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Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase...

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Detalles Bibliográficos
Autores principales: Lageix, Sebastien, Rothenburg, Stefan, Dever, Thomas E., Hinnebusch, Alan G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014428/
https://www.ncbi.nlm.nih.gov/pubmed/24811037
http://dx.doi.org/10.1371/journal.pgen.1004326
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author Lageix, Sebastien
Rothenburg, Stefan
Dever, Thomas E.
Hinnebusch, Alan G.
author_facet Lageix, Sebastien
Rothenburg, Stefan
Dever, Thomas E.
Hinnebusch, Alan G.
author_sort Lageix, Sebastien
collection PubMed
description The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn(−) substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd(−) substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd(−) substitutions enhance YKD-KD interactions in vitro, whereas Gcn(−) substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd(−) substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD.
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spelling pubmed-40144282014-05-14 Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells Lageix, Sebastien Rothenburg, Stefan Dever, Thomas E. Hinnebusch, Alan G. PLoS Genet Research Article The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn(−) substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd(−) substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd(−) substitutions enhance YKD-KD interactions in vitro, whereas Gcn(−) substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd(−) substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD. Public Library of Science 2014-05-08 /pmc/articles/PMC4014428/ /pubmed/24811037 http://dx.doi.org/10.1371/journal.pgen.1004326 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
spellingShingle Research Article
Lageix, Sebastien
Rothenburg, Stefan
Dever, Thomas E.
Hinnebusch, Alan G.
Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells
title Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells
title_full Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells
title_fullStr Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells
title_full_unstemmed Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells
title_short Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells
title_sort enhanced interaction between pseudokinase and kinase domains in gcn2 stimulates eif2α phosphorylation in starved cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014428/
https://www.ncbi.nlm.nih.gov/pubmed/24811037
http://dx.doi.org/10.1371/journal.pgen.1004326
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