The goal of the intended research is to understand the role of the JNK signaling pathway in determination of cell fate in the Drosophila eye. The preliminary results from a screen that I am currently conducting suggest that the JNK pathway may play a more significant role in Drosophila eye development than has previously been realized. This screen has already identified a mutation in the gene that encodes a Drosophila homologue of the POSH protein in mice. The mouse homologue was identified in a screen for proteins which interacted with the small GTPase Racl. Racl is known to be involved in the activation of the JNK signaling pathway. Our identification of a Drosophila homologue of the POSH protein in a screen for mutations affecting eye development suggests that this protein may also be involved in JNK signaling during eye development. I will perform a detailed genetic and biochemical analysis on the Posh gene in Drosophila. I will determine the domains of POSH that are required for signaling by performing site-directed mutagenesis on conserved residues in the SH3 and Rac-binding domains of POSH. These mutated constructs will be tranformed back into Drosophila to determine which residues are necessary for POSH function. In addition, protein partners of POSH will be identified by combining the results of immunoprecipitation assays and GST-POSH fusion protein pull-down experiments performed on Drosophila embryonic cell culture extracts. The results of the genetic and biochemical experiments should provide us with an understanding of the role of Posh in JNK signaling during Drosophila eye development.
