The long-term objectives of this proposal are to identify and characterize proteins that function in signal transduction pathways and discover how these proteins act together to regulate cell fates during development. This information will be important for understanding fundamental aspects of animal development and developing new diagnostic and therapeutic strategies for human diseases that result from abnormalities of signaling pathways, such as birth defects and cancer. The proposed research focuses on an evolutionarily conserved signaling pathway that includes a receptor tyrosine kinase, Ras, mitogen activated protein (MAP) kinase, and ETS transcription factors. These pathways mediate many different cell fate decisions during development, and mutations that affect these proteins are a common cause of human tumors. MAP kinase function will be investigated by characterizing two docking sites that enable MAP kinases to interact with substrate proteins, identifying additional docking sites, determining how docking sites affect the selection of phosphorylation sites in substrate proteins, and identifying the region of MAP kinase that interacts with docking sites. Information about docking sites will be used to investigate how phosphorylation affects protein function, develop peptide inhibitors of MAP kinase and predict new substrates of MAP kinase. Candidate substrates will be evaluated in vitro and in vivo. These studies should illuminate how MAP kinase activity causes different cell fates in different developmental contexts. ETS transcription factors are conserved mediators of MAP kinase signaling, including the LIN-1 protein in Caenorhabditis elegans. LIN-1 regulation will be investigated by characterizing DNA binding, transcriptional regulation, and protein stability, and determining how these properties are regulated by MAP kinase activity. Target genes that are regulated by LIN-1 will be identified using molecular and genetic approaches, and a network of genes that interact with LIN-1 will be identified and characterized. These studies will illuminate how MAP kinases identify and interact with substrate proteins, how substrate proteins mediate different cell fates during animal development, how ETS transcription factors are regulated by MAP kinase and how ETS proteins regulate cell fates during development.
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