This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, L.
Right arrow Articles by Ardehali, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, L.
Right arrow Articles by Ardehali, H.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, February 2008, p. 1007-1017, Vol. 28, No. 3
0270-7306/08/$08.00+0     doi:10.1128/MCB.00224-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Glucose Phosphorylation and Mitochondrial Binding Are Required for the Protective Effects of Hexokinases I and II{triangledown} ,{dagger}

Lin Sun, Shetha Shukair, Tejaswitha Jairaj Naik, Farzad Moazed, and Hossein Ardehali*

Feinberg Cardiovascular Institute, Northwestern University School of Medicine, Chicago, Illinois

Received 6 February 2007/ Returned for modification 5 April 2007/ Accepted 5 November 2007


arrow
ABSTRACT
 
Alterations in glucose metabolism have been demonstrated for diverse disorders ranging from heart disease to cancer. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. HKI and II can bind to mitochondria through their N-terminal hydrophobic regions, and their overexpression in tissue culture protects against cell death. In order to determine the relative contributions of mitochondrial binding and glucose-phosphorylating activities of HKs to their overall protective effects, we expressed full-length HKI and HKII, their truncated proteins lacking the mitochondrial binding domains, and catalytically inactive proteins in tissue culture. The overexpression of full-length proteins resulted in protection against cell death, decreased levels of reactive oxygen species, and possibly inhibited mitochondrial permeability transition in response to H2O2. However, the truncated and mutant proteins exerted only partial effects. Similar results were obtained with primary neonatal rat cardiomyocytes. The HK proteins also resulted in an increase in the phosphorylation of voltage-dependent anion channel (VDAC) through a protein kinase C{varepsilon} (PKC{varepsilon})-dependent pathway. These results suggest that both glucose phosphorylation and mitochondrial binding contribute to the protective effects of HKI and HKII, possibly through VDAC phosphorylation by PKC{varepsilon}.


arrow
INTRODUCTION
 
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 {alpha} 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 ({Delta}{Psi}m) (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 C{varepsilon} (PKC{varepsilon}) interacts with and phosphorylates VDAC and that incubation of recombinant PKC{varepsilon} with isolated mitochondria results in a significant decrease in mPTP opening (11). Thus, VDAC is phosphorylated by GSK-3β and possibly PKC{varepsilon}, 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 PKC{varepsilon}-dependent pathway.


arrow
MATERIALS AND METHODS
 
Construction of full-length, truncated, and mutant HKI and HKII. Plasmids containing the HKI and HKII cDNAs were kindly provided by Daryl Granner (Vanderbilt University, Nashville, TN). The constructs for this study are shown in Fig. 1A, and the details are available in the supplemental material.


Figure 1
View larger version (30K):
[in this window]
[in a new window]

  		FIG. 1. Overexpression of various HK proteins in HEK293 cells. (A) Plasmid constructs used in our studies. All protein products contain GFP at their C termini. Since only the C-terminal half of HKI (C-HKI) is catalytically active, a mutation (D657A) was introduced in that half to make the enzyme inactive. Both halves of HKII have catalytic activity; thus, mutations in both halves (D209A and D657A) were made. The truncated proteins lack the N-terminal 21-amino-acid hydrophobic region and are unable to bind to the mitochondria. Each individual half of HKI and HKII was also expressed as a GFP fusion protein. M, mitochondrial binding domain; N, N-terminal half; Mito binding, mitochondrial binding; Glc phos, glucose phosphorylation; Trunc, truncated; Mut, mutant. (B) Western blots of extracts from transfected cells. Endogenous protein is at 100 kDa, while exogenously expressed GFP fusion proteins are at 120 kDa. The top panel was probed with HKI, and the bottom panel was probed with HKII antibody. M-HKI and M-HKII, mutant HKI and HKII, respectively; Tr-HKI and Tr-HKII, truncated HKI and HKII, respectively. (C) Confocal images of cells transfected with HK constructs and stained with TMRE. Full-length and mutant HKs localize mostly to mitochondria, while truncated proteins are cytoplasmic. Control cells were sham-transfected cells.

Transfection of the constructs into HEK293 cells. The constructs were transfected into the HEK293 cells by the Invitrogen Lipofectamine Plus kit. About 3 h after transfection, medium plus serum was added and the cells were incubated for 2 days before cell viability was measured. The medium was changed to serum-free medium 24 h prior to the viability studies. The overexpression of HK proteins was confirmed by Western blotting. Since the exogenous HKs are fused to green fluorescent protein (GFP), they are larger than the native HKs and can be distinguished. To establish stable transfectants, cells were treated with 2 mg/ml of G418 on the first day followed by 500 µg/ml.

Viability studies. Viability studies are described in the supplemental material.

Measurement of the MPT in live cells. To measure MPT, we used 10 µM of CellTrace calcein blue acetoxymethyl ester (AM), which is the blue version of calcein AM. This form of calcein was used in place of green calcein to avoid interference with GFP fluorescence. Tetramethylrhodamine ethyl ester (TMRE) at 100 nM was also added to delineate the mitochondria. Cells were monitored at baseline and 15- and 30-min intervals after the addition of H2O2 by use of confocal microscopy.

IP of VDAC and its phosphorylated form. For PKC{varepsilon}, we used PKC{varepsilon} inhibitor peptide (Santa Cruz Biotechnology) at a final concentration of 750 nM (46). To isolate mitochondria, cells were washed in phosphate-buffered saline and incubated for 30 min on ice in lysis buffer (68 mmol/liter sucrose, 200 mmol/liter mannitol, 50 mmol/liter KCl, 1 mmol/liter EGTA, 1 mmol/liter EDTA, 1 mmol/liter dithiothreitol, and one Complete Mini protease inhibitor tablet [Roche]). Cells were then homogenized twice with an ultrasound sonicator and centrifuged at 4°C (500 x g) to remove nuclei, unbroken cells, and debris. The supernatant was centrifuged at 14,000 x g for 15 min. Immunoprecipitation (IP) studies were performed using a Protein G IP kit (Sigma). For phospho-VDAC, VDAC antibody was used as the IP antibody and phosphothreonine antibody was used to probe the membranes.

Preparation of neonatal rat cardiomyocytes (NRCM) and adenoviruses. NRCM preparation and adenoviral construction were performed as described previously (3) and in the supplemental material.


arrow
RESULTS
 
Overexpression of HK constructs in HEK293 cells. In order to characterize the relative contributions of glucose phosphorylation and mitochondrial binding of HKs to their overall protective effects, we cloned full-length, truncated (lacking the N-terminal mitochondrial binding domain), and mutant HKI and HKII cDNAs into a GFP-containing plasmid (Fig. 1A) and transfected them into HEK293 cells. The full-length enzymes can bind to mitochondria and can phosphorylate glucose. Although the truncated HKI and HKII proteins cannot bind to mitochondria, they contain the catalytic domains and maintain their glucose-phosphorylating activity. The catalytically inactive enzymes contain the mitochondrial binding sequence but do not phosphorylate glucose. Western blot analysis of extracts from transfected cells demonstrated the expression of GFP fusion proteins of full-length, truncated, and mutant HKs in these cells (120-kDa band in Fig. 1B). Furthermore, our results demonstrate the expression of endogenous HKI and HKII proteins in HEK293 cells (100-kDa band in Fig. 1B). Confocal analysis of transfected cells demonstrated that the majority of the overexpressed full-length and mutant proteins localize to the mitochondria, while the truncated protein is mostly cytoplasmic (Fig. 1C). It should be noted that a portion of cells transfected with the full-length and mutant proteins also displayed green fluorescence throughout the cell (data not shown). Transfection efficiency in our experiments is shown in Fig. S1 in the supplemental material.

Full-length HKs result in full protection, while truncated and mutant proteins exert partial protection. Hydrogen peroxide was used to induce cell death. Concentration curve and time course studies demonstrated that a 60-min treatment of HEK293 cells with 1 mM H2O2 resulted in an ~35 to 40% reduction in TMRE uptake (see Fig. S2A and B in the supplemental material). This H2O2 concentration and treatment time course were used for subsequent viability studies.

Forty-eight hours after transfection, cells were treated with the mitochondrial marker TMRE and were analyzed under confocal microscopy at baseline and 15 min after treatment with H2O2. While the full-length proteins led to the retention of TMRE in response to H2O2, the overexpression of the truncated or mutant proteins resulted in only partial protection (Fig. 2A). Cells treated with TMRE were also subjected to flow cytometry, and the data show that full-length proteins exert complete protection, while the mutant and truncated proteins partially protect against H2O2-induced cell death (Fig. 2B). Similar results were obtained when trypan blue exclusion was used as an additional measure of cell viability (Fig. 2C). Constructs lacking mitochondrial binding and glucose phosphorylation activity did not exert any protective effects. As shown in Fig. 2D and E, the protective effects of the HK constructs persisted for at least 6 h after the addition of H2O2. The HK constructs did not exert any protection in the absence of glucose in the medium (see Fig. S3 in the supplemental material).


Figure 2
View larger version (37K):
[in this window]
[in a new window]

  		FIG. 2. Full-length HKI and HKII protect against H2O2-induced cell death, while the truncated and mutant proteins exert partial protection. (A) Confocal images of cells transfected with various HK constructs and loaded with TMRE at baseline and 15 min after the addition of H2O2. The first image in each series is a merged image of green and red fluorescence (i.e., TMRE and GFP). While the full-length proteins maintained TMRE in the mitochondria after H2O2 treatment, truncated and mutant proteins led to only partial effects. (B) Flow cytometry of transfected cells treated with TMRE showed that H2O2 treatment leads to about 35% cell death in GFP-transfected cells. Full-length proteins led to significant protection, while the mutant and truncated proteins exerted only partial protection. Truncated and mutant proteins (i.e., TM-HKI and TM-HKII) did not protect against cell death. (C) Summary of trypan blue exclusion studies of cells transfected with various HK constructs. In accordance with TMRE results, full-length HK proteins protected against cell death, while the mutant and truncated proteins exerted partial protection. (D and E) Time course of cell death in the presence of various HKI and HKII constructs in response to H2O2 as assessed by trypan blue staining. Data are represented as mean ± standard error of the mean (SEM). n ≥ 6 in each group, except for TM-HKI and -HKII (n = 3). Abbreviations: Tr, truncated; TM, truncated and mutant; M, mutant F, full length.

In addition to performing the TMRE and trypan blue experiments, we also studied the effects of overexpression of HK proteins on cyto c release and Bax translocation into the mitochondria. The overexpression of full-length HKs led to a significant increase in the mitochondrial cyto c levels, suggesting a decrease in cyto c release into the cytoplasm. Similarly, full-length HKI and HKII decreased the mitochondrial Bax level, indicating a decrease in Bax translocation into the mitochondria. However, cells transfected with both truncated and mutant HKI and HKII displayed a lower degree of change in these parameters (Fig. 3A). Thus, in accordance with the TMRE and trypan blue studies, these results suggest that both glucose phosphorylation and mitochondrial binding contribute to the overall protective effects of HKs. We also assessed the effects of overexpression of HK constructs on cyto c distribution within the cells by confocal microscopy. HKI and II constructs prevented cyto c release into the cytoplasm in response to H2O2 (Fig. 3B).


