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MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblasts and epithelial cells
N Rivard1, M J Boucher, C Asselin
1Groupe du Conseil de Recherches Médicales sur le Développement Fonctionnel et la Physiopathologie du Tube Digestif, Département d'Anatomie et Biologie Cellulaire, Quebec J1H 5N4, Canada. nrivard@courrier.usherb.ca
Abstract:
The present report delineates the critical pathway in the G(1) phase involved in downregulation of p27(Kip1), a cyclin-dependent kinase inhibitor, which plays a pivotal role in controlling entry into the S phase of the cell cycle. In resting CCL39 fibroblasts and IEC-6 intestinal epithelial cells, protein levels of p27(Kip1) were elevated but dramatically decreased on serum stimulation, along with hyperphosphorylation of pRb and increased CDK2 activity. In both cell types, expression of ras resulted in an increase of basal and serum-stimulated E2F-dependent transcriptional activity and a reduction in p27(Kip1) protein levels as well. The role of the mitogen-activated protein (MAP) kinase cascade in p27(Kip1) reduction and S phase reentry was reinforced by the blockades of serum-induced E2F-dependent transcriptional activity and p27(Kip1) downregulation with the MKK-1/2 inhibitor PD-98059. In both cell lines, downregulation of p27(Kip1) was associated with a repression of its synthesis, an event mediated by the p42/p44 MAP kinase pathway. Using an antisense approach, we demonstrated that p27(Kip1) may control cell cycle exit in both cell types. These data indicate that activation of the MAP kinase cascade is required for S phase entry and p27(Kip1) downregulation in fibroblasts and epithelial cells.
Insights
The mitogen-activated protein (MAP) kinase cascade is essential for cell cycle progression. This pathway downregulates p27(Kip1) (a cell cycle inhibitor), promoting entry into S phase in fibroblasts and epithelial cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- p27(Kip1) is a cyclin-dependent kinase inhibitor crucial for controlling cell cycle entry into S phase.
- Elevated p27(Kip1) levels are observed in resting cells, inhibiting proliferation.
Purpose of the Study:
- To elucidate the critical pathway regulating p27(Kip1) downregulation during G1 phase.
- To investigate the role of the MAP kinase cascade in p27(Kip1) reduction and cell cycle reentry.
Main Methods:
- Utilized CCL39 fibroblasts and IEC-6 intestinal epithelial cells.
- Measured protein levels of p27(Kip1), pRb phosphorylation, and CDK2 activity.
- Investigated the effects of ras expression and MKK-1/2 inhibitor PD-98059.
- Employed an antisense approach to study p27(Kip1) function.
Main Results:
- Serum stimulation decreased p27(Kip1) levels and increased pRb phosphorylation and CDK2 activity.
- Ras expression reduced p27(Kip1) levels and enhanced E2F-dependent transcription.
- PD-98059 blocked serum-induced E2F activity and p27(Kip1) downregulation.
- p42/p44 MAP kinase pathway mediated the repression of p27(Kip1) synthesis.
Conclusions:
- Activation of the MAP kinase cascade is a prerequisite for S phase entry.
- MAP kinase signaling is required for the downregulation of p27(Kip1) in fibroblasts and epithelial cells.
- p27(Kip1) plays a role in controlling cell cycle exit.