Mechanism of interferon action: Phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosome-associated kinase processing site specificity similar to hemin-regulated rabbit reticulocyte kinase

Abstract

The phosphorylation of purified protein synthesis factors catalyzed by protein kinase preparations isolated from interferon-treated human amnion cells was examined. Ribosomal salt-wash fractions prepared from interferon-treated human cells contained a protein kinase that catalyzed the [.gamma.-32P]ATP-mediated phosphorylation of the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2.alpha.); this kinase activity was significantly enhanced in interferon-treated as compared to untreated cells. The tryptic [32P]phosphopeptide pattern obtained for eIF-2.alpha. phosphorylated by the interferon-mediated human kinase was indistinguishable from the pattern obtained for eIF-2.alpha. phosphorylated by the hemin-regulated rabbit reticulocyte kinase when analyzed by TLC with 3 different solvent systems and by high-voltage electrophoresis. O-[32P]phosphoserine was liberated by partial acid hydrolysis from eIF-2.alpha. phosphorylated by the human or the rabbit kinase. In addition to the phosphorylation of eIF-2.alpha., interferon treatment of human cells enhanced the phosphorylation of 2 additional ribosome-associated proteins designated Pl and Pf. The major phosphoester linkage observed for the human and murine phosphoprotein Pl was O-phosphoserine. The interferon-mediated phosphorylation of eIF-2.alpha. and protein Pl was dependent on the presence of RNA with double-stranded character; Pf phosphorylation was not affected by double-stranded [reovirus] RNA. Apparently the interferon-mediated ribosome-associated human protein kinase catalyzes the phosphorylation of eIF-2.alpha. in a site-specific manner that is apparently identical with the reaction catalyzed by the hemin-regulated rabbit reticulocyte kinase; hence, the phosphorylation of eIF-2 may play a role in regulating the initiation of transplantation in interferon-treated cells. [These findings have relevance to the anti-viral activity of interferon.].