title: Specific binding of the WASP N-terminal domain to Btk is critical for TLR2 signaling in macrophages.
abstract: Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we revealed that WASP is involved in lipopolysaccharide-TLR4 signaling in macrophages by association of Bruton's tyrosine kinase (Btk) with the WASP N-terminal domain. Btk has been shown to play important roles in the signaling of several TLRs and to modulate the inflammatory response in macrophages. In this study, we evaluated the importance of the interaction between Btk and WASP in TLR2 signaling by using bone marrow-derived macrophage cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) or VL single-domain intrabodies. In this Tg bone marrow-derived macrophages, specific interaction between WASP and Btk were strongly inhibited by masking of the binding site in the WASP N-terminal domain. There was impairment of gene expression of TNF-α, IL-6, and IL-1β and phosphorylation of inhibitor of κB α/β (IKKα/β) and nuclear factor (NF)-κB upon stimulation with TLR2 ligands. Furthermore, tyrosine phosphorylation of WASP following TLR2-ligand stimulation was severely inhibited in the Tg bone marrow-derived macrophages, as shown by the impairment in WASP tyrosine phosphorylation following lipopolysaccharide stimulation. These results strongly suggest that the association between the WASP N-terminal domain and Btk plays an important role in the TLR2-signaling pathway in macrophages.
authors.affiliation: Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Chisato
authors.initials: C
authors.lastname: Sakuma
authors.affiliation: Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan. Electronic address: mitsuru.sato@affrc.go.jp.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Sato
authors.affiliation: Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Takato
authors.initials: T
authors.lastname: Takenouchi
authors.affiliation: Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kitani
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D051550:I-kappa B Kinase / Q000378:metabolism; D055666:Lipopeptides / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008089:Listeria monocytogenes; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology; D008562:Membrane Glycoproteins / Q000378:metabolism; D008822:Mice, Transgenic; D016328:NF-kappa B / Q000378:metabolism; D010587:Phagocytosis; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D057127:Single-Chain Antibodies / Q000378:metabolism; D013329:Structure-Activity Relationship; D051195:Toll-Like Receptor 2 / Q000378:metabolism*; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D016207:Cytokines; D055666:Lipopeptides; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; C508075:Pam(3)CSK(4) peptide; D017472:Receptors, Interleukin-1; D057127:Single-Chain Antibodies; C499045:TIRAP protein, mouse; D051195:Toll-Like Receptor 2; D051316:Wiskott-Aldrich Syndrome Protein; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625951:Btk protein, mouse; D051550:I-kappa B Kinase
keywords: Bruton's tyrosine kinase (Btk); Intrabody; Macrophage; Toll-like receptor 2 (TLR2); Toll/IL-1 receptor domain-containing adaptor protein (TIRAP); Wiskott–Aldrich syndrome protein (WASP)