Partial reaction of peptide initiation inhibited by phosphorylation of either initiation factor eIF-2 or 40S ribosomal proteins.

Kramer, Gisela; Henderson, A. B.; Pinphanichakarn, Pairoh; Wallis, Mary H.; Hardesty, Boyd

doi:10.1073/pnas.74.4.1445

Cited by 60 publications

(28 citation statements)

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“…Gross and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 Fractionated Protein-Synthesizing System Assay. The fractionated cell-free system was that described by Hardesty et al (14).…”

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confidence: 99%

Multistep regulatory system for activation of a cyclic AMP-independent eukaryotic initiation factor 2 kinase.

Henderson¹,

Miller²,

Hardesty³

1979

Proc. Natl. Acad. Sci. U.S.A.

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Three functionally related components that block peptide initiation have been identified in lysates of rabbit reticulocytes. The components function consecutively in a cascade type sequence of reactions to cause phosphorylation of eukaryotic peptide initiation factor 2 (eIF-2). The eIF-2 kinase activated as part of this sequence has been tentatively identified as the same protein kinase that is activated by heme deficiency as part of the hemin-controlled repressor (HCR) system. The first component in the sequence is heat stable and can be reversibly activated by heat or pressure. It activates a second, heat-labile, component that in turn directly or indirectly activates the hemin-controlled eIF-2 kinase. This heat-labile component appears to function through proteolysis. This reaction sequence is not detectably affecte by heme or cyclic AMP and thus appears to provide an alternative mechanism, independent of heme, for activation of the cyclic AMP-independent eIF-2 kinase of the HCR system.

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confidence: 99%

Multistep regulatory system for activation of a cyclic AMP-independent eukaryotic initiation factor 2 kinase.

Henderson¹,

Miller²,

Hardesty³

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…The inhibitor has been identified as a cyclic AMPindependent protein kinase that specifically phosphorylates the 38,000-dalton polypeptide (a subunit) of eIF-2 (21)(22)(23)(24)(25). This phosphorylation of eIF-2 prevents the binding of Met-tRNAf to the 40S ribosomal subunit in crude and in partially purified systems (24,(26)(27)(28). Recent evidence indicates that the formation of the ternary complex [eIF-2-Met-tRNAf-GTP] and its subsequent binding to 40S subunits may involve other factors in addition to eIF-2 (29)(30)(31)(32) and that the phosphorylation of eIF-2 may inhibit its interaction with these factors (28,32).…”

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confidence: 99%

Inhibition of protein synthesis initiation by oxidized glutathione: Activation of a protein kinase that phosphorylates the α subunit of eukaryotic initiation factor 2

Ernst

Levin

London

1978

Proc. Natl. Acad. Sci. U.S.A.

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Oxidized glutathione (GSSG) (0.02-0.5 mM) inhibits reticulocyte lysates by a mechanism similar to that observed in heme deficiency. Incubation of hemin-supplemented postribosomal supernates with GSSG results in the activation of a translational inhibitor [I(GSSG)J. The activation of I(GSSG) is enhanced by the presence of an energy-regenerating system. The simultaneous addition of 1 mM dithiothreitol blocks the activation of the GSSG-induced inhibitor; however, once inhibitor is formed, its activity is not affected by 1 mM dithiothreitol. GSSG-treated postribosomal supernates and partially purified preparations of I(GSSG) inhibit protein synthesis in emin-supplemented lysates with biphasic kinetics. Inhibition by I(GSSG) is blocked by cyclic AMP (2-10 mM) and is potentiated by ATP (2 mM). The inhibition is also blocked or reversed by eukaryotic initiation factor eIF-2. The activation of I(GSSG) is accompanied by an increased cyclic AMP-independent protein kinase activity which phosphorylates the 38,000da ton component (a subunit) of eIF-2; however, GSSG treatment of supernates does not alter the activity of the cyclic AMP-independent protein kinase activity that phosphorylates the 49,000-dalton polypeptide component (ft subunit) of eIF-2.These data indicate that GSSG treatment of reticulocyte lysates results in the activation of a protein kinase with inhibitory and phosphorylation properties similar to those of the heme-regulated cyclic AMP-independent protein kinase which is activated in heme deficiency.

