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. 1991 Mar 1;274(Pt 2):433–438. doi: 10.1042/bj2740433

Counter-regulation by insulin and isoprenaline of a prominent fat-associated phosphoprotein doublet in rat adipocytes.

R A Mooney 1, K L Bordwell 1
PMCID: PMC1150157  PMID: 1848760

Abstract

1. In the adipocyte, phosphorylation/dephosphorylation of regulatory proteins is a common mechanism of metabolic regulation. We have observed a very prominent phosphoprotein doublet of 61 kDa and 63 kDa in rat adipocytes that is markedly responsive to hormones. The 63 kDa band was the predominant phosphoprotein in the cell in response to 0.1 microM-isoprenaline, whereas the 61 kDa band was nearly absent. Insulin alone did not alter 32P incorporation into the doublet, but partially counteracted the effects of isoprenaline, decreasing label in the 63 kDa band by as much as 50% and resulting in the reappearance of the 61 kDa band. 2. Subcellular fractionation demonstrated that both phosphoprotein bands were fat-associated. Neither insulin nor isoprenaline altered this localization. Peptide maps (one-dimensional) of the 61/63 kDa bands demonstrated close sequence similarity. Amino acid analysis revealed the presence of phosphoserine and phosphothreonine. The latter was more prominent in the 61 kDa band. Isoprenaline caused an absolute increase in both phosphoamino acids. 3. Permeabilization of 32P-labelled isoprenaline-treated cells with digitonin initiated rapid dephosphorylation of the 63 kDa band, with reappearance of the 61 kDa band. Insulin increased the rate of dephosphorylation by 2-3-fold when present with isoprenaline before permeabilization. 4. In permeabilized adipocytes, cyclic AMP (1 microM-1 mM) increased phosphorylation of the 61/63 kDa doublet by 4-10-fold in the presence of [gamma-32P]ATP, but insulin had no effect. 5. We conclude that this prominent phosphoprotein, migrating as a 61/63 kDa doublet, is coupled to the cyclic AMP-dependent protein kinase and is associated with an insulin-stimulated phosphoprotein phosphatase activity. This fat-associated phosphoprotein, which is under counter-regulatory hormonal control, may play a role in hormone-dependent lipid metabolism.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Beebe S. J., Redmon J. B., Blackmore P. F., Corbin J. D. Discriminative insulin antagonism of stimulatory effects of various cAMP analogs on adipocyte lipolysis and hepatocyte glycogenolysis. J Biol Chem. 1985 Dec 15;260(29):15781–15788. [PubMed] [Google Scholar]
  3. 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]
  4. Eckerskorn C., Mewes W., Goretzki H., Lottspeich F. A new siliconized-glass fiber as support for protein-chemical analysis of electroblotted proteins. Eur J Biochem. 1988 Oct 1;176(3):509–519. doi: 10.1111/j.1432-1033.1988.tb14308.x. [DOI] [PubMed] [Google Scholar]
  5. Elks M. L., Manganiello V. C. Antilipolytic action of insulin: role of cAMP phosphodiesterase activation. Endocrinology. 1985 May;116(5):2119–2121. doi: 10.1210/endo-116-5-2119. [DOI] [PubMed] [Google Scholar]
  6. Hepp K. D. Adenylate cyclase and insulin action. Effect of insulin, nonsuppressible insulin-like material, and diabetes on adenylate-cyclase activity in mouse liver. Eur J Biochem. 1972 Dec 4;31(2):266–276. doi: 10.1111/j.1432-1033.1972.tb02529.x. [DOI] [PubMed] [Google Scholar]
  7. Heyworth C. M., Rawal S., Houslay M. D. Guanine nucleotides can activate the insulin-stimulated phosphodiesterase in liver plasma membranes. FEBS Lett. 1983 Apr 5;154(1):87–91. doi: 10.1016/0014-5793(83)80880-1. [DOI] [PubMed] [Google Scholar]
  8. Londos C., Honnor R. C., Dhillon G. S. cAMP-dependent protein kinase and lipolysis in rat adipocytes. III. Multiple modes of insulin regulation of lipolysis and regulation of insulin responses by adenylate cyclase regulators. J Biol Chem. 1985 Dec 5;260(28):15139–15145. [PubMed] [Google Scholar]
  9. Loten E. G., Sneyd J. G. An effect of insulin on adipose-tissue adenosine 3':5'-cyclic monophosphate phosphodiesterase. Biochem J. 1970 Nov;120(1):187–193. doi: 10.1042/bj1200187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Martensen T. M. Chemical properties, isolation, and analysis of O-phosphates in proteins. Methods Enzymol. 1984;107:3–23. doi: 10.1016/0076-6879(84)07003-8. [DOI] [PubMed] [Google Scholar]
  11. Mooney R. A., Anderson D. L. Phosphorylation of the insulin receptor in permeabilized adipocytes is coupled to a rapid dephosphorylation reaction. J Biol Chem. 1989 Apr 25;264(12):6850–6857. [PubMed] [Google Scholar]
  12. Mooney R. A., Ebersohl R. D., McDonald J. M. Novel approach to the study of the regulation of hormone-sensitive lipase in rat adipocytes. Permeabilization of cells with digitonin. Eur J Biochem. 1983 Nov 15;136(3):603–608. doi: 10.1111/j.1432-1033.1983.tb07783.x. [DOI] [PubMed] [Google Scholar]
  13. Parker P. J., Embi N., Caudwell F. B., Cohen P. Glycogen synthase from rabbit skeletal muscle. State of phosphorylation of the seven phosphoserine residues in vivo in the presence and absence of adrenaline. Eur J Biochem. 1982 May;124(1):47–55. doi: 10.1111/j.1432-1033.1982.tb05904.x. [DOI] [PubMed] [Google Scholar]
  14. RODBELL M. METABOLISM OF ISOLATED FAT CELLS. I. EFFECTS OF HORMONES ON GLUCOSE METABOLISM AND LIPOLYSIS. J Biol Chem. 1964 Feb;239:375–380. [PubMed] [Google Scholar]
  15. Roach P. J., Rosell-Perez M., Larner J. Muscle glycogen synthase in vivo state: Effects of insulin administration on the chemical and kinetic properties of the purified enzyme. FEBS Lett. 1977 Aug 1;80(1):95–98. doi: 10.1016/0014-5793(77)80414-6. [DOI] [PubMed] [Google Scholar]
  16. Strålfors P., Björgell P., Belfrage P. Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3317–3321. doi: 10.1073/pnas.81.11.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Walkenbach R. J., Hazen R., Larner J. Reversible inhibition of cyclic AMP-dependent protein kinase by insulin. Mol Cell Biochem. 1978 Feb 24;19(1):31–41. doi: 10.1007/BF00231232. [DOI] [PubMed] [Google Scholar]
  18. Walseth T. F., Johnson R. A. The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP. Biochim Biophys Acta. 1979 Mar 28;562(1):11–31. doi: 10.1016/0005-2787(79)90122-9. [DOI] [PubMed] [Google Scholar]

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