Raf-1 kinase activation is uncoupled from downstream MEK/ERK pathway in cells treated with Src tyrosine kinase inhibitor PP2.
Recently we demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, markedly enhanced Ras-independent activation of Raf-1 by the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H(2)O(2)). We report here that Raf-1 knockdown cells were significantly more sensitive to treatment of PP2 than control cells. This PP2-induced growth inhibition was found to be linked to decreased ERK and p38 activity. Interestingly, the growth of Sprouty knockdown cells appeared to be inhibited at earlier time points of PP2 treatment when compared with control cells. Unexpectedly, siRNA-mediated knockdown of Spry2, which is known to modulate the Ras/Raf/MAPK signal through feedback regulation, resulted in decreased Raf-1 kinase activity. PP2 had limited effect on the ability of PMA/H(2)O(2) to induce significant phosphorylation of MEK/ERK proteins in both Spry2 knockdown and control cells, indicating that PP2-mediated activation of Raf-1 did not potentiate signaling through the downstream MEK/ERK pathway. Taken together our results suggest that Raf-1 signaling may be bypassed in PP2-treated cells by uncoupling from downstream MEK/ERK pathway.
Lee
Michael
M
Department of Biology, College of Natural Sciences, University of Incheon, 177 Dowha-dong, Nam-gu, Incheon 402-749, Republic of Korea. mikelee@incheon.ac.kr
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D041681:NIH 3T3 Cells; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology*; D034622:RNA Interference; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors*
C412373:AG 1879; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D019061:src-Family Kinases; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases