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

Biochem Biophys Res Commun. 2010 Sep 17;400(2):265-70. doi: 10.1016/j.bbrc.2010.08.055. Epub 2010 Aug 20.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Animals
  • Cell Line
  • Interleukin-10 / antagonists & inhibitors
  • Interleukin-10 / biosynthesis*
  • Interleukin-6 / antagonists & inhibitors
  • Interleukin-6 / biosynthesis*
  • Macrophages / enzymology
  • Macrophages / immunology*
  • Mice
  • Phosphoproteins / metabolism*
  • Protein Array Analysis
  • Proteomics
  • Toll-Like Receptor 4 / metabolism*
  • Transcription, Genetic
  • Tyrosine / metabolism


  • Adaptor Proteins, Signal Transducing
  • Interleukin-6
  • Phosphoproteins
  • Pik3ap1 protein, mouse
  • Tlr4 protein, mouse
  • Toll-Like Receptor 4
  • Interleukin-10
  • Tyrosine