Molecular and Cellular Biochemistry

, Volume 442, Issue 1–2, pp 47–58 | Cite as

Purification and characterization of skeletal muscle pyruvate kinase from the hibernating ground squirrel, Urocitellus richardsonii: potential regulation by posttranslational modification during torpor

Article

Abstract

Ground squirrel torpor during winter hibernation is characterized by numerous physiological and biochemical changes, including alterations to fuel metabolism. During torpor, many tissues switch from carbohydrate to lipid catabolism, often by regulating key enzymes within glycolytic and lipolytic pathways. This study investigates the potential regulation of pyruvate kinase (PK), a key member of the glycolytic pathway, within the skeletal muscle of hibernating ground squirrels. PK was purified from the skeletal muscle of control and torpid Richardson’s ground squirrels, and PK kinetics, structural stability, and posttranslational modifications were subsequently assessed. Torpid PK displayed a nearly threefold increase in K m PEP as compared to control PK when assayed at 5 °C. ProQ Diamond phosphoprotein staining as well as phospho-specific western blots indicated that torpid PK was significantly more phosphorylated than the euthermic control. PK from the torpid condition was also shown to possess nearly twofold acetyl content as compared to control PK. In conclusion, skeletal muscle PK from the Richardson’s ground squirrel may be regulated posttranslationally between the euthermic and torpid states, and this may inhibit PK functioning during torpor in accordance with the decrease in glycolytic rate during dormancy.

Keywords

Hibernation Urocitellus richardsonii Glycolysis Reversible protein phosphorylation Acetylation 

