Membrane localization of Raf assists engagement of downstream effectors.
We have previously described a small molecule-directed protein dimerization strategy, using coumermycin to juxtapose Raf fusion proteins containing the coumermycin-binding domain GyrB. Oligomerization of cytoplasmically localized Raf-GyrB fusion proteins leads to an increase in the kinase activity of both Raf and its substrate Mek. Surprisingly, more distal targets, such as Erk1 and Erk2, are not activated using this approach. Here we report that coumermycin-induced oligomerization of a membrane-localized Raf-GyrB fusion protein potently activated Erk1 and Erk2, up-regulated Fos protein levels, and induced expression of many immediate-early response genes. Thus, both membrane localization and oligomerization of Raf-GyrB are required to target Raf signals to downstream effectors. The ability to activate the entire Raf signal transduction cascade conditionally, using coumermycin-induced oligomerization, should prove useful for dissecting Raf-mediated effects on gene expression and cellular differentiation.
Merck Research Laboratories, Rahway, New Jersey 07065, USA. farra005@tc.umn.edu
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Farrar
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Tian
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Perlmutter
D016475:3T3 Cells; D049933:Aminocoumarins; D000818:Animals; D002273:Carcinogens; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism*; D003374:Coumarins / Q000494:pharmacology; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017874:Immediate-Early Proteins / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009675:Novobiocin / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation
D049933:Aminocoumarins; D002273:Carcinogens; D003374:Coumarins; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D016760:Proto-Oncogene Proteins c-fos; D011993:Recombinant Fusion Proteins; D009675:Novobiocin; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D013755:Tetradecanoylphorbol Acetate; C004628:coumermycin