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.
Loh
J E
JE
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510.
Schindler
C
C
Ziemiecki
A
A
Harpur
A G
AG
Wilks
A F
AF
Flavell
R A
RA
D015153:Blotting, Western; D002451:Cell Compartmentation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007370:Interferon Type I / Q000494:pharmacology*; D050832:Interferon-Stimulated Gene Factor 3; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D007371:Interferon-gamma / Q000494:pharmacology*; D053614:Janus Kinase 2; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction; D053634:TYK2 Kinase; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; C494227:IRF9 protein, human; D007370:Interferon Type I; D050832:Interferon-Stimulated Gene Factor 3; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D007371:Interferon-gamma; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D053634:TYK2 Kinase; C070139:TYK2 protein, human