2009 Fiscal Year Final Research Report
Functional analysis of novel tyrosine-phosphorylated protein in LPS-stimulated macrophages
| Project/Area Number |
20790713
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Infectious disease medicine
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| Research Institution | National Institute of Infectious Diseases |
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Principal Investigator |
MATSUMURA Takayuki National Institute of Infectious Diseases, 免疫部, 研究員 (50434379)
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| Project Period (FY) |
2008 – 2009
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| Keywords | 感染症防御学 / 自然免疫 / プロテオミクス |
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| Research Abstract |
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>) negatively regulates the TLR4 signaling-mediated production of IL-6 and IL-10, most likely via the Syk-BCAP_<-L>-PI3K-PLCγ2-NF_<-κ>B pathway. BCAP_<-L> is likely to tightly control TLR signaling to prevent undesired or persistent stimulation that might be harmful to the host. Our study will provide new insights into the molecular mechanisms of the regulatory system with regard to cytokine production in innate immune responses, which may help us to design novel strategies for the prevention of infectious diseases.
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