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 (RfI) 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 H(2)O(2) may be required for the signalling pathway leading to Raf-1 activation. This H(2)O(2)-induced Raf-1 kinase activation was also blocked by inhibition of Ras--PI3K signalling pathway using alpha-hydroxyfarnesylphosphonic acid and wortmannin. Taken together, these results indicate that S1P-induced Raf-1 kinase activation is mediated by H(2)O(2) stimulation of Ras--PI3K pathway, and is enhanced by ChoP in the presence of ATP.
Lee
Michael
M
Toxicology Research Center, Korea Research Institute of Chemical Technology, P.O. Box 107, Yusong, 305-600, Taejon, South Korea. mikelee@krict.re.kr
Han
Sang Seop
SS
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D004357:Drug Synergism; D004789:Enzyme Activation; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008246:Lysophospholipids; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010767:Phosphorylcholine / Q000494:pharmacology*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*
D008246:Lysophospholipids; D010767:Phosphorylcholine; C060506:sphingosine 1-phosphate; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D019869:Phosphatidylinositol 3-Kinases; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras); D013110:Sphingosine