Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts.
The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.
D000650:Amnion / Q000201:enzymology; D000818:Animals; D015852:Eukaryotic Initiation Factor-2; D005260:Female; D006801:Humans; D007370:Interferon Type I / Q000494:pharmacology*; D007700:Kinetics; D007739:L Cells / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D010448:Peptide Initiation Factors / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011247:Pregnancy; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism*; D013045:Species Specificity; D013379:Substrate Specificity
D015852:Eukaryotic Initiation Factor-2; D007370:Interferon Type I; D010448:Peptide Initiation Factors; D011506:Proteins; D012330:RNA, Double-Stranded; D011494:Protein Kinases; D008274:Magnesium; D011188:Potassium