Figure 1.
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.
Figure 2.
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.
Figure 3.
MALT1 undergoes auto-proteolysis in vitro.
A) Features of F-STII-MALT1 and LZ-MALT1. F: Flag epitope, STII: StrepII-tag, 6H: 6-Histidine tag, LZ: Leuzine zipper. B) Top: In vitro cleavage of the fluorogenic tetratpeptide substrate Ac-LVSR-AMC (50 µM) by F-STII-MALT1 in increasing concentrations of the cosmotropic salt NH4-citrate (0.2, 0.4, 0.6, and 0.8 M M), by F-STII-MALT1 in 0.8M NH4-citrate buffer in the presence of the MALT1 protease inhibitors z-VRPR-fmk (10 µM) and z-LVSR-fmk (10 µM) and by LZ-MALT1 or LZ-MALT1-C464A in 0.8 M NH M NH4-citrate buffer. The barchart shows cleavage activity as Fluorescence Units (FU) increase/min. Results are expressed as means ± SD (n = 3). Bottom: enzymatic reactions were analysed by immunoblotting with a-MALT1-C, a-p76 neo-epitope and a-MALT1-N.
Figure 4.
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.
Figure 5.
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.
Figure 6.
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.
Figure 7.
MALT1 auto-proteolysis is required for NF-κB transcriptional activity in Jurkat T cells.
A) Relative IL-2 and CSF2 production of indicated Jurkat T cell lines, stimulated for 18 hrs with PMA hrs with PMA/ionomycin, measured via ELISA. Data shown as mean +/- S.D. (n = 3). B) Jurkat T cells expressing MALT1 and the JΔM-CA, JΔM-RA, and JΔM cells were stimulated for the indicated times with P/I and IL-2 and CSF2 transcript levels were determined via qRT-PCR. C) Jurkat T cells with ectopic expression of MALT1 and JΔM-CA, JΔM-RA and JΔM were electroporated (Amaxa, Nucleofection) with Luciferase reporter constructs driven by the IL-2 promoter or the Igκ3-ConA promoter, and 24 hrs later stimulated with P hrs later stimulated with P/I for 18 hrs before Luciferase activity was measured. Data shown as mean +/- S.D. (n = 3). D) GSEA showing a significant enrichment of NF-κB-target genes (FDR q<0,001) in the pre-ranked down-regulated genes from JΔM-CA, JΔM-RA and RACA cells stimulated for 18 hrs with P hrs with P/I. Gene list depicted at the right side are NF-κB target genes down-regulated in JΔM-CA, JΔM-RA and RACA cells after 3 and 18 hrs stimulation with P hrs stimulation with P/I.
Figure 8.
Model for MALT1 functions in TCR-mediated NF-κB1 activation.
A) The adaptor function of MALT1 is required for TCR-mediated activation of the IKK complex. Via formation of the CARMA1/BCL10/MALT1 complex MALT1 controls TRAF6-mediated K63 poly-ubiquitination of the gamma subunit of the IKK complex. Concurrent phosphorylation of IKKβ activates the IKK complex that phosphorylates the NF-κB inhibitor IκB, induces its proteasomal degradation and allows nuclear translocation of NF-κB complexes consisting of p50, p65 and REL. B) Parallel induction of MALT1 protease activity prevents de-ubiquitination of IKKγ and possibly other substrates via A20 cleavage and facilitates DNA binding of p65- or REL-containing NF-κB complexes via RELB cleavage. C) MALT1 auto-proteolysis represents a third level of MALT1 regulation that controls in a TRAF6-dependent and BCL10-independent manner the transcriptional activation of nuclear NF-κB complexes via a yet unknown mechanism.