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. 1982 Oct 15;208(1):221–229. doi: 10.1042/bj2080221

The effects of glucagon, phenylephrine and insulin on the phosphorylation of cytoplasmic, mitochondrial and membrane-bound proteins of intact liver cells from starved rats.

A M Vargas, A P Halestrap, R M Denton
PMCID: PMC1153949  PMID: 6760856

Abstract

1. The effects of glucagon, insulin and phenylephrine on the phosphorylation of cytoplasmic, mitochondrial and membrane proteins were studied in intact hepatocytes from 24 h-starved rats incubated with [32P]Pi. A rapid cell-fractionation technique was used, followed by radioautography of the proteins separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. Glucagon consistently caused a significant increase in the phosphorylation of four readily separable cytoplasmic phosphoproteins, of Mr 93000, 50000, 46000 and 20000, and a decrease in phosphorylation of a phosphoprotein of Mr 22000. Phosphorylation of the protein of Mr 46000 was also enhanced by both phenylephrine and insulin, and that of Mr 93000 by phenylephrine. 3. The phosphoprotein of Mr 22000 was not precipitated by boiling for 5 min, and had a mobility identical with that of similar protein whose phosphorylation is enhanced in the adipocyte by insulin [Belsham & Denton (1980) Biochem. Soc. Trans. 8, 382-383]. 4. Glucagon, but not phenylephrine or insulin, enhanced the phosphorylation of a mitochondrial protein of Mr 35000 and of four plasma- or microsomal-membrane proteins of Mr 50000, 30000, 23000 and 19000. 5. Mitochondria from glucagon-treated animals or hepatocytes phosphorylated a protein of Mr 30000 when incubated in vitro with [32P]Pi and ADP. Phosphorylation of this protein did not occur with mitochondria from control, phenylephrine- or insulin-treated cells. 6. The significance of these hormonally induced changes in protein phosphorylation is discussed.

