Multiple mechanisms ensure the correct spatial and temporal activity of LET-23/EGFR and LIN-12/Notch signaling during vulval fate patterning. We report here that SEL-10/Fbw7, an F-box and WD repeat containing protein that promotes ubiquitination and proteasome-mediated degradation of phosphorylated substrates, regulates the activity of the two main signaling pathways that specify vulval fate pattern.
Previous work showed that
sel-10 inhibits
lin-12 activity during C. elegans vulval fate patterning and that LIN-12/Notch proteins are conserved substrates of SEL-10/Fbw7. We now find that
sel-10 also negatively regulates the activity of the Ras pathway through
lin-45, a C. elegans B-Raf and key component of the EGFR signal transduction pathway. Loss of
sel-10 suppresses defects resulting from partial loss of
lin-45 activity. The hypomorphic alleles
lin-45(
n2506) and
lin-45(
n2018) greatly reduce
lin-45 activity; most mutant larvae die during L1 with a rod-like morphology. This rod-like lethality is efficiently suppressed by
sel-10(
ar41). In addition, loss of
sel-10 enhances vulval defects caused by a hyperactive
lin-45. The transgene
lin-45(ED) carries mutations that render LIN-45 constitutively active. Tissue-specific expression of LIN-45(ED) in vulva precursor cells (VPCs) results in a Multivulva (Muv) phenotype with large ventral protrusions characteristic of excessive Ras/Raf/MAPK signaling. The Muv phenotype of animals carrying
lin-45(ED) is enhanced by
sel-10(
ar41).
We have also analyzed the sequences mediating SEL-10 regulation of LIN-12 and LIN-45. Previous work showed that SEL-10 binds the C-terminal PEST sequence of intracellular LIN-12. We have found that both LIN-12 and LIN-45 proteins contain peptide sequences that match reported Fbw7 binding sites in human proteins. Although VPC-specific expression of LIN-12(intra) does not produce vulval defects, we found that mutation of the predicted SEL-10 binding site, which lies within the PEST sequence of LIN-12(intra), results in a Muv phenotype characteristic of hyperactive
lin-12(d) alleles. In LIN-45, the predicted SEL-10 binding site lies within a mostly unconserved region; however, the corresponding residues in mammalian Raf proteins are highly phosphorylated. We found that mutation of the predicted SEL-10 binding site within LIN-45(ED) enhances the severity and penetrance of the resulting Muv phenotype. Given the effects on signaling in this important paradigm, we are now investigating the developmental regulation and role of
sel-10.