ABSTRACT
Signal transduction typically displays a so-called bow-tie topology: Multiple receptors lead to multiple cellular responses but the signals all pass through a narrow waist of central signaling nodes. One such signaling node for several inflammatory and oncogenic signaling pathways is the CARD-CC/BCL10/MALT1 (CBM) complexes, which get activated by protein kinase C (PKC)-mediated phosphorylation of the caspase activation and recruitment domain (CARD)-coiled-coil domain (CC) component. In humans, there are four CARD-CC family proteins (CARD9, CARD10, CARD11, and CARD14) and 9 true PKC isozymes (α to ι). At this moment, less than a handful of PKC::CARD-CC relationships are known. In order to explore the biologically relevant combinatorial space out of all 36 potential permutations in this two-component signaling event, we made use of CARD10-deficient human embryonic kidney 293T cells for subsequent pairwise cotransfections of all CARD-CC family members and all activated PKCs. Upon analysis of NF-κB-dependent reporter gene expression, we could define specific PKC::CARD-CC relationships. Surprisingly, as many as 21 PKC::CARD-CC functional combinations were identified. CARD10 was responsive to most PKCs, while CARD14 was mainly activated by PKCδ. The CARD11 activation profile was most similar to that of CARD9. We also discovered the existence of mixed protein complexes between different CARD-CC proteins, which was shown to influence their PKC response profile. Finally, multiple PKCs were found to use a common phosphorylation site to activate CARD9, while additional phosphorylation sites contribute to CARD14 activation. Together, these data reveal the combinatorial space of PKC::CARD-CC signal transduction nodes, which will be valuable for future studies on the regulation of CBM signaling.
Subject(s)
B-Cell CLL-Lymphoma 10 Protein/genetics , CARD Signaling Adaptor Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/genetics , NF-kappa B/genetics , Protein Kinase C/genetics , Amino Acid Sequence , Animals , B-Cell CLL-Lymphoma 10 Protein/metabolism , Binding Sites , CARD Signaling Adaptor Proteins/classification , CARD Signaling Adaptor Proteins/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/metabolism , NF-kappa B/metabolism , Phosphorylation , Phylogeny , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Isoforms/classification , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase C/classification , Protein Kinase C/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , TransfectionABSTRACT
The NOD-like receptor (NLR) family of proteins is involved in the regulation of innate immune responses and cell death pathways. Recent findings show that the NLR family member NLRC4 (also known as IPAF) has important roles in innate immune responses to Gram-negative bacteria. Macrophages infected with Legionella pneumophila, Salmonella typhimurium, Shigella flexneri, or Pseudomonas aeruginosa activate caspase-1 in an NLRC4-dependent manner leading to macrophage cell death and the release of proinflammatory cytokines. This review will discuss these findings as well as the role of bacterial type III and type IV secretion systems and flagellin in NLRC4-mediated caspase-1 activation.