Raf-1/MEK/MAPK pathway is necessary for the G2/M transition induced by nocodazole.
The dynamic balance between polymerization and depolymerization of microtubules is critical for cells to enter and exit mitosis, and drugs that disrupt this balance, such as taxol, colchicine, and nocodazole, arrest the cell cycle in mitosis. Although the Raf/MEK/MAPK pathway can be activated by these drugs, its role in mitosis has not been addressed. Here, we characterize activation of Raf/MEK/MAPK by nocodazole when mitosis is induced. We find that at early time points (up to 3 h) in nocodazole induction, Raf/MEK/MAPK is activated, and inhibition of MAPK activation by a MEK inhibitor, PD98059 or U0126, reduces the number of cells entering mitosis by creating a block at G(2). At later time points and in mitosis, activation of MEK/MAPK is severely inhibited, even though Raf-1 activity remains high and can be further increased by growth factor. This inhibition is reversed when cells are released from metaphase and enter G(0)/G(1) phase. In addition, we find that binding of Raf-1 to 14-3-3 is progressively induced by nocodazole, reaching a maximum in mitosis, and that this binding is necessary to maintain mitotic Raf-1 activity. Our present study indicates that activation of the Raf/MEK/MAPK pathway is necessary for the G(2)/M progression.
Hayne
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Diabetes and Metabolism Research Unit, Section of Endocrinology, Evans Department of Medicine and the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Tzivion
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G
Luo
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D048948:14-3-3 Proteins; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D019556:COS Cells; D004789:Enzyme Activation / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008938:Mitosis / Q000187:drug effects*; D009570:Nitriles / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D048948:14-3-3 Proteins; D002070:Butadienes; D005419:Flavonoids; D009570:Nitriles; C113580:U 0126; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D015739:Nocodazole; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one