Functional And Mechanistic Studies Of STAT2-T404 Phosphorylation Modifications In Antiviral And Antitumor

STAT2 plays a critical role in resisting pathogen invasion and tumor cell proliferation due to its ability to mediate the transduction of typeⅠinterferon（IFN-Ⅰ）and typeⅢ interferon（IFN-Ⅲ）signaling.In the classical pathway,IFN-Ⅰand IFN-Ⅲ bind to the heterodimer receptors and phosphorylate STAT2 and STAT1 via JAK1 and TYK2.Phosphorylated STAT1 and STAT2 combine with IRF9 to form the transcription factor complex ISGF3,which enters into nucleus and binds to the interferon stimulated response element of a specific gene to regulate the expression of ISGs（IFN stimulated genes）and inhibit the proliferation of pathogens and tumor cells though multiple mechanisms.During the process,the activity of STAT2 was regulated by various post translational modifications（PTMs）,which is critical for the body to rapidly respond to pathogen invasion,inhibit tumor cell proliferation,and maintain the homeostasis of the immune system.However,the functional research on the post translational modification of STAT2 in antiviral and anti-tumor processes is not comprehensive.Previously,our research group discovered the phosphorylation modification at the highly conserved STAT2 T404 site in different species.However,the function and mechanism of this phosphorylation modification in antiviral and anti-tumor processes still remains elusive.Therefore,this study aims to reveal the critical functions of STAT2 T404phosphorylation modification via in vitro and in vivo studies.Firstly,in the U6A（STAT2 null）cell model,wild-type STAT2（WT）,simulated unphosphorylated T404A（T404A）,and simulated phosphorylated T404E（T404E）mutants were stably expressed,and treated with IFN-β.The results shown that T404phosphorylation positively regulates the IFN-I signaling pathway and enhances the antiviral potential of cells.Considering the conservation of this site in different species,Stat2 T403A^-/-（same as human T404）transgenic mice were constructed.Subsequently,WT and Stat2 T403A^-/-mice derived mouse primary MEFs,BMDM,and BMDC were treated with m IFN-β.The results showed that the T403A mutation reduced the expression of ISGs and increased the susceptibility of cells to viral infection.To further explore the effect of STAT2 T403 phosphorylation modification on the expression of ISGs.RNA derived from WT and T403A^-/- BMDM after IFN-βtreatment were used to performed the RNA-seq.The results showed that STAT2 T403 phosphorylation increases the expression of most ISGs,and several unknown ISGs were discovered.Those above results indicated that T404 or T403 phosphorylation modification of STAT2 can enhance the antiviral activity of IFN-I.Secondly,WT and T403A^-/-mice were infected with VSV（RNA virus）and HSV（DNA virus）,respectively.The results showed that the mortality,weight loss,and viral load in tissues of T403A^-/-mice were significantly higher than those of WT mice.Histopathology analysis also showed that T403A^-/-mice were sensitive to viral infection.The above results indicate that STAT2T403 phosphorylation modification can upregulate the expression of ISGs and thus enhance the antiviral activity of IFN-I in vivo.Next,by analyzing the amino acid sequences,we found that the inhibitorκB kinase（IKK）family could recognize this amino acid sequence.Exogenous expression of TBK1 and IKKεinduced the STAT2T404 phosphorylation.The TBK1 and IKKεinhibitor and sh RNA treatment both downregulated the enhancement effect of STAT2 T404 phosphorylation on the IFN-Ⅰsignaling pathway.These results suggested that TBK1 and IKKεdirectly bind to STAT2and catalyze the STAT2 T404 phosphorylation.Finally,by simulating the conformation of STAT1 and STAT2 heterodimer,we found that the T404 site is located on the binding surface of the heterodimer.Based on the findings of the research group members,we hypothesized that STAT2 T404 phosphorylation positively regulates IFN-I mediated antiviral activity by accelerating the conformational change of STAT2 and STAT1dimers.Considering that STAT2 can either inhibit or promote tumorigenesis depending on the unique environment presented by each type of cancer through a variety of mechanisms,we continue to explore the role of STAT2 T403 phosphorylation in tumorigenesis.By establishing DSS and AOM/DSS induced colitis and colorectal cancer models in WT and T403A^-/-mice respectively,we found that DSS administration induced the STAT2 T403 phosphorylation.Moreover,STAT2 T403 phosphorylation could reduce the susceptibility of mice to DSS,thus decreasing the occurrence of colitis and colorectal cancer.High-throughput sequencing of RNA derived from WT and T403A^-/-mice after AOM/DSS treatment revealed that STAT2 T403 phosphorylation reduced the activity of DSS induced Wnt and PI3K signaling pathways.Growing evidences have shown that abnormally activated Wnt/PI3K signaling is closely related to the initiation and progression of several malignancies,including colorectal cancer,and the disorder of the Wnt signaling pathway is an important driving factor and one of the most representative pathological signaling pathway in colorectal cancer.These results suggest that STAT2 T403 phosphorylation may inhibit the occurrence of colitis and colorectal cancer by down-regulating DSS-induced Wnt and PI3K signaling pathways activities.In conclusion,this study found the phosphorylation modification at the highly conserved STAT2 T404 site in different species.Moreover,in vitro and in vivo studies have shown that T404 phosphorylation enhanced the potential of cells and mice to resist viral infection by up-regulating the activity of the IFN-Ⅰsignaling pathway.In addition,STAT2 T404 phosphorylation also reduced the initiation and progression of DSS-induced colitis and colorectal cancer.Finally,the activated threonine kinases TBK1 and IKKεduring viral infection and tumor progression directly bind STAT2 and catalyze the STAT2 T404 phosphorylation.Considering the important role of STAT2 in viral infection and tumor progression,these studies are expected to provide new therapeutic targets and theoretical basis for the subsequent treatment of infectious diseases and tumor related diseases.