Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1984 May;3(5):953–959. doi: 10.1002/j.1460-2075.1984.tb01913.x

Purification to homogeneity of protein kinase C from bovine brain--identity with the phorbol ester receptor.

P J Parker, S Stabel, M D Waterfield
PMCID: PMC557457  PMID: 6329748

Abstract

The calcium- and phospholipid-dependent kinase activity (protein kinase C) was isolated from bovine brains by a combination of DEAE-cellulose chromatography, gel filtration and hydrophobic chromatography on octyl-Sepharose and phenyl-Sepharose. The phorbol ester receptor co-purifies with the protein kinase C throughout the procedure yielding a homogeneous protein of 79 500 daltons on SDS-polyacrylamide gels. The purified kinase incorporated approximately 5000 nmol phosphate into substrate/min/mg protein at saturating concentrations of Ca2+ and phosphatidyl serine. Reciprocal plots of protein kinase activity at varying phosphatidyl serine concentrations were biphasic and yielded two apparent Ka values for phosphatidyl serine of 0.6-2 and 35-80 micrograms/ml). These apparent Ka values were reduced 2- to 3-fold by either diolein (20 micrograms/ml) or phorbol-12,13-dibutyrate (10 micrograms/ml). The protein binds [3H]phorbol-12,13-dibutyrate ( [3H]PDB) with high affinity (Ka = 15 nM) in a phosphatidyl serine-dependent manner. At saturating phosphatidyl serine concentrations 0.89 mol [3H]PDB are bound per mol protein. The identification of protein kinase C as the phorbol ester receptor is discussed with respect to the function and regulation of this protein.

