Ceramide stimulates epidermal growth factor receptor phosphorylation in A431 human epidermoid carcinoma cells: Evidence that ceramide may mediate sphingosine action

Tzipora Goldkorn, Kenneth A. Dressler, Josephia Muindi, Norman S. Radin, John Mendelsohn, David Menaldino, Dennis Liotta, Richard N. Kolesnick

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Abstract

Recent studies suggest the existence of a signal transduction pathway involving sphingomyelin and derivatives (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies compare effects of ceramide, sphingosine, and N,N-dimethylsphingosine on epidermal growth factor (EGF) receptor phosphorylation in A431 human epidermoid carcinoma cells. To increase ceramide solubility, a ceramide containing octanoic acid at the second position (C8-cer) was synthesized. C8-cer induced time- and concentration-dependent EGF receptor phosphorylation. This event was detectable by 2 min and maximal by 10 min. As little as 0.1 μM C8-cer was effective, and 3 μM C8-cer induced maximal phosphorylation to 1.9-fold of control. EGF (20 nM) increased phosphorylation to 2.1-fold of control. Sphingosine stimulated receptor phosphorylation over the same concentration range (0.03-3 μM) and to the same extent (1.8-fold of control) as ceramide. The effects of C8-cer and sphingosine were similar by three separate criteria, phosphoamino acid analysis, anti-phosphotyrosine antibody immunoblotting, and phosphopeptide mapping by high performance liquid chromatography. Phosphorylation occurred specifically on threonine residues. N,N-Dimethylsphingosine, a potential derivative of sphingosine, was less effective. Since sphingosine and ceramide are interconvertible, the level of each compound was measured under conditions sufficient for EGF receptor phosphorylation. C8-cer (0.1-1 μM) induced dose-responsive elevation of cellular ceramide from 132 to 232 pmol·106 cells-1. In contrast, cellular sphingosine levels did not rise. This suggests that C8-cer acts without conversion to sphingosine. Exogenous sphingosine (0.1-1 μM) also increased cellular ceramide levels to 227 pmol·106 cells-1, but did not increase its own cellular level of 12 pmol·106 cells-1. Higher sphingosine concentrations that induced no further increase in EGF receptor phosphorylation produced very large elevations in cellular sphingosine. Hence, at ef-fective concentrations, both compounds elevated cellular ceramide but not sphingosine levels. Additional studies performed with [3H]sphingosine demonstrated that cells contain substantially less N,N-dimethylsphingosine than free sphingosine and, during short term incubation, convert less than 5% of added sphingosine to N,N-dimethylsphingosine. These studies provide evidence that ceramide may have bioeffector properties and suggest sphingosine may act in part by conversion to ceramide.

Original languageEnglish (US)
Pages (from-to)16092-16097
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number24
StatePublished - 1991
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Goldkorn, T., Dressler, K. A., Muindi, J., Radin, N. S., Mendelsohn, J., Menaldino, D., Liotta, D., & Kolesnick, R. N. (1991). Ceramide stimulates epidermal growth factor receptor phosphorylation in A431 human epidermoid carcinoma cells: Evidence that ceramide may mediate sphingosine action. Journal of Biological Chemistry, 266(24), 16092-16097.