Advertisement

Protein Kinase C Regulation by Diacylglycerols: Structure-Function Relationships and Mechanism

  • Robert M. Bell
  • Barry R. Ganong
  • Carson R. Loomis
  • Yusuf A. Hannun
Part of the NATO ASI Series book series (NSSA, volume 116)

Summary

A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids, diacylglycerols, and diacylglycerol analogues. Triton X-100 micelles containing 8 mol% phosphatidylserine (PS) and 2.5 mol% sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1. Protein kinase C activity was almost totally dependent on diC18:1 in the mixed micellar assay. At 8 mol% PS, diC18:1 stimulated maximally at 1 mol%. At 2.5 mol% diC18:1, PS did not activate until 3 mol% and then did so cooperatively with maximal stimulation occurring at 6–8 mol%. Molecular sieve chromatography demonstrated that monomeric protein kinase C interacts with Triton X-100 micelles in a PS and Ca2+ dependent manner. Interpretations follow: 1) a single molecule of diC18:1 activates monomeric protein kinase C; 2) a phospholipid bilayer is not required; 3) four or more molecules of PS are required. In addition, several diacylglycerol analogues were synthesized to determine the exact structural features required for activation. The data suggest that both carbonyls of the oxygen esters and the 3-hydroxyl are required. A model of protein kinase C activation by PS and diacylglycerol was formulated.

Keywords

Protein Kinase Mixed Micelle Epidermal Growth Factor Binding Stimulate Protein Kinase Molecular Sieve Chromatography 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Y. Nishizuka, The role of protein kinase C in cell surface signal transduction and tumour promotion, Nature (London) 308: 693 (1984).CrossRefGoogle Scholar
  2. 2.
    Y. Nishizuka, Turnover of inositol phospholipids and signal transduction, Science 225: 1365 (1984).PubMedCrossRefGoogle Scholar
  3. 3.
    M. Castagna, Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa, and Y. Nishizuka, Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters, J. Biol. Chem. 257: 7847 (1982).PubMedGoogle Scholar
  4. 4.
    J.E. Niedel, L.J. Kuhn, and G.R. Vandenbark, Phorbol diester receptor copurifies with protein kinase C, Proc. Natl. Acad. Sci. U.S.A. 80: 36 (1983)PubMedCrossRefGoogle Scholar
  5. 5.
    K.L. Leach, M.L. James, and P.M. Blumberg, Characterization of a specific phorbol ester aporeceptor in mouse brain cytosol, Proc. Natl. Acad. Sci. U.S.A. 80: 4208 (1983).PubMedCrossRefGoogle Scholar
  6. 6.
    Y.A. Hannun, C.R. Loomis, and R.M. Bell, Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine, J. Biol. Chem. 260: 10039 (1985).PubMedGoogle Scholar
  7. 7.
    B.R. Ganong, C.R. Loomis, Y.A. Hannun, and R.M. Bell, Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols, Proc. Natl. Acad. Sci. U.S.A. (in press).Google Scholar
  8. 8.
    E.G. Lapetina, B. Reep, B.R. Ganong, and R.M. Bell, Exogenous sn-1,2diacylglycerols containing saturated fatty acids function as bioregulators of protein kinase C in human platelets, J. Biol. Chem. 260: 1358 (1985).PubMedGoogle Scholar
  9. 9.
    R.J. Davis, B.R. Ganong, R.M. Bell, and M.P. Czech, Structural requirements for diacylglycerols to mimic tumor-promoting phorbol diester action on the epidermal growth factor receptor, J. Biol. Chem. 260: 5315 (1985).PubMedGoogle Scholar
  10. 10.
    P.M. Conn, B.R. Ganong, J. Ebeling, D. Staley, J.E. Niedel, and R.M. Bell, Diacylglycerols release LH: Structure-activity relations reveal a role for protein kinase C, Biochem. Biophys. Res. Comm. 126: 532 (1985).PubMedCrossRefGoogle Scholar
  11. 11.
    J.G. Ebeling, G.R. Vandenbark, L.J. Kuhn, B.R. Ganong, R.M. Bell, and J.E. Niedel, Diacylglycerols mimic phorbol diester induction of leukemic cell differentiation, Proc. Natl. Acad. Sci. U.S.A. 82: 815 (1985).PubMedCrossRefGoogle Scholar
  12. 12.
    A.M. Jetten, B.R. Ganong, G.R. Vandenbark, J.E. Shirley, and R.M. Bell, Role of protein kinase C in diacylglycerol-mediated induction of ornithine decarboxylase and reduction of epidermal growth factor binding, Proc. Natl. Acad. Sci. U.S.A. 82: 1941 (1985).PubMedCrossRefGoogle Scholar
  13. 13.
    T. Mori, Y. Takai, B. Yu, J. Takahashi, Y. Nishizuka, and T. Fujikura, Specificity of the fatty acyl moieties of diacylglycerol for the activation of calcium-activated, phospholipid-dependent protein kinase, J. Biochem. (Tokyo) 91: 427 (1982).Google Scholar
  14. 14.
    U. Kikkawa, Y. Takai, Y. Tanaka, R. Miyake, and Y. Nishizuka, Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters, J. Biol. Chem. 258: 11442 (1983).PubMedGoogle Scholar
  15. 15.
    R.R. Rando and N. Young, The stereospecific activation of protein kinase C, Biochem. Biophys. Res. Comm. 122: 818 (1984).PubMedCrossRefGoogle Scholar
  16. 16.
    A. Kishimoto, N. Kajikawa, M. Shiota, and Y. Nishizuka, Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease, J. Biol. Chem. 258: 1156 (1983).PubMedGoogle Scholar
  17. 17.
    K.B. Delclos, E. Yeh, and P.M. Blumberg, 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. 80: 3054 (1983).PubMedCrossRefGoogle Scholar
  18. 18.
    R.H. Kretsinger and D.J. Nelson, Calcium in biological systems, Coord. Chem. Rev. 18: 29 (1976).CrossRefGoogle Scholar
  19. 19.
    Y. Takai, A. Kishimoto, Y. Iwasa, Y. Kawahara, T. Mori, and Y. Nishizuka, Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids, J. Biol. Chem. 254: 3692 (1979).PubMedGoogle Scholar

Copyright information

© Plenum Press, New York 1986

Authors and Affiliations

  • Robert M. Bell
    • 1
  • Barry R. Ganong
    • 1
  • Carson R. Loomis
    • 1
  • Yusuf A. Hannun
    • 1
  1. 1.Department of BiochemistryDuke University Medical CenterDurhamUSA

Personalised recommendations