ABSTRACT
MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4(+) T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation.
Subject(s)
Alternative Splicing/genetics , CD4-Positive T-Lymphocytes/immunology , Caspases/genetics , Lymphocyte Activation/immunology , Neoplasm Proteins/genetics , Signal Transduction , Animals , Caspases/metabolism , Down-Regulation , Enzyme Activation , Exons/genetics , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoprotein U/metabolism , Humans , Interleukin-2/biosynthesis , JNK Mitogen-Activated Protein Kinases/metabolism , Jurkat Cells , Mice, Inbred C57BL , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , NF-kappa B/metabolism , Neoplasm Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , TNF Receptor-Associated Factor 6/metabolism , Th17 Cells/immunology , Up-RegulationABSTRACT
Survival of activated B cell-subtype (ABC) of diffuse large B cell lymphoma (DLBCL) is driven by chronic B cell receptor (BCR) signaling that activates the canonical NF-κB pathway. Inhibition of BTK by Ibrutinib has been shown to kill ABC DLBCL cells that carry activating mutations in the BCR adaptor CD79. However, mutations in BTK or in downstream components such as CARMA1/CARD11 can render lymphomas Ibrutinib resistant. Therefore, we assessed here the simultaneous inhibition of BTK and the protease MALT1 that acts downstream of CARMA1 and is essential for ABC DLBCL tumor growth. We show that in CD79 mutant cells BTK is a crucial upstream regulator of MALT1, but dispensable in CARMA1 mutant ABC DLBCL. Combined inhibition of BTK by Ibrutinib and MALT1 by S-Mepazine additively impaired MALT1 cleavage activity and expression of NF-κB pro-survival factors. Thereby, combinatorial Ibrutinib and S-Mepazine treatment enhanced killing of CD79 mutant ABC DLBCL cells. Moreover, while expression of oncogenic CARMA1 in CD79 mutant cells conferred Ibrutinib resistance, double mutant cells were still sensitive to MALT1 inhibition by S-Mepazine. Thus, based on the genetic background combinatorial BTK and MALT1 inhibition may improve effectiveness of therapeutic treatment and reduce the chances for the development of drug resistances.
Subject(s)
CD79 Antigens/metabolism , Caspases/metabolism , Mutation , Neoplasm Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase , Apoptosis/drug effects , Apoptosis/genetics , Blotting, Western , CARD Signaling Adaptor Proteins/genetics , CARD Signaling Adaptor Proteins/metabolism , CD79 Antigens/genetics , Caspases/genetics , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Drug Synergism , Guanylate Cyclase/genetics , Guanylate Cyclase/metabolism , Humans , Jurkat Cells , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , NF-kappa B/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Phenothiazines/chemistry , Phenothiazines/pharmacology , Piperidines , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Pyrazoles/pharmacology , Pyrimidines/pharmacology , StereoisomerismABSTRACT
MALT1 paracaspase is activated upon antigen receptor stimulation to promote lymphocyte activation. In addition, deregulated MALT1 protease activity drives survival of distinct lymphomas such as the activated B cell type of diffuse large B cell lymphoma (ABC-DLBCL). Here, we designed fluorophore or biotin-coupled activity based-probes (ABP) that covalently modify the active center of MALT1. MALT1-ABPs are exclusively labeling an active modified full length form of MALT1 upon T cell stimulation. Further, despite the CARMA1 requirement for initial MALT1 activation, the MALT1-ABPs show that protease activity is not confined to the high-molecular CARMA1-BCL10-MALT1 (CBM) complex. Using biotin-coupled ABPs, we developed a robust assay for sensitive and selective detection of active MALT1 in cell lines, primary lymphocytes, and DLBCL tumor biopsies. Taken together, MALT1-ABPs represent powerful chemical tools to measure cellular MALT1 activation, determine efficacy of small molecule inhibitors, and classify lymphomas based on MALT1 activity status.
