This was followed by incubation with salmon sperm DNA/protein A agarose slurry for 1?h, then samples were sequentially washed with low salt buffer, high salt buffer, LiCl buffer, and twice with TE buffer

This was followed by incubation with salmon sperm DNA/protein A agarose slurry for 1?h, then samples were sequentially washed with low salt buffer, high salt buffer, LiCl buffer, and twice with TE buffer. modulate TLR2-mediated IFN- production in macrophages. Introduction Toll-like receptors (TLR) play a critical role in the early innate immune response to pathogens by recognizing pathogen-associated molecular patterns (PAMPs) and are involved in sensing endogenous danger signals1, 2. TLR2, which is usually expressed on monocytes, mature macrophages and dendritic cells, and mast cells, specifically recognizes components of Gram-positive bacteria, including bacterial lipoproteins, lipomannans and lipoteichoic acids3. TLR2 can form a heterodimer with either TLR1 to recognize triacylated lipopeptides, such as the synthetic ligand Pam3CSK4, or TLR6 to recognize diacylated lipopeptides like MALP-21, 3C5. The dimerization of these TLRs allows for recognition of a more specific and wider array of microbial components6. Upon stimulation with TLR2 ligands, MyD88 cis-Urocanic acid recruits IL-1 receptor-associated kinase-4 (IRAK-4) to TLR2 through conversation of the death domains of both molecules. IRAK-1 is usually cis-Urocanic acid activated by phosphorylation and associates with TRAF6, thereby activating the IKK complex cis-Urocanic acid and leading to activation of MAP kinases and NF-B3, 7C9. These signaling pathways are critical for TLR2-mediated pro- and/or anti-inflammatory cytokine production. Tollip and IRAK-M interact with IRAK-1 and negatively regulate TLR-mediated signaling pathways10, cis-Urocanic acid 11. Recent data indicate that localization of TLR2 ligands within endosomal compartments regulates TLR2-mediated induction of type I interferons (IFNs)12C15, suggesting a possible role of endocytic pathways in TLR2 signaling. However, the regulatory factor and/or signaling pathways that lead to TLR2-mediated type I IFN induction are unclear. Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) is an evolutionarily conserved protein tyrosine phosphatase with a widespread expression pattern16. It contains two N-terminal SH domains, a classic protein-tyrosine phosphatase domain name at the C-terminal end and two important tyrosine resides (Tyr542 and Tyr580) in the C-terminal tail16, 17. SHP2 positively regulates cytokine and growth factor signaling, but negatively regulates the activation of T and B lymphocytes and IFN- signaling18C21. These regulatory functions of SHP2 have been proposed to occur in either a phosphatase activity-dependent or -impartial manner; e.g., SHP2 inhibits TLR3- and TLR4-activated IFN- production in a phosphatase activity-independent manner by binding to TANK-binding kinase 1 (TBK1)21. SHP2 activity is usually regulated by several molecules, ZAP70 and PKA, in multiple signaling pathways22C25. In the CXADR present study, we demonstrate that SHP2 and Gsk3 reciprocally regulate TLR2-mediated IFN- production, thereby identifying SHP2 and Gsk3 as a negative and a positive regulator, respectively, of the TLR2 signaling pathway. Results SHP2 is a negative regulator of IFN- induction by TLR2 ligand SHP2 negatively regulates TRIF-dependent pro-inflammatory cytokines and type I IFN induction in TLR3 and TLR4 signaling21. Although SHP2 did not affect TLR2-mediated pro-inflammatory cytokine production, no previous report investigated whether SHP2 regulates TLR2-mediated IFN- induction. TLR2 agonist Pam3CSK4 could trigger IFN- expression at both mRNA and protein levels in macrophages (Supplementary Fig.?S1). We next examined the effects of the SHP2 inhibitor NSC87877 on IFN- expression induced by the Pam3CSK4 in macrophages. Pharmacological inhibition of SHP2 potentiated Pam3CSK4-induced IFN- mRNA expression (Fig.?1a). SHP2 undergoes rapid phosphorylation of the tyrosine residue Y542 upon Pam3CSK4 stimulation (Supplementary Fig.?S2), suggesting that a TLR2 signaling pathway involving SHP2 regulates IFN- expression. In the canonical type I IFN-induced signaling pathway, IFN- activates Janus kinase 1 and tyrosine kinase 2, which phosphorylate the cytoplasmic transcription factors signal transducer and activator of transcription 1 (STAT1) and STAT226, 27. Phosphorylation of STAT1 upon Pam3CSK4.