Nfar1 Stability Of Il-2 Mrna In The Upstream Regulatory Mechanism

The interleukin-2 mRNA is a labile transcript containing AU-rich elements(ARE)that is transiently stabilized by CD28 receptor signaling. The ARE-binding proteins(AUBPs)bind to mRNAs containing an ARE in their 3' untranslated region and promote or repress their deadenylation and rapid degradation.Independent signal transduction pathways have been reported to stabilize ARE-containing transcripts by a process thought to involve phosphorylation of ARE-binding proteins.NFARI is an AUBPwhich binds and stabilizes IL-2 mRNA.Inspection of the coding sequence of NFAR-1 revealed the presence of one AKT consensus phosphorylation sites-Serine 647.Here we report that AKT associates with NFARI in vivo with colocalization in the nucleus of the cell.Experiments in vitro and in vivo shows AKT phosphorylates NFARI on Serine 647.To evaluate the physiology of AKT phosphorylating NFAR-I,we observed the difference of IL-2 stabilization between NFARI,NFARI-S647A and NFAR1-S647E,which mocked S647 is phosphoried.The wild type stabilized IL-2 mRNA as described before,while NFARI-S647A IL-2 mRNA decayed just like cmv-Myc expressed.When we transfected the NFARI-S647E plasmid by the same procedure,IL-2mRNA was even more stable than NFAR1 was transfected.Otherwise,we found IL-2 mRNA t_1/2of wild type and AKT co-transfected is longer than NFARIS647A and AKT co-transfected. Furthermore,NFARI stabilzing IL-2 mRNA is sensitive to LY290042,the inhibitor of PI3-K.But for NFAR-IS647A,LY290042 seems to have no effect on IL-2 mRNA stabilization.From these data,we concluded that AKT phosphorylates NFARI on S647 for IL-2 mRNA stabilization.It has been well reported that CD28 costimulation leads to a dramatic up-regulation in IL-2 expression mediated by both enhanced transcription and induction of mRNA stabilization.We were curious to know whether CD28 costimulation is concerned with NFARl phosphorylation by AKT and the role of NFARI S647 phosphorylation in CD28-induced T cell activation.We observed treatment with anti-CD28 induced distinct increase in phorsphorylation of NFARI at Ser647 and AKT phosphorylation was up-regulated coincidently.Coincubation anti-CD28 with the LY294002 prevented CD28-mediated increase of AKT activity and phosphorylation of NFARI.By Radioimmunoaseey,we found NFAR1 and NFARI-S647A had distinct influence on posttranscription regulation.While NFAR1 stabilized CD28-mediated IL-2 mRNA and promoted the secretion of IL-2 protein,there was no stabilization of IL-2 mRNA observed as NFAR1S647A is concerned. This result illuminates NFARI S647A phosphorylation is the key point in NFARI stabilizing IL-2 mRNA by CD28 costimulation,which is consistent with the data above.