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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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Targeting mp-MALT1 to DRMs induces its proteolysis at R149 in 293T cells.A) Features of mp-MALT1 (mp: myristoylation-palmitoylation sequence) and Ub-p76 (Ubiquitin-p76 fusion protein). R149: MALT1 cleavage site. Flag: Flag epitope, DD: Death Domain, Ig: immunoglobulin-like domain, p20: caspase p20-like domain, C464: MALT1 catalytic cysteine, T6-Ig and T6-C: TRAF6 binding site in second Ig domain and C-terminus, respectively. Ub: Ubiquitin. B) NF-κB-reporter assays of 293T cells transiently expressing wild-type MALT1, mp-MALT1 or empty vector (mock). NF-κB-dependent luciferase activity is shown as fold induction of vector-transfected cells and represents the mean +/- S.D. of at least three independent experiments (n = 3). Cell lysates were immunoblotted with a-Flag, a non-specific band was used as loading control (LC). C) Lysates of 293T cells transiently transfected with mp-MALT1 were subjected to sucrose density gradient centrifugation and aliquots of the serial fractions (1-12 from top to bottom) were immunoblotted with a-MALT1-N, a-Lck, a kinase residing in the Detergent Resistant Membrane (DRM) fractions, and a-GAPDH, a cytosolic marker. D-E) Immunoblot of lysates of 293T cells transiently expressing wild-type MALT1, mp-MALT1 and its mutants or Ubiquitin-p76 as specified with indicated antibodies. eMALT1: endogenous MALT1. β-actin (D) and LC: non-specific band (E) are loading controls. Arrows (panel C, D, E)) indicate the N-terminal p19 or the C-terminal p76 cleavage fragment respectively. All molecular mass standards are in kDa.
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pone-0103774-g001: Targeting mp-MALT1 to DRMs induces its proteolysis at R149 in 293T cells.A) Features of mp-MALT1 (mp: myristoylation-palmitoylation sequence) and Ub-p76 (Ubiquitin-p76 fusion protein). R149: MALT1 cleavage site. Flag: Flag epitope, DD: Death Domain, Ig: immunoglobulin-like domain, p20: caspase p20-like domain, C464: MALT1 catalytic cysteine, T6-Ig and T6-C: TRAF6 binding site in second Ig domain and C-terminus, respectively. Ub: Ubiquitin. B) NF-κB-reporter assays of 293T cells transiently expressing wild-type MALT1, mp-MALT1 or empty vector (mock). NF-κB-dependent luciferase activity is shown as fold induction of vector-transfected cells and represents the mean +/- S.D. of at least three independent experiments (n = 3). Cell lysates were immunoblotted with a-Flag, a non-specific band was used as loading control (LC). C) Lysates of 293T cells transiently transfected with mp-MALT1 were subjected to sucrose density gradient centrifugation and aliquots of the serial fractions (1-12 from top to bottom) were immunoblotted with a-MALT1-N, a-Lck, a kinase residing in the Detergent Resistant Membrane (DRM) fractions, and a-GAPDH, a cytosolic marker. D-E) Immunoblot of lysates of 293T cells transiently expressing wild-type MALT1, mp-MALT1 and its mutants or Ubiquitin-p76 as specified with indicated antibodies. eMALT1: endogenous MALT1. β-actin (D) and LC: non-specific band (E) are loading controls. Arrows (panel C, D, E)) indicate the N-terminal p19 or the C-terminal p76 cleavage fragment respectively. All molecular mass standards are in kDa.

Mentions: T or B cell receptor stimulation induces CARMA1-mediated recruitment of BCL10 and MALT1 to the lipid raft membrane fractions, which is essential for NF-κB activation [14], [22], [43]. To test whether lipid raft targeting promotes MALT1 activation, we generated a fusion of MALT1 to the N-terminal myristoylation-palmitoylation signal sequence of Lck (mp-MALT1, Figure 1A), which can target proteins into glycosphingolipid-enriched membranes [44]. When ectopically expressed in 293T cells, this construct was highly active, while MALT1 alone was unable to activate an NF-κB reporter ([45] and Figure 1B). We demonstrated previously through subcellular fractionation via sucrose density gradient centrifugation that ectopic MALT1 in 293T cells is merely cytosolic [5]. Subcellular fractionation of mp-MALT1 showed that it, as expected, also resides in the detergent resistant membrane (DRM) fractions, the latter marked by the presence of the kinase Lck (Figure 1C, lane 4 and 5). With the MALT1 antibody used (MALT1-N), which recognizes the N-terminus of MALT1, we further detected a 19 kDa fragment (p19) in the DRM fractions, (Figure 1C, lane 4–5). This N-terminal p19 fragment was also detectable in lysates of 293T cells expressing mp-MALT1, though not in lysates of cells expressing wild-type MALT1 (Figure 1D, lane 1–2). Expression of a catalytically inactive form of MALT1 (mp-MALT1-C464A) failed to generate the p19 fragment (Figure 1D, lane 3), suggesting that MALT1 protease activity is required for the generation of this N-terminal fragment.


