title: Electrophilic Modification of PKM2 by 4-Hydroxynonenal and 4-Oxononenal Results in Protein Cross-Linking and Kinase Inhibition.
abstract: Rapidly proliferating cells require an increased rate of metabolism to allow for the production of nucleic acids, amino acids, and lipids. Pyruvate kinase catalyzes the final step in the glycolysis pathway, and different isoforms display vastly different catalytic efficiencies. The M2 isoform of pyruvate kinase (PKM2) is strongly expressed in cancer cells and contributes to aerobic glycolysis in what is commonly termed the Warburg effect. Here, we show that PKM2 is covalently modified by the lipid electrophiles 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE modify multiple sites on PKM2 in vitro, including Cys424 and His439, which play a role in protein-protein interactions and fructose 1,6-bis-phosphate binding, respectively. Modification of these sites results in a dose-dependent decrease in enzymatic activity. In addition, high concentrations of the electrophile, most notably in the case of ONE, result in substantial protein-protein cross-linking in vitro and in cells. Exposure of RKO cells to electrophiles results in modification of monomeric PKM2 in a dose-dependent manner. There is a concomitant decrease in PKM2 activity in cells upon ONE exposure, but not HNE exposure. Together, our data suggest that modification of PKM2 by certain electrophiles results in kinase inactivation.
authors.forename: Jeannie M
authors.identifier: 0000-0001-9699-0931
authors.initials: JM
authors.lastname: Camarillo
authors.forename: Jody C
authors.initials: JC
authors.lastname: Ullery
authors.forename: Kristie L
authors.initials: KL
authors.lastname: Rose
authors.affiliation: A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Chemistry, and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.
authors.forename: Lawrence J
authors.identifier: 0000-0002-7834-6285
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D000447:Aldehydes / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D057930:Click Chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007659:Ketones / Q000494:pharmacology*; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000447:Aldehydes; D004791:Enzyme Inhibitors; D007659:Ketones; C575062:4-oxopentanal; D011770:Pyruvate Kinase; C027576:4-hydroxy-2-nonenal