Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1993 Mar 15;290(Pt 3):827–832. doi: 10.1042/bj2900827

Circular dichroism analysis of ligand-induced conformational changes in protein kinase C. Mechanism of translocation of the enzyme from the cytosol to the membranes and its implications.

L Boscá 1, F Morán 1
PMCID: PMC1132356  PMID: 8457212

Abstract

The structural changes following the binding to protein kinase C (PKC) of activators that promote its translocation to lipid environments were studied by far-u.v. c.d. and intrinsic fluorescence measurements of the protein. In the absence of activators, PKC contained 40% alpha-helix, with an average size of 13 amino acids per alpha-helix segment, and 12% beta-structure as deduced from c.d. spectral analysis while fitting a set of model proteins of known structure. Ligands that promote translocation and activation of the enzyme, such as Ca2+ ions and phorbol esters, produced drastic changes in the c.d. spectra which may be interpreted as a reduction in the average number of consecutive amino acids in the alpha-helix. Most of the total alpha-helix structure was conserved and an increase in beta-structure was produced by active phorbol esters. These activators differentially affected the fluorescence of PKC: phorbol esters shifted the emission maximum to the red, whereas Ca2+ produced a marked decrease in the intensity of the fluorescence emission, suggesting in both cases that tryptophan residues were exposed to increased polar environments after binding of the ligands.

Full text

PDF
827

Images in this article

829Figure 1
on p.829

Selected References

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

  1. Bazzi M. D., Nelsestuen G. L. Extensive segregation of acidic phospholipids in membranes induced by protein kinase C and related proteins. Biochemistry. 1991 Aug 13;30(32):7961–7969. doi: 10.1021/bi00246a013. [DOI] [PubMed] [Google Scholar]
  2. Bell R. M., Burns D. J. Lipid activation of protein kinase C. J Biol Chem. 1991 Mar 15;266(8):4661–4664. [PubMed] [Google Scholar]
  3. Bell R. M. Protein kinase C activation by diacylglycerol second messengers. Cell. 1986 Jun 6;45(5):631–632. doi: 10.1016/0092-8674(86)90774-9. [DOI] [PubMed] [Google Scholar]
  4. Berridge M. J. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem. 1987;56:159–193. doi: 10.1146/annurev.bi.56.070187.001111. [DOI] [PubMed] [Google Scholar]
  5. Bolotina I. A., Chekhov V. O., Lugauskas V. Iu, Ptitsyn O. B. Opredelenie vtorichnoi struktury belkov iz spektrov krugovog dikhroizma. II. Uchet vklada beta-izgibov. Mol Biol (Mosk) 1980 Jul-Aug;14(4):902–909. [PubMed] [Google Scholar]
  6. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chang C. T., Wu C. S., Yang J. T. Circular dichroic analysis of protein conformation: inclusion of the beta-turns. Anal Biochem. 1978 Nov;91(1):13–31. doi: 10.1016/0003-2697(78)90812-6. [DOI] [PubMed] [Google Scholar]
  9. Chen Y. H., Yang J. T., Chau K. H. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974 Jul 30;13(16):3350–3359. doi: 10.1021/bi00713a027. [DOI] [PubMed] [Google Scholar]
  10. Clark J. D., Lin L. L., Kriz R. W., Ramesha C. S., Sultzman L. A., Lin A. Y., Milona N., Knopf J. L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell. 1991 Jun 14;65(6):1043–1051. doi: 10.1016/0092-8674(91)90556-e. [DOI] [PubMed] [Google Scholar]
  11. Gibrat J. F., Garnier J., Robson B. Further developments of protein secondary structure prediction using information theory. New parameters and consideration of residue pairs. J Mol Biol. 1987 Dec 5;198(3):425–443. doi: 10.1016/0022-2836(87)90292-0. [DOI] [PubMed] [Google Scholar]
  12. Gopalakrishna R., Barsky S. H., Thomas T. P., Anderson W. B. Factors influencing chelator-stable, detergent-extractable, phorbol diester-induced membrane association of protein kinase C. Differences between Ca2+-induced and phorbol ester-stabilized membrane bindings of protein kinase C. J Biol Chem. 1986 Dec 15;261(35):16438–16445. [PubMed] [Google Scholar]
  13. Hannun Y. A., Loomis C. R., Bell R. M. Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine. J Biol Chem. 1985 Aug 25;260(18):10039–10043. [PubMed] [Google Scholar]
  14. Hannun Y. A., Loomis C. R., Bell R. M. Protein kinase C activation in mixed micelles. Mechanistic implications of phospholipid, diacylglycerol, and calcium interdependencies. J Biol Chem. 1986 Jun 5;261(16):7184–7190. [PubMed] [Google Scholar]
  15. Huang K. P., Chan K. F., Singh T. J., Nakabayashi H., Huang F. L. Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase. J Biol Chem. 1986 Sep 15;261(26):12134–12140. [PubMed] [Google Scholar]
  16. Huang K. P. The mechanism of protein kinase C activation. Trends Neurosci. 1989 Nov;12(11):425–432. doi: 10.1016/0166-2236(89)90091-x. [DOI] [PubMed] [Google Scholar]
  17. Johnson W. C., Jr Secondary structure of proteins through circular dichroism spectroscopy. Annu Rev Biophys Biophys Chem. 1988;17:145–166. doi: 10.1146/annurev.bb.17.060188.001045. [DOI] [PubMed] [Google Scholar]
  18. Kabsch W., Sander C. How good are predictions of protein secondary structure? FEBS Lett. 1983 May 8;155(2):179–182. doi: 10.1016/0014-5793(82)80597-8. [DOI] [PubMed] [Google Scholar]
  19. Kikkawa U., Kishimoto A., Nishizuka Y. The protein kinase C family: heterogeneity and its implications. Annu Rev Biochem. 1989;58:31–44. doi: 10.1146/annurev.bi.58.070189.000335. [DOI] [PubMed] [Google Scholar]
  20. Kikkawa U., Nishizuka Y. The role of protein kinase C in transmembrane signalling. Annu Rev Cell Biol. 1986;2:149–178. doi: 10.1146/annurev.cb.02.110186.001053. [DOI] [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. 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]
  23. Lee M. H., Bell R. M. The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium- and phosphatidylserine-dependent phorbol diester binding activity. J Biol Chem. 1986 Nov 15;261(32):14867–14870. [PubMed] [Google Scholar]
  24. McFadden P. N., Mandpe A., Koshland D. E., Jr Calcium- and lipid-independent protein kinase C autophosphorylation. Activation by low pH. J Biol Chem. 1989 Aug 5;264(22):12765–12771. [PubMed] [Google Scholar]
  25. Menéndez-Arias L., Gómez-Gutiérrez J., García-Ferrández M., García-Tejedor A., Morán F. A BASIC microcomputer program to calculate the secondary structure of proteins from their circular dichroism spectrum. Comput Appl Biosci. 1988 Nov;4(4):479–482. doi: 10.1093/bioinformatics/4.4.479. [DOI] [PubMed] [Google Scholar]
  26. Murzin A. G., Finkelstein A. V. General architecture of the alpha-helical globule. J Mol Biol. 1988 Dec 5;204(3):749–769. doi: 10.1016/0022-2836(88)90366-x. [DOI] [PubMed] [Google Scholar]
  27. Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
  28. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  29. Pace C. N. Conformational stability of globular proteins. Trends Biochem Sci. 1990 Jan;15(1):14–17. doi: 10.1016/0968-0004(90)90124-t. [DOI] [PubMed] [Google Scholar]
  30. Parker P. J., Coussens L., Totty N., Rhee L., Young S., Chen E., Stabel S., Waterfield M. D., Ullrich A. The complete primary structure of protein kinase C--the major phorbol ester receptor. Science. 1986 Aug 22;233(4766):853–859. doi: 10.1126/science.3755547. [DOI] [PubMed] [Google Scholar]
  31. Provencher S. W., Glöckner J. Estimation of globular protein secondary structure from circular dichroism. Biochemistry. 1981 Jan 6;20(1):33–37. doi: 10.1021/bi00504a006. [DOI] [PubMed] [Google Scholar]
  32. Rando R. R., Young N. The stereospecific activation of protein kinase C. Biochem Biophys Res Commun. 1984 Jul 31;122(2):818–823. doi: 10.1016/s0006-291x(84)80107-2. [DOI] [PubMed] [Google Scholar]
  33. Rooman M. J., Wodak S. J. Identification of predictive sequence motifs limited by protein structure data base size. Nature. 1988 Sep 1;335(6185):45–49. doi: 10.1038/335045a0. [DOI] [PubMed] [Google Scholar]
  34. Shah J., Shipley G. G. Circular dichroic studies of protein kinase C and its interactions with calcium and lipid vesicles. Biochim Biophys Acta. 1992 Feb 13;1119(1):19–26. doi: 10.1016/0167-4838(92)90228-6. [DOI] [PubMed] [Google Scholar]
  35. Toumadje A., Alcorn S. W., Johnson W. C., Jr Extending CD spectra of proteins to 168 nm improves the analysis for secondary structures. Anal Biochem. 1992 Feb 1;200(2):321–331. doi: 10.1016/0003-2697(92)90473-k. [DOI] [PubMed] [Google Scholar]
  36. Woodgett J. R., Hunter T. Isolation and characterization of two distinct forms of protein kinase C. J Biol Chem. 1987 Apr 5;262(10):4836–4843. [PubMed] [Google Scholar]
  37. Wooten M. W., Vandenplas M., Nel A. E. Rapid purification of protein kinase C from rat brain. A novel method employing protamine-agarose affinity column chromatography. Eur J Biochem. 1987 Apr 15;164(2):461–467. doi: 10.1111/j.1432-1033.1987.tb11079.x. [DOI] [PubMed] [Google Scholar]
  38. Yang J. T., Wu C. S., Martinez H. M. Calculation of protein conformation from circular dichroism. Methods Enzymol. 1986;130:208–269. doi: 10.1016/0076-6879(86)30013-2. [DOI] [PubMed] [Google Scholar]

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

RESOURCES