Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Aug 1;341(Pt 3):839–845.

Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A.

S Resjö 1, A Oknianska 1, S Zolnierowicz 1, V Manganiello 1, E Degerman 1
PMCID: PMC1220425  PMID: 10417351

Abstract

Phosphodiesterase type 3B (PDE3B) has been shown to be activated and phosphorylated in response to insulin and hormones that increase cAMP. In order to study serine/threonine protein phosphatases involved in the regulation of rat adipocyte PDE3B, we investigated the phosphorylation and activation of PDE3B in vivo in response to phosphatase inhibitors and the dephosphorylation and deactivation of PDE3B in vitro by phosphatases purified from rat adipocyte homogenates. Okadaic acid and calyculin A induced dose- and time-dependent activation of PDE3B. Maximal effects were obtained after 30 min using 1 microM okadaic acid (1.8-fold activation) and 300 nM calyculin A (4-fold activation), respectively. Tautomycin and cyclosporin A did not induce activation of PDE3B. Incubation of adipocytes with 300 nM calyculin A inhibited protein phosphatase (PP) 1 and PP2A completely. Okadaic acid (1 microM) reduced PP2A activity by approx. 50% but did not affect PP1 activity, and 1 microM tautomycin reduced PP1 activity by approx. 60% but PP2A activity by only 11%. This indicates an important role for PP2A in the regulation of PDE3B. Furthermore, rat adipocyte PDE3B phosphatase activity co-purified with PP2A but not with PP1 during MonoQ chromatography. As compared with insulin, okadaic acid and calyculin A induced phosphorylation of PDE3B by 2.8- and 14-fold respectively, whereas tautomycin and cyclosporin A had no effect. Both calyculin A and okadaic acid induced phosphorylation on serine 302, the site known to be phosphorylated on PDE3B in response to insulin and isoproterenol (isoprenaline), as well as on sites not identified previously. In summary, PP2A seems to be involved in the regulation of PDE3B in vivo and can act as a PDE3B phosphatase in vitro. In comparison with insulin, calyculin A induced a dramatic activation of PDE3B and both calyculin A and okadaic acid induced phosphorylation on additional sites, which could have a role in signalling pathways not yet identified.

Full Text

The Full Text of this article is available as a PDF (212.4 KB).

