title: Identification of a Regulatory Acidic Motif as the Determinant of Membrane Localization of TICAM-2.
abstract: TLR4 triggers LPS signaling through the adaptors Toll/IL-1R domain-containing adaptor molecule (TICAM)-2 (also called TRAM) and TICAM-1 (also called TRIF), together with Toll/IL-1R domain-containing adaptor protein (TIRAP) and MyD88. The MyD88 pathway mediates early phase responses to LPS on the plasma membrane, whereas the TICAM pathway mediates late-phase responses, which induce the production of type I IFN and activation of inflammasomes. TICAM-2 bridges TLR4 and TICAM-1 for LPS signaling in the endosome. Recently, we identified an acidic motif, E87/D88/D89 in TICAM-2, that provides the interaction surfaces between TICAM-2 and TICAM-1. In the present study, we found additional D91/E92 in TICAM-2, conserved across species, that is crucial for TICAM-1 activation. The D91A/E92A mutant protein was distributed largely to the cytosol, despite myristoylation, suggesting its importance for assistance of membrane localization of TICAM-2. An ectopically expressed D91A/E92A mutant per se failed to activate TICAM-1, unlike its wild-type counterpart that forms self-aggregation, but it still retained the ability to pass LPS-mediated IFN regulatory factor (IRF)3 activation. In a TICAM-2 knockout human cell line expressing TLR4/MD-2 with or without CD14, overexpression of the D91A/E92A mutant did not activate IRF3, but upon LPS stimulation, it induced sufficient TLR4-mediated IRF3 activation with high coefficient colocalization. Hence, the D91/E92 motif guides TICAM-2 membrane localization and self-activation for signaling. Our results suggest the presence of two distinct steps underlying endosomal LPS signaling on TICAM-2 for TICAM-1 activation: TICAM-2 assembling in TLR4 and/or TICAM-2 self-activation. D91A/E92A of TICAM-2 selectively associates the TLR4-dependent TICAM-2 assembling, but not cytosolic TICAM-2 self-aggregation, to activate TICAM-1.
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan; and.
authors.forename: Kenji
authors.initials: K
authors.lastname: Funami
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan; and.
authors.forename: Misako
authors.initials: M
authors.lastname: Matsumoto
authors.affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Enokizono
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan; and.
authors.forename: Noriko
authors.initials: N
authors.lastname: Ishii
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan; and.
authors.forename: Megumi
authors.initials: M
authors.lastname: Tatematsu
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan; and.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Oshiumi
authors.affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan; and seya-tu@pop.med.hokudai.ac.jp.
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Seya
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism; D007370:Interferon Type I / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D007370:Interferon Type I; C495377:LY96 protein, human; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D053594:Myeloid Differentiation Factor 88; C470409:TICAM1 protein, human; C480228:TICAM2 protein, human; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4