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
. 1983 Nov;80(22):6858–6862. doi: 10.1073/pnas.80.22.6858

Purified rat brain calcium- and phospholipid-dependent protein kinase phosphorylates ribosomal protein S6.

C J Le Peuch, R Ballester, O M Rosen
PMCID: PMC390085  PMID: 6417656

Abstract

The Ca2+-phospholipid-regulated protein kinase has been purified to homogeneity from a 100,000 X g supernatant fluid of rat brain homogenate by a procedure that includes DEAE-cellulose chromatography and successive filtrations on Ultrogel AcA 34 in EGTA and in phosphatidylserine and Ca2+. A more rapid purification consisting of DEAE-cellulose chromatography, Ultrogel AcA 34 gel filtration chromatography, and DEAE-trisacryl chromatography, all in the presence of EGTA, was also developed. Although the enzyme obtained by the latter procedure is not homogeneous, it exhibits properties similar to those of the pure enzyme and is more stable. In addition, the DEAE-trisacryl step permitted resolution of a contaminating Ca2+-inhibitable protein kinase that can interfere with studies of the Ca2+-phospholipid-stimulated enzyme. The homogeneous enzyme, purified about 300-fold, was estimated to have a Mr of 84,000. Its activity was 20- to 30-fold higher in the presence of phospholipid and Ca2+ than in the presence of phospholipid and EGTA, EGTA, or Ca2+ alone. The specific activity of the activated kinase was 852 nmol of P incorporated into histone per min/mg at 20 degrees C. The pure enzyme underwent autophosphorylation in a Ca2+- and phospholipid-dependent manner. This reaction was inhibited in the presence of histones without affecting the kinetic properties of the enzyme. Under optimal assay conditions, the homogeneous enzyme was activated 10-20% by either 10 microM diolein or 100 nM phorbol 12-myristate 13-acetate. Activation of the purified enzyme by diolein or the phorbol ester was far greater (3- to 4-fold) when aggregated instead of freshly sonicated phospholipids were used, suggesting that these compounds affect the interaction of the enzyme with phospholipids and Ca2+. The purified enzyme catalyzed the phosphorylation of the 40S ribosomal subunit protein S6. The Km for S6 was approximately equal to 1 microM and it was estimated that 2 mol of phosphate were incorporated per mol of S6. The observation that protein S6 can be phosphorylated by the purified Ca2+-phospholipid-dependent protein kinase may link recent reports that phorbol ester tumor promoters activate the Ca2+-phospholipid-dependent protein kinase in vitro and stimulate phosphorylation of the ribosomal protein S6 in vivo.

