Mechanism of translational control by partial phosphorylation of the α subunit of eukaryotic initiation factor 2

John Siekierka, V. Manne, S. Ochoa

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Abstract

Catalysis of ternary complex formation by the GDP exchange factor (GEF), in the presence of Mg2+, is blocked by phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF-2). We proposed earlier that this phosphorylation interferes with the interaction between eIF-2 and GEF (then termed ESP). If so, inhibition should be related to the extent of phosphorylation. However, work in other laboratories indicated that in fully inhibited, heme-deficient lysates only 20-40% of the EIF-2 is phosphorylated. To understand the nature of the molecular lesion in eIF-2-α phosphorylation we used a system of pure components in which the rate of exchange of eIF-2-bound [3H]GDP with unlabeled GDP (via the reaction eIF + GEF ⇆ eIF-2·GEF + GDP) was measured by using mixtures of eIF-2(αP)·[3H]GDP and eIF-2·[3H]GDP in different proportions at constant concentration of eIF-2·GEF. If, for example, the ratio of eIF-2·GEF to total (phosphorylated and unphosphorylated) eIF-2·[3H]GDP was 0.25, the exchange was found to be maximally inhibited when the proportion of eIF-2(αP)·[3H]GDP in the mixture reached 25%. This suggests that the reaction stops because the available GEF is trapped in an inactive complex with eIF-2(αP). In the absence of free GEF, eIF-2 would not be able to recycle and initiation would come to a standstill when the available eIF-2 is tied up as eIF-2·GDP. The trapping of GEF by eIF-2(αP) is strongly supported by the following observation. Incubation of eIF-2·GEF with excess [3H]GDP leads to the formation of eIF-2·[3H]GDP and free GEF and, if eIF-2(α32P)·GDP is also present, all of the GEF is converted to eIF-2(α32P)·GEF. This suggests that, whereas the equilibrium of the reaction eIF-2·GEF + GDP ⇆ eIF-2·GDP + GEF favors the formation of free GEF, the equilibrium of the reaction eIF-2(αP)·GDP + GEF ⇆ eIF-2(αP)·GEF + GDP is in favor of the association of GEF to eIF-2(αP).

Original languageEnglish
Pages (from-to)352-356
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume81
Issue number2 I
DOIs
StatePublished - 1 Jan 1984

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Eukaryotic Initiation Factor-2
Guanine Nucleotide Exchange Factors
Phosphorylation

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@article{18106493efcf40928fa8fd6414c182cf,
title = "Mechanism of translational control by partial phosphorylation of the α subunit of eukaryotic initiation factor 2",
abstract = "Catalysis of ternary complex formation by the GDP exchange factor (GEF), in the presence of Mg2+, is blocked by phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF-2). We proposed earlier that this phosphorylation interferes with the interaction between eIF-2 and GEF (then termed ESP). If so, inhibition should be related to the extent of phosphorylation. However, work in other laboratories indicated that in fully inhibited, heme-deficient lysates only 20-40{\%} of the EIF-2 is phosphorylated. To understand the nature of the molecular lesion in eIF-2-α phosphorylation we used a system of pure components in which the rate of exchange of eIF-2-bound [3H]GDP with unlabeled GDP (via the reaction eIF + GEF ⇆ eIF-2·GEF + GDP) was measured by using mixtures of eIF-2(αP)·[3H]GDP and eIF-2·[3H]GDP in different proportions at constant concentration of eIF-2·GEF. If, for example, the ratio of eIF-2·GEF to total (phosphorylated and unphosphorylated) eIF-2·[3H]GDP was 0.25, the exchange was found to be maximally inhibited when the proportion of eIF-2(αP)·[3H]GDP in the mixture reached 25{\%}. This suggests that the reaction stops because the available GEF is trapped in an inactive complex with eIF-2(αP). In the absence of free GEF, eIF-2 would not be able to recycle and initiation would come to a standstill when the available eIF-2 is tied up as eIF-2·GDP. The trapping of GEF by eIF-2(αP) is strongly supported by the following observation. Incubation of eIF-2·GEF with excess [3H]GDP leads to the formation of eIF-2·[3H]GDP and free GEF and, if eIF-2(α32P)·GDP is also present, all of the GEF is converted to eIF-2(α32P)·GEF. This suggests that, whereas the equilibrium of the reaction eIF-2·GEF + GDP ⇆ eIF-2·GDP + GEF favors the formation of free GEF, the equilibrium of the reaction eIF-2(αP)·GDP + GEF ⇆ eIF-2(αP)·GEF + GDP is in favor of the association of GEF to eIF-2(αP).",
author = "John Siekierka and V. Manne and S. Ochoa",
year = "1984",
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doi = "10.1073/pnas.81.2.352",
language = "English",
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journal = "Proceedings of the National Academy of Sciences of the United States of America",
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Mechanism of translational control by partial phosphorylation of the α subunit of eukaryotic initiation factor 2. / Siekierka, John; Manne, V.; Ochoa, S.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 81, No. 2 I, 01.01.1984, p. 352-356.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Mechanism of translational control by partial phosphorylation of the α subunit of eukaryotic initiation factor 2

AU - Siekierka, John

AU - Manne, V.

