Site-Specific Phosphorylation of the α Subunit of Eukaryotic Initiation Factor eIF-2 by the Heme-Regulated and Double-Stranded RNA-Activated eIF-2α Kinases from Rabbit Reticulocyte Lysates

The site specificity of phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α) by the heme-regulated and double-stranded RNA-activated eIF-2α kinases were compared by phosphopeptide mapping. eIF-2α was maximally phosphorylated in vitro with [γ-³²P]ATP and either crude or partially purified preparations of the kinases. ³²P-Labeled eIF-2α was isolated by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. The fixed, stained, and dried polypeptide band was excised and then exhaustively digested directly in the gel slice with one of several proteases (trypsin, chymotrypsin, subtilisin, or thermolysin); the resultant [³²P]phosphopeptides were analyzed by one-dimensional chromatography or by two-dimensional chromatography and high-voltage electrophoresis. In addition, limited proteolysis of [³²P]eIF-2α contained in fixed, dried, and stained gel slices was achieved with Staphylococcus aureus protease V8, chymotrypsin, or subtilisin, and the partial ³²P-labeled cleavage products were analyzed by gel electrophoresis. Each protease produced distinct and reproducible [³²P]phosphopeptide profiles after partial or exhaustive proteolysis of [³²P]eIF-2α. With a given protease, identical [³²P]phosphopeptide patterns were obtained whether eIF-2α was phosphorylated by the heme-regulated or the double-stranded RNA-activated kinase. These data indicate that, in vitro, the kinases phosphorylate sites on eIF-2α that are identical or proximally located in the primary sequence. In this report we also provide preliminary evidence that the two eIF-2α kinases activated in lysates by heme deficiency or double-stranded RNA phosphorylate site(s) of endogenous eIF-2α that are similar, if not identical, to the sites phosphorylated in vitro with partially purified eIF-2α kinase(s) and eIF-2.