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Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by
D Woods1, D Parry, H Cherwinski
1Department of Cell Signaling, DNAX Research Institute, Palo Alto, California 94304, USA.
Abstract:
The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.
Insights
Raf kinases control cell cycle entry. Different Raf forms cause either cell cycle progression or arrest, with p21Cip1 induction mediating arrest, suggesting Raf
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Raf family of protein kinases are key regulators of intracellular signaling pathways.
- Their role in cell cycle progression, particularly entry into the S phase, is complex and context-dependent.
Purpose of the Study:
- To investigate the differential effects of various Raf kinases (deltaA-Raf, deltaRaf-1, deltaB-Raf) on NIH 3T3 cell cycle progression.
- To elucidate the molecular mechanisms underlying Raf-mediated cell cycle control, focusing on cyclin-dependent kinase inhibitors (CKIs).
Main Methods:
- Conditional activation of engineered Raf kinases (Raf:ER) in NIH 3T3 cells and primary mouse embryo fibroblasts (MEFs).
- Analysis of cell cycle phase distribution (G1 arrest, S phase entry).
- Quantification of cell cycle regulatory proteins (cyclins D1, E, p27Kip1, p21Cip1) via Western blotting or similar techniques.
- Studies using wild-type and p21Cip1 knockout MEFs.
Main Results:
- DeltaA-Raf:ER promoted cell cycle progression, while deltaRaf-1:ER and deltaB-Raf:ER induced G1 arrest.
- All Raf forms increased cyclin D1 and E, but only deltaRaf-1:ER and deltaB-Raf:ER induced p21Cip1, correlating with arrest.
- Raf-induced p21Cip1 expression in MEFs was p53-independent; p21Cip1 knockout MEFs showed robust proliferation upon Raf activation.
Conclusions:
- Raf kinase activity level dictates cell cycle outcome: low activity promotes progression, high activity induces arrest via p21Cip1.
- The induction of p21Cip1 is a critical mechanism for Raf-mediated cell cycle arrest, analogous to yeast alpha-factor arrest.
- Raf kinases play dual roles in mammalian cell proliferation and differentiation, modulated by signaling strength and duration.