A role for casein kinase II phosphorylation in the regulation of IRF-1 transcriptional activity.
The Interferon Regulatory Factors (IRFS) play an important role in the transcriptional control of growth regulatory and immunoregulatory genes. The inducibility and availability of IRF-1 and IRF-2 are influenced by external stimuli, such as virus infection or interferon treatment. In the present study, we sought to examine the potential modulatory role of phosphorylation on IRF-1 transcriptional activity. During the purification of IRF recombinant proteins, a kinase activity copurified with IRF-1 (and IRF-2) from baculovirus infected Sf9 insect cell extracts, but not from E. coli extracts. The kinase activity was also identified in Jurkat T cells, specifically interacted with IRF proteins in GST affinity chromatography, and phosphorylated IRF-1 with high specificity in vitro. Using an in gel kinase assay with recombinant IRF-1 as substrate, two molecular weight forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Furthermore, far western analysis of protein-protein interactions demonstrated that casein kinase II directly interacted with IRF-1 protein. Deletion mutation analysis of IRF-1 revealed that IRF-1 was phosphorylated at two clustered sites, one located between amino acids 138-150, the other in the C-terminal acidic activation domain between amino acids 219-231. Cotransfection studies comparing wild type and point mutated forms of IRF-1 demonstrated that mutations of the four phosphoaceptor residues in the C-terminal transactivation domain, significantly decreased transactivation by IRF-1, indicating that casein kinase II may be involved in the regulation of IRF-1 function. Strikingly, the casein kinase II clusters in IRF-1 resemble the sites identified in the C-terminal PEST domain of IkappaBalpha. The present experiments, together with previously published studies with IkappaBalpha, c-Jun and other proteins, indicate a broad role for casein kinase II phosphorylation in the regulation of transcription factor activity.
Lin
R
R
Lady Davis Institute for Medical Research, and Department of Medicine, McGill University, Montreal, Quebec, Canada.
Hiscott
J
J
D015153:Blotting, Western; D047390:Casein Kinase II; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D050856:Interferon Regulatory Factor-1; D007370:Interferon Type I / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D007370:Interferon Type I; D010750:Phosphoproteins; D014157:Transcription Factors; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases