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MEK kinase activity is not necessary for Raf-1 function
M Hüser1, J Luckett, A Chiloeches
1Department of Biochemistry, MRC Toxicology Unit and Division of Biomedical Services, University of Leicester, University Road, Leicester LE1 7RH, UK.
Abstract:
Raf-1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf-1 is achieved by RAS:GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a 'knockout' of the raf-1 gene in mice as well as a rafFF mutant version of endogenous Raf-1 with Y340FY341F mutations. Raf-1(-/-) mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf-1 from cells derived from raf-1(FF/FF) mice has no detectable activity towards MEK in vitro, and yet raf-1(FF/FF) mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf-1(-/-) and raf-1(FF/FF) mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf-1 is not essential for normal mouse development and that Raf-1 plays a key role in preventing apoptosis.
Insights
Raf-1 kinase is crucial for preventing apoptosis during mouse development, but its MEK kinase activity is not essential. Gene targeting revealed distinct roles for Raf-1 beyond cell proliferation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Developmental Biology
Background:
- Raf-1 protein kinase is a key component of the Ras/Raf/MEK/ERK signaling pathway.
- Raf-1 activation involves RAS:GTP binding and tyrosine phosphorylation at residues 340/341.
Purpose of the Study:
- To investigate the essential functions of Raf-1 during mouse development.
- To determine the role of Raf-1's MEK kinase activity in vivo.
- To elucidate the contribution of Raf-1 to apoptosis and cell proliferation.
Main Methods:
- Gene targeting in mice to create a raf-1 knockout (Raf-1(-/-)).
- Generation of a raf-1 mutant mouse with Y340FY341F substitutions (raf-1(FF/FF)).
- Analysis of embryonic lethality, vascular defects, apoptosis, cell proliferation, and ERK activation.
Main Results:
- Raf-1(-/-) mice exhibit embryonic lethality with vascular defects and increased apoptosis.
- raf-1(FF/FF) mice are viable and fertile, with normal phenotypes and ERK activation.
- Cells from raf-1(FF/FF) mice show no detectable MEK kinase activity in vitro.
Conclusions:
- Raf-1's MEK kinase activity is dispensable for normal mouse development.
- Raf-1 plays a critical role in inhibiting apoptosis during embryogenesis.
- The study highlights distinct functions of Raf-1 beyond its canonical kinase activity.