title: Association of Lyn kinase with membrane rafts determines its negative influence on LPS-induced signaling.
abstract: Lipopolysaccharide (LPS) is the component of Gram-negative bacteria that activates Toll-like receptor 4 (TLR4) to trigger proinflammatory responses. We examined the involvement of Lyn tyrosine kinase in TLR4 signaling of macrophages, distinguishing its catalytic activity and intermolecular interactions. For this, a series of Lyn-GFP constructs bearing point mutations in particular domains of Lyn were overexpressed in RAW264 macrophage-like cells or murine peritoneal macrophages, and their influence on LPS-induced responses was analyzed. Overproduction of wild-type or constitutively active Lyn inhibited production of TNF-α and CCL5/RANTES cytokines and down-regulated the activity of NFκB and IRF3 transcription factors in RAW264 cells. The negative influence of Lyn was nullified by point mutations of Lyn catalytic domain or Src homology 2 (SH2) or SH3 domains or of the cysteine residue that undergoes LPS-induced palmitoylation. Depending on the cell type, overproduction of those mutant forms of Lyn could even up-regulate LPS-induced responses, and this effect was reproduced by silencing of endogenous Lyn expression. Simultaneously, the Lyn mutations blocked its LPS-induced accumulation in the raft fraction of RAW264 cells. These data indicate that palmitoylation, SH2- and SH3-mediated intermolecular interactions, and the catalytic activity of Lyn are required for its accumulation in rafts, thereby determining the negative regulation of TLR4 signaling.
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw 02-093, Poland.
authors.forename: Kinga
authors.initials: K
authors.lastname: Borzęcka-Solarz
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw 02-093, Poland.
authors.forename: Justyna
authors.initials: J
authors.lastname: Dembińska
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw 02-093, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Hromada-Judycka
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw 02-093, Poland.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Traczyk
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw 02-093, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Ciesielska
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw 02-093, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Ziemlińska
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw 02-093, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Świątkowska
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw 02-093, Poland k.kwiatkowska@nencki.gov.pl.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kwiatkowska
mesh_terms: D000818:Animals; D002460:Cell Line; D018946:Chemokine CCL5 / Q000378:metabolism; D049452:Green Fluorescent Proteins; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000378:metabolism; D008297:Male; D021962:Membrane Microdomains / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D018946:Chemokine CCL5; D050838:Interferon Regulatory Factor-3; C494233:Irf3 protein, mouse; D008070:Lipopolysaccharides; D016328:NF-kappa B; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases