Characterization of the catalytic subunit of casein kinase II expressed in Escherichia coli and regulation of activity.
The catalytic (alpha) subunit of casein kinase II from Drosophila, cloned and expressed in Escherichia coli (Saxena, A., Padmanabha, R., and Glover, C. V. C., (1987) Mol. Cell. Biol. 7, 3409-3417), has been purified and characterized, and the properties have been compared to those of the holoenzyme. The catalytic subunit exhibits protein kinase activity with casein as substrate and is autophosphorylated. The specific activity of the purified subunit is 6% of the activity of the holoenzyme from reticulocytes or from Drosophila. The alpha subunit is a monomer, eluting at Mr = 40,000 upon gel filtration in high salt, but as part of an aggregate in low salt. The alpha subunit has been purified to apparent homogeneity by sequential chromatography on DEAE-cellulose, Mono S, and Mono Q. A single band, Mr = 37,000, is detected by silver staining following polyacrylamide gel electrophoresis. The isolated alpha subunit displays apparent Km values for beta casein, ATP, and GTP similar to those of the holoenzyme. The activity of the alpha subunit is inhibited by heparin with an I50 of 0.1-0.3 micrograms/ml, a value similar to that observed for the holoenzyme; autophosphorylation is also inhibited by heparin. Polylysine has no stimulatory effect on the activity of the catalytic subunit, as measured with casein and by autophosphorylation, but stimulates both activities with the holoenzyme. When physiological substrates for casein kinase II are examined, glycogen synthase and eukaryotic initiation factor 3 (eIF-3) (p120) are phosphorylated by the alpha subunit at a rate equivalent to that of the holoenzyme, while phosphorylation of eIF-3 (p67) is reduced 9-fold and eIF-2 beta is not modified. From these data, it can be concluded that the alpha subunit of casein kinase II is sufficient for catalysis, is autophosphorylated, and can be directly inhibited by heparin, whereas the beta subunit mediates the effects of basic stimulatory compounds and is involved in recognition and/or binding to specific physiological substrates.
Lin
W J
WJ
Department of Biochemistry, University of California, Riverside 92521.
Tuazon
P T
PT
Traugh
J A
JA
D000818:Animals; D047388:Casein Kinases; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D006493:Heparin; D007700:Kinetics; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism
D006493:Heparin; D004247:DNA; D011494:Protein Kinases; D047388:Casein Kinases