Figure 3
View larger version (50K):
[in this window]
[in a new window]

  		FIG. 3. HKI and HKII inhibit cyto c release, Bax translocation into the mitochondria, and the generation of ROS. (A) cyto c release and Bax translocation into the mitochondria in response to H2O2 in cells transfected with various HK constructs. Mitochondrial extracts from cells were isolated and membranes were probed with cyto c and Bax antibodies. The levels of mitochondrial cyto c were significantly reduced in GFP-transfected cells with H2O2 treatment, while mitochondrial Bax was increased under those conditions. However, the overexpression of full-length HKI and HKII reversed those effects. Truncated and mutant HKI and HKII had partial effects. An antibody against ATP synthase was used as an internal control. (B) Immunofluorescence of control cells and cells overexpressing GFP or various HK constructs stained with cyto c antibody in the presence and absence of H2O2. While treatment of control and GFP-transfected cells resulted in cyto c redistribution within the cells, the overexpression of HKs (at least partially) reversed this process. (C) Confocal images of cells treated with various HK constructs and treated with an ROS marker, mitoSOX. While GFP-transfected cells showed an increase in the levels of intracellular ROS in the presence of H2O2, this was significantly reduced in cells transfected with HK constructs. The bar graph represents a summary of flow cytometry experiments from mitoSOX studies. Data are represented as mean ± SEM. n = 3 in each group. Abbreviations: Cont., control; FL, full-length; Tr, truncated; M, mutant.

Studies by da-Silva et al. suggested that mitochondrially bound HK plays a role in the rate of endogenous ROS generation, possibly through an ADP-recycling process (21). Thus, we assessed whether the overexpression of HKs changed the levels of cellular ROS in the presence of exogenously added H2O2. MitoSOX, a fluorescent marker of ROS, was added to transfected cells followed by the addition of H2O2. As shown in Fig. 3C, full-length proteins led to a significant reduction in the levels of ROS in cells treated with H2O2. The truncated and mutant proteins also resulted in a significant reduction in ROS levels but slightly less than that seen for the full-length protein (and did not reach statistical significance).

N- and C-terminal halves of HKI and HKII are partially protective against H2O2-induced cell death. HKI and HKII consist of two homologous 50-kDa halves. While the C-terminal half of HKI contains the only catalytically active site of the protein, both halves of HKII are fully capable of phosphorylating glucose. In order to better characterize the protective effects of HKI and HKII proteins, we overexpressed each half of these enzymes separately in HEK293 cells. Western blot analysis of transfected cellular extracts demonstrated the presence of 70-kDa proteins, representing each half of the proteins fused to GFP (Fig. 4A). The cells were then treated with TMRE in the presence and absence of H2O2 followed by flow cytometry. As shown in Fig. 4B, the overexpression of individual halves of the HKI and HKII proteins resulted in only partial protection against H2O2-induced cell death. Similar results were obtained when trypan blue exclusion was used to assess cell death (Fig. 4C). Although the N-terminal half of HKII can both bind to the mitochondria and phosphorylate glucose, its overexpression did not lead to full protection.


Figure 4
View larger version (34K):
[in this window]
[in a new window]

  		FIG. 4. Overexpression of individual HK halves results in partial protection. (A) Western blot of extracts from cells overexpressing the N- and C-terminal halves of HKI (N-HKI and C-HKI, respectively) and HKII. The GFP fusion protein of each half yields a protein of about 68 kDa. The left panels represent extracts from cells treated with N- and C-terminal halves of HKI, and the right panels show extracts from cells transfected with N- and C-terminal halves of HKII. (B) Flow cytometry of cells transfected with HKI and HKII halves and loaded with TMRE. The overexpression of each half led to partial protection against H2O2-induced cell death. (C) Similar results were obtained when trypan blue staining was used to measure cell death. Data are represented as mean ± SEM. n ≥ 6 in the HK groups.

HKI and HKII are protective in primary cardiomyocyte cultures. The studies reported thus far were performed in HEK293 cells, which are an immortalized transformed cell line. Thus, it is essential to extend the studies to a primary cell line in order to better assess the physiological significance of these findings. Since both HKI and HKII are expressed in cardiac cells, we chose NRCM for our studies. NRCM do not take up DNA when transfected with routine techniques. Thus, full-length and truncated HKI and HKII adenoviruses were made to introduce these DNAs into the cell. Full-length HKI virus resulted in an increase in HK activity in cell extracts in a dose-dependent manner (see Fig. S4 in the supplemental material). As shown in Fig. 5A, the transfection of NRCM with different HK adenoviruses yielded green fluorescence in >90% of the cells. Since the HK and GFP cDNAs are under different promoters, the generated HKs are similar in size to the endogenous proteins. Western blot analysis showed significantly high levels of both HKI and HKII proteins in cells transfected with HK adenoviruses compared to GFP-transfected cells (Fig. 5B). In accordance with results obtained with HEK293 cells, the overexpression of full-length proteins resulted in significant protection, while truncated proteins exerted only partial protection (Fig. 5C).


Figure 5
View larger version (32K):
[in this window]
[in a new window]

  		FIG. 5. Full-length HKI and HKII protect against H2O2-induced cell death in NRCM. (A) Confocal images of NRCM transduced with full-length and truncated HKI and HKII adenoviruses. More than 90% of the cells displayed green fluorescence when the adenovirus was added. (B) Western blots of cells transduced with GFP and various HK adenoviruses. The HK adenoviruses contain GFP and the HK cDNAs under different promoters; thus, the overexpressed HKs are the same size as the endogenous proteins. There are a significantly higher levels of HK in cells transduced with HK adenoviruses compared to GFP-only adenovirus. The top and bottom panels were probed with HKI and HKII antibodies, respectively. (C) Summary data of flow cytometry of NRCM treated with various HK constructs and loaded with TMRE. Data are represented as mean ± SEM. n = 3 in each group. Abbreviations: FL, full-length; Tr, truncated.

Full-length HKI and HKII inhibit MPT. The mechanism for the protective effects of HK binding to mitochondria is not totally understood, and both mPTP-dependent and -independent pathways have been proposed. In the next series of experiments, we attempted to study whether HK binding to mitochondria can cause inhibition of MPT in intact cells and isolated mitochondria. Calcein AM can diffuse across the membrane and is trapped inside the cell after it is converted to a pentavalent acid form. Given the relatively impermeable nature of the mitochondrial membrane to this dye, it does not enter the mitochondria for hours. However, the induction of MPT in response to injurious insults allows the leakage of the dye into the mitochondria. In our experiments, we used the blue form of calcein, and the mitochondria were simultaneously stained with TMRE. This allowed us to delineate the mitochondria and cytoplasm in green fluorescence positive cells. As shown in Fig. 6, while the treatment of control GFP-positive cells with H2O2 resulted in a significant mitochondrial permeability after 15 min, full-length HKI and HKII reversed this effect. The overexpression of the truncated proteins also led to a partial reversal to a degree significantly less than that seen for the full-length proteins. Thus, the induction of MPT by oxidant injury may be reversed by HK overexpression, and this mechanism is dependent on mitochondrial binding of these proteins.


Figure 6
View larger version (78K):
[in this window]
[in a new window]

  		FIG. 6. Full-length HKs possibly protect against MPT in response to H2O2. Confocal images of HEK293 cells transfected with HK constructs and loaded with CellTrace calcein blue AM and TMRE. Transfected cells were treated with H2O2, and confocal images were obtained at baseline and at 15- and 30-min intervals after the addition of H2O2. Results suggest that full-length HKI and HKII may prevent MPT, while the truncated proteins exert a less significant effect. Abbreviations: FL, full-length; Tr, truncated.

HK overexpression increases VDAC phosphorylation in a PKC{varepsilon}-dependent pathway. Since HK binding to mitochondria is dependent on the phosphorylation state of VDAC, we hypothesized that HKs regulate their mitochondrial binding by modulating VDAC phosphorylation. In order to test this hypothesis, we measured VDAC phosphorylation in response to the overexpression of HK proteins. Extracts of cells transfected with various HK constructs were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and their membranes were probed with VDAC antibody. We then measured phospho-VDAC by immunoprecipitating total VDAC and probing with a phosphothreonine-reactive antibody. As shown in Fig. 7A, full-length HKI and HKII resulted in about a 5-to 10-fold increase in VDAC phosphorylation without significant effects on the amount of total VDAC. The truncated and mutant proteins also increased VDAC phosphorylation, but to a lesser degree than the full-length proteins. These results suggest that the activation of the protein kinase that phosphorylates VDAC probably requires both the glucose phosphorylation and mitochondrial binding properties of HKs.


Figure 7
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 7. Full-length HKI and HKII increase VDAC phosphorylation in a PKC{varepsilon}-dependent pathway. (A) Western blots of extracts from HEK293 cells transfected with various HK constructs. The extracts were loaded on a column with VDAC antibody, and the eluate was probed with a phosphothreonine antibody, thus representing phosphorylated VDAC (top). The relative intensity of phosphorylated VDAC to total VDAC in each lane is represented in the bar graph next to the blots. Full-length HKI and HKII resulted in a significant increase in VDAC phosphorylation, while the truncated and mutant forms led to a smaller response. (B) Western blot of cell extracts treated with full-length HKI or HKII in the presence or absence of a PKC{varepsilon} inhibitor peptide. The addition of the PKC{varepsilon} inhibitor peptide resulted in a significant decrease in VDAC phosphorylation both in the presence and in the absence of HK overexpression, suggesting that VDAC phosphorylation is mediated through PKC{varepsilon}. The membranes in each panel were probed for phospho-VDAC (top) or total VDAC (bottom). The bar graphs next to each membrane represent the relative intensity of phosphorylated to total VDAC. Abbreviations: FL, full-length; M, mutant; Tr, truncated; In, inhibitor.

In order to characterize the protein kinases involved in HK-induced VDAC phosphorylation, we focused our studies on PKC{varepsilon}. This protein is shown to physically interact with VDAC in cardiomyocytes (12). The addition of a PKC{varepsilon} inhibitor peptide to HEK293 cells resulted in a significant decrease in VDAC phosphorylation at baseline and in the presence of HKI and HKII overexpression (Fig. 7B). These results suggest that PKC{varepsilon} is a mediator of VDAC phosphorylation in response to HKI and HKII overexpression.


arrow
DISCUSSION
 
HKs play a critical role in ATP production by catalyzing the first step in glucose metabolism and maintaining the gradient for glucose entry into the cell (13, 24, 33, 42). In the past several years, HKI and HKII have also been implicated as playing a role in cell death and survival. The overexpression of these enzymes in tissue culture protects cells from oxidant-induced cell death (2, 17). Some studies have implied that the increase in ATP production as a result of glucose metabolism may be the underlying mechanism for the protective effects of HKs (17). However, others have proposed that the binding of HKI and HKII to the mitochondria may lead to the closure of the proapoptotic channel mPTP, leading to cellular protection (9, 37). In this paper, we studied the mechanism for the protective effects of HKI and HKII. We overexpressed full-length HKI and HKII, truncated proteins (lacking the N-terminal mitochondrial binding domain), and catalytically inactive forms of HKI and HKII in HEK293 cells. The truncated proteins can phosphorylate glucose but cannot bind to mitochondria, while the mutant enzymes can bind to mitochondria but cannot phosphorylate glucose. We then exposed the cells to H2O2 and assessed cellular injury by several methods: TMRE uptake, trypan blue exclusion studies, Bax translocation into the mitochondria, and cyto c release into the cytoplasm. These studies demonstrated that full-length proteins can protect against H2O2-induced cell death, while the mutant and truncated proteins exert only partial protection. Similar results were obtained with primary cardiac cells. Thus, we conclude that both mitochondrial binding and glucose phosphorylation contribute to and are needed for the protective effects of HKs. The full-length proteins also decreased the levels of intracellular ROS, suggesting an antioxidant role for these proteins. We then determined that HKI and HKII overexpression lead to an increase in VDAC phosphorylation in a PKC{varepsilon}-dependent pathway.

The phosphorylation of glucose maintains the downhill gradient for glucose metabolism and extramitochondrial glucose flux through the pentose phosphate pathway. Induction of the pentose phosphate pathway and the generation of NADPH may also contribute to protection against cell death. In oocytes, maintenance of the pentose phosphate pathway through a continuous supply of G6P prolongs the survival of the germ cells, and this process was shown to be through calcium/calmodulin-dependent protein kinase II (36). Thus, the protective effects of glucose phosphorylation by HKs may be through both enhanced ATP production and induction of the pentose phosphate pathway.

The HK binding to the mitochondria and its glucose-phosphorylating activity may play an important role in the development of various disorders ranging from cancer to heart disease. The levels of HKI and HKII are markedly elevated in many cancer cells (37). For example, the levels of HKII are >100-fold higher than normal levels in some tumors (41). Although it is not expressed in normal liver tissue, HKII becomes the predominant HK isoform in liver cancer (18). The levels of HKI are also upregulated in some tumors but to a lesser degree relative to HKII (23, 47). In fact, this unique feature of cancer cells is used to detect rapidly growing cells by use of positron emission tomography (40, 51). In addition, evidence now suggests that a large pool of HKs is bound to the mitochondria in cancer cells (37, 41). For the heart, it has been shown that mitochondrial HK binding increases with insulin treatment and ischemia in an isoform-specific manner (48). Taken together, these data suggest a major role for mitochondrial binding and glucose-phosphorylating activities of HKI and HKII in the progression of a number of diseases and identify them as a target for therapy.

The ATP that is generated through the process of oxidative phosphorylation is transported out of the mitochondrial matrix by ANT. In the intermembrane space, ATP may get consumed by enzymes such as mitochondrial creatine kinase or adenylate kinase, or it may get shuttled into the cytoplasm through VDAC. It was originally believed that the mitochondrially bound HK would have preferential access to this highly localized concentration of ATP (31, 41, 56). Furthermore, the ADP that is produced by glucose phosphorylation can be effectively delivered to ANT for transport to the matrix and ATP synthase. This hypothesis has been challenged by observations that the ATP concentration in the cytoplasm is usually in the 3 to 5 mM range, which is well above the KmATP of HKs. Furthermore, through the actions of mitochondrial and cytosolic creatine kinases, ATP is effectively shuttled from the mitochondria to the cytoplasm. If HK binding to mitochondria does in fact increase their glucose phosphorylation activity, then the mitochondrial binding of HKs may reduce apoptosis by both inhibition of MPT and more-efficient glucose metabolism due to their better access to mitochondrially generated ATP.

While the C-terminal half of HKI contains the only active site of the enzyme (54), both halves of HKII are catalytically active. Thus, the N-terminal half of HKII can both bind to the mitochondria and phosphorylate glucose (5). Yet, it exerted only partial protection in our experiments. One implication of this observation is that both halves of HKs may be needed for the full protective effects of these enzymes. Several studies have demonstrated a functional interaction between the two halves of HKI and HKII. G6P regulation of the C-terminal half of HKI is believed to be through its binding to the N-terminal half (53, 55), and glucose binding by the N-terminal half of HKII causes the activity of the C-terminal half to be regulated by significantly lower concentrations of G6P (4). Thus, the physical and functional interaction between the two halves of HKI and HKII may also influence the antiapoptotic effects of these enzymes. Furthermore, it is believed that HKs may form multimers (56, 57), and their intermolecular interactions may have physiological implications both in terms of regulation of glucose phosphorylation and apoptotic activities of these proteins. Thus, it is also possible that some of our overexpressed HK proteins may interact with endogenous HKs and disrupt the multimer formation of these proteins. This phenomenon could potentially explain some of the effects we have observed in our overexpression experiments.

The phosphorylation of VDAC may play a role in regulating its ability to bind to HKII and the opening of the mPTP. Pastorino et al. showed that GSK-3β can phosphorylate VDAC and that this phosphorylation decreases the ability of HKII to bind to this protein (38). Another study has demonstrated that PKC{varepsilon} can interact with and phosphorylate VDAC and reduce Ca2+-induced mitochondrial swelling (12). Our results show that the overexpression of HKI and II increases VDAC phosphorylation in a PKC{varepsilon}-dependent pathway. It is unlikely that HKs directly activate PKC{varepsilon}, and other mediators probably play a role in this process. It remains unclear what signaling molecules lead to the activation of PKC{varepsilon} and its possible translocation into the mitochondria and to VDAC phosphorylation.

Another controversy in the HK field is related to the question of whether HK binding to VDAC leads to its closure or to its opening. Rostovtseva et al. have suggested that VDAC closure, not opening, results in mitochondrial swelling and cell death (43). This is probably due to a reduced mitochondrial metabolite exchange, which would lead to significant functional implications for a cell, especially in the brain and heart. This hypothesis was supported by Majewski et al., who showed that HK dislocation from mitochondria could lead to the closure of VDAC and subsequent mitochondrial swelling and cell death (28). However, Azoulay-Zohar et al. showed that HK binding to VDAC causes its closure rather than its opening (9). Although our data do not resolve this controversy, we favor the former model, which is suggested by Rostovtseva et al. (43).

In summary, our results indicate that both glucose phosphorylation and mitochondrial binding activities of HKI and HKII are required for their protective effects. Furthermore, HKI and HKII overexpression increases VDAC phosphorylation in a PKC{varepsilon}-dependent pathway. These studies suggest that targeting HK enzyme activity and its cellular distribution may provide potential therapeutic options in various disorders ranging from cancer to heart disease.


arrow
ACKNOWLEDGMENTS
 
H.A. is supported by NIH grant K08 HL079387, R01 HL087149, the Schweppe Foundation, the American Heart Association, and a grant from the American Cancer Society, Illinois chapter.

