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. 1991 Nov 15;88(22):10009–10013. doi: 10.1073/pnas.88.22.10009

Characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor alpha.

S Mathias 1, K A Dressler 1, R N Kolesnick 1
PMCID: PMC52856  PMID: 1946418

Abstract

Recent investigations have identified a signal-transduction system involving sphingomyelin and derivatives. In this paradigm, sphingomyelin hydrolysis by a sphingomyelinase generates ceramide, which may be converted to the protein kinase C inhibitor sphingosine or to ceramide 1-phosphate. Ceramide may have second-messenger function because it induces epidermal growth factor receptor phosphorylation, presumably on Thr-669 in A-431 cells. The present studies describe a kinase that may mediate ceramide action. With a 19-amino acid epidermal growth factor receptor peptide containing Thr-669, a membrane-bound activity that phosphorylated the peptide was detected in A-431 cells. Activity was linearly related to ATP (0.3-300 microM) and peptide concentration (0.02-1 mg/ml), possessed a physiologic pH optimum (pH 7.0-7.4), and was Mg(2+)-dependent. Other cations--Ca2+, Mn2+, and Zn(2+)--were ineffective. Natural and synthetic ceramide induced time- and concentration-dependent enhancement of kinase activity. Ceramide (0.5 microM) increased kinase activity 2-fold by 30 s, and activity remained elevated for at least 15 min. As little as 0.001 microM ceramide was effective, and 1 microM ceramide induced maximal phosphorylation. Sphingosine was similarly effective. Because tumor necrosis factor (TNF) alpha rapidly induces sphingomyelin hydrolysis to ceramide during monocytic differentiation of HL-60 cells, its effects on kinase activity were assessed. Kinase activity was increased 1.5-fold at 5 min and 2-fold at 2 hr in membranes derived from TNF-stimulated cells. The effective concentration range was 3 pM-30 nM TNF. Exogenous ceramide induced a similar effect. In sum, these studies demonstrate the existence of an unusual Mg(2+)-dependent ceramide-activated protein kinase that may mediate some aspects of TNF-alpha function.

