Modulation of TLR signalling by the C-terminal Src kinase (Csk) in macrophages.
In macrophages and monocytes, lipopolysaccharide (LPS) triggers the production of pro-inflammatory cytokine through Toll-like receptor (TLR) 4. Although major TLR signalling pathways are mediated by serine or threonine kinases including IKK, TAK1, p38 and JNKs, a number of reports suggested that tyrosine phosphorylation of intracellular proteins is involved in LPS signalling. Here, we identified several tyrosine-phosphorylated proteins using mass spectrometric analysis in response to LPS stimulation. Among these proteins, we characterized C-terminal Src kinase (Csk), which negatively regulates Src-like kinases in RAW 264.7 cells using RNAi knockdown technology. Unexpectedly, LPS-induced CD40 activation and the secretion of pro-inflammatory cytokine such as IL-6 and TNF-alpha, was down-regulated in Csk knockdown cells. Furthermore, overall cellular tyrosine phosphorylation and TLR4-mediated activation of IkappaB-alpha, Erk and p38 but not of JNK, were also down-regulated in Csk knockdown cells. The protein expression levels of a tyrosine kinase, Fgr, were reduced in Csk knockdown cells, suggesting that Csk is a critical regulator of TLR4-mediated signalling by modifying the levels of Src-like kinases.
Aki
Daisuke
D
Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi, Fukuoka 812-8582, Japan.
Mashima
Ryuichi
R
Saeki
Kazuko
K
Minoda
Yasumasa
Y
Yamauchi
Moriyasu
M
Yoshimura
Akihiko
A
D000818:Animals; D019013:CD40 Antigens / Q000378:metabolism; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002853:Chromatography, Liquid; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D015850:Interleukin-6 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism / Q000502:physiology*; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D051197:Toll-Like Receptor 4; D015534:Trans-Activators / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases
D019013:CD40 Antigens; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D008070:Lipopolysaccharides; D011971:Receptors, Immunologic; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; C494080:Stat1 protein, mouse; C494087:Stat3 protein, mouse; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases