FIGURE 5.

Priming phosphorylation of IFNAR1 contributes to regulation of the extent of IFNα/β signaling. A, control (Con) human Huh7 cells and those expressing the HCV replicon were analyzed for IFNAR1 levels by immunoprecipitation-immunoblotting (upper panel). The lower three panels depict the experiments where gel loading was normalized to achieve comparable levels of immunopurified IFNAR1 in each lane. Phosphorylation of IFNAR1 on Ser⁵³² and Ser⁵³⁵ was determined by immunoblotting using the indicated antibodies. B, control human Huh7 cells and those expressing the HCV replicon were transfected with FLAG-tagged STAT1 alone with empty vector (Vec) or FLAG-IFNAR1 (wild type (WT) or S532A mutant) and were untreated or treated with IFNα (60 IU/ml for 30 min) as indicated. Lysates of these cells were immunoprecipitated (IP) using anti-FLAG antibody and analyzed by immunoblotting using antibodies against phospho-STAT1, total STAT1, and IFNAR1. C, mouse embryo fibroblasts from IFNAR1-null animals were reconstituted with murine FLAG-IFNAR1 (wild type or S523A mutant, which is a mouse analogue of human S532A mutant). The cells were treated with indicated doses of murine IFNβ for 1 h, incubated for 8 h in fresh medium, and then infected with VSV (multiplicity of infection of 0.1). Expression of VSV-M protein was analyzed 16 h later by immunoblotting. Levels of β-actin were also determined (lower panel). D, model for ligand-dependent and ligand-independent ubiquitination and degradation of IFNAR1. Both pathways converge at the level of degron phosphorylation (Ser(P)⁵³⁵, pS535). Signaling induced by IFN and dependent on the activity of Tyk2 does not require either CK1α (24) or priming phosphorylation (this study). Ligand-independent pathway initiated by inducers of UPR does not need either ligand or Tyk2 activity but requires CK1α (26) and PERK-dependent priming phosphorylation (this study).