Full text

PDF
953

Images in this article

955Fig. 3.
on p.955
956Fig. 5.
on p.956

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. Protein kinase activity associated with a phorbol ester receptor purified from mouse brain. Cancer Res. 1983 Sep;43(9):4333–4337. [PubMed] [Google Scholar]
  2. Ashendel C. L., Staller J. M., Boutwell R. K. Solubilization, purification, and reconstitution of a phorbol ester receptor from the particulate protein fraction of mouse brain. Cancer Res. 1983 Sep;43(9):4327–4332. [PubMed] [Google Scholar]
  3. Boynton A. L., Whitfield J. F., Isaacs R. J. Calcium-dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor-promoting phorbol ester (PMA). J Cell Physiol. 1976 Jan;87(1):25–32. doi: 10.1002/jcp.1040870105. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [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. Cohen P. The role of cyclic-AMP-dependent protein kinase in the regulation of glycogen metabolism in mammalian skeletal muscle. Curr Top Cell Regul. 1978;14:117–196. doi: 10.1016/b978-0-12-152814-0.50008-3. [DOI] [PubMed] [Google Scholar]
  7. Cohen P. The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature. 1982 Apr 15;296(5858):613–620. doi: 10.1038/296613a0. [DOI] [PubMed] [Google Scholar]
  8. Dicker P., Rozengurt E. Stimulation of DNA synthesis by tumour promoter and pure mitogenic factors. Nature. 1978 Dec 14;276(5689):723–726. doi: 10.1038/276723a0. [DOI] [PubMed] [Google Scholar]
  9. Frantz C. N., Stiles C. D., Scher C. D. The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate enhances the proliferative response of Balb/c-3T3 cells to hormonal growth factors. J Cell Physiol. 1979 Sep;100(3):413–424. doi: 10.1002/jcp.1041000305. [DOI] [PubMed] [Google Scholar]
  10. Greenebaum E., Nicolaides M., Eisinger M., Vogel R. H., Weinstein I. B. Binding of phorbol dibutyrate and epidermal growth factor to cultured human epidermal cells. J Natl Cancer Inst. 1983 Mar;70(3):435–441. [PubMed] [Google Scholar]
  11. HOKIN M. R., HOKIN L. E. Enzyme secretion and the incorporation of P32 into phospholipides of pancreas slices. J Biol Chem. 1953 Aug;203(2):967–977. [PubMed] [Google Scholar]
  12. Inoue M., Kishimoto A., Takai Y., Nishizuka Y. Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. II. Proenzyme and its activation by calcium-dependent protease from rat brain. J Biol Chem. 1977 Nov 10;252(21):7610–7616. [PubMed] [Google Scholar]
  13. Jaken S., Tashjian A. H., Jr, Blumberg P. M. Relationship between biological responsiveness to phorbol esters and receptor levels in GH4C1 rat pituitary cells. Cancer Res. 1981 Dec;41(12 Pt 1):4956–4960. [PubMed] [Google Scholar]
  14. Kaibuchi K., Sano K., Hoshijima M., Takai Y., Nishizuka Y. Phosphatidylinositol turnover in platelet activation; calcium mobilization and protein phosphorylation. Cell Calcium. 1982 Oct;3(4-5):323–335. doi: 10.1016/0143-4160(82)90020-3. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Kikkawa U., Takai Y., Tanaka Y., Miyake R., Nishizuka Y. Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters. J Biol Chem. 1983 Oct 10;258(19):11442–11445. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Laszlo A., Radke K., Chin S., Bissell M. J. Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6241–6245. doi: 10.1073/pnas.78.10.6241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  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. Nishizuka Y. Calcium, phospholipid turnover and transmembrane signalling. Philos Trans R Soc Lond B Biol Sci. 1983 Jul 5;302(1108):101–112. doi: 10.1098/rstb.1983.0043. [DOI] [PubMed] [Google Scholar]
  24. O'Brien T. G., Lewis M. A., Diamond L. Ornithine decarboxylase activity and DNA synthesis after treatment of cells in culture with 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 1979 Nov;39(11):4477–4480. [PubMed] [Google Scholar]
  25. O'Brien T. G., Simsiman R. C., Boutwell R. K. Induction of the polyamine-biosynthetic enzymes in mouse epidermis by tumor-promoting agents. Cancer Res. 1975 Jul;35(7):1662–1670. [PubMed] [Google Scholar]
  26. Qi D. F., Schatzman R. C., Mazzei G. J., Turner R. S., Raynor R. L., Liao S., Kuo J. F. Polyamines inhibit phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases. Biochem J. 1983 Aug 1;213(2):281–288. doi: 10.1042/bj2130281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rozengurt E., Legg A., Pettican P. Vasopressin stimulation of mouse 3T3 cell growth. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1284–1287. doi: 10.1073/pnas.76.3.1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schatzman R. C., Raynor R. L., Fritz R. B., Kuo J. F. Purification to homogeneity, characterization and monoclonal antibodies of phospholipid-sensitive Ca2+-dependent protein kinase from spleen. Biochem J. 1983 Feb 1;209(2):435–443. doi: 10.1042/bj2090435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sivak A. Induction of cell division: role of cell membrane sites. J Cell Physiol. 1972 Oct;80(2):167–173. doi: 10.1002/jcp.1040800203. [DOI] [PubMed] [Google Scholar]
  30. Solanki V., Slaga T. J. Specific binding of phorbol ester tumor promoters to intact primary epidermal cells from Sencar mice. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2549–2553. doi: 10.1073/pnas.78.4.2549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Takai Y., Kishimoto A., Iwasa Y., Kawahara Y., Mori T., Nishizuka Y. Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids. J Biol Chem. 1979 May 25;254(10):3692–3695. [PubMed] [Google Scholar]
  32. 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]
  33. Takai Y., Minakuchi R., Kikkawa U., Sano K., Kaibuchi K., Yu B., Matsubara T., Nishizuka Y. Membrane phospholipid turnover, receptor function and protein phosphorylation. Prog Brain Res. 1982;56:287–301. doi: 10.1016/S0079-6123(08)63780-2. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Wu V. S., Byus C. V. The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells. Life Sci. 1981 Nov 2;29(18):1855–1863. doi: 10.1016/0024-3205(81)90516-6. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES