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ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin

Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase e...

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Autores principales: Piserchio, Andrea, Long, Kimberly J., Browning, Luke S., Bohanon, Amanda L., Isiorho, Eta A., Dalby, Kevin N., Ghose, Ranajeet
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10151598/
https://www.ncbi.nlm.nih.gov/pubmed/37068230
http://dx.doi.org/10.1073/pnas.2300902120
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author Piserchio, Andrea
Long, Kimberly J.
Browning, Luke S.
Bohanon, Amanda L.
Isiorho, Eta A.
Dalby, Kevin N.
Ghose, Ranajeet
author_facet Piserchio, Andrea
Long, Kimberly J.
Browning, Luke S.
Bohanon, Amanda L.
Isiorho, Eta A.
Dalby, Kevin N.
Ghose, Ranajeet
author_sort Piserchio, Andrea
collection PubMed
description Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase eukaryotic elongation factor 2 (eEF-2) resulting from its specific phosphorylation by the calmodulin (CaM)-activated α–kinase eEF-2 kinase (eEF-2K). It has been suggested that the eEF-2K response to reduced cellular energy levels is indirect and mediated by the universal energy sensor AMP-activated protein kinase (AMPK) through direct stimulatory phosphorylation and/or downregulation of the eEF-2K-inhibitory nutrient-sensing mTOR pathway. Here, we provide structural, biochemical, and cell-biological evidence of a direct energy-sensing role of eEF-2K through its stimulation by ADP. A crystal structure of the nucleotide-bound complex between CaM and the functional core of eEF-2K phosphorylated at its primary stimulatory site (T348) reveals ADP bound at a unique pocket located on the face opposite that housing the kinase active site. Within this basic pocket (BP), created at the CaM/eEF-2K interface upon complex formation, ADP is stabilized through numerous interactions with both interacting partners. Biochemical analyses using wild-type eEF-2K and specific BP mutants indicate that ADP stabilizes CaM within the active complex, increasing the sensitivity of the kinase to CaM. Induction of energy stress through glycolysis inhibition results in significantly reduced enhancement of phosphorylated eEF-2 levels in cells expressing ADP-binding compromised BP mutants compared to cells expressing wild-type eEF-2K. These results suggest a direct energy-sensing role for eEF-2K through its cooperative interaction with CaM and ADP.
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spelling pubmed-101515982023-10-17 ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin Piserchio, Andrea Long, Kimberly J. Browning, Luke S. Bohanon, Amanda L. Isiorho, Eta A. Dalby, Kevin N. Ghose, Ranajeet Proc Natl Acad Sci U S A Biological Sciences Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase eukaryotic elongation factor 2 (eEF-2) resulting from its specific phosphorylation by the calmodulin (CaM)-activated α–kinase eEF-2 kinase (eEF-2K). It has been suggested that the eEF-2K response to reduced cellular energy levels is indirect and mediated by the universal energy sensor AMP-activated protein kinase (AMPK) through direct stimulatory phosphorylation and/or downregulation of the eEF-2K-inhibitory nutrient-sensing mTOR pathway. Here, we provide structural, biochemical, and cell-biological evidence of a direct energy-sensing role of eEF-2K through its stimulation by ADP. A crystal structure of the nucleotide-bound complex between CaM and the functional core of eEF-2K phosphorylated at its primary stimulatory site (T348) reveals ADP bound at a unique pocket located on the face opposite that housing the kinase active site. Within this basic pocket (BP), created at the CaM/eEF-2K interface upon complex formation, ADP is stabilized through numerous interactions with both interacting partners. Biochemical analyses using wild-type eEF-2K and specific BP mutants indicate that ADP stabilizes CaM within the active complex, increasing the sensitivity of the kinase to CaM. Induction of energy stress through glycolysis inhibition results in significantly reduced enhancement of phosphorylated eEF-2 levels in cells expressing ADP-binding compromised BP mutants compared to cells expressing wild-type eEF-2K. These results suggest a direct energy-sensing role for eEF-2K through its cooperative interaction with CaM and ADP. National Academy of Sciences 2023-04-17 2023-04-25 /pmc/articles/PMC10151598/ /pubmed/37068230 http://dx.doi.org/10.1073/pnas.2300902120 Text en Copyright © 2023 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Piserchio, Andrea
Long, Kimberly J.
Browning, Luke S.
Bohanon, Amanda L.
Isiorho, Eta A.
Dalby, Kevin N.
Ghose, Ranajeet
ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin
title ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin
title_full ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin
title_fullStr ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin
title_full_unstemmed ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin
title_short ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin
title_sort adp enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10151598/
https://www.ncbi.nlm.nih.gov/pubmed/37068230
http://dx.doi.org/10.1073/pnas.2300902120
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