Regulation and cytoprotective role of hexokinase III.
BACKGROUND
Hexokinases (HKs) catalyze the first step in glucose metabolism. Of the three mammalian 100-kDa HK isoforms, HKI and II can bind to mitochondria and protect against cell death. HKIII does not bind mitochondria, and little is known about its regulation or cytoprotective effects. We studied the regulation of HKIII at the transcriptional and protein levels and investigated its role in cellular protection.


RESULTS
We show that like HKII, HKIII expression is regulated by hypoxia, but other factors that regulate HKII expression have no effect on HKIII levels. This transcriptional regulation is partially dependent on hypoxia-inducible factor (HIF) signaling. We also demonstrate regulation at the protein level, as mutations in putative N-terminal substrate binding residues altered C-terminal catalytic activity, suggesting that HKIII activity is governed, in part, by interactions between these two domains. Overexpression of HKIII reduced oxidant-induced cell death, increased ATP levels, decreased the production of reactive oxygen species (ROS), and preserved mitochondrial membrane potential. HKIII overexpression was also associated with higher levels of transcription factors that regulate mitochondrial biogenesis, and greater total mitochondrial DNA content. Attempts to target HKIII to the mitochondria by replacing its N-terminal 32-amino-acid sequence with the mitochondrial-targeting sequence of HKII led to protein aggregation, suggesting that this region is necessary to maintain proper protein folding and solubility.


CONCLUSIONS
These results suggest that HKIII is regulated by hypoxia and there are functional interactions between its two halves. Furthermore, HKIII exerts protective effects against oxidative stress, perhaps by increasing ATP levels, reducing oxidant-induced ROS production, preserving mitochondrial membrane potential, and increasing mitochondrial biogenesis.
Division of Cardiology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.
Eugene
E
Wyatt
Rongxue
R
Wu
Wael
W
Rabeh
Hee-Won
HW
Park
Mohsen
M
Ghanefar
Hossein
H
Ardehali
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D019610:Cytoprotection / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006593:Hexokinase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D018613:Microscopy, Confocal; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
D051793:Hypoxia-Inducible Factor 1; D007527:Isoenzymes; D016877:Oxidants; D017382:Reactive Oxygen Species; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D006593:Hexokinase