AU - Ochoa, S.

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N2 - Catalysis of ternary complex formation by the GDP exchange factor (GEF), in the presence of Mg2+, is blocked by phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF-2). We proposed earlier that this phosphorylation interferes with the interaction between eIF-2 and GEF (then termed ESP). If so, inhibition should be related to the extent of phosphorylation. However, work in other laboratories indicated that in fully inhibited, heme-deficient lysates only 20-40% of the EIF-2 is phosphorylated. To understand the nature of the molecular lesion in eIF-2-α phosphorylation we used a system of pure components in which the rate of exchange of eIF-2-bound [3H]GDP with unlabeled GDP (via the reaction eIF + GEF ⇆ eIF-2·GEF + GDP) was measured by using mixtures of eIF-2(αP)·[3H]GDP and eIF-2·[3H]GDP in different proportions at constant concentration of eIF-2·GEF. If, for example, the ratio of eIF-2·GEF to total (phosphorylated and unphosphorylated) eIF-2·[3H]GDP was 0.25, the exchange was found to be maximally inhibited when the proportion of eIF-2(αP)·[3H]GDP in the mixture reached 25%. This suggests that the reaction stops because the available GEF is trapped in an inactive complex with eIF-2(αP). In the absence of free GEF, eIF-2 would not be able to recycle and initiation would come to a standstill when the available eIF-2 is tied up as eIF-2·GDP. The trapping of GEF by eIF-2(αP) is strongly supported by the following observation. Incubation of eIF-2·GEF with excess [3H]GDP leads to the formation of eIF-2·[3H]GDP and free GEF and, if eIF-2(α32P)·GDP is also present, all of the GEF is converted to eIF-2(α32P)·GEF. This suggests that, whereas the equilibrium of the reaction eIF-2·GEF + GDP ⇆ eIF-2·GDP + GEF favors the formation of free GEF, the equilibrium of the reaction eIF-2(αP)·GDP + GEF ⇆ eIF-2(αP)·GEF + GDP is in favor of the association of GEF to eIF-2(αP).

AB - Catalysis of ternary complex formation by the GDP exchange factor (GEF), in the presence of Mg2+, is blocked by phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF-2). We proposed earlier that this phosphorylation interferes with the interaction between eIF-2 and GEF (then termed ESP). If so, inhibition should be related to the extent of phosphorylation. However, work in other laboratories indicated that in fully inhibited, heme-deficient lysates only 20-40% of the EIF-2 is phosphorylated. To understand the nature of the molecular lesion in eIF-2-α phosphorylation we used a system of pure components in which the rate of exchange of eIF-2-bound [3H]GDP with unlabeled GDP (via the reaction eIF + GEF ⇆ eIF-2·GEF + GDP) was measured by using mixtures of eIF-2(αP)·[3H]GDP and eIF-2·[3H]GDP in different proportions at constant concentration of eIF-2·GEF. If, for example, the ratio of eIF-2·GEF to total (phosphorylated and unphosphorylated) eIF-2·[3H]GDP was 0.25, the exchange was found to be maximally inhibited when the proportion of eIF-2(αP)·[3H]GDP in the mixture reached 25%. This suggests that the reaction stops because the available GEF is trapped in an inactive complex with eIF-2(αP). In the absence of free GEF, eIF-2 would not be able to recycle and initiation would come to a standstill when the available eIF-2 is tied up as eIF-2·GDP. The trapping of GEF by eIF-2(αP) is strongly supported by the following observation. Incubation of eIF-2·GEF with excess [3H]GDP leads to the formation of eIF-2·[3H]GDP and free GEF and, if eIF-2(α32P)·GDP is also present, all of the GEF is converted to eIF-2(α32P)·GEF. This suggests that, whereas the equilibrium of the reaction eIF-2·GEF + GDP ⇆ eIF-2·GDP + GEF favors the formation of free GEF, the equilibrium of the reaction eIF-2(αP)·GDP + GEF ⇆ eIF-2(αP)·GEF + GDP is in favor of the association of GEF to eIF-2(αP).

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JF - Proceedings of the National Academy of Sciences of the United States of America

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