Skip to main content
NCBI 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
. 1984 Jan;81(2):607–610. doi: 10.1073/pnas.81.2.607

Competitive inhibition by diacylglycerol of specific phorbol ester binding.

N A Sharkey, K L Leach, P M Blumberg
PMCID: PMC344728  PMID: 6320198

Abstract

Emerging evidence suggests that the phorbol ester receptor in brain may be the same as the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Since protein kinase C activity is stimulated by unsaturated diacylglycerol and the phorbol esters can substitute for diacylglycerol in this stimulation, we have examined the effect of diacylglycerols on phorbol ester binding. Assays were carried out with the mouse brain cytosolic phorbol ester aporeceptor, which requires phospholipids for activity. In the presence of phosphatidylserine at 0.96 mg/ml, diolein inhibited specific binding of [3H]phorbol 12,13-dibutyrate ( [3H]PBt2) in a dose-dependent fashion to less than 10% of control levels. The inhibition curves fit the curve expected for a competitive inhibitor and yielded a Ki of 3.6 +/- 0.8 micrograms/ml (n = 5) [0.38% (wt/wt) the concentration of phosphatidylserine]. Scatchard analysis confirmed the competitive nature of the inhibition. At constant phospholipid concentrations, the Ki determined for diolein was independent of the diolein concentrations over the range of 1.5-80 micrograms/ml, suggesting that the inhibition did not arise simply by perturbation of the phospholipid bilayers. The Ki of diolein was approximately proportional to the absolute phospholipid concentration. With phosphatidylserine at 4.8 micrograms/ml, for example, the Ki was 52 ng/ml (1.1% of phosphatidylserine). In addition to diolein, the short-chain saturated diacylglycerol derivatives dicaprylin and dicaproin also inhibited [3H]PBt2 binding, whereas the long-chain saturated derivatives dipalmitin and distearin were much less active. Our results suggest (i) that diacylglycerol may act as an endogenous ligand for the phorbol ester receptor and (ii) that variation in lipid composition provides a mechanism for modulating phorbol ester receptor affinity.

