Purification, cloning, and expression of a murine phosphoprotein that binds the kappa B motif in vitro identifies it as the homolog of the human heterogeneous nuclear ribonucleoprotein K protein. Description of a novel DNA-dependent phosphorylation process.
The kappa B enhancer element regulates expression of many genes involved in immune responses and other processes. kappa B motif binds a number of proteins, some but not all, are related to the NF-kappa B family of transcription factors. We have previously identified a 65-kDa phosphoprotein that is specifically recognized by the kappa B motif (Ostrowski, J., Sims, J. E., Sibley, C. H., Valentine, M. A., Dower, S. K., Meier, K. E., and Bomsztyk, K. (1991) J. Biol. Chem. 266, 12722-12733). This protein is closely associated with a serine/threonine kinase that is responsive to treatment of cells with interleukin-1 and other agents. We report here purification, cloning, and expression of this kappa B motif-binding phosphoprotein. The primary structure deduced from the isolated murine cDNA, identifies the protein as the homolog of the human heterogeneous nuclear ribonucleoprotein K protein. Antipeptide antibodies and expression of the cloned cDNA in Escherichia coli, demonstrated that the K protein is the authentic phosphoprotein that binds the kappa B motif in vitro. We also demonstrate that the in vitro phosphorylation of the natural and the recombinant K proteins by the associated kinase is stimulated by the kappa B motif.
Ostrowski
J
J
Department of Medicine, University of Washington, Seattle 98195.
Van Seuningen
I
I
Seger
R
R
Rauch
C T
CT
Sleath
P R
PR
McMullen
B A
BA
Bomsztyk
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D047390:Casein Kinase II; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D034441:Heterogeneous-Nuclear Ribonucleoproteins; D016328:NF-kappa B; D010750:Phosphoproteins; D012261:Ribonucleoproteins; D004247:DNA; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases