STAT-phosphorylation-independent induction of interferon regulatory factor-9 by interferon-beta.
Type I interferon (IFN)-dependent STAT1 and STAT2 activation requires specific tyrosine residues (337Y and 512Y) located in the cytoplasmic domain of IFNAR-2c, the beta-subunit of the human type I IFN receptor. To identify STAT activation-independent induction of ISGs, we used a mutant cell line in which both 337Y and 512Y were substituted with phenylalanine (337F512F or FF mutant). In these cells, type I IFN failed to activate STAT1, STAT2, and STAT3 did not induce well-characterized ISGs and did not exert antiviral or antiproliferative effects. Using Oligonucleotide array (Affymetrix) analysis, we showed that interferon regulatory factor-9 (IRF-9) was the only gene induced by IFN-beta in FF cells. Transient transfection analysis using an IRF-9 promoter-reporter luciferase construct in FF cells confirmed induction of the IRF-9 transcription unit by IFN-beta. EMSA analysis using an IFN-stimulated response element (ISRE)-like sequence on the IRF-9 promoter detected 2 novel DNA-binding complexes induced in nuclear extracts of IFN-beta-treated FF cells. Supershift experiments identified the proteins IRF-1 and C/EBP-beta in the complex. These studies provide the first evidence that signaling pathways leading to gene transcription are activated by IFN-beta independent of STAT phosphorylation.
Rani
M R Sandhya
MR
Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Croze
Ed
E
Wei
Tao
T
Shrock
Jennifer
J
Josyula
Anupama
A
Kalvakolanu
Dhananjaya V
DV
Ransohoff
Richard M
RM
D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit / Q000378:metabolism*; D016899:Interferon-beta / Q000235:genetics / Q000494:pharmacology*; D046228:Microarray Analysis; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D050791:STAT Transcription Factors; D015398:Signal Transduction
D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D050791:STAT Transcription Factors; D016899:Interferon-beta