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An analysis of Mek1 signaling in cell proliferation and transformation
1Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA. heidi@biosun.harvard.edu
Abstract:
The Mek1 dual specificity protein kinase phosphorylates and activates the mitogen-activated protein kinases Erk1 and Erk2 in response to mitogenic stimulation. The molecular events downstream of Mek and Erk necessary to promote cell cycle entry are largely undefined. In order to study signals emanating from Mek independent of upstream proteins capable of activating multiple signaling pathways, we fused the hormone-binding domain of the estrogen receptor (ER) to the C terminus of constitutively activated Mek1 phosphorylation site mutants. Although 4-OH-tamoxifen stimulation of NIH-3T3 cells expressing constitutively activated Mek-ER resulted in only a small increase in specific activity of the fusion protein, a 5-10 fold increase in total cellular Mek activity was observed over a period of 1-2 days due to an accumulation of fusion protein. Induction of constitutively activated Mek-ER in NIH-3T3 cells resulted in accelerated S phase entry, proliferation in low serum, morphological transformation, and anchorage independent growth. Endogenous Erk1 and Erk2 were phosphorylated with kinetics similar to the elevation of Mek-ER activity. However, elevated Mek-ER activity attenuated subsequent stimulation of Erk1 and Erk2 by serum. 4-OH-tamoxifen stimulation of Mek-ER-expressing fibroblasts also resulted in up-regulation of cyclin D1 expression and down-regulation of p27(Kip1) expression, establishing a direct link between Mek1 and the cell cycle machinery.
Insights
Constitutively activated Mek1, fused to the estrogen receptor (ER), drives cell cycle entry and proliferation. This Mek1-ER fusion protein directly links Mek1 signaling to cell cycle regulators like cyclin D1 and p27(Kip1).
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Mek1 (MAPK/ERK Kinase 1) activates Erk1 and Erk2 kinases in response to mitogens.
- Downstream signaling events from Mek1 and Erk1/Erk2 that promote cell cycle entry are not fully understood.
Purpose of the Study:
- To investigate Mek1 signaling independently of upstream activators.
- To establish a direct link between Mek1 activity and cell cycle progression.
Main Methods:
- Constructed a fusion protein: constitutively active Mek1 (phosphorylation site mutants) linked to the estrogen receptor (ER) ligand-binding domain.
- Utilized 4-OH-tamoxifen to induce Mek1-ER activity in NIH-3T3 cells.
- Assessed Mek activity, Erk1/Erk2 phosphorylation, cell proliferation, and gene expression (cyclin D1, p27Kip1).
Main Results:
- Mek1-ER fusion protein accumulation led to a 5-10 fold increase in total cellular Mek activity.
- Induced Mek1-ER activity accelerated S phase entry, promoted proliferation in low serum, and caused morphological transformation.
- Elevated Mek1-ER activity led to Erk1/Erk2 phosphorylation but attenuated serum-induced stimulation.
- Upregulation of cyclin D1 and downregulation of p27(Kip1) were observed following Mek1-ER activation.
Conclusions:
- Constitutively active Mek1, when activated by tamoxifen, drives cell cycle progression.
- Mek1 signaling directly influences cell cycle regulators, including cyclin D1 and p27(Kip1).
- This study establishes a direct link between Mek1 kinase activity and the control of cell proliferation.