During IFN-α signaling, the activated STAT1 dimer and ISGF3 (STAT1:STAT2:p48) complex each acquires exposed nuclear localization signals. Importin-α/β complex recognizes these signals and induces translocation of the STAT complexes into the nucleus 35, 38, 39. However, no mechanisms governing the nuclear localization Nivolumab of STAT6 have been identified

yet, except that the translocation of STAT6 depends on its phosphorylation on Y641 39. In this regard, we have noted that the cytoplasmic retention complex containing pY-STAT6 did not interact with importin-α (Supporting Information Fig. S3, left panel). Interestingly, with increased association of pY-STAT6 with pY-STAT2 and p48 during IFN-α treatment from 0.5 to 4 h, there is a decreased interaction of pY-STAT1 with pY-STAT2 and p48 (Supporting Selleck Tyrosine Kinase Inhibitor Library Information Fig. S3, right panel). The observation raises a possibility that by the action of IFN-α-induced factors during 4 h treatment, pY-STAT1 gradually dissociates from the ISGF3 complex, which is then replaced with IL-4-activated pY-STAT6. This would result in the sequestration of STAT6 from the translocatable STAT6 homodimer to form the putative pY-STAT6:pY-STAT2:p48 complex incapable of importin binding and nuclear translocation, which is then retained in the cytosol. On the other hand,

it is also possible that pY-STAT6 is accumulated in the cytoplasm upon the inhibition of translocation mediated by IFN-α-induced factors, which then interacts with pY-STAT2 and p48. Several post-translational modifications other than tyrosine phosphorylation may be involved in the formation of the STAT complex retained in the cytosol, since STAT6 and/or STAT2 are thought to undergo serine/threonine

phosphorylation, acetylation, and sumoylation. Yet, in our experimental system, we have observed no Celecoxib detectable changes in these modifications on STAT6 or STAT2 by IFN-α and IL-4, which suggests that such post-translational modifications may not play a role in the molecular interaction and cytosolic accumulation of the STAT complex. As shown by the inhibition of the IL-4-induced CD23 expression and the IFN-α-induced IRF7 expression, a novel feature of the IFN-α and IL-4-induced cross-signaling found in the present study is the cytoplasmic co-retention of activated STATs (Figs. 3, and 4). By coimmunoprecipitation experiments, we have verified the molecular interaction among pY-STAT6, pY-STAT2, and p48 induced in cells upon treatment with IL-4 and IFN-α (Fig. 5A), which strongly suggests a possibility that these proteins are present in a molecular complex. To further examine the possibility that the inhibition by IFN-α and IL-4 is mediated via the formation and cytoplasmic retention of the pY-STAT6:pY-STAT2:p48, the effect of STAT over-expression was analyzed.