Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Aug 1;325(Pt 3):787–791. doi: 10.1042/bj3250787

Modulation of protein kinase C by endogenous sphingosine: inhibition of phorbol dibutyrate binding in Niemann-Pick C fibroblasts.

C Rodriguez-Lafrasse 1, R Rousson 1, S Valla 1, P Antignac 1, P Louisot 1, M T Vanier 1
PMCID: PMC1218624  PMID: 9271101

Abstract

The abnormal and variable increase in levels of free sphingoid bases recently described in fibroblasts from Niemann-Pick C patients allowed us to investigate the modulation of protein kinase C in vivo by endogenous sphingosine. The specific binding of [20-3H]phorbol 12, 13-dibutyrate to the regulatory domain of membrane-bound protein kinase C was significantly decreased in fibroblasts from patients compared with controls. A pronounced difference between the two groups (P<0.0001) was demonstrated in low-density lipoprotein-supplemented medium, i.e. under conditions known to disclose abnormal mobilization of unesterified cholesterol in Niemann-Pick C fibroblasts. Furthermore the degree of impairment of [3H]phorbol 12,13-dibutyrate binding was highly correlated (r=0.95) with the sphingosine levels measured in fibroblasts from those patients. Scatchard analysis of the binding data indicated that Niemann-Pick C and control fibroblasts contained almost the same number of binding sites per cell. A 8-34-fold increase in Kd was measured in Niemann-Pick C fibroblasts with at least a 5-fold increase in sphingosine levels. Removal, by cell fractionation, of membrane-bound protein kinase C from the bulk of sphingosine induced a normalization of Kd values. The overall results suggest that protein kinase C inhibition is directly related to sphingosine accumulation.

Full Text

The Full Text of this article is available as a PDF (313.3 KB).

Selected References

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

  1. Boneh A., Bach G. Inhibition of protein kinase C activity in mucolipidosis type-4: a model for a new pathogenetic mechanism in inborn errors of metabolism. Biochim Biophys Acta. 1993 Aug 4;1182(1):64–68. doi: 10.1016/0925-4439(93)90154-s. [DOI] [PubMed] [Google Scholar]
  2. Boneh A. Possible role for protein kinase C in the pathogenesis of inborn errors of metabolism. J Cell Biochem. 1995 Sep;59(1):27–32. doi: 10.1002/jcb.240590104. [DOI] [PubMed] [Google Scholar]
  3. Boneh A. Protein kinase C activity, phosphate uptake and endogenous substrate phosphorylation are altered in Zellweger syndrome. J Inherit Metab Dis. 1996;19(5):661–666. doi: 10.1007/BF01799843. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Goldin E., Roff C. F., Miller S. P., Rodriguez-Lafrasse C., Vanier M. T., Brady R. O., Pentchev P. G. Type C Niemann-Pick disease: a murine model of the lysosomal cholesterol lipidosis accumulates sphingosine and sphinganine in liver. Biochim Biophys Acta. 1992 Aug 19;1127(3):303–311. doi: 10.1016/0005-2760(92)90236-o. [DOI] [PubMed] [Google Scholar]
  6. Goldstein J. L., Basu S. K., Brown M. S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260. doi: 10.1016/0076-6879(83)98152-1. [DOI] [PubMed] [Google Scholar]
  7. Goodwin B. J., Weinberg J. B. Receptor-mediated modulation of human monocyte, neutrophil, lymphocyte, and platelet function by phorbol diesters. J Clin Invest. 1982 Oct;70(4):699–706. doi: 10.1172/JCI110665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hannun Y. A., Bell R. M. Lysosphingolipids inhibit protein kinase C: implications for the sphingolipidoses. Science. 1987 Feb 6;235(4789):670–674. doi: 10.1126/science.3101176. [DOI] [PubMed] [Google Scholar]
  9. Hannun Y. A., Bell R. M. Regulation of protein kinase C by sphingosine and lysosphingolipids. Clin Chim Acta. 1989 Dec 15;185(3):333–345. doi: 10.1016/0009-8981(89)90224-6. [DOI] [PubMed] [Google Scholar]
  10. Hannun Y. A., Loomis C. R., Merrill A. H., Jr, Bell R. M. Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J Biol Chem. 1986 Sep 25;261(27):12604–12609. [PubMed] [Google Scholar]
  11. Hartree E. F. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972 Aug;48(2):422–427. doi: 10.1016/0003-2697(72)90094-2. [DOI] [PubMed] [Google Scholar]
  12. Kobayashi T., Mitsuo K., Goto I. Free sphingoid bases in normal murine tissues. Eur J Biochem. 1988 Mar 15;172(3):747–752. doi: 10.1111/j.1432-1033.1988.tb13952.x. [DOI] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Mendez A. J., Oram J. F., Bierman E. L. Protein kinase C as a mediator of high density lipoprotein receptor-dependent efflux of intracellular cholesterol. J Biol Chem. 1991 Jun 5;266(16):10104–10111. [PubMed] [Google Scholar]
  15. Merrill A. H., Jr, Sereni A. M., Stevens V. L., Hannun Y. A., Bell R. M., Kinkade J. M., Jr Inhibition of phorbol ester-dependent differentiation of human promyelocytic leukemic (HL-60) cells by sphinganine and other long-chain bases. J Biol Chem. 1986 Sep 25;261(27):12610–12615. [PubMed] [Google Scholar]
  16. Merrill A. H., Jr, Wang E., Mullins R. E., Jamison W. C., Nimkar S., Liotta D. C. Quantitation of free sphingosine in liver by high-performance liquid chromatography. Anal Biochem. 1988 Jun;171(2):373–381. doi: 10.1016/0003-2697(88)90500-3. [DOI] [PubMed] [Google Scholar]
  17. Möllers C., Drobnik W., Resink T., Schmitz G. High-density lipoprotein and low-density lipoprotein-mediated signal transduction in cultured human skin fibroblasts. Cell Signal. 1995 Sep;7(7):695–707. doi: 10.1016/0898-6568(95)00041-m. [DOI] [PubMed] [Google Scholar]
  18. Perng M. D., Cheng T. J., Chen C. M., Lai Y. K. Induction of aggregation and augmentation of protein kinase-mediated phosphorylation of purified vimentin intermediate filaments by withangulatin A. Mol Pharmacol. 1994 Oct;46(4):612–617. [PubMed] [Google Scholar]
  19. Rodriguez-Lafrasse C., Rousson R., Pentchev P. G., Louisot P., Vanier M. T. Free sphingoid bases in tissues from patients with type C Niemann-Pick disease and other lysosomal storage disorders. Biochim Biophys Acta. 1994 May 25;1226(2):138–144. doi: 10.1016/0925-4439(94)90021-3. [DOI] [PubMed] [Google Scholar]
  20. Roff C. F., Goldin E., Comly M. E., Cooney A., Brown A., Vanier M. T., Miller S. P., Brady R. O., Pentchev P. G. Type C Niemann-Pick disease: use of hydrophobic amines to study defective cholesterol transport. Dev Neurosci. 1991;13(4-5):315–319. doi: 10.1159/000112179. [DOI] [PubMed] [Google Scholar]
  21. Scott-Burden T., Resink T. J., Hahn A. W., Baur U., Box R. J., Bühler F. R. Induction of growth-related metabolism in human vascular smooth muscle cells by low density lipoprotein. J Biol Chem. 1989 Jul 25;264(21):12582–12589. [PubMed] [Google Scholar]
  22. 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]
  23. Sokol J., Blanchette-Mackie J., Kruth H. S., Dwyer N. K., Amende L. M., Butler J. D., Robinson E., Patel S., Brady R. O., Comly M. E. Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol. J Biol Chem. 1988 Mar 5;263(7):3411–3417. [PubMed] [Google Scholar]
  24. Spiegel S., Olivera A., Carlson R. O. The role of sphingosine in cell growth regulation and transmembrane signaling. Adv Lipid Res. 1993;25:105–129. [PubMed] [Google Scholar]
  25. Steinschneider A., McLean M. P., Billheimer J. T., Azhar S., Gibori G. Protein kinase C catalyzed phosphorylation of sterol carrier protein 2. Endocrinology. 1989 Jul;125(1):569–571. doi: 10.1210/endo-125-1-569. [DOI] [PubMed] [Google Scholar]
  26. Vanier M. T., Rousson R., Garcia I., Bailloud G., Juge M. C., Revol A., Louisot P. Biochemical studies in Niemann-Pick disease. III. In vitro and in vivo assays of sphingomyelin degradation in cultured skin fibroblasts and amniotic fluid cells for the diagnosis of the various forms of the disease. Clin Genet. 1985 Jan;27(1):20–32. doi: 10.1111/j.1399-0004.1985.tb00180.x. [DOI] [PubMed] [Google Scholar]
  27. Wang E., Norred W. P., Bacon C. W., Riley R. T., Merrill A. H., Jr Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. J Biol Chem. 1991 Aug 5;266(22):14486–14490. [PubMed] [Google Scholar]
  28. Yamamoto T., Tokoro T., Eto Y. The attenuated elevation of cytoplasmic calcium concentration following the uptake of low density lipoprotein in type C Niemann-Pick fibroblasts. Biochem Biophys Res Commun. 1994 Jan 28;198(2):438–444. doi: 10.1006/bbrc.1994.1064. [DOI] [PubMed] [Google Scholar]

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

RESOURCES