The Ras-stimulated Raf/MEK/ERK kinase cascade plays critical roles in normal animal development and cancer. Multiple poorly understood mechanisms ensure that this cascade is active only when and where it should be. Increasing evidence suggests that compartmentalization of signaling proteins by scaffolds is an important component of such regulation. Our research uses C. elegans as a genetic model system to identify new regulators of Raf/MEK/ERK signaling and understand how they function. During the previous funding period (04/01/00 to 3/31/05), we demonstrated the essential and widespread role of the scaffold KSR in MEK/ERK activation, and we showed that the scaffold CNK promotes a specific step of Raf activation. We also identified multiple other conserved, but previously unstudied, gene products that cooperate with KSR-1 to promote certain downstream responses to this signaling cascade. In the next funding period, we will focus on several sets of these gene products to understand how compartmentalization of signaling proteins controls signal propagation and specificity. In Aim 1, we will test if inherent functional differences between the KSR-1 and KSR-2 paralogs contribute to tissue-specific responses. In Aim 2, we will determine the role of the scaffold CNK-1 and its binding partner KIC-1 in Raf activation and localization. Membrane translocation and endocytosis are key steps in Raf activation and de-activation, and we will test the model that CNK-1 and KIC-1 are involved in these processes. In Aim 3, we will determine the role of a conserved ubiquitin conjugating (E2) enzyme in Ras signaling and scaffold function. We will test the model that this E2 regulates trafficking and localization of the KSR or CNK scaffolds, or of other signaling proteins, and we will identify the mechanism by which it acts. In Aim 4, we will continue both forward genetic and RNAi-based screens for new Ras pathway regulators, and choose for further study those mostly likely to function with KSR, CNK and/or our E2 protein. Our studies are expected to reveal new aspects of Ras pathway regulation and clarify the mechanisms and biological relevance of signaling compartmentalization. Aberrant signaling by Ras or Raf is 1 of the most frequent causes of human cancers. In the long term, a detailed understanding of Raf/MEK/ERK regulation will allow the rational design of therapeutic approaches to treat such cancers.