A novel pathway for tumor necrosis factor-alpha and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3-kinase.
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.
Hanna
A N
AN
Signal Transduction Laboratories, and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
Chan
E Y
EY
Xu
J
J
Stone
J C
JC
Brindley
D N
DN
D000818:Animals; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D004789:Enzyme Activation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D002518:Ceramides; D014409:Tumor Necrosis Factor-alpha; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D016283:Proto-Oncogene Proteins p21(ras)