Association of Lyn kinase with membrane rafts determines its negative influence on LPS-induced signaling

• Published in: Molecular biology of the cell Vol. 28; no. 8; pp. 1147 - 1159
• Main Authors: Borzęcka-Solarz, Kinga, Dembińska, Justyna, Hromada-Judycka, Aneta, Traczyk, Gabriela, Ciesielska, Anna, Ziemlińska, Ewelina, Świątkowska, Anna, Kwiatkowska, Katarzyna
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 15-04-2017
• Subjects: Animals; Cell Line; Chemokine CCL5 - metabolism; Green Fluorescent Proteins; Interferon Regulatory Factor-3 - metabolism; Lipopolysaccharides - pharmacology; Macrophages - metabolism; Macrophages, Peritoneal - metabolism; Male; Membrane Microdomains - enzymology; Membrane Microdomains - metabolism; Mice; Mice, Inbred C57BL; NF-kappa B - metabolism; Phosphorylation; Protein-Tyrosine Kinases - metabolism; Signal Transduction; src-Family Kinases - genetics; src-Family Kinases - metabolism; Toll-Like Receptor 4 - genetics; Toll-Like Receptor 4 - metabolism; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.E16-09-0632

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.