Role of protein phosphorylation in activation of interferon-stimulated gene factors.
Possible involvement of protein phosphorylation in interferon (IFN)-mediated activation of IFN-stimulated gene factor 3 (ISGF3) was investigated. For this purpose, in vivo experiments with specific inhibitors of protein kinases and in vitro experiments with protein phosphatases were carried out. In HeLaM cells, 2-aminopurine, an inhibitor of double-stranded RNA-dependent protein kinase, blocked the induction of ISGF3 gamma subunit but not the activation of ISGF3 alpha subunit. A series of experiments using combinations of protein and RNA synthesis inhibitors and 2-aminopurine indicated that the block elicited by 2-aminopurine was at the level of ISGF3 gamma mRNA synthesis. Activation of ISGF3 alpha, although insensitive to 2-aminopurine, was completely blocked by 10 nM staurosporine, an inhibitor of protein kinase C. On the other hand, even 500 nM staurosporine did not block the induction of ISGF3 gamma. Incubation of cytoplasmic or nuclear extracts of IFN-treated HeLaM cells in vitro with alkaline phosphatase completely eliminated their ability to form the ISGF3 complex but not the ISGF1 complex. Treatment with acid phosphatase, on the other hand, changed the electrophoretic mobility of the ISGF3 complex but did not obliterate it. Complementation experiments revealed that ISGF3 alpha was the alkaline phosphatase-sensitive component of the complex. These results suggest that a protein kinase C-mediated phosphorylation step is involved in ISGF3 alpha activation and a 2-aminopurine-sensitive component is involved in ISGF3 gamma mRNA induction.
Bandyopadhyay
S K
SK
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195-5285.
Sen
G C
GC
D015075:2-Aminopurine / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D001483:Base Sequence; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D050832:Interferon-Stimulated Gene Factor 3; D050833:Interferon-Stimulated Gene Factor 3, alpha Subunit; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D016898:Interferon-alpha / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology*; D046911:Macromolecular Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*
D000470:Alkaloids; D004268:DNA-Binding Proteins; C494227:IRF9 protein, human; D050832:Interferon-Stimulated Gene Factor 3; D050833:Interferon-Stimulated Gene Factor 3, alpha Subunit; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D016898:Interferon-alpha; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D014157:Transcription Factors; D015075:2-Aminopurine; D007371:Interferon-gamma; D003513:Cycloheximide; D011494:Protein Kinases; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate