Limits...
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.

Show MeSH

Related in: MedlinePlus

Figure 5. MALT1 auto-proteolysis in activated B cells.A) ABC-DLBCL cell lines HBL-1 and OCI-Ly3 were treated with 50 µM z-VRPR-fmk (36 hrs hrs) and lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments with a-MALT1, a-Cleaved BCL10 and a-Tubulin (loading control). B-C) The GCB-DLBCL cell lines BJAB and Raji were left untreated or stimulated with PMA/ionomycin (30 min min) with or without pre-treatment with 50 µM z-VRPR-fmk (30 min min). Lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments, for p-ERK (activation control) and tubulin (loading control). D) Immunoblot of lysates of SSK41 cells and SSK41 cells with ectopic expression of the API2-MALT1 fusion variants A7M3 and A7M8, or the L232LI mutant of Card11 (C11m) respectively, with antibodies against the MALT1 C-terminus, the p76 neo-epitope, the CYLD C-terminus, the NIK C-terminus and Flag (ectopic A7M3, A7M8 and C11m). Numbers below blots depict band intensities of MALT1, p76 and the CYLD p70 fragment relative to lane 1. *  =  non-specific band. LC: loading control, a non-specific band obtained with the p76 neo-epitope antibody was used.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126661&req=5

pone-0103774-g005: Figure 5. MALT1 auto-proteolysis in activated B cells.A) ABC-DLBCL cell lines HBL-1 and OCI-Ly3 were treated with 50 µM z-VRPR-fmk (36 hrs hrs) and lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments with a-MALT1, a-Cleaved BCL10 and a-Tubulin (loading control). B-C) The GCB-DLBCL cell lines BJAB and Raji were left untreated or stimulated with PMA/ionomycin (30 min min) with or without pre-treatment with 50 µM z-VRPR-fmk (30 min min). Lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments, for p-ERK (activation control) and tubulin (loading control). D) Immunoblot of lysates of SSK41 cells and SSK41 cells with ectopic expression of the API2-MALT1 fusion variants A7M3 and A7M8, or the L232LI mutant of Card11 (C11m) respectively, with antibodies against the MALT1 C-terminus, the p76 neo-epitope, the CYLD C-terminus, the NIK C-terminus and Flag (ectopic A7M3, A7M8 and C11m). Numbers below blots depict band intensities of MALT1, p76 and the CYLD p70 fragment relative to lane 1. *  =  non-specific band. LC: loading control, a non-specific band obtained with the p76 neo-epitope antibody was used.

Mentions: Next we investigated the occurrence of MALT1 proteolysis in cell lines derived from DLBCL. The activated B-cell (ABC)-subtype of DLBCL is addicted to NF-κB signalling [49] and has constant MALT1 protease activity [37], [38]. Consequently, cell lines derived from such lymphomas, such as HBL-1 and OCI-Ly3, show a constitutive presence of cleaved BCL10 that can be detected with an antibody specifically recognizing the processed form of this protein [38] (Figure 5A). Likewise, the MALT1 p19 fragment was detected in lysates of these cells, and both MALT1 and BCL10 cleavage could be prevented by treating the cells with the MALT1 protease inhibitor z-VRPR-fmk (Figure 5A). In contrast, no MALT1 processing was detectable in the B cell lymphoma cell line BJAB, which is derived from the germinal center B-cell (GCB) subtype of DLBCL and has no steady MALT1 protease activity (Figure 5B). MALT1 processing was also undetectable in the EBV-transformed B-cell line Raji (Figure 5C). However, stimulation of these cells with PMA and ionomycin (P/I) induced MALT1 protease activity and the appearance of cleaved BCL10 and the MALT1 p19 fragment, which again could be blocked by addition of z-VRPR-fmk (Figure 5, B and C).


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)

Figure 5. MALT1 auto-proteolysis in activated B cells.A) ABC-DLBCL cell lines HBL-1 and OCI-Ly3 were treated with 50 µM z-VRPR-fmk (36 hrs hrs) and lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments with a-MALT1, a-Cleaved BCL10 and a-Tubulin (loading control). B-C) The GCB-DLBCL cell lines BJAB and Raji were left untreated or stimulated with PMA/ionomycin (30 min min) with or without pre-treatment with 50 µM z-VRPR-fmk (30 min min). Lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments, for p-ERK (activation control) and tubulin (loading control). D) Immunoblot of lysates of SSK41 cells and SSK41 cells with ectopic expression of the API2-MALT1 fusion variants A7M3 and A7M8, or the L232LI mutant of Card11 (C11m) respectively, with antibodies against the MALT1 C-terminus, the p76 neo-epitope, the CYLD C-terminus, the NIK C-terminus and Flag (ectopic A7M3, A7M8 and C11m). Numbers below blots depict band intensities of MALT1, p76 and the CYLD p70 fragment relative to lane 1. *  =  non-specific band. LC: loading control, a non-specific band obtained with the p76 neo-epitope antibody was used.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103774-g005: Figure 5. MALT1 auto-proteolysis in activated B cells.A) ABC-DLBCL cell lines HBL-1 and OCI-Ly3 were treated with 50 µM z-VRPR-fmk (36 hrs hrs) and lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments with a-MALT1, a-Cleaved BCL10 and a-Tubulin (loading control). B-C) The GCB-DLBCL cell lines BJAB and Raji were left untreated or stimulated with PMA/ionomycin (30 min min) with or without pre-treatment with 50 µM z-VRPR-fmk (30 min min). Lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments, for p-ERK (activation control) and tubulin (loading control). D) Immunoblot of lysates of SSK41 cells and SSK41 cells with ectopic expression of the API2-MALT1 fusion variants A7M3 and A7M8, or the L232LI mutant of Card11 (C11m) respectively, with antibodies against the MALT1 C-terminus, the p76 neo-epitope, the CYLD C-terminus, the NIK C-terminus and Flag (ectopic A7M3, A7M8 and C11m). Numbers below blots depict band intensities of MALT1, p76 and the CYLD p70 fragment relative to lane 1. *  =  non-specific band. LC: loading control, a non-specific band obtained with the p76 neo-epitope antibody was used.
Mentions: Next we investigated the occurrence of MALT1 proteolysis in cell lines derived from DLBCL. The activated B-cell (ABC)-subtype of DLBCL is addicted to NF-κB signalling [49] and has constant MALT1 protease activity [37], [38]. Consequently, cell lines derived from such lymphomas, such as HBL-1 and OCI-Ly3, show a constitutive presence of cleaved BCL10 that can be detected with an antibody specifically recognizing the processed form of this protein [38] (Figure 5A). Likewise, the MALT1 p19 fragment was detected in lysates of these cells, and both MALT1 and BCL10 cleavage could be prevented by treating the cells with the MALT1 protease inhibitor z-VRPR-fmk (Figure 5A). In contrast, no MALT1 processing was detectable in the B cell lymphoma cell line BJAB, which is derived from the germinal center B-cell (GCB) subtype of DLBCL and has no steady MALT1 protease activity (Figure 5B). MALT1 processing was also undetectable in the EBV-transformed B-cell line Raji (Figure 5C). However, stimulation of these cells with PMA and ionomycin (P/I) induced MALT1 protease activity and the appearance of cleaved BCL10 and the MALT1 p19 fragment, which again could be blocked by addition of z-VRPR-fmk (Figure 5, B and C).

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