The Ras/MAPK signaling pathway determines several developmental processes in Caenorhabditis elegans, including determination of the excretory duct cell fate during embryogenesis and vulval cell fates during larval stages. Scaffolding proteins KSR-1 and KSR-2 mediate the formation of signaling complexes and are redundantly required for proper excretory duct and vulval cell fates. Without an excretory duct cell, early stage larvae are incapable of excreting waste fluid and die with a phenotypically distinct rod-like lethality.
ksr-1 null mutants are fundamentally wild-type, but are highly sensitive to any decreases in Ras/MAPK signaling. In a mutagenesis screen for enhancers of
ksr-1 lethality, we identified the
vh20 mutation as having a ~50% rod-like lethal progeny in the
ksr-1(
n2526) background. A combination of SNP mapping and whole genome sequencing identified a missense mutation in Y39G10AR.7 as a candidate for
vh20. Interestingly, we had previously identified Y39G10AR.7 as a weak enhancer of
ksr-1 (6% rod-like lethality) in a genome-wide RNAi screen for ekl genes (Rocheleau and Sundaram, unpublished). BLASTp analysis only identifies Y39G10AR.7 homologs in other Caenorhabditis species. Protein alignments revealed a cluster of eight consensus Erk phosphorylation sites and four Erk docking sites (D-box and FXFP) strongly suggesting that Y39G10AR.7 might be a downstream target of MPK-1 Erk activity. The
vh20 E422K mutation disrupts stretch of negatively charged residues in the C-terminus suggesting this domain may be functionally important. We are currently performing transgenic rescue of
vh20 with a genomic clone of Y39G10AR.7. We will investigate the genetic relationship between
vh20 and other mutations in the Ras/MAPK pathway and downstream MPK-1 Erk substrates LIN-1 and EOR-1.