We thank Daryl Granner for critical evaluation of the manuscript and providing the HKI and HKII cDNA plasmids.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Tarry 12-725, 303 East Chicago Ave., Chicago, IL 60611. Phone: (312) 503-2296. Fax: (312) 503-0137. E-mail: h-ardehali{at}northwestern.edu Back

{triangledown} Published ahead of print on 26 November 2007. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back


arrow
REFERENCES
 
    1
  1. Aflalo, C., and H. Azoulay. 1998. Binding of rat brain hexokinase to recombinant yeast mitochondria: effect of environmental factors and the source of porin. J. Bioenerg. Biomembr. 30:245-255.[CrossRef][Medline]
    2. 2
  2. Ahmad, A., S. Ahmad, B. K. Schneider, C. B. Allen, L. Y. Chang, and C. W. White. 2002. Elevated expression of hexokinase II protects human lung epithelial-like A549 cells against oxidative injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 283:L573-L584.[Abstract/Free Full Text]
    3. 3
  3. Ardehali, H., B. O'Rourke, and E. Marban. 2005. Cardioprotective role of the mitochondrial ATP-binding cassette protein 1. Circ. Res. 97:740-742.[Abstract/Free Full Text]
    4. 4
  4. Ardehali, H., R. L. Printz, R. R. Whitesell, J. M. May, and D. K. Granner. 1999. Functional interaction between the N- and C-terminal halves of human hexokinase II. J. Biol. Chem. 274:15986-15989.[Abstract/Free Full Text]
    5. 5
  5. Ardehali, H., Y. Yano, R. L. Printz, S. Koch, R. R. Whitesell, J. M. May, and D. K. Granner. 1996. Functional organization of mammalian hexokinase II. Retention of catalytic and regulatory functions in both the NH2- and COOH-terminal halves. J. Biol. Chem. 271:1849-1852.[Abstract/Free Full Text]
    6. 6
  6. Arora, K. K., C. R. Filburn, and P. L. Pedersen. 1991. Glucose phosphorylation. Site-directed mutations which impair the catalytic function of hexokinase. J. Biol. Chem. 266:5359-5362.[Abstract/Free Full Text]
    7. 7
  7. Arora, K. K., C. R. Filburn, and P. L. Pedersen. 1993. Structure/function relationships in hexokinase. Site-directed mutational analyses and characterization of overexpressed fragments implicate different functions for the N- and C-terminal halves of the enzyme. J. Biol. Chem. 268:18259-18266.[Abstract/Free Full Text]
    8. 8
  8. Arora, K. K., and P. L. Pedersen. 1988. Functional significance of mitochondrial bound hexokinase in tumor cell metabolism. Evidence for preferential phosphorylation of glucose by intramitochondrially generated ATP. J. Biol. Chem. 263:17422-17428.[Abstract/Free Full Text]
    9. 9
  9. Azoulay-Zohar, H., A. Israelson, S. Abu-Hamad, and V. Shoshan-Barmatz. 2004. In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death. Biochem. J. 377:347-355.[CrossRef][Medline]
    10. 10
  10. Baijal, M., and J. E. Wilson. 1992. Functional consequences of mutation of highly conserved serine residues, found at equivalent positions in the N- and C-terminal domains of mammalian hexokinases. Arch. Biochem. Biophys. 298:271-278.[CrossRef][Medline]
    11. 11
  11. Baines, C. P., R. A. Kaiser, N. H. Purcell, N. S. Blair, H. Osinska, M. A. Hambleton, E. W. Brunskill, M. R. Sayen, R. A. Gottlieb, G. W. Dorn, J. Robbins, and J. D. Molkentin. 2005. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 4:658-662.[CrossRef]
    12. 12
  12. Baines, C. P., C. X. Song, Y. T. Zheng, G. W. Wang, J. Zhang, O. L. Wang, Y. Guo, R. Bolli, E. M. Cardwell, and P. Ping. 2003. Protein kinase Cepsilon interacts with and inhibits the permeability transition pore in cardiac mitochondria. Circ. Res. 92:873-880.[Abstract/Free Full Text]
    13. 13
  13. Bell, G. I., C. F. Burant, J. Takeda, and G. W. Gould. 1993. Structure and function of mammalian facilitative sugar transporters. J. Biol. Chem. 268:19161-19164.[Free Full Text]
    14. 14
  14. BeltrandelRio, H., and J. E. Wilson. 1992. Coordinated regulation of cerebral glycolytic and oxidative metabolism, mediated by mitochondrially bound hexokinase dependent on intramitochondrially generated ATP. Arch. Biochem. Biophys. 296:667-677.[CrossRef][Medline]
    15. 15
  15. BeltrandelRio, H., and J. E. Wilson. 1991. Hexokinase of rat brain mitochondria: relative importance of adenylate kinase and oxidative phosphorylation as sources of substrate ATP, and interaction with intramitochondrial compartments of ATP and ADP. Arch. Biochem. Biophys. 286:183-194.[CrossRef][Medline]
    16. 16
  16. BeltrandelRio, H., and J. E. Wilson. 1992. Interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation and creatine kinase. Arch. Biochem. Biophys. 299:116-124.[CrossRef][Medline]
    17. 17
  17. Bryson, J. M., P. E. Coy, K. Gottlob, N. Hay, and R. B. Robey. 2002. Increased hexokinase activity, of either ectopic or endogenous origin, protects renal epithelial cells against acute oxidant-induced cell death. J. Biol. Chem. 277:11392-11400.[Abstract/Free Full Text]
    18. 18
  18. Bustamante, E., H. P. Morris, and P. L. Pedersen. 1981. Energy metabolism of tumor cells. Requirement for a form of hexokinase with a propensity for mitochondrial binding. J. Biol. Chem. 256:8699-8704.[Abstract/Free Full Text]
    19. 19
  19. Casadio, R., I. Jacoboni, A. Messina, and V. De Pinto. 2002. A 3D model of the voltage-dependent anion channel (VDAC). FEBS Lett. 520:1-7.[CrossRef][Medline]
    20. 20
  20. Colombini, M., E. Blachly-Dyson, and M. Forte. 1996. VDAC, a channel in the outer mitochondrial membrane. Ion Channels 4:169-202.[Medline]
    21. 21
  21. da-Silva, W. S., A. Gomez-Puyou, M. T. de Gomez-Puyou, R. Moreno-Sanchez, F. G. De Felice, L. de Meis, M. F. Oliveira, and A. Galina. 2004. Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria. J. Biol. Chem. 279:39846-39855.[Abstract/Free Full Text]
    22. 22
  22. Fiek, C., R. Benz, N. Roos, and D. Brdiczka. 1982. Evidence for identity between the hexokinase-binding protein and the mitochondrial porin in the outer membrane of rat liver mitochondria. Biochim. Biophys. Acta 688:429-440.[Medline]
    23. 23
  23. Golestani, A., and M. Nemat-Gorgani. 2000. Hexokinase ‘binding sites’ of normal and tumoral human brain mitochondria. Mol. Cell. Biochem. 215:115-121.[CrossRef][Medline]
    24. 24
  24. Gould, G. W., and G. D. Holman. 1993. The glucose transporter family: structure, function and tissue-specific expression. Biochem. J. 295:329-341.[Medline]
    25. 25
  25. Halestrap, A. P., G. P. McStay, and S. J. Clarke. 2002. The permeability transition pore complex: another view. Biochimie 84:153-166.[Medline]
    26. 26
  26. Linden, M., P. Gellerfors, and B. D. Nelson. 1982. Pore protein and the hexokinase-binding protein from the outer membrane of rat liver mitochondria are identical. FEBS Lett. 141:189-192.[CrossRef][Medline]
    27. 27
  27. Magnani, M., M. Bianchi, A. Casabianca, V. Stocchi, A. Daniele, F. Altruda, M. Ferrone, and L. Silengo. 1992. A recombinant human ‘mini’-hexokinase is catalytically active and regulated by hexose 6-phosphates. Biochem. J. 285:193-199.[Medline]
