Association Of Shp2 With Phosphorylated IL-22R1 Is Required For Interleukin-22-induced Activation Of MAP Kinases

Interleukin-22 (IL-22) is a member of IL-10 family of cytokines produced by activated T cells and is involved in several tissue responses. IL-22 signals through a heterodimeric receptor composed of IL-22 receptor 1 (IL-22R1) and IL-10R2, and the intracellular signaling pathways mediated by IL-22 receptor are not completely known. This report shows that IL-22 induces IL-22R1 phosphorylation, and Src homology 2-containing protein-tyrosine phosphatase (Shp2) is recruited to the tyrosine phosphorylated IL-22R1 upon IL-22 stimulation. Furthermore, Tyr-251 and Tyr-301 of IL-22R1 are required for Shp2 binding to the IL-22R1. Shp2 binding to IL-22R1 and Shp2 protein tyrosine phosphatase activity are required for activation of MAP kinases and signal transducers and activators of transcription 3 (Stat3) phosphorylation by IL-22. These results reveal a critical role of Shp2 in IL-22 mediated signal transduction pathways.Methods1. Plasmid construction2. Cell culture and transfection3. Construction of stable-expressed Flp-In-293 cell lines4. Detection of protein expression on cell surface5. Immunoprecipitation and immunoblot analysis6. Cell proliferation assay Results1. plasmids pcDNA3.1-IL22R1-flag, pcDNA3.1-IL-22R1-Y251F-flag, pcDNA3.1-IL-22R1-Y301F-flag, pcDNA3.1-IL-22R1-FF-flag, pcDNA5/FRT-IL22R2-Flag, pcDNA5/FRT-IL22R1-FF-Flag have been constructed.2. Flp-In-293/IL-22R1 and Flp-In-293/IL-22R1-FF have been constructed.3. IL-22 induces Src, Erk1/2 and STAT3 phosphorylation,4. IL-22 stimulates Shp2 binding to IL-22R15. Tyr-251 and Tyr-301 of IL-22R1 are required for Shp2 binding to IL-22R1 and for IL-22-stimulated Erk2 and Stat3 activation6. The protein tyrosine phosphatase activity of Shp2 is required for IL-22-induced Erk2 activation7. Effects of Shp2 binding defective IL-22R1 receptor on IL-22-induced activation of cell proliferationIn summary, we first presented evidence that Shp2 is recruited to IL-22R1 upon stimulation with IL-22. Furthermore, both Tyrosine 251 and Tyrosine 301 of IL-22R1 are required for Shp2 binding to IL-22R1 and for IL-22-stimulated Erk2 activation. Moreover, Shp2-IL-22R1 interaction contributes to tyrosine and serine phosphorylation of STAT3 and cell proliferation in response to IL-22.