Selected References

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

  1. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahmad F., Gao G., Wang L. M., Landstrom T. R., Degerman E., Pierce J. H., Manganiello V. C. IL-3 and IL-4 activate cyclic nucleotide phosphodiesterases 3 (PDE3) and 4 (PDE4) by different mechanisms in FDCP2 myeloid cells. J Immunol. 1999 Apr 15;162(8):4864–4875. [PubMed] [Google Scholar]
  3. Andersen C. B., Roth R. A., Conti M. Protein kinase B/Akt induces resumption of meiosis in Xenopus oocytes. J Biol Chem. 1998 Jul 24;273(30):18705–18708. doi: 10.1074/jbc.273.30.18705. [DOI] [PubMed] [Google Scholar]
  4. Andjelković M., Jakubowicz T., Cron P., Ming X. F., Han J. W., Hemmings B. A. Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5699–5704. doi: 10.1073/pnas.93.12.5699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barford D. Molecular mechanisms of the protein serine/threonine phosphatases. Trends Biochem Sci. 1996 Nov;21(11):407–412. doi: 10.1016/s0968-0004(96)10060-8. [DOI] [PubMed] [Google Scholar]
  6. Beavo J. A. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev. 1995 Oct;75(4):725–748. doi: 10.1152/physrev.1995.75.4.725. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chen M. X., McPartlin A. E., Brown L., Chen Y. H., Barker H. M., Cohen P. T. A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus. EMBO J. 1994 Sep 15;13(18):4278–4290. doi: 10.1002/j.1460-2075.1994.tb06748.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cohen P. T. Novel protein serine/threonine phosphatases: variety is the spice of life. Trends Biochem Sci. 1997 Jul;22(7):245–251. doi: 10.1016/s0968-0004(97)01060-8. [DOI] [PubMed] [Google Scholar]
  10. Cohen P., Alemany S., Hemmings B. A., Resink T. J., Strålfors P., Tung H. Y. Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol. 1988;159:390–408. doi: 10.1016/0076-6879(88)59039-0. [DOI] [PubMed] [Google Scholar]
  11. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  12. Cohen P. The subunit structure of rabbit-skeletal-muscle phosphorylase kinase, and the molecular basis of its activation reactions. Eur J Biochem. 1973 Apr 2;34(1):1–14. doi: 10.1111/j.1432-1033.1973.tb02721.x. [DOI] [PubMed] [Google Scholar]
  13. Conti M., Nemoz G., Sette C., Vicini E. Recent progress in understanding the hormonal regulation of phosphodiesterases. Endocr Rev. 1995 Jun;16(3):370–389. doi: 10.1210/edrv-16-3-370. [DOI] [PubMed] [Google Scholar]
  14. Degerman E., Belfrage P., Manganiello V. C. Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3). J Biol Chem. 1997 Mar 14;272(11):6823–6826. doi: 10.1074/jbc.272.11.6823. [DOI] [PubMed] [Google Scholar]
  15. Degerman E., Smith C. J., Tornqvist H., Vasta V., Belfrage P., Manganiello V. C. Evidence that insulin and isoprenaline activate the cGMP-inhibited low-Km cAMP phosphodiesterase in rat fat cells by phosphorylation. Proc Natl Acad Sci U S A. 1990 Jan;87(2):533–537. doi: 10.1073/pnas.87.2.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ekholm D., Hemmer B., Gao G., Vergelli M., Martin R., Manganiello V. Differential expression of cyclic nucleotide phosphodiesterase 3 and 4 activities in human T cell clones specific for myelin basic protein. J Immunol. 1997 Aug 1;159(3):1520–1529. [PubMed] [Google Scholar]
  17. Eriksson H., Ridderstråle M., Degerman E., Ekholm D., Smith C. J., Manganiello V. C., Belfrage P., Tornqvist H. Evidence for the key role of the adipocyte cGMP-inhibited cAMP phosphodiesterase in the antilipolytic action of insulin. Biochim Biophys Acta. 1995 Apr 6;1266(1):101–107. doi: 10.1016/0167-4889(94)00237-9. [DOI] [PubMed] [Google Scholar]
  18. Eriksson H., Tornqvist H. Specific inhibition of the cGMP-inhibited cAMP phosphodiesterase blocks the insulin-like antilipolytic effect of growth hormone in rat adipocytes. Mol Cell Biochem. 1997 Apr;169(1-2):37–42. doi: 10.1023/a:1006886509892. [DOI] [PubMed] [Google Scholar]
  19. Favre B., Turowski P., Hemmings B. A. Differential inhibition and posttranslational modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic acid, and tautomycin. J Biol Chem. 1997 May 23;272(21):13856–13863. doi: 10.1074/jbc.272.21.13856. [DOI] [PubMed] [Google Scholar]
  20. Fisher D. A., Smith J. F., Pillar J. S., St Denis S. H., Cheng J. B. Isolation and characterization of PDE8A, a novel human cAMP-specific phosphodiesterase. Biochem Biophys Res Commun. 1998 May 29;246(3):570–577. doi: 10.1006/bbrc.1998.8684. [DOI] [PubMed] [Google Scholar]
  21. Hastie C. J., Cohen P. T. Purification of protein phosphatase 4 catalytic subunit: inhibition by the antitumour drug fostriecin and other tumour suppressors and promoters. FEBS Lett. 1998 Jul 24;431(3):357–361. doi: 10.1016/s0014-5793(98)00775-3. [DOI] [PubMed] [Google Scholar]
  22. Holm C., Langin D., Manganiello V., Belfrage P., Degerman E. Regulation of hormone-sensitive lipase activity in adipose tissue. Methods Enzymol. 1997;286:45–67. doi: 10.1016/s0076-6879(97)86004-1. [DOI] [PubMed] [Google Scholar]
  23. Honnor R. C., Dhillon G. S., Londos C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior. J Biol Chem. 1985 Dec 5;260(28):15122–15129. [PubMed] [Google Scholar]
  24. Huang X., Honkanen R. E. Molecular cloning, expression, and characterization of a novel human serine/threonine protein phosphatase, PP7, that is homologous to Drosophila retinal degeneration C gene product (rdgC). J Biol Chem. 1998 Jan 16;273(3):1462–1468. doi: 10.1074/jbc.273.3.1462. [DOI] [PubMed] [Google Scholar]
  25. Ishihara H., Martin B. L., Brautigan D. L., Karaki H., Ozaki H., Kato Y., Fusetani N., Watabe S., Hashimoto K., Uemura D. Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem Biophys Res Commun. 1989 Mar 31;159(3):871–877. doi: 10.1016/0006-291x(89)92189-x. [DOI] [PubMed] [Google Scholar]