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)

Targeting mp-MALT1 to DRMs induces its proteolysis at R149 in 293T cells.A) Features of mp-MALT1 (mp: myristoylation-palmitoylation sequence) and Ub-p76 (Ubiquitin-p76 fusion protein). R149: MALT1 cleavage site. Flag: Flag epitope, DD: Death Domain, Ig: immunoglobulin-like domain, p20: caspase p20-like domain, C464: MALT1 catalytic cysteine, T6-Ig and T6-C: TRAF6 binding site in second Ig domain and C-terminus, respectively. Ub: Ubiquitin. B) NF-κB-reporter assays of 293T cells transiently expressing wild-type MALT1, mp-MALT1 or empty vector (mock). NF-κB-dependent luciferase activity is shown as fold induction of vector-transfected cells and represents the mean +/- S.D. of at least three independent experiments (n = 3). Cell lysates were immunoblotted with a-Flag, a non-specific band was used as loading control (LC). C) Lysates of 293T cells transiently transfected with mp-MALT1 were subjected to sucrose density gradient centrifugation and aliquots of the serial fractions (1-12 from top to bottom) were immunoblotted with a-MALT1-N, a-Lck, a kinase residing in the Detergent Resistant Membrane (DRM) fractions, and a-GAPDH, a cytosolic marker. D-E) Immunoblot of lysates of 293T cells transiently expressing wild-type MALT1, mp-MALT1 and its mutants or Ubiquitin-p76 as specified with indicated antibodies. eMALT1: endogenous MALT1. β-actin (D) and LC: non-specific band (E) are loading controls. Arrows (panel C, D, E)) indicate the N-terminal p19 or the C-terminal p76 cleavage fragment respectively. All molecular mass standards are in kDa.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103774-g001: Targeting mp-MALT1 to DRMs induces its proteolysis at R149 in 293T cells.A) Features of mp-MALT1 (mp: myristoylation-palmitoylation sequence) and Ub-p76 (Ubiquitin-p76 fusion protein). R149: MALT1 cleavage site. Flag: Flag epitope, DD: Death Domain, Ig: immunoglobulin-like domain, p20: caspase p20-like domain, C464: MALT1 catalytic cysteine, T6-Ig and T6-C: TRAF6 binding site in second Ig domain and C-terminus, respectively. Ub: Ubiquitin. B) NF-κB-reporter assays of 293T cells transiently expressing wild-type MALT1, mp-MALT1 or empty vector (mock). NF-κB-dependent luciferase activity is shown as fold induction of vector-transfected cells and represents the mean +/- S.D. of at least three independent experiments (n = 3). Cell lysates were immunoblotted with a-Flag, a non-specific band was used as loading control (LC). C) Lysates of 293T cells transiently transfected with mp-MALT1 were subjected to sucrose density gradient centrifugation and aliquots of the serial fractions (1-12 from top to bottom) were immunoblotted with a-MALT1-N, a-Lck, a kinase residing in the Detergent Resistant Membrane (DRM) fractions, and a-GAPDH, a cytosolic marker. D-E) Immunoblot of lysates of 293T cells transiently expressing wild-type MALT1, mp-MALT1 and its mutants or Ubiquitin-p76 as specified with indicated antibodies. eMALT1: endogenous MALT1. β-actin (D) and LC: non-specific band (E) are loading controls. Arrows (panel C, D, E)) indicate the N-terminal p19 or the C-terminal p76 cleavage fragment respectively. All molecular mass standards are in kDa.
Mentions: T or B cell receptor stimulation induces CARMA1-mediated recruitment of BCL10 and MALT1 to the lipid raft membrane fractions, which is essential for NF-κB activation [14], [22], [43]. To test whether lipid raft targeting promotes MALT1 activation, we generated a fusion of MALT1 to the N-terminal myristoylation-palmitoylation signal sequence of Lck (mp-MALT1, Figure 1A), which can target proteins into glycosphingolipid-enriched membranes [44]. When ectopically expressed in 293T cells, this construct was highly active, while MALT1 alone was unable to activate an NF-κB reporter ([45] and Figure 1B). We demonstrated previously through subcellular fractionation via sucrose density gradient centrifugation that ectopic MALT1 in 293T cells is merely cytosolic [5]. Subcellular fractionation of mp-MALT1 showed that it, as expected, also resides in the detergent resistant membrane (DRM) fractions, the latter marked by the presence of the kinase Lck (Figure 1C, lane 4 and 5). With the MALT1 antibody used (MALT1-N), which recognizes the N-terminus of MALT1, we further detected a 19 kDa fragment (p19) in the DRM fractions, (Figure 1C, lane 4–5). This N-terminal p19 fragment was also detectable in lysates of 293T cells expressing mp-MALT1, though not in lysates of cells expressing wild-type MALT1 (Figure 1D, lane 1–2). Expression of a catalytically inactive form of MALT1 (mp-MALT1-C464A) failed to generate the p19 fragment (Figure 1D, lane 3), suggesting that MALT1 protease activity is required for the generation of this N-terminal fragment.

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