Notes

Acknowledgements

The research was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (OPG6793) to K.B. Storey and by an NSERC CGS postgraduate scholarship to R.A.V. Bell.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Ljungstrom O, Hjelmquist G, Engstrom L (1974) Phosphorylation of purified rat liver pyruvate kinase by cyclic-3′,5′-AMP-stimulated protein kinase. Biochem Biophys Acta 358:289–298Google Scholar
  2. 2.
    Riou JP, Claus TH, Pilkis SJ (1978) Stimulation of glucagon of an in vivo phosphorylation of rat hepatic pyruvate kinase. J Biol Chem 253:656–659PubMedGoogle Scholar
  3. 3.
    Storey KB (1987) Regulation of liver metabolism by enzyme phosphorylation during mammalian hibernation. J Biol Chem 262(4):1670–1673PubMedGoogle Scholar
  4. 4.
    Carey HV, Andrews MT, Martin SL (2003) Mammalian hibernation: cellular and molecular response to depressed metabolism and low temperature. Physiol Rev 83:1153–1181CrossRefPubMedGoogle Scholar
  5. 5.
    Zatzman ML (1984) Renal and cardiovascular effects of hibernation and hypothermia. Cryobiology 21(6):593–614CrossRefPubMedGoogle Scholar
  6. 6.
    McArthur MD, Milsom WK (1991) Changes in ventilation and respiratory sensitivity associated with hibernation in Columbian (Spermophilus columbianus) and golden-mantled (Spermophilus lateralis) ground squirrels. Physiol Zool 64:940–959CrossRefGoogle Scholar
  7. 7.
    Van Breukelen F, Sonenberg N, Martin SL (2004) Seasonal and state-dependent changes of eIF4E and 4E-BP1 during mammalian hibernation: implications for the control of translation during torpor. Am J Physiol Regul Integr Comp Physiol 287:349–353CrossRefGoogle Scholar
  8. 8.
    MacDonald JA, Storey KB (1999) Regulation of ground squirrel Na + K + ATPase activity by reversible phosphorylation during hibernation. Biochem Biophys Res Commun 254:424–429CrossRefPubMedGoogle Scholar
  9. 9.
    South FE, House WA (1967) Energy metabolism in hibernation. In: Fisher KC, Dawe AR, Lyman CP, Schonbaum E, South FE (eds) Mammalian hibernation III. Oliver and Boyd, Ltd., Edinburgh, pp 305–324Google Scholar
  10. 10.
    Tashima LS, Adelstein SJ, Lyman CP (1970) Radioglucose utilization by active, hibernating, and arousing ground squirrels. Am J Physiol 218:303–309PubMedGoogle Scholar
  11. 11.
    Brooks SBJ, Storey KB (1992) Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis. J Comp Physiol B 162:23–28CrossRefGoogle Scholar
  12. 12.
    Brooks SPJ (1992) A simple computer program with statistical tests for the analysis of enzyme kinetics. Biotechniques 13:906–911PubMedGoogle Scholar
  13. 13.
    Brooks SPJ (1994) A program for analyzing enzyme rate data obtained from a microplate reader. Biotechniques 17:1155–1161Google Scholar
  14. 14.
    Geiser F (1988) Reduction of metabolism during hibernation and daily torpor in mammals and birds: temperature effect or physiological inhibition? J Comp Phys B 158:25–37CrossRefGoogle Scholar
  15. 15.
    Hochachka PW, Guppy M (1987) Metabolic arrest and the control of biological time. Harvard University Press, CambridgeCrossRefGoogle Scholar
  16. 16.
    Hachimi ZE, Tijane M, Boissonnet G, Benjouad A, Desmadril M, Yon JM (1990) Regulation of the skeletal muscle metabolism during hibernation of Jaculus orientalis. Comp Biochem Physiol B 96(3):457–459CrossRefPubMedGoogle Scholar
  17. 17.
    Abnous K, Storey KB (2008) Skeletal muscle hexokinase: regulation in mammalian hibernation. Mol Cell Biochem 319:41–50CrossRefPubMedGoogle Scholar
  18. 18.
    Hall ER, Cottam GL (1978) Isozymes of pyruvate kinase in vertebrates: their physical, chemical kinetic and immunological properties. Int J Biochem 9:785–794CrossRefPubMedGoogle Scholar
  19. 19.
    English TE, Storey KB (2000) Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog Cynomys leucurus. Arch Biochem Biophys 376:91–100CrossRefPubMedGoogle Scholar
  20. 20.
    Dohm GL, Patel VK, Kasperek GJ (1986) Regulation of muscle pyruvate metabolism during exercise. Biochem Med Metabol Biol 35(3):260–266CrossRefGoogle Scholar
  21. 21.
    Storey KB, Kelly DA (1995) Glycolysis and energetics in organs of hibernating mice (Zapus hudsonius). Can J Zool 73:202–207CrossRefGoogle Scholar
  22. 22.
    Larson TM, Laughlin LT, Holden HM, Rayment I, Reed GH (1994) Structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate. Biochemisty 33(20):6301–6309CrossRefGoogle Scholar
  23. 23.
    Frey PA, Hegeman AD (2007) Phosphotransfer and nucleotidyltransfer. Enzymatic reaction mechanisms. Oxford University Press, New York, pp 495–496Google Scholar
  24. 24.
    MacDermott M (1990) The intracellular concentration of free magnesium in extensor digitorum longus muscles of the rat. Exp Physiol 75:763–769CrossRefPubMedGoogle Scholar
  25. 25.
    Bijlani RL, Manjunatha S (2010) Membrane potential at rest and during activity. In: Understanding medical physiology: a textbook for medical students. Jaypee Brothers Medical Publishers Ltd. New Delhi, India p 118Google Scholar
  26. 26.
    Willis JS, Goldman SS, Foster RF (1971) Tissue K concentration in relation to the role of the kidney in hibernation and the cause of periodic arousal. Comp Biochem Physiol A Physiol 39(3):437–445CrossRefGoogle Scholar
  27. 27.
    Jurica MS, Mesecar A, Heath PJ, Shi W, Nowak T, Stoddard BL (1998) The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate. Structure 6(2):195–210CrossRefPubMedGoogle Scholar
  28. 28.
    Carbonell J, Marco R, Feliu JE, Sols A (1973) Pyruvate kinase: classes of regulatory isozymes in mammalian tissues. FEBS J 37:148–156Google Scholar
  29. 29.