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Selected References

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

  1. Aitken A., Holmes C. F., Campbell D. G., Resink T. J., Cohen P., Leung C. T., Williams D. H. Amino acid sequence at the site on protein phosphatase inhibitor-2, phosphorylated by glycogen synthase kinase-3. Biochim Biophys Acta. 1984 Nov 9;790(3):288–291. doi: 10.1016/0167-4838(84)90034-7. [DOI] [PubMed] [Google Scholar]
  2. Bajjalieh S. M., Martin T. F., Floor E. Synaptic vesicle ceramide kinase. A calcium-stimulated lipid kinase that co-purifies with brain synaptic vesicles. J Biol Chem. 1989 Aug 25;264(24):14354–14360. [PubMed] [Google Scholar]
  3. Bowen S., Stanley K., Selva E., Davis R. J. Constitutive phosphorylation of the epidermal growth factor receptor blocks mitogenic signal transduction. J Biol Chem. 1991 Jan 15;266(2):1162–1169. [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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Breitman T. R., Selonick S. E., Collins S. J. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sci U S A. 1980 May;77(5):2936–2940. doi: 10.1073/pnas.77.5.2936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brenner D. A., O'Hara M., Angel P., Chojkier M., Karin M. Prolonged activation of jun and collagenase genes by tumour necrosis factor-alpha. Nature. 1989 Feb 16;337(6208):661–663. doi: 10.1038/337661a0. [DOI] [PubMed] [Google Scholar]
  7. Carpenter G., Cohen S. Epidermal growth factor. J Biol Chem. 1990 May 15;265(14):7709–7712. [PubMed] [Google Scholar]
  8. Chaplinski T. J., Niedel J. E. Cyclic nucleotide-induced maturation of human promyelocytic leukemia cells. J Clin Invest. 1982 Nov;70(5):953–964. doi: 10.1172/JCI110707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  10. Countaway J. L., Northwood I. C., Davis R. J. Mechanism of phosphorylation of the epidermal growth factor receptor at threonine 669. J Biol Chem. 1989 Jun 25;264(18):10828–10835. [PubMed] [Google Scholar]
  11. Davis R. J., Gironès N., Faucher M. Two alternative mechanisms control the interconversion of functional states of the epidermal growth factor receptor. J Biol Chem. 1988 Apr 15;263(11):5373–5379. [PubMed] [Google Scholar]
  12. Donato N. J., Gallick G. E., Steck P. A., Rosenblum M. G. Tumor necrosis factor modulates epidermal growth factor receptor phosphorylation and kinase activity in human tumor cells. Correlation with cytotoxicity. J Biol Chem. 1989 Dec 5;264(34):20474–20481. [PubMed] [Google Scholar]
  13. Dressler K. A., Kolesnick R. N. Ceramide 1-phosphate, a novel phospholipid in human leukemia (HL-60) cells. Synthesis via ceramide from sphingomyelin. J Biol Chem. 1990 Sep 5;265(25):14917–14921. [PubMed] [Google Scholar]
  14. Edelman A. M., Blumenthal D. K., Krebs E. G. Protein serine/threonine kinases. Annu Rev Biochem. 1987;56:567–613. doi: 10.1146/annurev.bi.56.070187.003031. [DOI] [PubMed] [Google Scholar]
  15. Faucher M., Gironès N., Hannun Y. A., Bell R. M., Davis R. J. Regulation of the epidermal growth factor receptor phosphorylation state by sphingosine in A431 human epidermoid carcinoma cells. J Biol Chem. 1988 Apr 15;263(11):5319–5327. [PubMed] [Google Scholar]
  16. Glazer A. N. Light guides. Directional energy transfer in a photosynthetic antenna. J Biol Chem. 1989 Jan 5;264(1):1–4. [PubMed] [Google Scholar]
  17. Goldkorn T., Dressler K. A., Muindi J., Radin N. S., Mendelsohn J., Menaldino D., Liotta D., Kolesnick R. N. Ceramide stimulates epidermal growth factor receptor phosphorylation in A431 human epidermoid carcinoma cells. Evidence that ceramide may mediate sphingosine action. J Biol Chem. 1991 Aug 25;266(24):16092–16097. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Heisermann G. J., Gill G. N. Epidermal growth factor receptor threonine and serine residues phosphorylated in vivo. J Biol Chem. 1988 Sep 15;263(26):13152–13158. [PubMed] [Google Scholar]
  21. Heisermann G. J., Wiley H. S., Walsh B. J., Ingraham H. A., Fiol C. J., Gill G. N. Mutational removal of the Thr669 and Ser671 phosphorylation sites alters substrate specificity and ligand-induced internalization of the epidermal growth factor receptor. J Biol Chem. 1990 Aug 5;265(22):12820–12827. [PubMed] [Google Scholar]
  22. Kim M. Y., Linardic C., Obeid L., Hannun Y. Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell differentiation. J Biol Chem. 1991 Jan 5;266(1):484–489. [PubMed] [Google Scholar]
  23. Kolesnick R. N. 1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells. J Biol Chem. 1987 Dec 15;262(35):16759–16762. [PubMed] [Google Scholar]
  24. Kolesnick R. N., Clegg S. 1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase. J Biol Chem. 1988 May 15;263(14):6534–6537. [PubMed] [Google Scholar]
  25. Kolesnick R. N., Hemer M. R. Characterization of a ceramide kinase activity from human leukemia (HL-60) cells. Separation from diacylglycerol kinase activity. J Biol Chem. 1990 Nov 5;265(31):18803–18808. [PubMed] [Google Scholar]
  26. Kolesnick R. N. Sphingomyelinase action inhibits phorbol ester-induced differentiation of human promyelocytic leukemic (HL-60) cells. J Biol Chem. 1989 May 5;264(13):7617–7623. [PubMed] [Google Scholar]
  27. Marino M. W., Feld L. J., Jaffe E. A., Pfeffer L. M., Han H. M., Donner D. B. Phosphorylation of the proto-oncogene product eukaryotic initiation factor 4E is a common cellular response to tumor necrosis factor. J Biol Chem. 1991 Feb 15;266(5):2685–2688. [PubMed] [Google Scholar]
  28. Marino M. W., Pfeffer L. M., Guidon P. T., Jr, Donner D. B. Tumor necrosis factor induces phosphorylation of a 28-kDa mRNA cap-binding protein in human cervical carcinoma cells. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8417–8421. doi: 10.1073/pnas.86.21.8417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Okazaki T., Bell R. M., Hannun Y. A. Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation. J Biol Chem. 1989 Nov 15;264(32):19076–19080. [PubMed] [Google Scholar]
  31. Okazaki T., Bielawska A., Bell R. M., Hannun Y. A. Role of ceramide as a lipid mediator of 1 alpha,25-dihydroxyvitamin D3-induced HL-60 cell differentiation. J Biol Chem. 1990 Sep 15;265(26):15823–15831. [PubMed] [Google Scholar]
  32. Robaye B., Hepburn A., Lecocq R., Fiers W., Boeynaems J. M., Dumont J. E. Tumor necrosis factor-alpha induces the phosphorylation of 28kDa stress proteins in endothelial cells: possible role in protection against cytotoxicity? Biochem Biophys Res Commun. 1989 Aug 30;163(1):301–308. doi: 10.1016/0006-291x(89)92135-9. [DOI] [PubMed] [Google Scholar]
  33. Schütze S., Scheurich P., Pfizenmaier K., Krönke M. Tumor necrosis factor signal transduction. Tissue-specific serine phosphorylation of a 26-kDa cytosolic protein. J Biol Chem. 1989 Feb 25;264(6):3562–3567. [PubMed] [Google Scholar]
  34. Shiroo M., Matsushima K. Enhanced phosphorylation of 65 and 74 kDa proteins by tumor necrosis factor and interleukin-1 in human peripheral blood mononuclear cells. Cytokine. 1990 Jan;2(1):13–20. doi: 10.1016/1043-4666(90)90038-u. [DOI] [PubMed] [Google Scholar]
  35. Vilcek J., Lee T. H. Tumor necrosis factor. New insights into the molecular mechanisms of its multiple actions. J Biol Chem. 1991 Apr 25;266(12):7313–7316. [PubMed] [Google Scholar]
  36. Wilson E., Olcott M. C., Bell R. M., Merrill A. H., Jr, Lambeth J. D. Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst. J Biol Chem. 1986 Sep 25;261(27):12616–12623. [PubMed] [Google Scholar]
  37. Zhang Y. H., Lin J. X., Yip Y. K., Vilcek J. Enhancement of cAMP levels and of protein kinase activity by tumor necrosis factor and interleukin 1 in human fibroblasts: role in the induction of interleukin 6. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6802–6805. doi: 10.1073/pnas.85.18.6802. [DOI] [PMC free article] [PubMed] [Google Scholar]

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