Mutant cell lines unresponsive to alpha/beta and gamma interferon are defective in tyrosine phosphorylation of ISGF-3 alpha components

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.

[1]  G. Stark,et al.  Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon‐alpha and ‐gamma signal transduction pathways. , 1993, The EMBO journal.

[2]  B. Mach,et al.  Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome) , 1993, Cell.

[3]  J. Dixon,et al.  In vitro activation of the transcription factor ISGF3 by interferon alpha involves a membrane-associated tyrosine phosphatase and tyrosine kinase. , 1993, The Journal of biological chemistry.

[4]  M. David,et al.  In vitro activation of the transcription factor gamma interferon activation factor by gamma interferon: evidence for a tyrosine phosphatase/kinase signaling cascade , 1993, Molecular and cellular biology.

[5]  Y. Koide,et al.  Inhibition of tyrosine phosphorylation prevents IFN-gamma-induced HLA-DR molecule expression. , 1993, Journal of immunology.

[6]  J. Darnell,et al.  Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein. , 1992, Science.

[7]  N. Reich,et al.  Tyrosine phosphorylation is required for activation of an alpha interferon-stimulated transcription factor. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. Aebersold,et al.  Proteins of transcription factor ISGF-3: one gene encodes the 91-and 84-kDa ISGF-3 proteins that are activated by interferon alpha. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[9]  R. Aebersold,et al.  The proteins of ISGF-3, the interferon alpha-induced transcriptional activator, define a gene family involved in signal transduction. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[10]  J. Darnell,et al.  Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. , 1992, Science.

[11]  Xin-Yuan Fu.  A transcription factor with SH2 and SH3 domains is directly activated by an interferon α-induced cytoplasmic protein tyrosine kinase(s) , 1992, Cell.

[12]  R. Flavell,et al.  Dissection of the interferon gamma‐MHC class II signal transduction pathway reveals that type I and type II interferon systems share common signalling component(s). , 1992, The EMBO journal.

[13]  A. Wilks,et al.  Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase , 1991, Molecular and cellular biology.

[14]  D. Lew.  Cytoplasmic activation ofGAF,an IFN-'y-regulated DNA-binding factor , 1991 .

[15]  I. Kerr,et al.  High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both α and γ interferons , 1991 .

[16]  D. Levy,et al.  Interferon-alpha regulates nuclear translocation and DNA-binding affinity of ISGF3, a multimeric transcriptional activator. , 1990, Genes & development.

[17]  J. Darnell,et al.  Interferon-dependent transcriptional activation: signal transduction without second messenger involvement? , 1990, The New biologist.

[18]  R. Margolskee,et al.  Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector , 1990, Molecular and cellular biology.

[19]  J. Darnell,et al.  Cytoplasmic activation of ISGF3, the positive regulator of interferon-alpha-stimulated transcription, reconstituted in vitro. , 1989, Genes & development.

[20]  J E Darnell,et al.  Interferon-induced nuclear factors that bind a shared promoter element correlate with positive and negative transcriptional control. , 1988, Genes & development.

[21]  S. de la Luna,et al.  Efficient transformation of mammalian cells with constructs containing a puromycin-resistance marker. , 1988, Gene.

[22]  T. Maniatis,et al.  Activation of the human beta-interferon gene requires an interferon-inducible factor , 1986, Molecular and cellular biology.

[23]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[24]  Induction of the Ly-6A/E gene by interferon a/,8 and y requires a DNA element to which a tyrosine-phosphorylated 91-kDa protein binds , 2022 .