SUMMARY Eukaryotic signaling pathways translate external cues in specific contexts into diverse biological outputs. Proper timing and duration of a signaling response requires careful regulation of stability, trafficking, activation, and deactivation of multiple components within a network. This overall coordination can involve positive feedback circuits to amplify signaling and/or negative feedback circuits to constrain signaling. The Ras signaling pathway is crucial in development to regulate proliferation, cell survival, cell fate, and patterning. Mutations in Ras or other components of the pathway that lead to increased Ras signaling cause developmental disorderes collectively known as “Rasopathies” such as Noonan's syndrome or Neurofibromatosis Type 1 (NF1). Using Drosophila, we discovered that Rabex-5 (an E3) downregulates Ras by promoting its mono- and di-ubiquitination. Specifically, we discovered that impairing Ras ubiquitination in vivo in Drosophila led to striking effects on cell proliferation, cell survival, developmental patterning, and organismal longevity, reflecting a role for Ras ubiquitination in development, tumor suppression, and survival. The power of Drosophila genetics and the well-established paradigm of studying Ras in Drosophila make this system ideally suited to characterize this phenomenon in a multi-cellular context. We more recently discovered that Rabex-5 targeting of Ras requires an N-terminal tyrosine in Ras, Tyrosine 4 (Y4). We hypothesize that phosphorylation of this tyrosine directs targeting of Ras by Rabex-5. Our hypothesis predicts that the kinase(s) and phosphatase(s) that regulate modification of Y4 are crucial regulators of Ras signaling. The goal of our application is to elucidate the role of tyrosine phosphorylation of Ras to direct its inhibitory Rabex-5- mediated ubiquitination and maintain pathway homeostasis. Specifically, we propose to further characterize the biological role of Y4 phosphorylation in Ras ubiquitination, to identify the Ras Y4 kinase(s) and phosphatase(s), and to characterize their roles in regulating Ras in development.