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 tran scriptional control of growth regulatory and immunoregulatory genes. The in ducibility and availability of IRF-1 and IRF-2 are influenced by external s timuli, such as virus infection or interferon treatment. In the present stu dy, we sought to examine the potential modulatory role of phosphorylation o n IRF-1 transcriptional activity. During the purification of IRF recombinan t proteins, a kinase activity copurified with IRF-1 (and IRF-2) from baculo virus infected Sf9 insect cell extracts, but not from E. coli extracts. The kinase activity was also identified in Jurkat T cells, specifically intera cted with IRF proteins in GST affinity chromatography, and phosphorylated I RF-1 with high specificity in vitro. Using an in gel kinase assay with reco mbinant IRF-1 as substrate, two molecular weight forms of the kinase (43 an d 38 kDa) were identified. Biochemical criteria identified the kinase activ ity as the alpha catalytic subunit of casein kinase II (CKII). Furthermore, far western analysis of protein-protein interactions demonstrated that cas ein kinase II directly interacted with IRF-1 protein. Deletion mutation ana lysis of IRF-1 revealed that IRF-1 was phosphorylated at two clustered site s, one located between amino acids 138-150, the other in the C-terminal aci dic activation domain between amino acids 219-231. Cotransfection studies c omparing wild type and point mutated forms of IRF-1 demonstrated that mutat ions of the four phosphoaceptor residues in the C-terminal transactivation domain, significantly decreased transactivation by IRF-1, indicating that c asein kinase II may be involved in the regulation of IRF-1 function. Striki ngly, the casein kinase LI clusters in IRF-1 resemble the sites identified in the C-terminal PEST domain of I kappa B alpha [129]. The present experim ents, together with previously published studies with I kappa B alpha, c-Ju n and other proteins, indicate a broad role for casein kinase II phosphoryl ation in the regulation of transcription factor activity.