    28. 28
  28. Majewski, N., V. Nogueira, P. Bhaskar, P. E. Coy, J. E. Skeen, K. Gottlob, N. S. Chandel, C. B. Thompson, R. B. Robey, and N. Hay. 2004. Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak. Mol. Cell 16:819-830.[CrossRef][Medline]
    29. 29
  29. Mannella, C. A. 1998. Conformational changes in the mitochondrial channel protein, VDAC, and their functional implications. J. Struct. Biol. 121:207-218.[CrossRef][Medline]
    30. 30
  30. Mannella, C. A., M. Forte, and M. Colombini. 1992. Toward the molecular structure of the mitochondrial channel, VDAC. J. Bioenerg. Biomembr. 24:7-19.[CrossRef][Medline]
    31. 31
  31. Mathupala, S. P., A. Rempel, and P. L. Pedersen. 1997. Aberrant glycolytic metabolism of cancer cells: a remarkable coordination of genetic, transcriptional, post-translational, and mutational events that lead to a critical role for type II hexokinase. J. Bioenerg. Biomembr. 29:339-343.[CrossRef][Medline]
    32. 32
  32. Middleton, R. J. 1990. Hexokinases and glucokinases. Biochem. Soc. Trans. 18:180-183.[Medline]
    33. 33
  33. Mueckler, M. 1994. Facilitative glucose transporters. Eur. J. Biochem. 219:713-725.[Medline]
    34. 34
  34. Nakagawa, T., S. Shimizu, T. Watanabe, O. Yamaguchi, K. Otsu, H. Yamagata, H. Inohara, T. Kubo, and Y. Tsujimoto. 2005. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 434:652-658.[CrossRef][Medline]
    35. 35
  35. Nemat-Gorgani, M., and J. E. Wilson. 1986. Rat brain hexokinase: location of the substrate nucleotide binding site in a structural domain at the C-terminus of the enzyme. Arch. Biochem. Biophys. 251:97-103.[CrossRef][Medline]
    36. 36
  36. Nutt, L. K., S. S. Margolis, M. Jensen, C. E. Herman, W. G. Dunphy, J. C. Rathmell, and S. Kornbluth. 2005. Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2. Cell 123:89-103.[CrossRef][Medline]
    37. 37
  37. Pastorino, J. G., and J. B. Hoek. 2003. Hexokinase II: the integration of energy metabolism and control of apoptosis. Curr. Med. Chem. 10:1535-1551.[CrossRef][Medline]
    38. 38
  38. Pastorino, J. G., J. B. Hoek, and N. Shulga. 2005. Activation of glycogen synthase kinase 3beta disrupts the binding of hexokinase II to mitochondria by phosphorylating voltage-dependent anion channel and potentiates chemotherapy-induced cytotoxicity. Cancer Res. 65:10545-10554.[Abstract/Free Full Text]
    39. 39
  39. Pastorino, J. G., N. Shulga, and J. B. Hoek. 2002. Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis. J. Biol. Chem. 277:7610-7618.[Abstract/Free Full Text]
    40. 40
  40. Patronas, N. J., G. Di Chiro, C. Kufta, D. Bairamian, P. L. Kornblith, R. Simon, and S. M. Larson. 1985. Prediction of survival in glioma patients by means of positron emission tomography. J. Neurosurg. 62:816-822.[Medline]
    41. 41
  41. Pedersen, P. L., S. Mathupala, A. Rempel, J. F. Geschwind, and Y. H. Ko. 2002. Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention. Biochim. Biophys. Acta 1555:14-20.[Medline]
    42. 42
  42. Printz, R. L., H. Osawa, H. Ardehali, S. Koch, and D. K. Granner. 1997. Hexokinase II gene: structure, regulation and promoter organization. Biochem. Soc. Trans. 25:107-112.[Medline]
    43. 43
  43. Rostovtseva, T. K., W. Tan, and M. Colombini. 2005. On the role of VDAC in apoptosis: fact and fiction. J. Bioenerg. Biomembr. 37:129-142.[CrossRef][Medline]
    44. 44
  44. Schinzel, A. C., O. Takeuchi, Z. Huang, J. K. Fisher, Z. Zhou, J. Rubens, C. Hetz, N. N. Danial, M. A. Moskowitz, and S. J. Korsmeyer. 2005. Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia. Proc. Natl. Acad. Sci. USA 102:12005-12010.[Abstract/Free Full Text]
    45. 45
  45. Schirch, D. M., and J. E. Wilson. 1987. Rat brain hexokinase: location of the substrate hexose binding site in a structural domain at the C-terminus of the enzyme. Arch. Biochem. Biophys. 254:385-396.[CrossRef][Medline]
    46. 46
  46. Shizukuda, Y., and P. M. Buttrick. 2001. Protein kinase C(epsilon) modulates apoptosis induced by beta-adrenergic stimulation in adult rat ventricular myocytes via extracellular signal-regulated kinase (ERK) activity. J. Mol. Cell. Cardiol. 33:1791-1803.[CrossRef][Medline]
    47. 47
  47. Smith, T. A. 2000. Mammalian hexokinases and their abnormal expression in cancer. Br. J. Biomed. Sci. 57:170-178.[CrossRef][Medline]
    48. 48
  48. Southworth, R., K. A. Davey, A. Warley, and P. B. Garlick. 2006. A re-evaluation of the roles of hexokinase I and II in the heart. Am. J. Physiol. Heart Circ. Physiol. 292:H378-H386.[CrossRef][Medline]
    49. 49
  49. Sui, D., and J. E. Wilson. 1997. Structural determinants for the intracellular localization of the isozymes of mammalian hexokinase: intracellular localization of fusion constructs incorporating structural elements from the hexokinase isozymes and the green fluorescent protein. Arch. Biochem. Biophys. 345:111-125.[CrossRef][Medline]
    50. 50
  50. Ureta, T. 1982. The comparative isozymology of vertebrate hexokinases. Comp. Biochem. Physiol. B 71:549-555.[CrossRef][Medline]
    51. 51
  51. Wagner, H. N., Jr. 1993. Oncology: a new engine for PET/SPECT. Highlights of the Society of Nuclear Medicine annual meeting. J. Nucl. Med. 34:13N-16N, 19N, 22N-29N.
    52. 52
  52. Weiss, J. N., P. Korge, H. M. Honda, and P. Ping. 2003. Role of the mitochondrial permeability transition in myocardial disease. Circ. Res. 93:292-301.[Abstract/Free Full Text]
    53. 53
  53. White, T. K., and J. E. Wilson. 1990. Binding of nucleoside triphosphates, inorganic phosphate, and other polyanionic ligands to the N-terminal region of rat brain hexokinase: relationship to regulation of hexokinase activity by antagonistic interactions between glucose 6-phosphate and inorganic phosphate. Arch. Biochem. Biophys. 277:26-34.[CrossRef][Medline]
    54. 54
  54. White, T. K., and J. E. Wilson. 1989. Isolation and characterization of the discrete N- and C-terminal halves of rat brain hexokinase: retention of full catalytic activity in the isolated C-terminal half. Arch. Biochem. Biophys. 274:375-393.[CrossRef][Medline]
    55. 55
  55. White, T. K., and J. E. Wilson. 1987. Rat brain hexokinase: location of the allosteric regulatory site in a structural domain at the N-terminus of the enzyme. Arch. Biochem. Biophys. 259:402-411.[CrossRef][Medline]
    56. 56
  56. Wilson, J. E. 1995. Hexokinases. Rev. Physiol. Biochem. Pharmacol. 126:65-198.[Medline]
    57. 57
  57. Xie, G., and J. E. Wilson. 1990. Tetrameric structure of mitochondrially bound rat brain hexokinase: a crosslinking study. Arch. Biochem. Biophys. 276:285-293.[CrossRef][Medline]
    58. 58
  58. Xie, G. C., and J. E. Wilson. 1988. Rat brain hexokinase: the hydrophobic N-terminus of the mitochondrially bound enzyme is inserted in the lipid bilayer. Arch. Biochem. Biophys. 267:803-810.[CrossRef][Medline]

