LIN-1 encodes a 441 amino acid protein that is likely to be a DNA-binding transcription factor, since it contains an N-terminal ETS domain.
lin-1(lf) mutations cause a multivulva phenotype, and
lin-1(gf) mutations cause a vulvaless phenotype, suggesting
lin-1 is a switch molecule and
lin-1 activity prevents Pn.p cells from adopting the primary vulval fate. Genes in the Ras signaling pathway promote the primary fate, and epistasis analyses indicate
lin-1 functions downstream of these genes, including
mpk-1 Erk MAP kinase, suggesting
lin-1 is negatively regulated by the action of this signaling pathway. To investigate whether LIN-1 is directly regulated by MAP kinase, we produced LIN-1 protein in E. coli and assayed partially purified protein for phosphorylation by murine Erk2 MAP kinase in vitro. Full-length LIN-1 protein was an excellent substrate with a Km of 0.2 uM, significantly lower that the 3.3 uM Km of myelin basic protein, a protein frequently used to assay Erk activity. The C-terminal region of LIN-1 (residues 281-441) was necessary for phosphorylation and sufficient to function as a good Erk2 substrate. The
lin-1(gf) mutations all affect the C-terminal region of LIN-1, suggesting this region is required for negative regulation of LIN-1. These observation indicate that phosphorylation of the C-terminal region of LIN-1 by Erk negatively regulates LIN-1 activity. We produced and assayed two mutant LIN-1 proteins that contained the alterations caused by two different
lin-1(gf) mutations. These proteins had Km values that were significantly higher the Km of wild-type LIN-1, suggesting the
lin-1(gf) mutations function by diminishing LIN-1 phosphorylation by Erk. The
lin-1(gf) mutations may affect a recognition motif for Erk MAP kinase.