Full text

PDF
6858

Images in this article

6861Image
on p.6861

Selected References

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

  1. Anderson W. M., Grundholm A., Sells B. H. Modification of ribosomal proteins during liver regeneration. Biochem Biophys Res Commun. 1975 Feb 3;62(3):669–676. doi: 10.1016/0006-291x(75)90451-9. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Ballinger D. G., Hunt T. Fertilization of sea urchin eggs is accompanied by 40 S ribosomal subunit phosphorylation. Dev Biol. 1981 Oct 30;87(2):277–285. doi: 10.1016/0012-1606(81)90151-2. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Decker S. Phosphorylation of ribosomal protein S6 in avian sarcoma virus-transformed chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4112–4115. doi: 10.1073/pnas.78.7.4112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dedman J. R., Potter J. D., Jackson R. L., Johnson J. D., Means A. R. Physicochemical properties of rat testis Ca2+-dependent regulator protein of cyclic nucleotide phosphodiesterase. Relationship of Ca2+-binding, conformational changes, and phosphodiesterase activity. J Biol Chem. 1977 Dec 10;252(23):8415–8422. [PubMed] [Google Scholar]
  7. Duncan R., McConkey E. H. Rapid alterations in initiation rate and recruitment of inactive RNA are temporally correlated with S6 phosphorylation. Eur J Biochem. 1982 Apr;123(3):539–544. doi: 10.1111/j.1432-1033.1982.tb06565.x. [DOI] [PubMed] [Google Scholar]
  8. Erlichman J., Sarkar D., Fleischer N., Rubin C. S. Identification of two subclasses of type II cAMP-dependent protein kinases. Neural-specific and non-neural protein kinases. J Biol Chem. 1980 Sep 10;255(17):8179–8184. [PubMed] [Google Scholar]
  9. Gressner A. M., Wool I. G. The phosphorylation of liver ribosomal proteins in vivo. Evidence that only a single small subunit protein (S6) is phosphorylated. J Biol Chem. 1974 Nov 10;249(21):6917–6925. [PubMed] [Google Scholar]
  10. Haselbacher G. K., Humbel R. E., Thomas G. Insulin-like growth factor: insulin or serum increase phosphorylation of ribosomal protein S6 during transition of stationary chick embryo fibroblasts into early G1 phase of the cell cycle. FEBS Lett. 1979 Apr 1;100(1):185–190. doi: 10.1016/0014-5793(79)81160-6. [DOI] [PubMed] [Google Scholar]
  11. Hidaka H., Sasaki Y., Tanaka T., Endo T., Ohno S., Fujii Y., Nagata T. N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4354–4357. doi: 10.1073/pnas.78.7.4354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hidaka H., Yamaki T., Naka M., Tanaka T., Hayashi H., Kobayashi R. Calcium-regulated modulator protein interacting agents inhibit smooth muscle calcium-stimulated protein kinase and ATPase. Mol Pharmacol. 1980 Jan;17(1):66–72. [PubMed] [Google Scholar]
  13. 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]
  14. Issinger O. G., Beier H., Speichermann N., Flokerzi V., Hofmann F. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Biochem J. 1980 Jan 1;185(1):89–99. doi: 10.1042/bj1850089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Katoh N., Wise B. C., Wrenn R. W., Kuo J. F. Inhibition by adriamycin of calmodulin-sensitive and phospholipid-sensitive calcium-dependent phosphorylation of endogenous proteins from heart. Biochem J. 1981 Jul 15;198(1):199–205. doi: 10.1042/bj1980199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Katoh N., Wise B. C., Wrenn R. W., Kuo J. F. Inhibition by adriamycin of calmodulin-sensitive and phospholipid-sensitive calcium-dependent phosphorylation of endogenous proteins from heart. Biochem J. 1981 Jul 15;198(1):199–205. doi: 10.1042/bj1980199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Katoh N., Wrenn R. W., Wise B. C., Shoji M., Kuo J. F. Substrate proteins for calmodulin-sensitive and phospholipid-sensitive Ca2+-dependent protein kinases in heart, and inhibition of their phosphorylation by palmitoylcarnitine. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4813–4817. doi: 10.1073/pnas.78.8.4813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kawahara Y., Takai Y., Minakuchi R., Sano K., Nishizuka Y. Possible involvement of Ca2+-activated, phospholipid-dependent protein kinase in platelet activation. J Biochem. 1980 Sep;88(3):913–916. doi: 10.1093/oxfordjournals.jbchem.a133048. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kraft A. S., Anderson W. B., Cooper H. L., Sando J. J. Decrease in cytosolic calcium/phospholipid-dependent protein kinase activity following phorbol ester treatment of EL4 thymoma cells. J Biol Chem. 1982 Nov 25;257(22):13193–13196. [PubMed] [Google Scholar]
