Further characterization of the protein kinase activity mediated by interferon in mouse and human cells.
Interferon-treated mouse and human cells show enhanced levels of a protein kinase activity which is manifested by the phosphorylation of endogenous Mr = 67,000 and 72,000 proteins, respectively. Such kinase activity can be assayed after its partial purification on poly(I) X poly(C)-Sepharose. Under these experimental conditions, the apparent km of the kinase for ATP is 1.0 X 10(-6) M and 2.5 X 10(-6) M in enzyme fractions from mouse L-929 and human HeLa cells, respectively. The Mr = 67,000 and 72,000 proteins are phosphorylated by their serine and threonine residues, the ratio of which is modified in preparations from interferon-treated cells. Both of these phosphoproteins are composed of several subspecies with similar isoelectric points (pIs) in the range of 7.2 to 8.2. This heterogeneity is due to the number of phosphate groups per molecule of protein. Accordingly, the pIs of highly phosphorylated proteins are at a less basic pH (7.2 to 7.5). Furthermore, highly phosphorylated proteins show an increase in their apparent molecular weights compared to partially phosphorylated ones. This corresponds to an increase of Mr = 1,500. Partial proteolysis of the 32P-labeled Mr = 67,000 and 72,000 proteins by Staphylococcus aureus V8 protease, alpha-chymotrypsin and thrombin, indicated that these phosphoproteins differ in their polypeptide structure. Phosphorylation of the Mr = 67,000 and 72,000 proteins in enzyme fractions from control L-929 and HeLa cells is enhanced by mixing with extracts from interferon-treated heterologous cells. Proteins, Mr = 67,000 and 72,000, therefore, may serve as suitable substrates for an exogenous kinase, thus indicating that the substrate in enzyme fractions from control cells is less phosphorylated because of a low level of kinase activity.
D000818:Animals; D002846:Chromatography, Affinity; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007370:Interferon Type I / Q000494:pharmacology*; D007372:Interferons / Q000502:physiology; D007700:Kinetics; D007739:L Cells / Q000201:enzymology; D051379:Mice; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011070:Poly I-C; D011494:Protein Kinases / Q000096:biosynthesis* / Q000302:isolation & purification
D007370:Interferon Type I; D010446:Peptide Fragments; D010748:Phosphopeptides; D007372:Interferons; D011494:Protein Kinases; D011070:Poly I-C