing MALT1, MALT1-R149A, MALT1-C464A or MALT1-RACA, either untreated (-) or stimulated for 18 hrs with PMA hrs with PMA/ionomycin (P/I). Data shown as mean +/- S.D. (n = 3). Inset: Immunoblot with a-MALT1-C and a-Flag showing expression of ectopic MALT1 and mutants relative to endogenous MALT1 (lane 1). Numbers indicate fold overexpression relative to endogenous MALT1. AS: a-specific band obtained with a-Flag that serves as loading control. C) Immunoblot of cell lysates (top) and bio-IPs (bottom) from Jurkat T cells and Jurkat T cells with stable expression of Avi-tagged MALT1 or MALT1 mutants R149A, C464A and RACA with indicated antibodies.
Mentions: Next, we investigated whether MALT1 auto-processing affects T-cell activation. Stimulation of Jurkat T cells induced MALT1 protease activity, as demonstrated by the appearance of cleaved BCL10 after 30 minutes of P/I stimulation (Figure 6A). Simultaneously, the MALT1 p19 fragment was detected in lysates of these cells, and both cleavage events could be prevented by treatment of the cells with the MALT1 protease inhibitor z-VRPR-fmk (Figure 6A). To assess the relevance of MALT1 cleavage in T-cell activation, we generated Jurkat T cells that overexpress wild-type MALT1, the cleavage insensitive R149A mutant, the catalytically inactive C464A mutant or the double R149A/C464A mutant (RACA). Stimulation of these cells with P/I showed that none of the mutants affected the levels of inducible phosphorylation of IκBα or JNK or the nuclear accumulation of NF-κB subunits (Figure S4), events that are known to depend on the scaffold function of MALT1. Moreover, and in contrast to the C464A and RACA mutants, the R149A mutant did not affect cleavage of the MALT1 substrates A20, CYLD or RELB (Figure S4). Similar observations were made in the C464A and R149A cells in which endogenous MALT1 was inactivated using TALENs that target exon 2 of MALT1 (Figure S5 and S6).

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