Full text

PDF
607

Selected References

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

  1. Ashendel C. L., Staller J. M., Boutwell R. K. Identification of a calcium- and phospholipid- dependent phorbol ester binding activity in the soluble fraction of mouse tissues. Biochem Biophys Res Commun. 1983 Feb 28;111(1):340–345. doi: 10.1016/s0006-291x(83)80157-0. [DOI] [PubMed] [Google Scholar]
  2. Blumberg P. M. In vitro studies on the mode of action of the phorbol esters, potent tumor promoters, part 2. Crit Rev Toxicol. 1981 Jun;8(3):199–234. doi: 10.3109/10408448109109658. [DOI] [PubMed] [Google Scholar]
  3. Blumberg P. M. In vitro studies on the mode of action of the phorbol esters, potent tumor promoters: part 1. Crit Rev Toxicol. 1980 Dec;8(2):153–197. doi: 10.3109/10408448009037493. [DOI] [PubMed] [Google Scholar]
  4. Boggs J. M. Intermolecular hydrogen bonding between lipids: influence on organization and function of lipids in membranes. Can J Biochem. 1980 Oct;58(10):755–770. doi: 10.1139/o80-107. [DOI] [PubMed] [Google Scholar]
  5. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
  6. Delclos K. B., Yeh E., Blumberg P. M. Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester. Proc Natl Acad Sci U S A. 1983 May;80(10):3054–3058. doi: 10.1073/pnas.80.10.3054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Diamond L., O'Brien T. G., Baird W. M. Tumor promoters and the mechanism of tumor promotion. Adv Cancer Res. 1980;32:1–74. doi: 10.1016/s0065-230x(08)60360-7. [DOI] [PubMed] [Google Scholar]
  8. Driedger P. E., Blumberg P. M. Specific binding of phorbol ester tumor promoters. Proc Natl Acad Sci U S A. 1980 Jan;77(1):567–571. doi: 10.1073/pnas.77.1.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dunn J. A., Blumberg P. M. Specific binding of [20-3H]12-deoxyphorbol 13-isobutyrate to phorbol ester receptor subclasses in mouse skin particulate preparations. Cancer Res. 1983 Oct;43(10):4632–4637. [PubMed] [Google Scholar]
  10. Dunphy W. G., Kochenburger R. J., Castagna M., Blumberg P. M. Kinetics and subcellular localization of specific [3H]phorbol 12, 13-dibutyrate binding by mouse brain. Cancer Res. 1981 Jul;41(7):2640–2647. [PubMed] [Google Scholar]
  11. Hitchings G. H., Burchall J. J. Inhibition of folate biosynthesis and function as a basis for chemotherapy. Adv Enzymol Relat Areas Mol Biol. 1965;27:417–468. doi: 10.1002/9780470122723.ch9. [DOI] [PubMed] [Google Scholar]
  12. Horowitz A. D., Greenebaum E., Weinstein I. B. Identification of receptors for phorbol ester tumor promoters in intact mammalian cells and of an inhibitor of receptor binding in biologic fluids. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2315–2319. doi: 10.1073/pnas.78.4.2315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaibuchi K., Takai Y., Nishizuka Y. Cooperative roles of various membrane phospholipids in the activation of calcium-activated, phospholipid-dependent protein kinase. J Biol Chem. 1981 Jul 25;256(14):7146–7149. [PubMed] [Google Scholar]
  14. Kikkawa U., Takai Y., Minakuchi R., Inohara S., Nishizuka Y. Calcium-activated, phospholipid-dependent protein kinase from rat brain. Subcellular distribution, purification, and properties. J Biol Chem. 1982 Nov 25;257(22):13341–13348. [PubMed] [Google Scholar]
  15. Kishimoto A., Takai Y., Mori T., Kikkawa U., Nishizuka Y. Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J Biol Chem. 1980 Mar 25;255(6):2273–2276. [PubMed] [Google Scholar]
  16. Kreibich G., Hecker E. Active principles of croton oil. X. Preparation of tritium labeled croton oil factor A1 and other tritium labeled phorbol derivatives. Z Krebsforsch. 1970;74(4):448–456. [PubMed] [Google Scholar]
  17. Kuo J. F., Andersson R. G., Wise B. C., Mackerlova L., Salomonsson I., Brackett N. L., Katoh N., Shoji M., Wrenn R. W. Calcium-dependent protein kinase: widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoperazine. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7039–7043. doi: 10.1073/pnas.77.12.7039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Leach K. L., James M. L., Blumberg P. M. Characterization of a specific phorbol ester aporeceptor in mouse brain cytosol. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4208–4212. doi: 10.1073/pnas.80.14.4208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lukasiewicz R. J., Bennett E. L. alpha-Bungarotoxin binding properties of a central nervous system nicotinic acetylcholine receptor. Biochim Biophys Acta. 1978 Dec 1;544(2):294–308. doi: 10.1016/0304-4165(78)90098-3. [DOI] [PubMed] [Google Scholar]
  20. Michell R. H. Inositol phospholipids and cell surface receptor function. Biochim Biophys Acta. 1975 Mar 25;415(1):81–47. doi: 10.1016/0304-4157(75)90017-9. [DOI] [PubMed] [Google Scholar]
  21. Mori T., Takai Y., Yu B., Takahashi J., Nishizuka Y., Fujikura T. Specificity of the fatty acyl moieties of diacylglycerol for the activation of calcium-activated, phospholipid-dependent protein kinase. J Biochem. 1982 Feb;91(2):427–431. doi: 10.1093/oxfordjournals.jbchem.a133714. [DOI] [PubMed] [Google Scholar]
  22. Niedel J. E., Kuhn L. J., Vandenbark G. R. Phorbol diester receptor copurifies with protein kinase C. Proc Natl Acad Sci U S A. 1983 Jan;80(1):36–40. doi: 10.1073/pnas.80.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rohrsehneider L. R., Boutwell R. K. Phorbol esters, fatty acids and tumour promotion. Nat New Biol. 1973 Jun 13;243(128):212–213. doi: 10.1038/newbio243212a0. [DOI] [PubMed] [Google Scholar]
  24. Sando J. J., Young M. C. Identification of high-affinity phorbol ester receptor in cytosol of EL4 thymoma cells: requirement for calcium, magnesium, and phospholipids. Proc Natl Acad Sci U S A. 1983 May;80(9):2642–2646. doi: 10.1073/pnas.80.9.2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shoyab M., Todaro G. J. Specific high affinity cell membrane receptors for biologically active phorbol and ingenol esters. Nature. 1980 Dec 4;288(5790):451–455. doi: 10.1038/288451a0. [DOI] [PubMed] [Google Scholar]
  26. Shoyab M., Warren T. C., Todaro G. J. Tissue and species distribution and developmental variation of specific receptors for biologically active phorbol and ingenol esters. Carcinogenesis. 1981;2(12):1273–1276. doi: 10.1093/carcin/2.12.1273. [DOI] [PubMed] [Google Scholar]
  27. Takai Y., Kishimoto A., Kikkawa U., Mori T., Nishizuka Y. Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system. Biochem Biophys Res Commun. 1979 Dec 28;91(4):1218–1224. doi: 10.1016/0006-291x(79)91197-5. [DOI] [PubMed] [Google Scholar]
  28. Wise B. C., Raynor R. L., Kuo J. F. Phospholipid-sensitive Ca2+-dependent protein kinase from heart. I. Purification and general properties. J Biol Chem. 1982 Jul 25;257(14):8481–8488. [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