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. 2004 Jan 15;377(Pt 2):347–355. doi: 10.1042/BJ20031465

In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death.

Heftsi Azoulay-Zohar 1, Adrian Israelson 1, Salah Abu-Hamad 1, Varda Shoshan-Barmatz 1
PMCID: PMC1223882  PMID: 14561215

Abstract

In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.

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Selected References

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  1. Adams J. M., Cory S. The Bcl-2 protein family: arbiters of cell survival. Science. 1998 Aug 28;281(5381):1322–1326. doi: 10.1126/science.281.5381.1322. [DOI] [PubMed] [Google Scholar]
  2. Aflalo C., Azoulay H. Binding of rat brain hexokinase to recombinant yeast mitochondria: effect of environmental factors and the source of porin. J Bioenerg Biomembr. 1998 Jun;30(3):245–255. doi: 10.1023/a:1020544803475. [DOI] [PubMed] [Google Scholar]
  3. Arora K. K., Pedersen P. L. Functional significance of mitochondrial bound hexokinase in tumor cell metabolism. Evidence for preferential phosphorylation of glucose by intramitochondrially generated ATP. J Biol Chem. 1988 Nov 25;263(33):17422–17428. [PubMed] [Google Scholar]
  4. Azoulay-Zohar H., Aflalo C. Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary physico-chemical determinants. Eur J Biochem. 2000 May;267(10):2973–2980. doi: 10.1046/j.1432-1033.2000.01313.x. [DOI] [PubMed] [Google Scholar]
  5. Babel D., Walter G., Götz H., Thinnes F. P., Jürgens L., König U., Hilschmann N. Studies on human porin. VI. Production and characterization of eight monoclonal mouse antibodies against the human VDAC "Porin 31HL" and their application for histotopological studies in human skeletal muscle. Biol Chem Hoppe Seyler. 1991 Dec;372(12):1027–1034. doi: 10.1515/bchm3.1991.372.2.1027. [DOI] [PubMed] [Google Scholar]
  6. Benz R. Permeation of hydrophilic solutes through mitochondrial outer membranes: review on mitochondrial porins. Biochim Biophys Acta. 1994 Jun 29;1197(2):167–196. doi: 10.1016/0304-4157(94)90004-3. [DOI] [PubMed] [Google Scholar]
  7. Beutner G., Rück A., Riede B., Brdiczka D. Complexes between porin, hexokinase, mitochondrial creatine kinase and adenylate translocator display properties of the permeability transition pore. Implication for regulation of permeability transition by the kinases. Biochim Biophys Acta. 1998 Jan 5;1368(1):7–18. doi: 10.1016/s0005-2736(97)00175-2. [DOI] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  9. Brdiczka D., Beutner G., Rück A., Dolder M., Wallimann T. The molecular structure of mitochondrial contact sites. Their role in regulation of energy metabolism and permeability transition. Biofactors. 1998;8(3-4):235–242. doi: 10.1002/biof.5520080311. [DOI] [PubMed] [Google Scholar]
  10. Bryson Jane M., Coy Platina E., Gottlob Kathrin, Hay Nissim, Robey R. Brooks. Increased hexokinase activity, of either ectopic or endogenous origin, protects renal epithelial cells against acute oxidant-induced cell death. J Biol Chem. 2001 Dec 18;277(13):11392–11400. doi: 10.1074/jbc.M110927200. [DOI] [PubMed] [Google Scholar]
  11. Bustamante E., Morris H. P., Pedersen P. L. Energy metabolism of tumor cells. Requirement for a form of hexokinase with a propensity for mitochondrial binding. J Biol Chem. 1981 Aug 25;256(16):8699–8704. [PubMed] [Google Scholar]
  12. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J. 1999 Jul 15;341(Pt 2):233–249. [PMC free article] [PubMed] [Google Scholar]
  13. Crompton M., Virji S., Doyle V., Johnson N., Ward J. M. The mitochondrial permeability transition pore. Biochem Soc Symp. 1999;66:167–179. doi: 10.1042/bss0660167. [DOI] [PubMed] [Google Scholar]
