lin-25 lies downstream of
let-60 ras in the genetic pathway for induction of vulval fates and encodes a novel 130 kDa protein. LIN-25 is expressed first in all six VPCs independently of genes in the ras-MAPK cascade but later becomes restricted to the descendents of cells adopting vulval fates, P5.p, P6.p and P7.p. In order to help elucidate how LIN-25 functions biochemically we sort to identify parts of the protein most important for its activity. To identify evolutionary conserved domains we cloned
lin-25 from the related nematode C. briggsae . We identified a fosmid clone, G05D12, by DNA hybrization which the C. briggsae sequencing consortium kindly sequenced for us. The C. briggsae
lin-25 gene is predicted by Genefinder to have a similar arrangement of exons and introns to that of C. elegans
lin-25 . The sequence of the predicted product of the C. briggsae gene has however, diverged significantly from that of C. elegans LIN-25 (57% identity). The amino acid identities are fairly evenly distributed throughout the entire length of the two proteins but three domains can be identified, the largest at the C-terminus, within which the identity is much higher. The C. briggsae gene appears to be an orthologue of C. elegans
lin-25 since it is able to rescue the C. elegans
lin-25 phenotype. 10 alleles of
lin-25 have been isolated to date, 7 that behave as null mutations by genetic criteria. Characterization of these alleles reveals that the C-terminus of LIN-25 is required for stability. The null allele,
ku78 , is predicted to encode a truncated protein lacking the last 121 amino acids (of 1139).
ku78 homozygotes contain only very low levels of LIN-25 protein. In extracts from WT worms, multiple polypeptides are detected with anti-LIN-25 antibodies that have lower apparent molecular weights than the full-length protein. Tagging of the WT protein with the HA epitope reveals that these smaller species result from removal of the C-terminus.
lin-25 's position in the genetic pathway suggested that the protein might be a target of the MPK-1 MAPK. To examine this possibility we generated mutants in vitro encoding proteins lacking the potential MAP kinase phosphorylation sites. A gene encoding a protein lacking all four sites rescues the
lin-25 phenotype with almost WT efficiency. Thus LIN-25 does not appear to be a direct target of MPK-1.