Regulatory Mechanism Of STING Tyrosine Phosphorylation And Its Role In Antiviral Innate Immune Response

ObjectivesSTING molecule plays a very important regulatory role in the process of antiviral innate immune response and is one of the key molecules of innate immunity.However,the current studies on the activation of STING molecule mainly focus on its serine/threonine phosphorylation.Recent studies have shown that STING tyrosine phosphorylation is also involved in the antiviral response,but its molecular mechanism remains to be fully clarified.In this study,we conducted an in-depth investigation of the effects and regulatory mechanisms of the tyrosine phosphorylation of STING in the antiviral immune response,which provided evidence and foundation for the thorough elucidation of the antiviral pathway mechanism and the efficient manipulation of virus infection related diseases.MethodsThrough STRING database screening and experimental verification,tyrosine kinases and phosphatases that may regulate STING molecules were identified,and their effects and mechanisms were further studied.The cellular model of inhibition of JAK2 signaling constructed by using Fedratinib,a specific inhibitor of JAK2 molecule,and JAK2-specific small interfering RNA.The effect of tyrosine kinase and phosphatase on STING tyrosine phosphorylation were investigated by western blot,real-time PCR,and further verified by the data from the mutant construct with the tyrosine phosphorylation mutantion or non-phosphorylation mutation.Through multi-species sequence alignment and conservation analysis of STING,tyrosine sites in STING protein with strong conservation were mutated from tyrosine to alanine one by one construct the STING mutant plamid for further investigation.Results1.Database screening and experimental verification reveals that JAK2 may be a kinase involved in the regulation of STING tyrosine phosphorylation.The correlation between the expression levels of candidate kinase and STING molecules was analyzed by the STRING database and the web-based software TIMER:Tumor IMmune Estimation Resource,which showed that the expression level of JAK2 and STING was significantly positively correlated.Fedratinib,the specific inhibitor of JAK2,is applied for the inhibition of JAK2.After the activity of JAK2 tyrosine kinase was inhibited by Fedratinib or siRNA against JAK2,the tyrosine phosphorylation level of STING molecule was significantly down-regulated,as well as the downstream signal molecules p-TBK1,P-IRF3.The production of IFN-β and expression of ISG genes were also significantly down-regulated,which indicated the positive regulation of STING signaling by JAK2 in the anti-viral immune response.2.PTPN2 induces dephosphorylation of STING and negatively regulats the antiviral immune response.The possible tyrosine phosphatase of STING was screened by the small interference sequences of PTPN family molecules and further verified by the experiments in the antiviral immune reaction.Our data showed that PTPN2 and PTPN12 could negatively regulate the tyrosine phosphorylation level of STING and the STING-mediated antiviral signaling pathway.In addition,PTPN2 could interact with STING,which suggested that PTPN2 might be the tyrosine phosphatase to induce tyrosine de-phosphorylation of STING during the antiviral immune response.3.The tyrosine phosphorylation of Y274 residue of STING was significantly involved in the STING-mediated antiviral immune response.(1)The sequence alignment of eight species of STING and the conservation analysis of the candidate tyrosine residues were carried out,and alanine scanning was performed for further functional verification of the candidate conserved tyrosine residues.The Y274 residue of STING was screened out as a candidate tyrosine phosphorylation site of STING during the antiviral immune response.(2)The 17 tyrosine site mutants of STING molecule were overexpressed,and then the activation of downstream pathway was compared with that of the wild type STING molecule,respectively.The data showed that the phosphorylation level of STING/Y274A mutant was significantly decreased compared with that of wild type STING.The tyrosine phosphorylation signal of STING/Y274A mutant was completely eliminated as detected by the STING/Y274 site-specific antibody.(3)STING/Y274A and STING-WT constructs were transfected into HEK293T cells and were directly activated by 2’,3’-cGAMP.dsDNA,HSV-1,ISD were used to activate cGAS,and the wild type STING and STING/Y274A mutants were indirectly activated by these stimulation.The STING/S162A mutant construct was sensitive to DMXAA(mouse STING specific agonist),and was further used for the verification of the effect of STING/Y274A mutant.All the above experimental data showed that STING/Y274 residue was phosphorylated and it further positively regulated the antiviral signaling pathway.The construction of the permanent STING/Y274 phosphorylation mutant and permanent STING/Y274 non-phosphorylation mutant of STING/Y274 further verified the tyrosine phosphorylation of STING/Y274 during antiviral immune response.Furthermore,the mutation of aspartate and alanine at the STING/Y274 site further verified the phosphorylation of this site and the positive regulation of antiviral signaling pathway.Conclusions and innovations1.This study revealed for the first time the regulatory effect of JAK2 on the tyrosine phosphorylation of STING,which further promote the activation of STING pathway in the antiviral process.2.This study revealed that STING molecule phosphorylation at tyrosine 274 and this site mediated antiviral immune response,which provided new targeted site for the clinical development of effective small molecule compounds and potential strategies for the manipulation of virus infection related diseases.3.This study revealed for the first time that PTPN2 negatively regulates the tyrosine phosphorylation level of STING and further attenuated the antiviral immune response.4.This project aims to develop a specific inhibitor of PTPN2 molecule,thus providing a potential therapeutic strategy for the manipulation of viral infection related diseases.Fedratinib,as a specific inhibitor of JAK2,has a strong clinical application potential to inhibit the excessive increased release of IFN-β caused by viral infection to avoid unnecessary inflammation and tissue damage.