enzymatic activity of these constructs was measured using the G6P/NADPH-coupling assay. Mutations in the C-terminal domain abolished glucose phosphorylation, which is consistent with previous reports indicating that the catalytic activity of HKIII is restricted to the C-terminal domain (data not shown) [8,19,33]. Mutations in the putative N-terminal glucose- and G6P-binding sites reduced the specific activity of the protein (Figure 2B), but the putative N-terminal ATP-binding site mutation did not. Collec- tively, these results suggest that inhibition of glucose or G6P binding, but not ATP binding, in the N-terminal domain reduces the catalytic activity of the C-terminal domain. Thus, there is functional interaction between the N- and C-halves of HKIII. HKIII overexpression protects against oxidant-induced cell death Previous studies have shown that HKI and II protect cells from oxidant-induced cell death [27,34]. We next studied the potential role of HKIII in cytoprotection, using a similar model of H2O2 induced cell death in HEK293 (HEK) cells [27]. Cells were transfected with plasmids coding for GFP or HKIII-GFP expression, and overexpression was confirmed via confocal fluorescent microscopy and Western blot (Figure 3A and B). Furthermore, overexpression of the HKIII-GFP construct resulted in a significant increase in total hexokinase activity compared to GFP (Figure 3C). Thirty-six to forty-eight hours after transfection, cells were serum starved overnight in basal MEM media, and then exposed to oxidative stress using exogenous H2O2. Cell viability was assessed using propidium iodide exclusion and Annexin V to HK3 Regulation and Protection evaluate necrotic and apoptotic cell death, respectively. Both assays demonstrate that survival was higher in cells transfected with the HKIII-GFP plasmid than in GFP transfected controls (Figure 4 A–D). These results indicate that HKIII protects against oxidant-induced cell death. HKIII overexpression increases ATP levels and reduces ROS production Glucose metabolism is necessary to maintain cellular ATP levels which is vital to cell viability [35]. To evaluate the effect of HKIII on cellular energy production, we measured ATP levels in HEK cells transfected with HKIII-GFP or GFP plasmid. Cells were serum starved overnight in basal MEM media, and then treated with 500 mM H2O2 before the assessment of ATP levels. Cells overexpressing HKIII-GFP had significantly higher ATP levels at baseline and after exposure to H2O2, compared with GFP controls (Figure 5A). Increased ROS production can also induce cell death, and we have previously shown that the overexpression of HKI or HKII reduces cellular ROS levels [27]. Here, we evaluated the potential influence of HKIII on ROS production. Under normal culture conditions, ROS levels in cells transfected with HKIII-GFP or GFP were not readily detected using the fluorescent mitochondrial superoxide indicator MitoSox (Figure 5B); however, ROS production after exposure to exogenous H2O2 was significantly lower in HKIII-GFP overexpressing cells compared to GFP controls (Figures 5B and C, S3A and B). Collectively, these results suggest that the reduction in cell death associated with HKIII overexpression could evolve from an increase in ATP levels or a decline in ROS production. Figure 3. Overexpression of HKIII in HEK293 cells. (A) Confocal microscopy images demonstrate successful transfection of HEK293 cells with the GFP and HKIII-GFP vector plasmids. HKIII-GFP does not appear cytosolic with no observed localization to any subcellular compartment. The cells were stained with TMRE (red) to identify mitochondria, and nuclei were labeled with Hoechst (blue). (B) Overexpression was confirmed by Western blots of cell lysates with a GFP antibody. (C) Total hexokinase activity was significantly increased in cells overexpressing HKIII compared to GFP control. doi:10.1371/journal.pone.0013823.g003 PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e13823