Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Oct 25;91(22):10630–10634. doi: 10.1073/pnas.91.22.10630

Phorbol ester-induced stimulation and phosphorylation of adenylyl cyclase 2.

O Jacobowitz 1, R Iyengar 1
PMCID: PMC45075  PMID: 7938004

Abstract

Adenylyl cyclase 2 was expressed in Sf9 cells by recombinant baculovirus infection. Phorbol 12-myristate 13-acetate (PMA) treatment of cells expressing adenylyl cyclase 2 (AC2) increased basal activity. This increase was blocked by staurosporine, a protein kinase C inhibitor. PMA treatment increased Vmax without affecting Km. Greatest increase in basal activity was seen at physiologically relevant Mg2+ concentrations. PMA treatment did not alter sensitivity to guanine nucleotide stimulatory factor (Gs) but enhanced stimulation at all concentrations of activated Gs alpha subunit tested. AC2 was tagged at the N terminus with an 8-amino acid epitope. Epitope-tagged AC2 was purified to apparent homogeneity in a single step by using an antiepitope antibody-affinity column. The eluate was resolved by SDS/PAGE. Silver staining of the gel showed a 106-kDa band. The purified protein was recognized by antipeptide antibody against a region common to all mammalian adenylyl cyclases. The epitope-tagged enzyme expressed in Sf9 cells was also stimulated by PMA. When cells were labeled with 32P and treated with PMA, a 3-fold increase in 32P incorporation of purified epitope-tagged AC2 was observed. We conclude that activation of protein kinase C results in phosphorylation and stimulation of AC2, a cell-surface G protein effector enzyme. Thus, covalent modification of cell-surface effectors may provide an independent mode for signal transmission through G protein pathways.