  21. Kraft A. S., Anderson W. B. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature. 1983 Feb 17;301(5901):621–623. doi: 10.1038/301621a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Lastick S. M., McConkey E. H. Control of ribosomal protein phosphorylation in HeLa cells. Biochem Biophys Res Commun. 1980 Aug 14;95(3):917–923. doi: 10.1016/0006-291x(80)91560-0. [DOI] [PubMed] [Google Scholar]
  25. Lastick S. M., McConkey E. H. HeLa ribosomal protein S6. Insulin and dibutyryl cyclic AMP affect different phosphopeptides. J Biol Chem. 1981 Jan 25;256(2):583–585. [PubMed] [Google Scholar]
  26. Levin R. M., Weiss B. Specificity of the binding of trifluoperazine to the calcium-dependent activator of phosphodiesterase and to a series of other calcium-binding proteins. Biochim Biophys Acta. 1978 May 3;540(2):197–204. doi: 10.1016/0304-4165(78)90132-0. [DOI] [PubMed] [Google Scholar]
  27. Limas C. J. Phosphorylation of cardiac sarcoplasmic reticulum by a calcium-activated, phospholipid-dependent protein kinase. Biochem Biophys Res Commun. 1980 Oct 16;96(3):1378–1383. doi: 10.1016/0006-291x(80)90103-5. [DOI] [PubMed] [Google Scholar]
  28. Martin-Pérez J., Thomas G. Ordered phosphorylation of 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells. Proc Natl Acad Sci U S A. 1983 Feb;80(4):926–930. doi: 10.1073/pnas.80.4.926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Minakuchi R., Takai Y., Yu B., Nishizuka Y. Widespread occurrence of calcium-activated, phospholipid-dependent protein kinase in mammalian tissues. J Biochem. 1981 May;89(5):1651–1654. doi: 10.1093/oxfordjournals.jbchem.a133362. [DOI] [PubMed] [Google Scholar]
  30. Mori T., Takai Y., Minakuchi R., Yu B., Nishizuka Y. Inhibitory action of chlorpromazine, dibucaine, and other phospholipid-interacting drugs on calcium-activated, phospholipid-dependent protein kinase. J Biol Chem. 1980 Sep 25;255(18):8378–8380. [PubMed] [Google Scholar]
  31. 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]
  32. Nielsen P. J., Manchester K. L., Towbin H., Gordon J., Thomas G. The phosphorylation of ribosomal protein S6 in rat tissues following cycloheximide injection, in diabetes, and after denervation of diaphragm. A simple immunological determination of the extent of S6 phosphorylation on protein blots. J Biol Chem. 1982 Oct 25;257(20):12316–12321. [PubMed] [Google Scholar]
  33. Nielsen P. J., Thomas G., Maller J. L. Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes. Proc Natl Acad Sci U S A. 1982 May;79(9):2937–2941. doi: 10.1073/pnas.79.9.2937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nilsen-Hamilton M., Hamilton R. T., Allen W. R., Potter-Perigo S. Synergistic stimulation of S6 ribosomal protein phosphorylation and DNA synthesis by epidermal growth factor and insulin in quiescent 3T3 cells. Cell. 1982 Nov;31(1):237–242. doi: 10.1016/0092-8674(82)90423-8. [DOI] [PubMed] [Google Scholar]
  35. Ringer D. P., Kizer D. E., King R. L., Jr Differences in the distribution of phosphate content in the ribosomal subunit proteins of free and membrane-bound ribosomes from normal and regenerating rat liver. Biochim Biophys Acta. 1981 Nov 27;656(1):62–68. doi: 10.1016/0005-2787(81)90027-7. [DOI] [PubMed] [Google Scholar]
  36. Schatzman R. C., Wise B. C., Kuo J. F. Phospholipid-sensitive calcium-dependent protein kinase: inhibition by antipsychotic drugs. Biochem Biophys Res Commun. 1981 Feb 12;98(3):669–676. doi: 10.1016/0006-291x(81)91166-9. [DOI] [PubMed] [Google Scholar]
  37. Smith C. J., Wejksnora P. J., Warner J. R., Rubin C. S., Rosen O. M. Insulin-stimulated protein phosphorylation in 3T3-L1 preadipocytes. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2725–2729. doi: 10.1073/pnas.76.6.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Speaker M. G., Sturgill T. W., Orlow S. J., Chia G. H., Pifko-Hirst S., Rosen O. M. The effects of trifluoperazine on the macrophage-like cell line, J774. Ann N Y Acad Sci. 1980;356:162–178. doi: 10.1111/j.1749-6632.1980.tb29609.x. [DOI] [PubMed] [Google Scholar]
  39. Spector T. Refinement of the coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0.5 to 50 microgram of protein. Anal Biochem. 1978 May;86(1):142–146. doi: 10.1016/0003-2697(78)90327-5. [DOI] [PubMed] [Google Scholar]
  40. Takai Y., Kaibuchi K., Sano K., Nishizuka Y. Counteraction of calcium-activated, phospholipid-dependent protein kinase activation by adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in platelets. J Biochem. 1982 Jan;91(1):403–406. doi: 10.1093/oxfordjournals.jbchem.a133700. [DOI] [PubMed] [Google Scholar]
  41. Takai Y., Kishimoto A., Inoue M., Nishizuka Y. Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J Biol Chem. 1977 Nov 10;252(21):7603–7609. [PubMed] [Google Scholar]
  42. Tanaka T., Ohmura T., Yamakado T., Hidaka H. Two types of calcium-dependent protein phosphorylations modulated by calmodulin antagonists. Naphthalenesulfonamide derivatives. Mol Pharmacol. 1982 Sep;22(2):408–412. [PubMed] [Google Scholar]
  43. Thomas G., Martin-Pérez J., Siegmann M., Otto A. M. The effect of serum, EGF, PGF2 alpha and insulin on S6 phosphorylation and the initiation of protein and DNA synthesis. Cell. 1982 Aug;30(1):235–242. doi: 10.1016/0092-8674(82)90029-0. [DOI] [PubMed] [Google Scholar]
  44. Thomas G., Siegmann M., Gordon J. Multiple phosphorylation of ribosomal protein S6 during transition of quiescent 3T3 cells into early G1, and cellular compartmentalization of the phosphate donor. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3952–3956. doi: 10.1073/pnas.76.8.3952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tran P. L., Ter-Minassian-Saraga L., Madelmont G., Castagna M. Tumor promoter 12-O-tetradecanoylphorbol 13-acetate alters state, fluidity and hydration of 1,2-diacyl-sn-glycero-3-phosphocholine bilayers. Biochim Biophys Acta. 1983 Jan 5;727(1):31–38. doi: 10.1016/0005-2736(83)90365-6. [DOI] [PubMed] [Google Scholar]
  46. Wettenhall R. E., Cohen P. Isolation and characterisation of cyclic AMP-dependent phosphorylation sites from rat liver ribosomal protein S6. FEBS Lett. 1982 Apr 19;140(2):263–269. doi: 10.1016/0014-5793(82)80910-1. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Witt J. J., Roskoski R., Jr Rapid protein kinase assay using phosphocellulose-paper absorption. Anal Biochem. 1975 May 26;66(1):253–258. doi: 10.1016/0003-2697(75)90743-5. [DOI] [PubMed] [Google Scholar]
  49. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  50. Wrenn R. W., Katoh N., Kuo J. F. Stimulation by phospholipid of calcium-dependent phosphorylation of endogenous proteins from mammalian tissues. Biochim Biophys Acta. 1981 Aug 17;676(2):266–269. doi: 10.1016/0304-4165(81)90195-1. [DOI] [PubMed] [Google Scholar]
  51. Wrenn R. W., Katoh N., Wise B. C., Kuo J. F. Stimulation by phosphatidylserine and calmodulin of calcium-dependent phosphorylation of endogenous proteins from cerebral cortex. J Biol Chem. 1980 Dec 25;255(24):12042–12046. [PubMed] [Google Scholar]
  52. Zabrenetzky V. S., Bruckwick E., Lovenberg W. Calcium stimulation of protein kinase C in the absence of added phospholipids. Biochem Biophys Res Commun. 1981 Sep 16;102(1):135–141. doi: 10.1016/0006-291x(81)91499-6. [DOI] [PubMed] [Google Scholar]
  53. Zasloff M., Ochoa S. Purification of eukaryotic initiation factor 1 (EIF1) from Artemia salina embryos. Methods Enzymol. 1974;30:197–206. doi: 10.1016/0076-6879(74)30022-5. [DOI] [PubMed] [Google Scholar]
  54. del Grande R. W., Traugh J. A. Phosphorylation of 40-S ribosomal subunits by cAMP-dependent, cGMP-dependent and protease-activated protein kinases. Eur J Biochem. 1982 Apr 1;123(2):421–428. doi: 10.1111/j.1432-1033.1982.tb19785.x. [DOI] [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