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Selected References

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  1. Alexander M. C., Kowaloff E. M., Witters L. A., Dennihy D. T., Avruch J. Purification of a hepatic 123,000-dalton hormone-stimulated 32P-peptide and its identification as ATP-citrate lyase. J Biol Chem. 1979 Aug 25;254(16):8052–8056. [PubMed] [Google Scholar]
  2. Avruch J., Leone G. R., Martin D. B. Effects of epinephrine and insulin on phosphopeptide metabolism in adipocytes. J Biol Chem. 1976 Mar 10;251(5):1511–1515. [PubMed] [Google Scholar]
  3. Avruch J., Witters L. A., Alexander M. C., Bush M. A. Effects of glucagon and insulin on cytoplasmic protein phosphorylation in hepatocytes. J Biol Chem. 1978 Jul 10;253(13):4754–4761. [PubMed] [Google Scholar]
  4. Behar-Bannelier M., Murray R. K. An electrophoretic study of endogenous phosphorylation in vitro of the polypeptides of microsomal membrane fractions of mouse liver. Biochem J. 1980 Apr 1;187(1):147–156. doi: 10.1042/bj1870147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Belsham G. J., Brownsey R. W., Denton R. M. Reversibility of the insulin-stimulated phosphorylation of ATP citrate lyase and a cytoplasmic protein of subunit Mr 22000 in adipose tissue. Biochem J. 1982 Apr 15;204(1):345–352. doi: 10.1042/bj2040345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Belsham G. J., Brownsey R. W., Hughes W. A., Denton R. M. Anti-insulin receptor antibodies mimic the effects of insulin on the activities of pyruvate dehydrogenase and acetylCoA carboxylase and on specific protein phosphorylation in rat epididymal fat cells. Diabetologia. 1980 Apr;18(4):307–312. doi: 10.1007/BF00251011. [DOI] [PubMed] [Google Scholar]
  7. Belsham G. J., Denton R. M., Tanner M. J. Use of a novel rapid preparation of fat-cell plasma membranes employing Percoll to investigate the effects of insulin and adrenaline on membrane protein phosphorylation within intact fat-cells. Biochem J. 1980 Nov 15;192(2):457–467. doi: 10.1042/bj1920457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Belsham G. J., Denton R. M. The effect of insulin and adrenaline on the phosphorylation of a 22 000-molecular weight protein within isolated fat cells; possible identification as the inhibitor-1 of the 'general phosphatase' [proceedings]. Biochem Soc Trans. 1980 Jun;8(3):382–383. doi: 10.1042/bst0080382. [DOI] [PubMed] [Google Scholar]
  9. Benjamin W. B., Singer I. Actions of insulin, epinephrine, and dibutyryl cyclic adenosine 5'-monophosphate on fat cell protein phosphorylations. Cyclic adenosine 5'-monophosphate dependent and independent mechanisms. Biochemistry. 1975 Jul 29;14(15):3301–3309. doi: 10.1021/bi00686a003. [DOI] [PubMed] [Google Scholar]
  10. Berry M. N., Friend D. S. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969 Dec;43(3):506–520. doi: 10.1083/jcb.43.3.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Blackshear P. J., Nemenoff R. A., Avruch J. Preliminary characterization of a heat-stable protein from rat adipose tissue whose phosphorylation is stimulated by insulin. Biochem J. 1982 Jun 15;204(3):817–824. doi: 10.1042/bj2040817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Blat C., Loeb J. E. Effect of glucagon on phosphorylation of some rat liver ribosomal proteins in vivo. FEBS Lett. 1971 Oct 15;18(1):124–126. doi: 10.1016/0014-5793(71)80425-8. [DOI] [PubMed] [Google Scholar]
  13. Brownsey R. W., Hughes W. A., Denton R. M. Adrenaline and the regulation of acetyl-coenzyme A carboxylase in rat epididymal adipose tissue. Inactivation of the enzyme is associated with phosphorylation and can be reversed on dephosphorylation. Biochem J. 1979 Oct 15;184(1):23–32. doi: 10.1042/bj1840023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Bygrave F. L., Tranter C. J. The subcellular location, maturation and response to increased plasma glucagon of ruthenium red-insensitive calcium-ion transport in rat liver. Biochem J. 1978 Sep 15;174(3):1021–1030. doi: 10.1042/bj1741021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Garrison J. C., Borland M. K., Florio V. A., Twible D. A. The role of calcium ion as a mediator of the effects of angiotensin II, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes. J Biol Chem. 1979 Aug 10;254(15):7147–7156. [PubMed] [Google Scholar]
  16. Garrison J. C. The effects of glucagon, catecholamines, and the calcium ionophore A23187 on the phosphorylation of rat hepatocyte cytosolic proteins. J Biol Chem. 1978 Oct 10;253(19):7091–7100. [PubMed] [Google Scholar]
  17. Gellerfors P., Nelson B. D. A rapid method for the isolation of intact mitochondria from isolated rat liver cells. Anal Biochem. 1979 Feb;93(1):200–203. [PubMed] [Google Scholar]
  18. Halestrap A. P., Scott R. D., Thomas A. P. Mitochondrial pyruvate transport and its hormonal regulation. Int J Biochem. 1980;11(2):97–105. doi: 10.1016/0020-711x(80)90241-4. [DOI] [PubMed] [Google Scholar]
