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.