Identification of BCAP-(L) as a negative regulator of the TLR signaling-induced production of IL-6 and IL-10 in macrophages by tyrosine phosphoproteomics.
Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-(L)) and an alternatively initiated or spliced (Bcap-(S)) mRNA, and little is known about the differential functions of the BCAP-(L) and BCAP-(S) proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-(L) enhanced IL-6 and IL-10 production but not TNF-α production in TLR ligand-stimulated macrophages. We propose that BCAP-(L) (but not BCAP-(S)) is a negative regulator of the TLR-mediated host defense system in macrophages.
Matsumura
Takayuki
T
Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041, Japan.
Oyama
Masaaki
M
Kozuka-Hata
Hiroko
H
Ishikawa
Kosuke
K
Inoue
Takafumi
T
Muta
Tatsushi
T
Semba
Kentaro
K
Inoue
Jun-Ichiro
J
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D016753:Interleukin-10 / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D015850:Interleukin-6 / Q000096:biosynthesis*; D008264:Macrophages / Q000201:enzymology / Q000276:immunology*; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D040081:Protein Array Analysis; D040901:Proteomics; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014158:Transcription, Genetic; D014443:Tyrosine / Q000378:metabolism; D000093242:Interleukin-6 Inhibitors
D048868:Adaptor Proteins, Signal Transducing; D016753:Interleukin-10; D015850:Interleukin-6; D010750:Phosphoproteins; D051197:Toll-Like Receptor 4; D014443:Tyrosine; D000093242:Interleukin-6 Inhibitors; C487978:Pik3ap1 protein, mouse; C493487:Tlr4 protein, mouse