Molecular and Cellular Biology, February 2008, p. 1007-1017, Vol. 28, No. 3
0270-7306/08/$08.00+0     doi:10.1128/MCB.00224-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Miyamoto, S., Rubio, M., Sussman, M. A. (2009). Nuclear and mitochondrial signalling Akts in cardiomyocytes. Cardiovasc Res 82: 272-285 [Abstract] [Full Text]  
  • Zhai, P., Sadoshima, J. (2008). Overcoming an Energy Crisis?: An Adaptive Role of Glycogen Synthase Kinase-3 Inhibition in Ischemia/Reperfusion. Circ. Res. 103: 910-913 [Full Text]  
  • Rengaraj, D., Kim, D. K., Zheng, Y. H., Lee, S. I., Kim, H., Han, J. Y. (2008). Testis-Specific Novel Transcripts in Chicken: In Situ Localization and Expression Pattern Profiling During Sexual Development. Biol. Reprod. 79: 413-420 [Abstract] [Full Text]  
  • Yuan, S., Fu, Y., Wang, X., Shi, H., Huang, Y., Song, X., Li, L., Song, N., Luo, Y. (2008). Voltage-dependent anion channel 1 is involved in endostatin-induced endothelial cell apoptosis. FASEB J. 22: 2809-2820 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, L.
Right arrow Articles by Ardehali, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, L.
Right arrow Articles by Ardehali, H.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»