Regulation and Cytoprotective Role of Hexokinase III Eugene Wyatt1, Rongxue Wu 1 , Wael Rabeh2, Hee-Won Park2,3, Mohsen Ghanefar1, Hossein Ardehali1* 1 Division of Cardiology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America, 2 Structural Genomics Consortium, University of Toronto, Toronto, Canada, 3 Department of Pharmacology, University of Toronto, Toronto, Canada Abstract 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. Methodology/Principal Findings: 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/Significance: 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. Citation: Wyatt E, Wu R, Rabeh W, Park H-W, Ghanefar M, et al. (2010) Regulation and Cytoprotective Role of Hexokinase III. PLoS ONE 5(11): e13823. doi:10.1371/ journal.pone.0013823 Editor: Stefan W o¨lfl, Universita¨t Heidelberg, Germany Received May 4, 2010; Accepted October 14, 2010; Published November 3, 2010 Copyright: 2010 Wyatt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: H.A. is supported by National Institutes of Health grants K08 HL079387 and R01 HL087149 http://grants.nih.gov/grants/oer.htm. E.W. and R.W. are supported by grants from the American Heart Association http://www.americanheart.org. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: h-ardehali@northwestern.edu Introduction Hexokinases (HKs or ATP:D-hexose 6-phosphotransferase [EC 2.7.1.1] in IUB nomenclature) transfer a phosphoryl moiety from ATP to the 6-hydroxyl of glucose to produce glucose-6-phosphate (G6P). The glucose phosphorylation reaction carried out by HKs maintains the concentration gradient necessary for facilitative glucose transporters (GLUTs) to move glucose into the cell [1,2,3,4]. There are 4 mammalian hexokinase isozymes: HKI, II, III, and IV; hexokinase IV is also known as glucokinase (GK). The molecular weights of HKI, II, and III are approximately 100-kDa, whereas HKIV has a molecular weight of 50-kDa. The three 100- kDa HK isozymes have extensive sequence homology and a high affinity for glucose (Km,1 mM), and are inhibited by the reaction product G6P; however, differences in tissue expression, cellular distribution, and regulation suggest that each isozyme has a distinct metabolic role [5,6,7]. HKI is ubiquitously expressed, while HKII is primarily expressed in insulin sensitive tissue such as skeletal and cardiac muscle. HKIII expression is relatively low in most tissue, with the highest levels reported in lung, kidney, and liver [7,8,9]. HKIV has a low affinity for glucose, is not inhibited by G6P, and is expressed mainly in pancreatic B cells and the liver, where it is believed to function as a ‘glucose sensor’ [8]. The 100-kDa HK isozymes are each composed of two 50-kDa domains and may have evolved from the genetic duplication and fusion of an ancestral 50-kDa HK. Early descriptions of these enzymes proposed that one of the 50-kDa subunits retained catalytic activity, and the other became the regulatory domain [7,8]. Only the C-terminal domain of HKI is catalytically active, but G6P binds to both domains [10,11,12,13,14,15,16]. In HKII, both domains are catalytically active and sensitive to G6P, and glucose binding to the N-terminal domain decreases the concentration of G6P required to regulate the activity of the C- terminal domain, suggesting that the two halves functionally interact [17,18]. Like HKI, the catalytic activity of HKIII is restricted to its C-terminal half [17,19,20]. HKI and HKII also contain a conserved 21-amino-acid sequence in the N-terminal domain that is predicted to form a hydrophobic a-helix and enables these proteins to bind to mitochondria [21,22]. HKIII does not contain the conserved mitochondrial-binding sequence and is cytoplasmic, although some investigations report perinuclear binding [23]. One conse- PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e13823