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High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells

We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly ass...

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Autores principales: Schmitt, Danielle L., Dranchak, Patricia, Parajuli, Prakash, Blivis, Dvir, Voss, Ty, Kohnhorst, Casey L., Kyoung, Minjoung, Inglese, James, An, Songon
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10403072/
https://www.ncbi.nlm.nih.gov/pubmed/37540718
http://dx.doi.org/10.1371/journal.pone.0289707
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author Schmitt, Danielle L.
Dranchak, Patricia
Parajuli, Prakash
Blivis, Dvir
Voss, Ty
Kohnhorst, Casey L.
Kyoung, Minjoung
Inglese, James
An, Songon
author_facet Schmitt, Danielle L.
Dranchak, Patricia
Parajuli, Prakash
Blivis, Dvir
Voss, Ty
Kohnhorst, Casey L.
Kyoung, Minjoung
Inglese, James
An, Songon
author_sort Schmitt, Danielle L.
collection PubMed
description We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells.
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spelling pubmed-104030722023-08-05 High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells Schmitt, Danielle L. Dranchak, Patricia Parajuli, Prakash Blivis, Dvir Voss, Ty Kohnhorst, Casey L. Kyoung, Minjoung Inglese, James An, Songon PLoS One Research Article We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells. Public Library of Science 2023-08-04 /pmc/articles/PMC10403072/ /pubmed/37540718 http://dx.doi.org/10.1371/journal.pone.0289707 Text en https://creativecommons.org/publicdomain/zero/1.0/This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication.
spellingShingle Research Article
Schmitt, Danielle L.
Dranchak, Patricia
Parajuli, Prakash
Blivis, Dvir
Voss, Ty
Kohnhorst, Casey L.
Kyoung, Minjoung
Inglese, James
An, Songon
High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells
title High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells
title_full High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells
title_fullStr High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells
title_full_unstemmed High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells
title_short High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells
title_sort high-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10403072/
https://www.ncbi.nlm.nih.gov/pubmed/37540718
http://dx.doi.org/10.1371/journal.pone.0289707
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