  14. Fanciulli M., Paggi M. G., Bruno T., Del Carlo C., Bonetto F., Gentile F. P., Floridi A. Glycolysis and growth rate in normal and in hexokinase-transfected NIH-3T3 cells. Oncol Res. 1994;6(9):405–409. [PubMed] [Google Scholar]
  15. Gincel D., Silberberg S. D., Shoshan-Barmatz V. Modulation of the voltage-dependent anion channel (VDAC) by glutamate. J Bioenerg Biomembr. 2000 Dec;32(6):571–583. doi: 10.1023/a:1005670527340. [DOI] [PubMed] [Google Scholar]
  16. Gincel D., Zaid H., Shoshan-Barmatz V. Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function. Biochem J. 2001 Aug 15;358(Pt 1):147–155. doi: 10.1042/0264-6021:3580147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gottlob K., Majewski N., Kennedy S., Kandel E., Robey R. B., Hay N. Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev. 2001 Jun 1;15(11):1406–1418. doi: 10.1101/gad.889901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Halestrap A. P., Doran E., Gillespie J. P., O'Toole A. Mitochondria and cell death. Biochem Soc Trans. 2000 Feb;28(2):170–177. doi: 10.1042/bst0280170. [DOI] [PubMed] [Google Scholar]
  19. Hashimoto M., Wilson J. E. Membrane potential-dependent conformational changes in mitochondrially bound hexokinase of brain. Arch Biochem Biophys. 2000 Dec 1;384(1):163–173. doi: 10.1006/abbi.2000.2085. [DOI] [PubMed] [Google Scholar]
  20. Haworth R. A., Hunter D. R. Allosteric inhibition of the Ca2+-activated hydrophilic channel of the mitochondrial inner membrane by nucleotides. J Membr Biol. 1980 Jun 15;54(3):231–236. doi: 10.1007/BF01870239. [DOI] [PubMed] [Google Scholar]
  21. Hodge T., Colombini M. Regulation of metabolite flux through voltage-gating of VDAC channels. J Membr Biol. 1997 Jun 1;157(3):271–279. doi: 10.1007/s002329900235. [DOI] [PubMed] [Google Scholar]
  22. Ichas F., Mazat J. P. From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to high-conductance state. Biochim Biophys Acta. 1998 Aug 10;1366(1-2):33–50. doi: 10.1016/s0005-2728(98)00119-4. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Lemasters J. J., Qian T., Bradham C. A., Brenner D. A., Cascio W. E., Trost L. C., Nishimura Y., Nieminen A. L., Herman B. Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death. J Bioenerg Biomembr. 1999 Aug;31(4):305–319. doi: 10.1023/a:1005419617371. [DOI] [PubMed] [Google Scholar]
  25. Lindén M., Gellerfors P., Nelson B. D. Pore protein and the hexokinase-binding protein from the outer membrane of rat liver mitochondria are identical. FEBS Lett. 1982 May 17;141(2):189–192. doi: 10.1016/0014-5793(82)80044-6. [DOI] [PubMed] [Google Scholar]
  26. Liu X., Kim C. N., Yang J., Jemmerson R., Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996 Jul 12;86(1):147–157. doi: 10.1016/s0092-8674(00)80085-9. [DOI] [PubMed] [Google Scholar]
  27. Novgorodov S. A., Gudz T. I., Jung D. W., Brierley G. P. The nonspecific inner membrane pore of liver mitochondria: modulation of cyclosporin sensitivity by ADP at carboxyatractyloside-sensitive and insensitive sites. Biochem Biophys Res Commun. 1991 Oct 15;180(1):33–38. doi: 10.1016/s0006-291x(05)81250-1. [DOI] [PubMed] [Google Scholar]
  28. Oltvai Z. N., Milliman C. L., Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993 Aug 27;74(4):609–619. doi: 10.1016/0092-8674(93)90509-o. [DOI] [PubMed] [Google Scholar]
  29. Pastorino John G., Shulga Nataly, Hoek Jan B. Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis. J Biol Chem. 2001 Dec 18;277(9):7610–7618. doi: 10.1074/jbc.M109950200. [DOI] [PubMed] [Google Scholar]
