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Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of act...

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Autores principales: Lageix, Sebastien, Zhang, Jinwei, Rothenburg, Stefan, Hinnebusch, Alan G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335047/
https://www.ncbi.nlm.nih.gov/pubmed/25695491
http://dx.doi.org/10.1371/journal.pgen.1004991
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author Lageix, Sebastien
Zhang, Jinwei
Rothenburg, Stefan
Hinnebusch, Alan G.
author_facet Lageix, Sebastien
Zhang, Jinwei
Rothenburg, Stefan
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. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd(-) phenotype), while other substitutions block kinase activation (Gcn(-) phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn(-) substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd(-) substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.
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spelling pubmed-43350472015-02-24 Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo Lageix, Sebastien Zhang, Jinwei Rothenburg, Stefan Hinnebusch, Alan G. PLoS Genet Research Article The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd(-) phenotype), while other substitutions block kinase activation (Gcn(-) phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn(-) substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd(-) substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction. Public Library of Science 2015-02-19 /pmc/articles/PMC4335047/ /pubmed/25695491 http://dx.doi.org/10.1371/journal.pgen.1004991 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
Zhang, Jinwei
Rothenburg, Stefan
Hinnebusch, Alan G.
Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo
title Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo
title_full Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo
title_fullStr Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo
title_full_unstemmed Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo
title_short Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo
title_sort interaction between the trna-binding and c-terminal domains of yeast gcn2 regulates kinase activity in vivo
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335047/
https://www.ncbi.nlm.nih.gov/pubmed/25695491
http://dx.doi.org/10.1371/journal.pgen.1004991
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