Nonreceptor Tyrosine Kinase C-Abl- And Arg-Mediated IRF3 Phosphorylation Regulates IFN-? Production

Sensing of pathogens by the innate immune system is important for host to initiate defensive responses.The host senses viral and bacterial pathogen invasion via the recognition of pathogen-associated molecular patterns(PAMPs)by pattern recognition receptors(PRRs),and then promotes the expression of type I interferons.Interferon regulatory factor 3(IRF3)is a key transcriptional regulator of type I interferon(IFN)-dependent immune responses against DNA and RNA viruses,which preexists in the cytoplasm of uninfected cells and translocates to the nucleus following viral infection.Upon viral infection,IRF3 is phosphorylated at its C-terminus on serine 385,386,396,398,402,405,and threonine 404,and the serine phosphorylation is important for the activation of IRF3 and regulation of the expression of IFN-?.Through associating with other proteins,non-receptor tyrosine kinase c-Abl functions in regulating cytoskeleton remodeling,cell adhesion,cell proliferation,response to oxidative stress and DNA damage,autophagy,cell transformation,apoptosis and cell migration.c-Abl also plays roles in regulating differentiation of thymocyte and promoting differentiation of adipocytes,and notably,in T-cell development and immune responses.However,the mechanisms of c-Abl involved in innate immunity regulation are poorly understood.The aim of this report is to study the function and mechanism of c-Abl in innate immunity.Co-immunoprecipitation(Co-IP)using anti-c-Abl antibody revealed that c-Abl associated with IRF3.By overexpression of the related proteins in the cells and following Co-IP,it was comfirmed that c-Abl associated with IRF3.Far-western demonstrated that c-Abl directly bound to IRF3 in vitro.It was also found that both SH2 and SH3 domain of c-Abl can interact with IRF3,and IRF3 preferred binding to SH2 domain,which suggested that IRF3 might be a substrate of c-Abl kinase.Immunoblotting of anti-Flag-IRF3 immunoprecipitates by anti-phosphorylated tyrosine antibody demonstrated that IRF3 was phosphorylated specifically by c-Abl and Abl related gene product(Arg).The phosphorylated sites were revealed by LC-MS/MS and IRF3 was was found phosphorylated by c-Abl mainly at Y292.Other uncovered tyrosine sites might also contribute to c-Abl-mediated IRF3 phosphorylation.Duo-luciferase analysis showed that when Y292 was mutated to F,IRF3-induecd transcription of IFN-? was significantly reduced.Through real-time PCR,it was confirmed that poly(dA:dT)-induced aim2 transcription was also significantly reduced by AMN107 treatment,which suggested that c-Abl may play important roles in AIM2 inflammasome activation.Cells were stimulated with dsDNA,DNA virus and Francisella tularensis subsp.holarctica live vaccine strain(Ft LVS).Results showed that the AIM2 inflammasome activation was reduced by the treatment of Abl inhibitor AMN107.In the Ft LVS infection mouse model,much higher mortality rate was observed in c-Abl/Arg siRNA-treated mice compared to negative control siRNA-treated ones.The expression of IFN-? and AIM2 was lower in c-Abl/Arg siRNA-treated mice than that in negative control siRNA-treated ones.The level of IL-18 was significantly lower in c-Abl/Arg siRNA-treated mice than that in negative control siRNA-treated ones.In concert,mice treated with Abl inhibitor AMN107 resulted in higher mortality in live Ft LVS infection mouse model.The expression of IFN-? and AIM2 in spleen was lower in AMN107-treated mice than that in untreated ones.The level of caspase-1 p20 in spleen was also significantly lower in AMN107-treated mice than that in untreated ones.The level of IL-18 was significantly lower in AMN107-treated mice than that in untreated ones.For the first time,this study reported the tyrosine phosphorylation of IRF3 by c-Abl and Arg and found that Y292 of IRF3 may be an important phosphorylation site.c-Abl and Arg was involved in the regulation of AIM2 inflammasome activation through mediating the expression of IFN-? in response to Ft LVS.This study provides a new insight into the function of c-Abl and Arg in regulating immune response and AIM2 inflammasome activation.