A novel pathway for tumor necrosis factor-alpha and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3-kinase

A N Hanna, E Y Chan, J Xu, J C Stone, D N Brindley

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Treatment of confluent rat2 fibroblasts with C2-ceramide (N-acetylsphingosine), sphingomyelinase, or tumor necrosis factor-alpha (TNFalpha) increased phosphatidylinositol (PI) 3-kinase activity by 3-6-fold after 10 min. This effect of C2-ceramide depended on tyrosine kinase activity and an increase in Ras-GTP levels. Increased PI 3-kinase activity was also accompanied by its translocation to the membrane fraction, increases in tyrosine phosphorylation of the p85 subunit, and physical association with Ras. Activation of PI 3-kinase by TNFalpha, sphingomyelinase, and C2-ceramide was inhibited by tyrosine kinase inhibitors (genistein and PP1). The stimulation of PI 3-kinase by sphingomyelinase and C2-ceramide was not observed in fibroblasts expressing dominant-negative Ras (N17) and the stimulation by TNFalpha was decreased by 70%. PI 3-kinase activation by C2-ceramide was not modified by inhibitors of acidic and neutral ceramidases, and it was not observed with the relatively inactive analog, dihydro-C2-ceramide. It is proposed that activation of Ras and PI 3-kinase by ceramide can contribute to signaling effects of TNFalpha that occur downstream of sphingomyelinase activation and result in increased fibroblasts proliferation.

Original languageEnglish
Pages (from-to)12722-9
Number of pages8
JournalJournal of Biological Chemistry
Issue number18
Publication statusPublished - 30 Apr 1999


  • animals
  • cell line
  • ceramides
  • enzyme activation
  • phosphatidylinositol 3-kinases
  • protein-tyrosine kinases
  • proto-oncogene proteins p21(ras)
  • rats
  • signal transduction
  • tumor necrosis factor-alpha

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