Kinase suppressor of Ras (KSR) is a scaffold protein that is required to localize components of the Ras/MAPK pathway to the plasma membrane to activate Raf kinase and to facilitate interactions between Raf and downstream kinases, MEK and ERK. C. elegans has two genes that encode for KSR proteins,
ksr-1 and
ksr-2, which are redundantly required for specification of the excretory duct cell. Less than 1% of
ksr-1 single mutants die as a "rod-like" lethal, but is strongly enhanced by removal of
ksr-2 or other positive regulators of the pathway, such as a scaffolding protein involved in Raf activation,
cnk-1. A genome-wide RNAi screen was conducted to find enhancers of
ksr-1 lethality (ekl genes) (Rocheleau et al. 2008). Most of the ekl genes identified in this screen encode regulators of gene expression and display pleiotropic phenotypes and were unlikely candidates to be direct regulators of Ras/MAPK signaling. Some of the ekl genes found like
ubc-25, an E2 ubiquitin conjugating enzyme, and
ekl-5, a novel uncharacterized gene, did not have pleiotropic phenotypes and might have a more direct role with
ksr-1 to facilitate signaling in the Ras/MAPK pathway. We predict that a forward genetic ekl screen would identify a different but partially overlapping set of ekl genes, mostly those without pleiotropic phenotypes and more likely to have a direct role in regulating Ras/MAPK signaling. I performed a forward genetic screen using EMS mutagenesis to isolate genes that enhance the "rod-like" lethal phenotype of
ksr-1 mutants. From this screen, I have isolated ten candidates, seven of which are confirmed enhancers. Single Nucleotide Polymorphism (SNP) mapping was used to map these mutants to a chromosomal location on the C. elegans genome. I have mapped three mutants to chromosome I, one mutant to chromosome III, and three mutants to chromosome X.
vh1 and
vh18, two of the mutants mapped to chromosome I, failed to complement
ubc-25.
ubc-25(
vh1) contains a nonsense mutation that causes a premature stop codon early in the gene and is likely a null allele.
ubc-25(
vh18) is a slightly less penetrant enhancer and contains a missense mutation that causes a Histidine to Arginine substitution adjacent to the UBC domain.
vh2,
vh22, and
vh23 were mapped to chromosome X close to
ekl-5 and but they all complement
ekl-5, suggesting that they might be mutations in new regulators of the Ras/MAPK pathway that function closely with KSR.