Identification of Canonical Tyrosine-dependent and Non-canonical Tyrosine-independent STAT3 Activation Sites in the Intracellular Domain of the Interleukin 23 Receptor

Abstract

Background: Activation of STAT3 is the major signaling pathway of IL-23. Results:The study provides detailed characterization of IL-23-dependent signal transduction with the focus on STAT3 phosphorylation. Conclusion: Canonical tyrosine-dependent and non-canonical tyrosine-independent STAT3 activation sites are located within the IL-23R. Significance: This may be the first step in elucidating the signal transduction of IL-23, an important cytokine for T H 17 cell development.

Non-Canonical Interleukin 23 Receptor Complex Assembly

“…Murine Ba/F3 cell lines have been described previously (22). FLAG polyclonal antibody was from Sigma-Aldrich (St. Louis, MO).…”

“…Mutations within p40, p19, or IL-12R␤1 were generated by PCR followed by DpnI digestion of methylated template DNA. cDNAs coding for murine IL-23R (gene ID 209590) and IL-12R␤1 (gene ID 16161) have been described previously (22). The extracellular domains of IL-23R (amino acids 1-315) comprising D1, D2, and D3 and IL-12R␤1 (amino acids 1-566) containing D1-D5 were C-terminally fused with human Fc tags (soluble (s)IL-23R and sIL-12R␤1).…”

“…22. Cells were cultured for 3 days in a final volume of 100 l in the presence of 10% cell culture supernatant of transiently transfected COS-7 cells secreting the indicated cytokines, the purified p40_D2D3-p19Fc, or defined concentrations of p40-p19-Fp, or cells were left unstimulated.…”

“…c-myc monoclonal antibody (9E10) was produced and purified as described in Ref. 22. Peroxidase-conjugated secondary antibodies and human Fc antibody were from Pierce.…”

“…Western Blotting-Equal amounts of proteins (cell culture supernatants, 50 l/lane; cell lysates, 15 l/lane; coimmunoprecipitates, 35 l/lane) were loaded and separated by SDS-PAGE, and Western blotting was performed as described previously (22).…”