HK3 Regulation and Protection Figure 8. Removal of the N-terminal 32 amino acids of HKIII causes protein aggregation. (A) Plasmids coding for the expression of the full- length HKII (HKII); full length HKIII (HKIII), a chimeric protein replacing the 32amino acid N-terminal region of HKIII (encoded by exon 1b) with the 21aa mitochondrial-targeting sequence of HKII (Mt-HKIII), and a truncated version that lacked the N-terminal 32aa region encoded by exon 1b (Tr- HKIII). Each construct also coded for GFP expression at the C-terminus. (B) Live cell confocal images of HEK cells after transfection with the indicated constructs. Overexpression of HKII resulted in co-localization with the mitochondria membrane marker TMRE (red). Co-localization was not observed with overexpression of the HKIII or Mt-HKIII constructs. Overexpression of Mt-HKIII or the truncated HKIII constructed resulted in aggregate formation (white arrows). Nuclei were labeled with Hoechst (blue). doi:10.1371/journal.pone.0013823.g008 for basal HKIII expression [9], but little else is known about the regulation of HKIII activity. Sequence analysis of the HKIII promoter identified several putative regulatory elements that may respond to cAMP, AMPK, or HIF signal transduction. Pharma- cological stimulation with cAMP or AMPK activators (forskolin and AICAR, respectively) had no measurable effect on HKIII mRNA expression, but cellular HKIII expression was significantly increased by hypoxia through a HIF-dependent pathway. These findings are the first to demonstrate that HKIII expression is regulated by a specific signaling pathway, and identifies a likely role for this isozyme in the cellular response to the metabolic stresses induced by hypoxia. Furthermore, the inducible expres- sion of HKII and III suggests that these enzymes have a role in adaptive metabolic responses to changes in the cellular environment. Previous reports also indicate that the two domains of HKI and HKII do not function independently. Here, we demonstrate that mutation of the glucose- or G6P-binding sites in the N-half of HKIII impairs catalysis in the C-half. Thus, the activity of HKIII appears to be governed, in part, by functional interactions between the two domains. Previous studies on HKI demonstrate a cooperative effect of glucose binding in the N-half to subsequent binding in the C-half [42,43]. It is possible that a similar intramolecular interaction occurs in HKIII, and reduction in N- half glucose binding results in diminished C-half binding, and an overall reduction in activity; however, other possibilities cannot be excluded. We also found that a mutation in the putative N- terminal G6P binding site reduced HKIII activity. This observa- tion (i.e., that mutation of an inhibitory site would decrease enzyme activity) was unexpected and may have been caused by a structural change that adversely affected glucose binding in the N- half. Furthermore, mutation of the N-terminal ATP binding site did not alter the catalytic activity of the enzyme, which suggests that either the mutated residue was not essential for N-terminal ATP binding, or ATP binding in the N-half of HKIII does not regulate the activity of the C-half. Further enzymatic analysis PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e13823