Effect of hemin on site-specific phosphorylation of eukaryotic initiation factor 2.

Abstract

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 ...

In situ phosphorylation of the α subunit of eukaryotic initiation factor 2 in reticulocyte lysates inhibited by heme deficiency, double-stranded RNA, oxidized glutathione, or the heme-regulated protein kinase

“…The phosphorylation of HRI in hemin-supplemented lysates inhibited by highly purified HRI was clearly detected in 10% gels (Fig. 4, tracks 9 20 ,uM hemin plus a crude CM-Sephadex preparation of N-ethylmaleimide-activated HRI (13, 14) (3.6 ,ug) (tracks 7,8 The inhibition of protein synthesis in heme-deficient lysates can be reversed by the delayed addition of hemin (4,29). In a typical heme-deficient lysate, protein synthesis declined abruptly at 5 min after the start of incubation (shut-off) (Fig.…”

“…There was a rapid phosphorylation of eIF-2a after each pulse in heme-deficient lysates, but not in hemin-supplemented lysates. The incorporation of 32p into eIF-2a in heme deficiency appeared to' increase for about '3 min after the 1-min pulse (tracks 2, 4, 6), the 4-min pulse (tracks 8,10,12), and the 9-min pulse (tracks 14,16,18). Within 4 min after each pulse the label in eIF-2a began to decline as the radioactivity 2120 Biochemistry: Ernst et in the endogenous nucleotide pool was correspondingly dihighly purified HRI and pulsed with [(y-32P]ATP indicates that minished (unpublished observations), which supports a a rapid phosphorylation of eIF-2a occurs that is similar to that mechanism involving a rapid phosphate turnover.…”

“…5 upper). The addition of hemin at 9 min resulted in a restoration of synthesis in this lysate after a lag period of about [6][7][8] min. We examined the effect of this phenomenon on the phosphorylation of lysate eIF-2a by using the one-dimensional NaDodSO4/polyacrylamide gel electrophoresis system.…”

“…Under all three conditions, inhibition is accompanied by the activation of cyclic AMPindependent protein kinases that phosphorylate the a subunit (38,000 daltons) of the eukaryotic initiation factor eIF-2 (for review, see ref. 7), designated eIF-2a (8). Each of the three protein kinases has been isolated and purified to various degrees (9)(10)(11)(12)(13)(14)(15); all three activities appear to be specific for eIF-2a.…”

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

“…Phosphorylated eIF-2 was analyzed by, discontinuous slab-gel electrophoresis in NaDodSO4/polyacrylamide (0.1% NaDodSO4/10% acrylamide/0.26% bisacrylamide) and autoradiography as described (11,15). In this gel system, eIF-2 migrates as three polypeptide components with molecular weights of 52,000 ('y subunit), 50,000 (f3 subunit), and 38,000 (a subunit) (15,30 In several experiments, samples were analyzed by two-dimensional chromatography and electrophoresis. In these experiments two samples were loaded at 6-cm intervals on each plate and then subjected to ascending chromatography in the first dimension as described above.…”