Choline phosphate potentiates sphingosine-1-phosphate-induced Raf-1 kinase activation dependent of Ras-phosphatidylinositol-3-kinase pathway

In NIH3T3 cells, sphingosine-1-phosphate (S1P) caused a significant increase of Raf-1 kinase activity as early as 2 min. Interestingly, choline phosphate (ChoP) produced synergistic increase of S1P-stimulated Raf-1 kinase activation in the presence of ATP while showing additive effect in the absence of ATP. However, Raf-1 kinase activation induced by S1P decreased in the presence of ATP when applied alone. The overexpression of N-terminal fragment of Raf-1 (Rf1) to inhibit Raf-Ras interaction caused the inhibition of S1P-induced Raf-1 kinase activation. Also, wortmannin, phosphatidylinositol-3-kinase (PI3K) inhibitor, exhibited inhibitory effects on S1P-induced activation of Raf-1 kinase. In addition, we demonstrated that the chemical antioxidant, N-acetylcysteine attenuated Raf-1 activation induced by S1P, suggesting that H2O2 may be required for the signalling pathway leading to Raf-1 activation. This H2O2-induced Raf-1 kinase activation was also blocked by inhibition of Ras-PI3K signalling pathway using alpha-hidroxyfarnesylphosphonic acid and wortmannin. Taken together, these results indicate that S1P-induced Raf-1 kinase activation is mediated by H2O2 stimulation of Ras-PI3K pathway, and is enhanced by ChoP in the presence of ATP. (C) 2002 Elsevier Science Inc. All rights reserved.