Characterisation of the Raf protein kinases by gene targeting

• Main Author: Hussain, Jahan Nurany
• Published: University of Leicester 2005
• Subjects: 612.01575
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431981

The role of B-Raf in cell growth and survival was investigated using primary MEFs lacking B-Raf. Cell growth and proliferation were found to be reduced and this coincided with alterations in the expression levels of various proteins involved in the G1 to S phase of the cell cycle. Analysis of B-raf cells upon serum withdrawal-induced apoptosis showed this was associated with decreased phosphorylation levels of phospho-T125-caspase 9. A B-raf allele containing a conditional kinase inactivating D594A mutation was generated. This was produced by designing a vector containing loxP sites flanking exons 15-18 of B-raf upstream of a second B-raf exon 15 containing the mutation. Upon gene targeting homologous recombination was successfully achieved for three ES clones and these were used to generate chimeric mice. Two of these targeted ES clones led to successful germline transmission. Mice carrying the mutation were mated with Cre-expressing mice, allowing the generation of mice heterozygous for the D594A mutation, B-raf+Lox-D594A. These mice were shown to have neoplasia due to splenomegaly and this was attributed to the elevated phospho-ERK levels observed. Intercrossing of B-raf+/Lox-D594A mice did not generate any viable B-rafLox-D594A/Lox-D594A mice, suggesting the D594A mutation was embryonic lethal. Analysis of B-rafLox-D594A/Lox-D594A MEFs, phospho-MEK and phospho-ERK levels were significantly elevated. This was attributed to increased C-Raf kinase activity observed within these cells. Further analysis indicated increased survival and increased cell growth and proliferation in these cells.