  19. Halestrap A. P. Stimulation of pyruvate transport in metabolizing mitochondria through changes in the transmembrane pH gradient induced by glucagon treatment of rats. Biochem J. 1978 Jun 15;172(3):389–398. doi: 10.1042/bj1720389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Halestrap A. P. The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. Biochem J. 1975 Apr;148(1):85–96. doi: 10.1042/bj1480085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Halestrap A. P. The nature of the stimulation of the respiratory chain of rat liver mitochondria by glucagon pretreatment of animals. Biochem J. 1982 Apr 15;204(1):37–47. doi: 10.1042/bj2040037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hopgood M. F., Clark M. G., Ballard F. J. Protein degradation in hepatocyte monolayers. Effects of glucagon, adenosine 3':5'-cyclic monophosphate and insulin. Biochem J. 1980 Jan 15;186(1):71–79. doi: 10.1042/bj1860071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Houslay M. D., Marchmont R. J. The insulin-stimulated cyclic AMP phosphodiesterase binds to a single class of protein sites on the liver plasma membrane. Biochem J. 1981 Sep 15;198(3):703–706. doi: 10.1042/bj1980703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hughes W. A., Brownsey R. W., Denton R. M. Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin. Biochem J. 1980 Nov 15;192(2):469–481. doi: 10.1042/bj1920469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hughes W. A., Halestrap A. P. The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation. Biochem J. 1981 May 15;196(2):459–469. doi: 10.1042/bj1960459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Janski A. M., Srere P. A., Cornell N. W., Veech R. L. Phosphorylation of ATP citrate lyase in response to glucagon. J Biol Chem. 1979 Oct 10;254(19):9365–9368. [PubMed] [Google Scholar]
  27. Katz A. M. Role of the contractile proteins and sarcoplasmic reticulum in the response of the heart to catecholamines: an historical review. Adv Cyclic Nucleotide Res. 1979;11:303–343. [PubMed] [Google Scholar]
  28. Kirk C. J., Michell R. H., Hems D. A. Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin. Biochem J. 1981 Jan 15;194(1):155–165. doi: 10.1042/bj1940155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kobayashi M., Ozawa T. Protein phosphorylation and dephosphorylation in liver plasma membrane. Effect of inorganic phosphate. J Biochem. 1981 Mar;89(3):723–730. doi: 10.1093/oxfordjournals.jbchem.a133252. [DOI] [PubMed] [Google Scholar]
  30. Le Peuch C. J., Le Peuch D. A., Demaille J. G. Phospholamban, activator of the cardiac sarcoplasmic reticulum calcium pump. Physicochemical properties and diagonal purification. Biochemistry. 1980 Jul 8;19(14):3368–3373. doi: 10.1021/bi00555a042. [DOI] [PubMed] [Google Scholar]
  31. Marchmont R. J., Houslay M. D. Insulin trigger, cyclic AMP-dependent activation and phosphorylation of a plasma membrane cyclic AMP phosphodiesterase. Nature. 1980 Aug 28;286(5776):904–906. doi: 10.1038/286904a0. [DOI] [PubMed] [Google Scholar]
  32. Mendes-Mourāo J., Halestrap A. P., Crisp D. M., Pogson C. I. The involvement of mitochondrial pyruvate transport in the pathways of gluconeogenesis from serine and alanine in isolated rat and mouse liver cells. FEBS Lett. 1975 Apr 15;53(1):29–32. doi: 10.1016/0014-5793(75)80674-0. [DOI] [PubMed] [Google Scholar]
  33. Mourão J. M., McGivan J. D., Chappell J. B. The effects L-leucine on the synthesis of urea, glutamate and glutamine by isolated rat liver cells. Biochem J. 1975 Feb;146(2):457–464. doi: 10.1042/bj1460457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rinaldi M. L., Le Peuch C. J., Demaille J. G. The epinephrine-induced activation of the cardiac slow Ca2+ channel is mediated by the cAMP-dependent phosphorylation of calciductin, a 23 000 Mr sarcolemmal protein. FEBS Lett. 1981 Jul 6;129(2):277–281. doi: 10.1016/0014-5793(81)80183-4. [DOI] [PubMed] [Google Scholar]
  35. Siess E. A., Fahimi F. M., Wieland O. H. Evidence that glucagon stabilizes rather than activates mitochondrial functions in rat liver. Hoppe Seylers Z Physiol Chem. 1981 Dec;362(12):1643–1651. doi: 10.1515/bchm2.1981.362.2.1643. [DOI] [PubMed] [Google Scholar]
  36. Siess E. A., Wieland O. H. Isolated hepatocytes as a model for the study of stable glucagon effects on mitochondrial respiratory functions. FEBS Lett. 1979 May 15;101(2):277–281. doi: 10.1016/0014-5793(79)81025-x. [DOI] [PubMed] [Google Scholar]
  37. Taylor W. M., Bygrave F. L., Blackmore P. F., Exton J. H. Stable enhancement of ruthenium red-insensitive calcium transport in an endoplasmic reticulum-rich fraction following the exposure of isolated rat liver cells to glucagon. FEBS Lett. 1979 Aug 1;104(1):31–34. doi: 10.1016/0014-5793(79)81079-0. [DOI] [PubMed] [Google Scholar]
  38. Thomas A. P., Halestrap A. P. Computer stimulation of the effects of alpha-cyano-4-hydroxycinnamate on gluconeogenesis from L-lactate in rat liver cells. Biochem J. 1981 Sep 15;198(3):561–564. doi: 10.1042/bj1980561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Walker J. H., Betts S. A., Manning R., Mayer R. J. Absorption of antisera for studies on specific enzyme turnover. Biochem J. 1976 Nov;159(2):355–362. doi: 10.1042/bj1590355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Waltenbaugh A. M., Friedmann N. Hormone sensitive calcium uptake by liver microsomes. Biochem Biophys Res Commun. 1978 May 30;82(2):603–608. doi: 10.1016/0006-291x(78)90917-8. [DOI] [PubMed] [Google Scholar]
  41. Witters L. A. Insulin stimulates the phosphorylation of acetyl-CoA carboxylase. Biochem Biophys Res Commun. 1981 May 29;100(2):872–878. doi: 10.1016/s0006-291x(81)80254-9. [DOI] [PubMed] [Google Scholar]
  42. Witters L. A., Kowaloff E. M., Avruch J. Glucagon regulation of protein phosphorylation. Identification of acetyl coenzyme A carboxylase as a substrate. J Biol Chem. 1979 Jan 25;254(2):245–248. [PubMed] [Google Scholar]
  43. Zahlten R. N., Hochberg A. A., Stratman F. W., Lardy H. A. Glucagon-stimulated phosphorylation of mitochondrial and lysosomal membranes of rat liver in vivo. Proc Natl Acad Sci U S A. 1972 Apr;69(4):800–804. doi: 10.1073/pnas.69.4.800. [DOI] [PMC free article] [PubMed] [Google Scholar]

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