Abstract
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.
Funder
American Association for Cancer Research
National Institute of General Medical Sciences
National Cancer Institute
National Center for Advancing Translational Sciences
Publisher
Public Library of Science (PLoS)
Reference57 articles.
1. Spatial Organization of Metabolic Enzyme Complexes in Cells;DL Schmitt;Biochemistry-Us,2017
2. Enzymatic complexes across scales;PL Kastritis;Essays Biochem,2018
3. Mapping of glycolytic enzyme-binding sites on human erythrocyte band 3;HY Chu;Biochem J,2006
4. Identification of a multienzyme complex for glucose metabolism in living cells;CL Kohnhorst;J Biol Chem,2017
5. Functional regulation of 4D metabolic network between multienzyme glucosome condensates and mitochondria in living cells;EL Kennedy;BioRxiv,2022
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