Mechanism of Interferon Action

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein Pl and the a 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 Pl/eIF-2a kinase was purified at least 1,500fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2a copurified with protein PI. The yield of Pl/eIF-2a 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 PI kinase was activated by dsRNA, a major phosphopeptide designated Xa. was phosphorylated; X& was not phosphorylated from PI which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNAdependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein PI by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the a subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the K, for ATP was determined to be 5 x lo-‘ M. Histones (HI, Ha, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF4B, 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 PI and eIF-Za phosphorylation by the purified kinase, whereas ethylene glycol bis(8-aminoethyl ether)NtNtN’,N’-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein PI or eIF-2a.