Full text

PDF
10632

Images in this article

10632Image
on p.10632
10633Image
on p.10633
10633Image
on p.10633

Selected References

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

  1. Bell J. D., Buxton I. L., Brunton L. L. Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Putative effect of C kinase on alpha s-GTP-catalytic subunit interaction. J Biol Chem. 1985 Mar 10;260(5):2625–2628. [PubMed] [Google Scholar]
  2. Bird I. M., Mason J. I., Oka K., Rainey W. E. Angiotensin-II stimulates an increase in cAMP and expression of 17 alpha-hydroxylase cytochrome P450 in fetal bovine adrenocortical cells. Endocrinology. 1993 Feb;132(2):932–934. doi: 10.1210/endo.132.2.8381079. [DOI] [PubMed] [Google Scholar]
  3. Bushfield M., Murphy G. J., Lavan B. E., Parker P. J., Hruby V. J., Milligan G., Houslay M. D. Hormonal regulation of Gi2 alpha-subunit phosphorylation in intact hepatocytes. Biochem J. 1990 Jun 1;268(2):449–457. doi: 10.1042/bj2680449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Camps M., Carozzi A., Schnabel P., Scheer A., Parker P. J., Gierschik P. Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits. Nature. 1992 Dec 17;360(6405):684–686. doi: 10.1038/360684a0. [DOI] [PubMed] [Google Scholar]
  5. Carty D. J., Padrell E., Codina J., Birnbaumer L., Hildebrandt J. D., Iyengar R. Distinct guanine nucleotide binding and release properties of the three Gi proteins. J Biol Chem. 1990 Apr 15;265(11):6268–6273. [PubMed] [Google Scholar]
  6. De Vivo M., Chen J., Codina J., Iyengar R. Enhanced phospholipase C stimulation and transformation in NIH-3T3 cells expressing Q209LGq-alpha-subunits. J Biol Chem. 1992 Sep 15;267(26):18263–18266. [PubMed] [Google Scholar]
  7. Federman A. D., Conklin B. R., Schrader K. A., Reed R. R., Bourne H. R. Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits. Nature. 1992 Mar 12;356(6365):159–161. doi: 10.1038/356159a0. [DOI] [PubMed] [Google Scholar]
  8. Iyengar R. Molecular and functional diversity of mammalian Gs-stimulated adenylyl cyclases. FASEB J. 1993 Jun;7(9):768–775. doi: 10.1096/fasebj.7.9.8330684. [DOI] [PubMed] [Google Scholar]
  9. Jacobowitz O., Chen J., Premont R. T., Iyengar R. Stimulation of specific types of Gs-stimulated adenylyl cyclases by phorbol ester treatment. J Biol Chem. 1993 Feb 25;268(6):3829–3832. [PubMed] [Google Scholar]
  10. Johnson J. A., Friedman J., Halligan R. D., Birnbaumer M., Clark R. B. Sensitization of adenylyl cyclase by P2 purinergic and M5 muscarinic receptor agonists in L cells. Mol Pharmacol. 1991 Oct;40(4):539–546. [PubMed] [Google Scholar]
  11. Katada T., Gilman A. G., Watanabe Y., Bauer S., Jakobs K. H. Protein kinase C phosphorylates the inhibitory guanine-nucleotide-binding regulatory component and apparently suppresses its function in hormonal inhibition of adenylate cyclase. Eur J Biochem. 1985 Sep 2;151(2):431–437. doi: 10.1111/j.1432-1033.1985.tb09120.x. [DOI] [PubMed] [Google Scholar]
  12. Kendler D. L., Brennan V., Davies T. F., Magnusson R. P. Expression of human thyroid peroxidase in insect cells using recombinant baculovirus. Mol Cell Endocrinol. 1993 Jun;93(2):199–206. doi: 10.1016/0303-7207(93)90124-3. [DOI] [PubMed] [Google Scholar]
  13. Lustig K. D., Conklin B. R., Herzmark P., Taussig R., Bourne H. R. Type II adenylylcyclase integrates coincident signals from Gs, Gi, and Gq. J Biol Chem. 1993 Jul 5;268(19):13900–13905. [PubMed] [Google Scholar]
  14. Naghshineh S., Noguchi M., Huang K. P., Londos C. Activation of adipocyte adenylate cyclase by protein kinase C. J Biol Chem. 1986 Nov 5;261(31):14534–14538. [PubMed] [Google Scholar]
  15. Peytremann A., Nicholson W. E., Brown R. D., Liddle G. W., Hardman J. G. Comparative effects of angiotensin and ACTH on cyclic AMP and steroidogenesis in isolated bovine adrenal cells. J Clin Invest. 1973 Apr;52(4):835–842. doi: 10.1172/JCI107247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Premont R. T., Iyengar R. Glucagon-induced desensitization of adenylyl cyclase in primary cultures of chick hepatocytes. Evidence for multiple pathways. J Biol Chem. 1988 Nov 5;263(31):16087–16095. [PubMed] [Google Scholar]
  17. Rozengurt E., Murray M., Zachary I., Collins M. Protein kinase C activation enhances cAMP accumulation in Swiss 3T3 cells: inhibition by pertussis toxin. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2282–2286. doi: 10.1073/pnas.84.8.2282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schubert D., LaCorbiere M., Whitlock C., Stallcup W. Alterations in the surface properties of cells responsive to nerve growth factor. Nature. 1978 Jun 29;273(5665):718–723. doi: 10.1038/273718a0. [DOI] [PubMed] [Google Scholar]
  19. Sibley D. R., Jeffs R. A., Daniel K., Nambi P., Lefkowitz R. J. Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes. Arch Biochem Biophys. 1986 Jan;244(1):373–381. doi: 10.1016/0003-9861(86)90126-8. [DOI] [PubMed] [Google Scholar]
  20. Simmoteit R., Schulzki H. D., Palm D., Mollner S., Pfeuffer T. Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters. FEBS Lett. 1991 Jul 8;285(1):99–103. doi: 10.1016/0014-5793(91)80734-k. [DOI] [PubMed] [Google Scholar]
  21. Sugden D., Vanecek J., Klein D. C., Thomas T. P., Anderson W. B. Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes. 1985 Mar 28-Apr 3Nature. 314(6009):359–361. doi: 10.1038/314359a0. [DOI] [PubMed] [Google Scholar]
  22. Takahashi J. S. Gene regulation. Circadian clocks à la CREM. Nature. 1993 Sep 23;365(6444):299–300. doi: 10.1038/365299a0. [DOI] [PubMed] [Google Scholar]
  23. Tang W. J., Gilman A. G. Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits. Science. 1991 Dec 6;254(5037):1500–1503. doi: 10.1126/science.1962211. [DOI] [PubMed] [Google Scholar]
  24. Taussig R., Quarmby L. M., Gilman A. G. Regulation of purified type I and type II adenylylcyclases by G protein beta gamma subunits. J Biol Chem. 1993 Jan 5;268(1):9–12. [PubMed] [Google Scholar]
  25. Wiener E., Scarpa A. Activation of protein kinase C modulates the adenylate cyclase effector system of B-lymphocytes. J Biol Chem. 1989 Mar 15;264(8):4324–4328. [PubMed] [Google Scholar]
  26. Wu D., LaRosa G. J., Simon M. I. G protein-coupled signal transduction pathways for interleukin-8. Science. 1993 Jul 2;261(5117):101–103. doi: 10.1126/science.8316840. [DOI] [PubMed] [Google Scholar]
  27. Yoshimasa T., Sibley D. R., Bouvier M., Lefkowitz R. J., Caron M. G. Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation. Nature. 1987 May 7;327(6117):67–70. doi: 10.1038/327067a0. [DOI] [PubMed] [Google Scholar]
  28. Yoshimura M., Cooper D. M. Type-specific stimulation of adenylylcyclase by protein kinase C. J Biol Chem. 1993 Mar 5;268(7):4604–4607. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES