Initiation factor 2 (eIF-2) is phosphorylated in vitro by two different cyclic nucleotide-independent protein kinases. As previously shown, a protein kinase activity that comigrates with the major casein kinase activity from rabbit reticulocytes phosphorylates eIF-2,B. In addition, a second protein kinase that specifically phosphorylates eIF-2a has been identified. Both protein kinase activities demonstrate cyclic nucleotide-independent activity and are not inhibited by the inhibitor protein diagnostic for cyclic AMP-regulated protein kinase activities. Phosphorylation of eIF-2a is almost completely inhibited by 20-35,uM hemin, whereas phosphorylation of eIF-2ft is only partially inhibited. Hemin acts by decreasing the rate of incorporation of phosphate into eIF-2a. The protein kinase activity that modifies eIF-2a has been shown to have inhibitory activity in the cell-free protein-synthesizing system, whereas the protein kinase for eIF-2Bt has no effect. The identity of the former enzyme with the hemin-controlled repressor and role of hemin in the control of initiation are discussed. Hemoglobin biosynthesis in both reticulocytes (1) and reticulocyte lysates (2-4) is under the direct control of hemin, which acts as a positive effector for globin synthesis. In the absence of added hemin, protein synthesis is inhibited in a reticulocyte lysate at chain initiation with a concomitant decrease in binding of Met-tRNAf to 40S ribosomal subunits (5-7). This inhibition is potentiated by addition of ATP (8,9). Inhibition produced by hemin deprivation is prevented in the reticulocyte lysate by high concentrations of GTP, cyclic AMP, and various purines (8-10). In addition, inhibition of globin synthesis coincides with the appearance of a repressor (11-13). The hemin-controlled repressor (HCR) is thought to be activated from a prorepressor at hemin concentrations suboptimal for translation (5-7, 12, 14-16). Supporting the concept that HCR inhibits initiation is the observation that the addition of initiation factor 2 (eIF-2), the factor that forms a ternary complex with Met-tRNAf and GTP and then binds to 40S subunits (17-23), will reverse inhibition due to hemin deprivation in the lysate system (24, 25).eIF-2 is composed of three subunits (25-31) designated a, p, and y according to a decreasing relative mobility in the gel electrophoresis system of Laemmli (32). Phosphorylation of eIF-2fl by a cyclic nucleotide-independent protein kinase has been described (26, 33). It has been reported that purified preparations of HCR possess a protein kinase activity that phosphorylates the small subunit of eIF-2 (29,(34)(35)(36). This modification may be correlated with the inhibition of protein synthesis resulting from hemin depletion (34,37,38). In these studies we have examined phosphorylation of the two subunits of eIF-2 by the two different protein kinases. 2-mercaptoethanol). Under these conditions, the protein kinase activity did not adhere to the resin. When the enzyme was rechromatographed in the presence of 0.25 M ...
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