  30. Pedersen Peter L., Mathupala Saroj, Rempel Annette, Geschwind J. F., Ko Young Hee. 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. 2002 Sep 10;1555(1-3):14–20. doi: 10.1016/s0005-2728(02)00248-7. [DOI] [PubMed] [Google Scholar]
  31. Rempel A., Bannasch P., Mayer D. Differences in expression and intracellular distribution of hexokinase isoenzymes in rat liver cells of different transformation stages. Biochim Biophys Acta. 1994 Nov 22;1219(3):660–668. doi: 10.1016/0167-4781(94)90225-9. [DOI] [PubMed] [Google Scholar]
  32. Rose I. A., Warms J. V., Kosow D. P. Specificity for the glucose-6-P inhibition site of hexokinase. Arch Biochem Biophys. 1974 Oct;164(2):729–735. doi: 10.1016/0003-9861(74)90086-1. [DOI] [PubMed] [Google Scholar]
  33. Rostovtseva T., Colombini M. VDAC channels mediate and gate the flow of ATP: implications for the regulation of mitochondrial function. Biophys J. 1997 May;72(5):1954–1962. doi: 10.1016/S0006-3495(97)78841-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sebastian S., Kenkare U. W. Insulin-like growth factor I induces tumor hexokinase RNA expression in cancer cells. Biochem Biophys Res Commun. 1997 Jun 18;235(2):389–393. doi: 10.1006/bbrc.1997.6797. [DOI] [PubMed] [Google Scholar]
  35. Shimizu S., Matsuoka Y., Shinohara Y., Yoneda Y., Tsujimoto Y. Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells. J Cell Biol. 2001 Jan 22;152(2):237–250. doi: 10.1083/jcb.152.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shinohara Y., Ishida T., Hino M., Yamazaki N., Baba Y., Terada H. Characterization of porin isoforms expressed in tumor cells. Eur J Biochem. 2000 Oct;267(19):6067–6073. doi: 10.1046/j.1432-1327.2000.01687.x. [DOI] [PubMed] [Google Scholar]
  37. Tsujimoto Yoshihide, Shimizu Shigeomi. The voltage-dependent anion channel: an essential player in apoptosis. Biochimie. 2002 Feb-Mar;84(2-3):187–193. doi: 10.1016/s0300-9084(02)01370-6. [DOI] [PubMed] [Google Scholar]
  38. Vieira H. L., Haouzi D., El Hamel C., Jacotot E., Belzacq A. S., Brenner C., Kroemer G. Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator. Cell Death Differ. 2000 Dec;7(12):1146–1154. doi: 10.1038/sj.cdd.4400778. [DOI] [PubMed] [Google Scholar]
  39. Vyssokikh Mikhail Y., Brdiczka Dieter. The function of complexes between the outer mitochondrial membrane pore (VDAC) and the adenine nucleotide translocase in regulation of energy metabolism and apoptosis. Acta Biochim Pol. 2003;50(2):389–404. [PubMed] [Google Scholar]
  40. Wilson J. E. Hexokinases. Rev Physiol Biochem Pharmacol. 1995;126:65–198. doi: 10.1007/BFb0049776. [DOI] [PubMed] [Google Scholar]
  41. Wilson J. E. Rapid purification of mitochondrial hexokinase from rat brain by a single affinity chromatography step on Affi-Gel blue. Prep Biochem. 1989;19(1):13–21. doi: 10.1080/10826068908544893. [DOI] [PubMed] [Google Scholar]
  42. Xie G. C., Wilson J. E. Rat brain hexokinase: the hydrophobic N-terminus of the mitochondrially bound enzyme is inserted in the lipid bilayer. Arch Biochem Biophys. 1988 Dec;267(2):803–810. doi: 10.1016/0003-9861(88)90090-2. [DOI] [PubMed] [Google Scholar]
  43. Xie G., Wilson J. E. Tetrameric structure of mitochondrially bound rat brain hexokinase: a crosslinking study. Arch Biochem Biophys. 1990 Jan;276(1):285–293. doi: 10.1016/0003-9861(90)90040-6. [DOI] [PubMed] [Google Scholar]
  44. Zamzami N., Kroemer G. The mitochondrion in apoptosis: how Pandora's box opens. Nat Rev Mol Cell Biol. 2001 Jan;2(1):67–71. doi: 10.1038/35048073. [DOI] [PubMed] [Google Scholar]
  45. Zoratti M., Szabò I. The mitochondrial permeability transition. Biochim Biophys Acta. 1995 Jul 17;1241(2):139–176. doi: 10.1016/0304-4157(95)00003-a. [DOI] [PubMed] [Google Scholar]

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