  26. KREBS E. G., FISCHER E. H. Phosphorylase activity of skeletal muscle extracts. J Biol Chem. 1955 Sep;216(1):113–120. [PubMed] [Google Scholar]
  27. Kremmer E., Ohst K., Kiefer J., Brewis N., Walter G. Separation of PP2A core enzyme and holoenzyme with monoclonal antibodies against the regulatory A subunit: abundant expression of both forms in cells. Mol Cell Biol. 1997 Mar;17(3):1692–1701. doi: 10.1128/mcb.17.3.1692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Liu J., Farmer J. D., Jr, Lane W. S., Friedman J., Weissman I., Schreiber S. L. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991 Aug 23;66(4):807–815. doi: 10.1016/0092-8674(91)90124-h. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. MacKintosh C., Klumpp S. Tautomycin from the bacterium Streptomyces verticillatus. Another potent and specific inhibitor of protein phosphatases 1 and 2A. FEBS Lett. 1990 Dec 17;277(1-2):137–140. doi: 10.1016/0014-5793(90)80828-7. [DOI] [PubMed] [Google Scholar]
  32. MacKintosh C., MacKintosh R. W. Inhibitors of protein kinases and phosphatases. Trends Biochem Sci. 1994 Nov;19(11):444–448. doi: 10.1016/0968-0004(94)90127-9. [DOI] [PubMed] [Google Scholar]
  33. Manganiello V. C., Murad F., Vaughan M. Effects of lipolytic and antilipolytic agents on cyclic 3',5'-adenosine monophosphate in fat cells. J Biol Chem. 1971 Apr 10;246(7):2195–2202. [PubMed] [Google Scholar]
  34. Manganiello V. C., Murata T., Taira M., Belfrage P., Degerman E. Diversity in cyclic nucleotide phosphodiesterase isoenzyme families. Arch Biochem Biophys. 1995 Sep 10;322(1):1–13. doi: 10.1006/abbi.1995.1429. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Rahn T., Ridderstråle M., Tornqvist H., Manganiello V., Fredrikson G., Belfrage P., Degerman E. Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes. Studies using the selective inhibitor wortmannin. FEBS Lett. 1994 Aug 22;350(2-3):314–318. doi: 10.1016/0014-5793(94)00797-7. [DOI] [PubMed] [Google Scholar]
  37. Rahn T., Rönnstrand L., Leroy M. J., Wernstedt C., Tornqvist H., Manganiello V. C., Belfrage P., Degerman E. Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol. J Biol Chem. 1996 May 10;271(19):11575–11580. doi: 10.1074/jbc.271.19.11575. [DOI] [PubMed] [Google Scholar]
  38. Rascón A., Degerman E., Taira M., Meacci E., Smith C. J., Manganiello V., Belfrage P., Tornqvist H. Identification of the phosphorylation site in vitro for cAMP-dependent protein kinase on the rat adipocyte cGMP-inhibited cAMP phosphodiesterase. J Biol Chem. 1994 Apr 22;269(16):11962–11966. [PubMed] [Google Scholar]
  39. Shibata H., Robinson F. W., Soderling T. R., Kono T. Effects of okadaic acid on insulin-sensitive cAMP phosphodiesterase in rat adipocytes. Evidence that insulin may stimulate the enzyme by phosphorylation. J Biol Chem. 1991 Sep 25;266(27):17948–17953. [PubMed] [Google Scholar]
  40. Smith C. J., Manganiello V. C. Role of hormone-sensitive low Km cAMP phosphodiesterase in regulation of cAMP-dependent protein kinase and lipolysis in rat adipocytes. Mol Pharmacol. 1989 Mar;35(3):381–386. [PubMed] [Google Scholar]
  41. Smith C. J., Vasta V., Degerman E., Belfrage P., Manganiello V. C. Hormone-sensitive cyclic GMP-inhibited cyclic AMP phosphodiesterase in rat adipocytes. Regulation of insulin- and cAMP-dependent activation by phosphorylation. J Biol Chem. 1991 Jul 15;266(20):13385–13390. [PubMed] [Google Scholar]
  42. Soderling S. H., Bayuga S. J., Beavo J. A. Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases. J Biol Chem. 1998 Jun 19;273(25):15553–15558. doi: 10.1074/jbc.273.25.15553. [DOI] [PubMed] [Google Scholar]
  43. Takai A., Sasaki K., Nagai H., Mieskes G., Isobe M., Isono K., Yasumoto T. Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein. Biochem J. 1995 Mar 15;306(Pt 3):657–665. doi: 10.1042/bj3060657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wera S., Hemmings B. A. Serine/threonine protein phosphatases. Biochem J. 1995 Oct 1;311(Pt 1):17–29. doi: 10.1042/bj3110017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wijkander J., Landström T. R., Manganiello V., Belfrage P., Degerman E. Insulin-induced phosphorylation and activation of phosphodiesterase 3B in rat adipocytes: possible role for protein kinase B but not mitogen-activated protein kinase or p70 S6 kinase. Endocrinology. 1998 Jan;139(1):219–227. doi: 10.1210/endo.139.1.5693. [DOI] [PubMed] [Google Scholar]
  46. Wood S. L., Emmison N., Borthwick A. C., Yeaman S. J. The protein phosphatases responsible for dephosphorylation of hormone-sensitive lipase in isolated rat adipocytes. Biochem J. 1993 Oct 15;295(Pt 2):531–535. doi: 10.1042/bj2950531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhao A. Z., Bornfeldt K. E., Beavo J. A. Leptin inhibits insulin secretion by activation of phosphodiesterase 3B. J Clin Invest. 1998 Sep 1;102(5):869–873. doi: 10.1172/JCI3920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhao A. Z., Zhao H., Teague J., Fujimoto W., Beavo J. A. Attenuation of insulin secretion by insulin-like growth factor 1 is mediated through activation of phosphodiesterase 3B. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):3223–3228. doi: 10.1073/pnas.94.7.3223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zolnierowicz S., Van Hoof C., Andjelković N., Cron P., Stevens I., Merlevede W., Goris J., Hemmings B. A. The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits. Biochem J. 1996 Jul 1;317(Pt 1):187–194. doi: 10.1042/bj3170187. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES