Glucose phosphorylation and mitochondrial binding are required for the protective effects of hexokinases I and II.

Mammalian hexokinases (HKs) convert glucose to glucose-6-phosphate (G6P), thus maintaining the downhill concentration gradient necessary to move glucose into the cell through glucose transporters (13, 24, 33, 42). Four isozymes of HKs are present in mammals: HKI, II, III, and IV (32, 50). HKIV is also known as glucokinase. The molecular masses of HKI, II, and III are about 100 kDa, whereas glucokinase has a molecular mass of 50 kDa. The protein and gene structures of the 100-kDa enzymes suggest that they evolved from gene duplication and fusion of an ancestral 50-kDa enzyme. In HKI and HKIII, only their C-terminal halves retained catalytic activity (6, 7, 10, 27, 35, 45, 54, 56), while both halves of HKII are catalytically active and sensitive to G6P (5). There is also functional interaction between the N- and C-terminal halves of HKII, as glucose binding by the N-terminal half causes the activity of the C-terminal half to be regulated at significantly lower concentrations of G6P (4). In addition to their role in glucose metabolism, HKI and HKII are also believed to play a role in regulating cell death (37). The levels of HKI and HKII are markedly elevated in many cancer cells (41), suggesting that their overexpression may play a role in dysregulated growth. The overexpression of HKI and HKII also leads to protection against oxidant-induced death in tissue culture. Bryson et al. showed that HKI overexpression in an established epithelial cell line leads to protection against oxidant-induced cell death (17). Furthermore, the induction of endogenous HK expression had similar effects. This protection was glucose dependent, suggesting that the increase in glucose phosphorylation may be the main mechanism for cellular protection by HKI. Ahmad et al. expressed HKII in lung epithelial cells and showed that the expression of this protein also protects against oxidant-induced cell death (2). Thus, the overexpression of both HKI and HKII protects cells against damage from oxidative stress. The mechanisms by which HKI and HKII protect against cell death are not clear, and two hypotheses have been proposed to explain the mechanism for this phenomenon: increase in glucose phosphorylation due to elevated HK activity and HK binding to mitochondria. HKI and HKII contain highly conserved 21-amino-acid sequences at their N termini that are predicted to form a hydrophobic α helix (49, 58). This structure is essential and sufficient for the binding of HKI and HKII to mitochondria. The mitochondrially bound HKs preferentially use mitochondrially generated ATP as a substrate (8, 14-16). The specificity of HKI and HKII binding to the mitochondria is believed to be due to its interaction with voltage-dependent anion channel (VDAC) (1, 9, 22, 26). VDAC is a 30-kDa protein in the outer membrane of the mitochondria that allows the transport of many solutes and metabolites in and out of the intermembrane space (19, 20, 29, 30). Although VDAC plays a major role in the physiological process of solute and metabolite transport, it is also believed to form part of a pathological channel known as the mitochondrial permeability transition pore (mPTP). The opening of mPTP allows an influx of solutes with molecular masses of <1,500 Da into the matrix and an abrupt dissipation of the mitochondrial membrane potential (ΔΨm) (25, 52). The exact molecular structure of mPTP has not been characterized, but it is thought to be a macromolecular protein complex (25, 52) consisting of cyclophilin D (CypD) in the matrix, adenine nucleotide translocator (ANT) in the inner membrane, and VDAC in the outer membrane. Whether HK binding to VDAC causes the closure of mPTP and the inhibition of the mitochondrial permeability transition (MPT) is not clear, and several studies have suggested both mPTP-dependent and -independent pathways for the protective effects of HK binding to mitochondria. Pastorino et al. showed that the displacement of HKII from the mitochondria promotes Bax translocation and cytochrome c (cyto c) release in the setting of a prodeath stimulus, while the overexpression of HKII had opposite effects (39). Since the opening of mPTP is believed to be independent of Bax (11, 34), these results suggest an mPTP-independent pathway. Majewski et al. recently showed that HK binding to the mitochondria inhibits apoptosis and that these protective effects are independent of Bax and Bak (28). Finally, Azoulay-Zohar et al. studied the effects of purified HKI protein on mitochondrial swelling in isolated mitochondria (9). They showed that HKI interacts with and closes VDAC and possibly mPTP in isolated mitochondria. However, they noted that in tissue culture the protective effects of HKI were reversed by staurosporine. Since mPTP opening is insensitive to staurosporine (11, 34, 44), these latter results suggest that HKI binding to the mitochondria may not influence mPTP opening. Thus, there is no clear evidence that HK binding to mitochondria inhibits MPT and the closure of mPTP. The binding of HKs to the mitochondria may be dependent on the phosphorylation state of VDAC. Recently, Pastorino et al. demonstrated that glycogen synthase kinase 3β (GSK-3β) can phosphorylate VDAC and that this phosphorylation affects HK binding to the mitochondria (38). The phosphorylation of VDAC in turn appears to regulate cell survival, as an increase in phosphorylation led to an increase in cell death. Baines et al. showed that protein kinase Cɛ (PKCɛ) interacts with and phosphorylates VDAC and that incubation of recombinant PKCɛ with isolated mitochondria results in a significant decrease in mPTP opening (11). Thus, VDAC is phosphorylated by GSK-3β and possibly PKCɛ, and its phosphorylation state may regulate HK binding to mitochondria, mPTP opening, and cell death. In this paper, we studied the relative contributions of glucose phosphorylation activity and mitochondrial binding of HKI and HKII to their overall protective effects and showed that both of these activities contribute to and are needed for this process. We also demonstrate that HKI and HKII reduce the intracellular levels of reactive oxygen species (ROS) and inhibit MPT. Furthermore, our results suggest that HK overexpression leads to VDAC phosphorylation through a PKCɛ-dependent pathway.