Identification of rgs19 as a target molecule of notch signaling in lipopolysaccharide-stimulated macrophages by phosphoproteomic

Abstract

Signaling between cell to cell depends on crosstalk of many intracellular signaling molecules such as phosphoproteins. Alterations in protein phosphorylation can be found in different signaling pathways. Aberration in this process may lead to abnormal cell behaviors such as cell proliferation, differentiation and cell death. Notch signaling is an evolutionarily well conserved signaling pathway which plays critical roles in regulating development and functions of immune cells. Recent evidences suggest the involvement of this signaling pathway in activation of macrophages which are innate immune cells, by corroboratively functioning with signaling cascades downstream of pattern recognition receptors such as Toll-like receptor pathway. In this study, the patterns of phosphorylated proteins in mouse macrophage cell line were compared using a proteomics approach. Cells were pre-treated with inhibitor of the Notch signaling (GSI) or vehicle control before subjecting to stimulation with lipopolysaccharide. Phosphoproteins were examined and compared by the two-dimensional gel electrophoresis. Gels were stained with phosphoprotein-specific dye, Pro-Q Diamond, and subsequently with Coomassie stain and silver stain for total proteins. The spots with statistical significant difference in phosphorylation between treatment with or without GSI were identified by LC/MS/MS. The results identified one of the spots as regulator of G-protein signaling 19 (RGS19) which showed decreased phosphorylation when Notch signaling is inhibited. The involvement of RGS19 in TLR4 and Notch signaling in macrophages was investigated by siRGS19 silencing approach. GSI treatment and silencing of RGS19 yielded similar results of increased activation of SAPK/JNK and decreased activation of Akt The results suggested that Notch signaling regulates production of cytokine TNF and IL-6 and expression of iNOS in LPS induced activation of TLR4 in macrophage via RGS19.