Subject(s)
Caspases/metabolism , Lymphoma, Large B-Cell, Diffuse/enzymology , Lymphoma, Large B-Cell, Diffuse/pathology , Molecular Probes/chemistry , Neoplasm Proteins/metabolism , T-Lymphocytes/enzymology , Biotin/chemistry , Blotting, Western , CARD Signaling Adaptor Proteins/metabolism , Cell Line , Click Chemistry , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Guanylate Cyclase/metabolism , Humans , Jurkat Cells , Molecular Probes/chemical synthesis , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Neoplasm Proteins/antagonists & inhibitors , RNA Interference , RNA, Small Interfering/metabolism , Rhodamines/chemistry , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
BACKGROUND: The CARMA1-BCL10-MALT1 (CBM) complex bridges T cell receptor (TCR) signaling to the canonical IκB kinase (IKK)/NF-κB pathway. The CBM complex constitutes a signaling cluster of more than 1 Mio Dalton. Little is known about factors that facilitate the rapid assembly and maintenance of this dynamic higher order complex. FINDINGS: Here, we report the novel interaction of the aryl hydrocarbon receptor (AHR) interacting protein (AIP) and the molecular scaffold protein CARMA1. In T cells, transient binding of CARMA1 and AIP enhanced formation of the CBM complex. Thereby, AIP promoted optimal IKK/NF-κB signaling and IL-2 production in response to TCR/CD28 co-stimulation. CONCLUSIONS: Our data demonstrate that AIP acts as a positive regulator of NF-κB signaling upon T cell activation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , CARD Signaling Adaptor Proteins/metabolism , Caspases/metabolism , Guanylate Cyclase/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , NF-kappa B/metabolism , Neoplasm Proteins/metabolism , Signal Transduction , T-Lymphocytes/metabolism , Animals , B-Cell CLL-Lymphoma 10 Protein , CD28 Antigens/metabolism , Cell Line, Tumor , Humans , I-kappa B Kinase/metabolism , Interleukin-2/metabolism , Lymph Nodes/cytology , Mice , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Multiprotein Complexes/metabolism , Primary Cell Culture , Spleen/cytologySubject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Neoplasm Proteins/antagonists & inhibitors , Phenothiazines/chemistry , Phenothiazines/pharmacology , Allosteric Regulation/drug effects , Caspases/chemistry , Caspases/metabolism , Cell Line , Humans , Models, Molecular , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolismABSTRACT
The Carma1-Bcl10-Malt1 (CBM) complex bridges T-cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF-κB pathway. NF-κB activation is triggered by PKCθ-dependent phosphorylation of Carma1 after TCR/CD28 co-stimulation. PKCθ-phosphorylated Carma1 was suggested to function as a molecular scaffold that recruits preassembled Bcl10-Malt1 complexes to the membrane. We have identified the serine-threonine protein phosphatase PP2A regulatory subunit Aα (PPP2R1A) as a novel interaction partner of Carma1. PPP2R1A is associated with Carma1 in resting as well as activated T cells in the context of the active CBM complex. By siRNA-mediated knockdown and in vitro dephosphorylation, we demonstrate that PP2A removes PKCθ-dependent phosphorylation of Ser645 in Carma1, and show that maintenance of this phosphorylation is correlated with increased T-cell activation. As a result of PP2A inactivation, we find that enhanced Carma1 S645 phosphorylation augments CBM complex formation, NF-κB activation and IL-2 or IFN-γ production after stimulation of Jurkat T cells or murine Th1 cells. Thus, our data define PP2A-mediated dephosphorylation of Carma1 as a critical step to limit T-cell activation and effector cytokine production.
Subject(s)
CARD Signaling Adaptor Proteins/metabolism , CD4-Positive T-Lymphocytes/physiology , Guanylate Cyclase/metabolism , Lymphocyte Activation/physiology , Multiprotein Complexes/metabolism , NF-kappa B/metabolism , Protein Phosphatase 2/metabolism , Animals , CARD Signaling Adaptor Proteins/genetics , DNA Primers/genetics , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Knockdown Techniques , Guanylate Cyclase/genetics , HEK293 Cells , Humans , Immunoprecipitation , Jurkat Cells , Luciferases , Mice , Mice, Transgenic , Phosphorylation , Reverse Transcriptase Polymerase Chain Reaction , Transduction, GeneticABSTRACT
The constant regeneration of the blood system during hematopoiesis requires tightly controlled lineage decisions of hematopoietic progenitor cells (HPCs). Because of technical limitations, differentiation of individual HPCs could not previously be analyzed continuously. It was therefore disputed whether cell-extrinsic cytokines can instruct HPC lineage choice or only allow survival of cells that are already lineage-restricted. Here, we used bioimaging approaches that allow the continuous long-term observation of individual differentiating mouse HPCs. We demonstrate that the physiological cytokines, macrophage colony-stimulating factor and granulocyte colony-stimulating factor, can instruct hematopoietic lineage choice.