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“…Inhibition may be dependent on the simultaneous phosphorylation of P55 and eIF-2a. Kramer et al [45] have suggested that the phosphorylation of both eIF-2a and 40-S ribosomal subunits is required for inhibition; however, this is unlikely since no increased phosphorylation of ribosomal proteins was observed in these studies.…”

Section: Discussionmentioning

confidence: 50%

Heme Deficiency and Phosphorylation of Ribosome‐Associated Proteins

Floyd

Traugh

1980

European Journal of Biochemistry

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Phosphorylation of proteins in the protein-synthesizing complex from rabbit reticulocyte lysates was examined under conditions of optimal protein synthesis and during inhibition of protein synthesis by hemin deprivation. A creatine phosphate -ATP -GTP -P, exchange system was used to maintain [ Y -~~P I A T P and [Y-~'P]GTP at high, constant specific activity. Phosphate incorporation into proteins in the 0.5 M KCl wash fraction and in salt-washed ribosomes was examined before and after inhibition of protein synthesis in hemin-deprived lysates and compared to phosphorylated proteins observed in hemin-supplemented lysates. Two of the phosphoproteins in the 0.5 M KCl wash fraction showed a twofold to fourfold increase in phosphate incorporation prior to inhibition of protein synthesis under conditions of hemin deprivation. One of these phosphoproteins was identified as the small subunit of initiation factor 2 (eIF-2), the initiation factor involved in the GTPdependent binding of Met-tRNAf to 40-S ribosomal subunits. A second protein, with a molecular weight of 55000, did not correspond to any of the highly purified initiation factors from reticulocytes, but was a major constituent of the salt-wash fraction. No changes in phosphate incorporation into the major phosphorylated 40-S ribosomal protein, S6, were observed; however, se\ era1 minor components, which were not completely removed by the high-salt wash, showed alterations in phosphorylation.Protein synthesis and phosphorylation were also examined in intact reticulocytu When iron and the iron-transporting protein, transferrin, were present, protein synthesis was lineal-for at least 2 h. When the cells were incubated in the nutritional media from which iron and transferrin had been omitted, inhibition occurred between 15 min and 30 min. Cells incubated in the absence of iron and transferrin showed increased phosphorylation of two proteins in the high-salt-wash fraction. These had niolecular weights of 38000 and 55000 and corresponded to the two proteins showing increased phosphorylation in the lysate system in the absence of hemin.Hemin has been identified as a translational regulator of globin synthesis in reticulocytes [1, 21 and reticulocyte lysates [3,4]. When lysates are incubated in the absence of hemin, formation of a hemincontrolled repressor occurs, which blocks peptide chain initiation [4 -71 resulting in a decrease in binding of Met-tRNAf to 40-S ribosomal subunits [8 -lo]. High concentrations of cIF-2. the initition factor involved in the GTP-de pendcnt binding o f met -t R N Af to 40-S ribosomal subunits [ l l -151, reverse inhibition produced by hemin deprivation [16,17] or by addition of the hemin-controlled repressor [17,18]. Several laboratories have identified a protein kinase activity associated with purified preparations of this repressor, which is highly specific for the cc subunit ( M , = 38000) Abbreviution. eIF-2 etc., eukaryotic initiation factor 2 etc of eIF-2 [19 -241. Phosphorylation of highly purified eIF-2 does not impair formation o...

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Partial reaction of peptide initiation inhibited by phosphorylation of either initiation factor eIF-2 or 40S ribosomal proteins.

Cited by 60 publications

References 35 publications

Multistep regulatory system for activation of a cyclic AMP-independent eukaryotic initiation factor 2 kinase.

Multistep regulatory system for activation of a cyclic AMP-independent eukaryotic initiation factor 2 kinase.

Inhibition of protein synthesis initiation by oxidized glutathione: Activation of a protein kinase that phosphorylates the α subunit of eukaryotic initiation factor 2

Heme Deficiency and Phosphorylation of Ribosome‐Associated Proteins

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