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Raf-1/MEK/MAPK pathway is necessary for the G2/M transition induced by nocodazole
1Diabetes 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.
Abstract:
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.
Insights
The Raf/MEK/MAPK pathway is essential for cell cycle progression into mitosis. Nocodazole treatment reveals this pathway
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubule dynamics are crucial for cell division (mitosis).
- Drugs like nocodazole disrupt microtubules, arresting cells in mitosis.
- The Raf/MEK/MAPK pathway's role in mitosis is not fully understood.
Purpose of the Study:
- To investigate the activation and role of the Raf/MEK/MAPK pathway during nocodazole-induced mitosis.
- To determine how this pathway influences cell cycle progression at G2/M.
Main Methods:
- Utilized nocodazole to induce mitosis in cells.
- Employed MEK inhibitors (PD98059, U0126) to block MAPK activation.
- Assessed Raf-1 and MEK/MAPK activity and localization.
- Analyzed Raf-1 binding to 14-3-3 proteins.
Main Results:
- Raf/MEK/MAPK pathway activation occurs early in nocodazole treatment, promoting G2/M transition.
- Inhibition of MAPK signaling causes a G2 block.
- Mitotic MAPK activation is inhibited, despite sustained Raf-1 activity.
- Raf-1 binding to 14-3-3 proteins increases during mitosis and is vital for Raf-1 activity.
Conclusions:
- Activation of the Raf/MEK/MAPK pathway is required for progression through G2 and into mitosis.
- 14-3-3 binding regulates Raf-1 activity during mitosis.