Interaction Between C-Abl And STAT1is Required For Full-Fledged Immunity Regulation Of IFNγ

The c-abl gene, which encodes a non-receptor protein tyrosine kinase, localizes on humanchromosome9. The gene was originally identified as the cellular homolog of the v-abl oncogeneof Abelson murine leukemia virus (A-MULV). The mammalian c-Abl protein, which was foundlocalized in multiple subcellular compartments, can be activated by many signals. c-Abl isimplicated in the regulation of many celluar processes such as cell proliferation, cytoskeletalreorganization, cell adhesion, oxidative and DNA damage stress responses, cell apoptosis, celltransformation, and cell migration. Targeted deletion of the c-abl gene in mice resulted inpleiotropic phenotypes including high perinatal lethality, immune deficiencies, splenic andthymic atrophy, lymphopenia, and increased susceptibility to infection. c-Abl kinases also havea role in the regulation of TCR and BCR-mediated signal transduction. The c-abl mutant miceexhibited impaired TCR-induced signaling, proliferation, and cytokine production. The BCRcoreceptor CD19is also a substrate of c-Abl. However, the molecular mechanisms by whichc-Abl was involved in the regulation of immune system have remained elusive.Our previous transcriptome data showed that the transcription level of inducible subunitMecl-1and regulatory subunit PA28α of immunoproteasome, which is mostly induced by IFN-γ,was decreased in c-Abl knockdown cells. Immunoproteasomes play very important role in theadaptive immune system. IFNγ, a type II IFN produced by activated T cells and natural killercells, is involved in anti-virus, immune surveillance, immunomodulatory and anti-tumor. Thebiological effects of IFNγ are elicited through activation of intracellular molecular signalingnetworks, the best characterized of which is the JAK–STAT pathway. After IFNγ binds withIFNγ receptors (IFNGR1and IFNGR2), JAK2is firstly autophosphorylated and then leads toJAK1transphosphorylation. Then the activated JAK1phosphorylates IFNGR1at Tyr440, andresult in the recruitment and activation of STAT1, probably through JAK2-mediatedphosphorylation at Tyr701. STAT1(Ser727) is also phosphorylated in the IFNγ signaltransduction. Phosphorylation of STAT1induces the formation of homodimer and releasingfrom the receptors. The dissociated STAT1homodimers enter the nucleus and bind to a GASconsensus sequence in the promoter of IFNγ responsive genes.In this study, we first validated transcriptome data by quantitative PCR. In the c-Ablknockdown cells, IFNγ can not effectively stimulate transcription of the inducible subunit Lmp2and regulatory subunit PA28α of the immunoproteasome. The transcription level of transporters associated with antigen processing (Tap1) was also found decreased in c-Abl deficient cells.Lmp2and Tap1genes are transcribed from a shared bidirectional promoter and regulated byINFγ. To assess the effect of the c-Abl on the IFNγ responsive genes promoter, the promoter inthe direction of Lmp2was cloned into the upstream of the luciferase gene. The luciferasereporter assay of wild type and mutated promoter showed that c-Abl regulated the transcriptionof Lmp2through GAS box, which is a target site for STAT1that are involved in the IFNγcanonical signaling pathway—JAK-STAT1pathway. These data suggested that c-Abl mayaffect the responsive gene expression of IFNγ through the regulation of STAT1.The c-Abl was collectively demonstrated interacted directly with STAT1byco-immunoprecipitation, GST-pull down, Far Western-blotting assay and proximity ligationassays (PLA). It was found that the SH2domain contribute to the interaction c-Abl with the Cterminus of STAT1. Further, we found that the interaction of c-Abl with STAT1was potentiatedby IFNγ in a dose dependent manner. The phosphorylation of STAT1(Tyr701) andSTAT1(Ser727) was found significantly decreased in c-Abl null MEFs compared to wild typecells. We then demonstrated that STAT1was a direct kinase-substrate of c-Abl by in vitrokinase assays, anti-phosphorylated tyrosine immunoblotting, as well as LC-MS/MS. By usingJAK2deficient cells or JAK1/2specific inhibitors, c-Abl was showed inducing STAT1phosphorylation in the absence of JAK kinases. c-Abl mediated phosphorylation of STAT1resulted in a increase of cellular STAT1level, homodimer formation and nucleus translocation.Consequently, c-Abl not only positively regulates Lmp2and Tap1transcription, but alsopromotes mRNA expression of other IFNγ-responsive genes such as Ip10and Isg15. Exposureof the JAWS II dendritic cell line to c-Abl selective inhibitor STI571reduced the ability of thecells to present OVA to a SIINFEKL-specific CD8+T-cell hybridoma. Moreover, c-Abl wasalso found modulated IFNγ-induced antiviral responses and virus proliferation in cultured cells.In the presense of IFNγ, WT MEFs were found more resistant to VSV induced cell death thanc-Abl knockdown MEFs or the cells treated with STI571. Further, viral proliferation wasinvestigated by GFP expressing NDV model. Compared with WT MEFs, siganificantly morecells were found infected by NDV by STI571treatment, while the overexpression of c-Abl caninhibit virus proliferation to a certain extent.In summary, we showed that c-Abl can specifically phosphorylate STAT1, and then regulatethe expression of IFNγ responsive genes, and finally play an importantly role in the antiviraleffect of IFNγ. To our knowedlege, it is the first time to show that a kinase other than JAK1/2was involved in the phosphorylation of STAT1.