Although the activation domains within early growth response gene protein 1 (Egr-1) have been mapped, little is known of the kinases which phosphorylate Egr-1 and how phosphorylation correlates with the transcriptional activity of Egr-1. In this study we report that casein kinase II (CKII) co-immunoprecipitates with Egr-1 from NIH 3T3 cell lysates. The association of Egr-1 and CKII requires the C terminus of Egr-1 and CKII phosphorylates Egr-1 in vitro. The in vitro phosphorylation of Egr-1 by

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... KII and that induced by serum in vivo was compared by examining the CNBr-digested fragments of the phosphorylated Egr-1. CKII strongly phosphorylates fragments 7 and 10 which cover part of the activation/nuclear localization and DNA binding domains of Egr-1. CKII also phosphorylates, albeit weakly, fragments 5 and 8 which cover part of activation domain and the entire repression domain of Egr-1, respectively. Strong phosphorylation on fragment 10 as well as fragment 5 was also observed in Egr-1 immunoprecipitated from serum-induced, 32 P-labeled cells. CKII phosphorylation of Egr-1 resulted in a decrease of its DNA binding as well as its transcriptional activities. egr-1 1 (1), also known as krox-24 (2), zif268 (3), Tis8 (4), and NGFI-A (5), is an immediate-early gene. It encodes a transcription factor with three zinc fingers and recognizes a GC-rich sequence, 5Ј-CGCCCCCGC-3Ј (6) which has been identified in the promoter regions of a number of genes. Egr-1 regulates these genes by binding to this consensus sequence. Besides inducing genes involved in cell proliferation, Egr-1 is also implicated in a number of differentiation processes in cardiac (7) , neural (1), osteoblast (8), and myeloid cells (9). It is likely that the different effects mediated by Egr-1 in various cells result from its phosphorylation and dephosphorylation (10). Reports that cells treated with kinase and phosphatase inhibitors (11, 12) show differences in their Egr-1 DNA binding ability support this hypothesis. However, little is known about which kinase(s) phosphorylates Egr-1 and if its functional domain(s) is phosphorylated. In addition, the identification of a repressor protein that associates with Egr-1 implies that protein-protein interactions can also regulate its transcriptional activity (13, 14) . The protein kinase CKII is an inducible kinase found in both the cytoplasmic and nuclear compartments, although recently CKII was reported to be a predominantly nuclear protein (15). It is a tetrameric Ser/Thr-specific protein kinase complex containing two catalytic (␣, 44 kDa, ␣Ј, 42 kDa) subunits and two regulatory (␤, 25 kDa) subunits (15). CKII is known to associate with a number of nuclear proteins such as DNA topoisomerase II (16) and FKBP25, the 25-kDa FK506-binding protein (17). CKII also phosphorylates a number of transcription factors and nuclear proteins such as the serum response factor (18), c-Jun (19) , and p53 (20) . Little is known of the physiological role of CKII or how it regulates the activity of transcription factors in vivo. In this report, we demonstrate that CKII associates with cellular Egr-1 both in vivo and in vitro and phosphorylates it in vitro. We have also identified the CKII phosphorylated CNBr peptides of Egr-1 and have studied the effect of CKII phosphorylation on Egr-1 DNA binding activity in vitro and its transcriptional activity in vivo.