Mutant Cell Lines Unresponsive to Alpha/Beta and Gamma Interferon Are Defective in Tyrosine Phosphorylation of ISGF-3α Components

Abstract

The 84-, 91-, and 113-kDa proteins of the ISGF-3α complex are phosphorylated on tyrosine residues upon alpha interferon (IFN-α) treatment and subsequently translocate to the nucleus together with a 48-kDa subunit. In this study, we investigated the presence and the functional status of ISGF-3α subunits and Tyk-2 and JAK1 tyrosine kinases in mutant HeLa cells defective in the IFN-α/β and -γ response. Stable cell fusion analysis revealed a single complementation group among one class (class B) of mutants. The class B mutants contain detectable level of mRNA and proteins of the 84-, 91-, and 113-kDa proteins, but neither the protein nor mRNA is inducible by IFN-α or -γ. The 91-kDa protein IFN-γ-activated factor fails to be activated into a DNA-binding state after IFN-α or -γ treatment. In addition, the 91-kDa protein is unable to localize in the nucleus after IFN-α and -γ treatment, and the 113-kDa protein fails to translocate after IFN-α treatment. Immunoprecipitation studies document a failure of phosphorylation of the 84- or 91-kDa proteins after IFN-α or -γ treatment. Similarly, no tyrosine-phosphorylated 113-kDa protein was detected after IFN-α treatment. The inability of class B mutants to phosphorylate the 84-, 91-, or 113-kDa protein on tyrosine residues correlated with the loss of biological response to IFN-α and -γ. The genetic defect appears to be the absence of the tyrosine kinase JAK1. Our data therefore confirm a recent report that JAK1 plays a critical early signaling role for both IFN-α/β and IFN-γ systems.