    Smith CR, Knowles VL, Plaxton WC (2000) Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures. Eur J Biochem 267:4477–4486CrossRefPubMedGoogle Scholar
  30. 30.
    Storey KB (1986) Aspartate activation of pyruvate kinase in anoxia tolerant molluscs. Comp Biochem Physiol B 83:807–812CrossRefGoogle Scholar
  31. 31.
    Storey KB (1989) Integrated control of metabolic rate depression via reversible phosphorylation of enzymes in hibernating mammals. In: Malan A, Canguilhem AB (eds) Living in the cold II Colloque INSERM. John Libbey Eurotext Ltd, Montrouge, pp 309–319Google Scholar
  32. 32.
    Whitwam RE, Storey KB (1991) Regulation of phosphofructokinase during estivation and anoxia in the land snail, Otala Lactea. Physiol Zool 64:595–610CrossRefGoogle Scholar
  33. 33.
    Ramnanan CJ, Storey KB (2006) Glucose-6-phosphate dehydrogenase regulation during hypometabolism. Biochem Biophys Res Commun 339:7–16CrossRefPubMedGoogle Scholar
  34. 34.
    Titanji VPK, Zetterqvist O, Engstrom L (1976) Regulation in vitro of rat liver pyruvate kina se by phosphorylation-dephosphorylation reactions, catalyzed by cyclic-AMP dependent protein kinases and a histone phosphatase. Biochim Biophys Acta 422(1):98–108CrossRefPubMedGoogle Scholar
  35. 35.
    Boivin P, Galand C, Estrada M (1980) Phosphorylation of human red cell and liver pyruvate kinase. Differences between liver and erythrocyte L-type subunits. Cell Mol Life Sci 36(8):900–901CrossRefGoogle Scholar
  36. 36.
    Fister P, Eigenbrodt E, Presek P, Reinacher M, Schoner W (1983) Pyruvate kinase type M2 is phosphorylated in the intact chicken liver cell. Biochem Biophys Res Commun 115(2):409–414CrossRefPubMedGoogle Scholar
  37. 37.
    MacDonald MJ, Kowluru A (1985) Evidence for calcium enhanced phosphorylation of pyruvate kinase by pancreatic islets. Mol Cell Biochem 68(2):107–114PubMedGoogle Scholar
  38. 38.
    Nakashima K, Fujii S, Kaku K, Kaneko T (1982) Calcium-calmodulin dependent phosphorylation of erythrocyte pyruvate kinase. Biochem Biophys Res Commun 104(1):285–289CrossRefPubMedGoogle Scholar
  39. 39.
    Weernink PA, Rijksen G, van der Heijden MC, Staal GE (1990) Phosphorylation of pyruvate kinase type K in human gliomas by a cyclic adenosine 5′-monophosphate-independent protein kinase. Cancer Res 50(15):4604–4610PubMedGoogle Scholar
  40. 40.
    Hitosugi T, Kang S, Vander Heiden MG, Chung T, Elf S, Lythgoe K, Dong S, Lonial S, Wang X, Chen GZ, Xie J, Gu T, Polakiewicz RD, Roesel JL, Boggon TJ, Khuri FR, Gilliland DG, Cantley LC, Kaufman J, Chen J (2009) Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth. Sci Signal 2(97):ra73CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Plaxton WC, Storey KB (1984) Phosphorylation in vivo of red-muscle pyruvate kinase from the channelled whelk, Busycotpus canaliculatum, in response to anoxic stress. Eur J Biochem 143:267–272CrossRefPubMedGoogle Scholar
  42. 42.
    Cowan KJ, Storey KB (1999) Reversible phosphorylation of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad, Scaphiopus couchii. Mol Cell Biochem 195:173–181CrossRefPubMedGoogle Scholar
  43. 43.
    Noguchi T, Inoue H, Tanaka T (1986) The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J Biol Chem 261:13807–13812PubMedGoogle Scholar
  44. 44.
    Gnad F, Ren S, Cox J, Olsen JV, Macek B, Oroshi M, Mann M (2007) PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites. Genome Biol 8:R250CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Blom N, Gammeltoft S, Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294:1351–1362CrossRefPubMedGoogle Scholar
  46. 46.
    Eigenbrodt E, Mostafa MA, Schoner W (1977) Inactivation of pyruvate kinase type M2 from chicken liver by phosphorylation, catalyzed by a cAMP-independent protein kinase. Hoppe Seylers Z Physiol Chem 358:1047–1055CrossRefPubMedGoogle Scholar
  47. 47.
    Presek P, Reinacher M, Eigenbrodt E (1988) Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus. FEBS Lett 242(1):194–198CrossRefPubMedGoogle Scholar
  48. 48.
    Zhao S, Xu W, Jiang W, Yu W, Lin Y, Zhang T, Yao J, Zhou L, Zeng Y, Li Y, Shi J, An W, Hancock SM, He F, Qin L, Chin J, Yang P, Chen X, Lei Q, Xiong Y, Guan K (2010) Regulation of cellular metabolism by protein lysine acetylation. Science 327:1000–1004CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Walter L, Bienz M (1998) Drosophila CBP represses the transcription factor TCF to antagonize Wingless signalling. Nature 395(6701):521–525CrossRefGoogle Scholar
  50. 50.
    Dhalluin C, Carlson JE, Zeng L, He C, Aggarwai AK, Zhou MM (1999) Structure and ligand of a histone acetyltransferase bromodomain. Nature 399(6735):491–496CrossRefPubMedGoogle Scholar
  51. 51.
    Caron C, Boyault C, Knochbin S (2005) Regulatory cross-talk between lysine acetylation and ubiquitination: role in the control of protein stability. Bioassays 27(4):408–415CrossRefGoogle Scholar
  52. 52.
    Jeong JW, Bae MK, Ahn MY, Kim SH, Sohn TK, Bae MH, Yoo MA, Song EJ, Lee KJ, Kim KW (2002) Regulation and destabilization of HIF-1alpha by ARD1-mediated acetylation. Cell 111(5):709–720CrossRefPubMedGoogle Scholar
  53. 53.
    Lv L, Li D, Zhao D, Lin R, Chu Y, Zhang H, Zha Z, Liu Y, Li Z, Xu Y, Wang G, Huang Y, Xoing Y, Guan KL, Lei QY (2011) Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth. Mol Cell 42(6):719–730CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  1. 1.Regenerative Medicine Program, Sprott Centre for Stem Cell Research, Ottawa Hospital Research InstituteThe Ottawa HospitalOttawaCanada
  2. 2.Institute of Biochemistry and Department of ChemistryCarleton UniversityOttawaCanada

Personalised recommendations