Nfya-1 functions as a substrate of ERK-MAP kinase during Caenorhabditis elegans vulval development

A common bridge between a linear cytoplasmic signal and broad nuclear regulation is the family of MAP kinases which can translocate to the nucleus upon activation by the cytoplasmic signal. One pathway which functions to activate the ERK family of MAP kinases is the Ras signaling pathway which functions at multiple times and locations during the development of Caenorhabditis elegans including the development of the excretory cell, germ cells, male tail, and vulva. It has been most extensively characterized during the development of the vulva which is formed from the vulval precursor cells (VPCs), a set of six equivalent, epithelial cells designated P3.p - P8.p. Although LIN-1 appears to be a primary target of ERK MAP kinase during vulval development, it is likely that other developmentally important molecules are also regulated by ERK-mediated phosphorylation. The identification of physiological substrates of MAP kinases has been aided by the identification of docking site domains in substrate proteins that contribute to high-affinity interactions with kinases. Our laboratory has identified the C. elegans protein, T08D10.1/NFYA-1, as a potential ERK MAP kinase substrate in this manner, and we have initiated a characterization of its role during Ras-mediated development. T08D10.1 possesses significant homology to the CCAAT-box DNA-binding domain of the vertebrate nuclear transcription factor-Y, alpha (NF-YA) family of proteins. NF-Y proteins act as part of a complex to regulate the transcription of a large number of genes, in particular, genes that function in the G1/S cell cycle transition. T08D10.1/NFYA-1 is predicted to code for a protein containing multiple potential phosphorylation sites for ERK MAP kinase and a D-domain docking site. We demonstrate through biochemical analysis of purified NFYA-1 protein that it can act in vitro as a high affinity substrate for activated ERK MAP kinase. Growth factor activation of the Ras pathway in a tissue culture system has negligible effect on the protein's transactivation potential, however, the DNA-binding activity of the protein is reduced after treatment with activated ERK-MAP kinase. We demonstrate through mutant analysis that nfya-1 acts to inhibit vulval development and functions downstream or in parallel to let-60/ras. Both the NF-Y complex and the Ras signaling pathway play a fundamental role in cell proliferation and oncogenesis and the connection between the two is an important insight into the mechanisms of cell fate specification and cellular response.