ABSTRACT
The 84-, 91-, and 113-kDa proteins of the ISGF-3 alpha complex are phosphorylated on tyrosine residues upon alpha interferon (IFN-alpha) 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-3 alpha subunits and Tyk-2 and JAK1 tyrosine kinases in mutant HeLa cells defective in the IFN-alpha/beta and -gamma 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-alpha or -gamma. The 91-kDa protein IFN-gamma-activated factor fails to be activated into a DNA-binding state after IFN-alpha or -gamma treatment. In addition, the 91-kDa protein is unable to localize in the nucleus after IFN-alpha and -gamma treatment, and the 113-kDa protein fails to translocate after IFN-alpha treatment. Immunoprecipitation studies document a failure of phosphorylation of the 84- or 91-kDa proteins after IFN-alpha or -gamma treatment. Similarly, no tyrosine-phosphorylated 113-kDa protein was detected after IFN-alpha 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-alpha and -gamma. 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-alpha/beta and IFN-gamma systems.
Subject(s)
DNA-Binding Proteins/metabolism , Interferon Type I/pharmacology , Interferon-gamma/pharmacology , Phosphoproteins/metabolism , Proto-Oncogene Proteins , Transcription Factors/metabolism , Blotting, Western , Cell Compartmentation , DNA-Binding Proteins/chemistry , Fluorescent Antibody Technique , Gene Expression Regulation , Genetic Complementation Test , HeLa Cells , Humans , In Vitro Techniques , Interferon-Stimulated Gene Factor 3 , Interferon-Stimulated Gene Factor 3, gamma Subunit , Janus Kinase 2 , Molecular Weight , Phosphoproteins/chemistry , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/physiology , Proteins/metabolism , RNA, Messenger/genetics , Signal Transduction , TYK2 Kinase , Transcription Factors/chemistryABSTRACT
We have used a herpes virus thymidine kinase (HSV-TK) based metabolic selection system to isolate mutants defective in the interferon gamma mediated induction of the MHC class II promoter. All the mutations act in trans and result in no detectable induction of MHC and invariant chain (Ii) gene expression. Scatchard analysis indicates that the mutants have a normal number of surface IFN gamma receptors with the same affinity constant. The mutants fall into two broad categories. One class of mutants is still able to induce MHC class I, IRF-1, 9-27, 1-8 and GBP genes by IFN gamma. A second class of mutants is defective for the IFN gamma induction of all the genes tested; surprisingly, the IFN alpha/beta induction of MHC class I, 9-27, ISG54 and ISG15 genes is also defective in these mutants, although different members of this class can be discriminated by the response of the GBP and IRF-1 genes to type I interferons. These data demonstrate that the signalling pathways of both type I and type II interferon systems share common signal transduction component(s). These mutants will be useful for the study of IFN gamma regulation of class II genes and Ii chain, and to elucidate molecular components of type I and type II interferon signal transduction.
Subject(s)
Genes, MHC Class II , Interferon Type I/physiology , Interferon-gamma/physiology , Mutagenesis , Signal Transduction/immunology , Base Sequence , DNA Probes , Ethyl Methanesulfonate/pharmacology , Genes, Viral , HLA-D Antigens/analysis , HLA-D Antigens/genetics , HeLa Cells , Humans , Major Histocompatibility Complex , Molecular Sequence Data , Oligodeoxyribonucleotides , Plasmids , Promoter Regions, Genetic , Restriction Mapping , Simplexvirus/enzymology , Simplexvirus/genetics , Thymidine Kinase/genetics , Transcription, Genetic , Transfection , Viral Structural Proteins/geneticsABSTRACT
We have investigated the role of interferon regulatory factor-1 (IRF-1), an interferon-gamma (IFN-gamma) inducible transcriptional activator, on major histocompatibility complex (MHC) class I gene transcription. IRF-1 alone is sufficient to trans-activate both transfected and endogenous class I genes and the effect of IRF-1 appears to be direct and sequence-specific. These data suggest that IRF-1 is involved in the IFN-gamma mediated induction of MHC class I expression.