KSR AND REGULATORS OF RAS SIGNALING IN C ELEGANS

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Our long term goal is to understand how Ras signaling controls animal growth and development. Ras can stimulate a complex web of signaling pathways, one of which is the Raf/MEK/ERK kinase cascade. Our specific goal is to understand the function of KSR (Kinase Suppressor of Ras), which I first identified as a positive regulator of Ras signaling in C. elegans. KSR may activate Raf or modulate signal propagation through Raf/MEK/ERK, or KSR may function in a separate signaling pathway. Since our data suggest that KSR binds to MEK yet functions in a common genetic pathway with Protein Phosphatase 2A (PP2A) and a Rac protein, we propose that KSR functions to coordinate two different Ras- stimulated pathways. To test this hypothesis, we will test the physical and regulatory relationship among KSR, PP2A and Rac and the known Raf/MEK/ERK cascade. In Aim l, we will test if KSR functions within the same or different cells as Ras (by mosaic analysis), define KSR's normal expression pattern (by immuno-localization) and test if KSR membrane localization is regulated by Ras, Rac or PP2A. In Aim 2, we will test if KSR colocalizes or physically interacts with Rac or PP2A, test if KSR can mediate interactions between Rac or PP2A and MEK (supporting a scaffold model), and test if Raf is the likely target of PP2A activity (supporting a Raf activation model). In Aim 3, we will test if ksr- 1 null mutant strains have alterations in MEK localization, protein complex formation, ERK phosphorylation or target gene expression. These are all activities that have been attributed to KSR based on overexpression studies. We will also conduct transgenic structure/function studies to identify sites important for KSR- 1 regulation and function, and we will test if KSR- 1 is the likely target of PP2A. In Aim 4, we will use genetic and reverse genetic approaches to identify additional genes that regulate Ras or Rac signaling, and we will characterize/clone three genes that we have already identified. Deregulated Ras signaling causes many human pathologies, including cancer. Our studies of KSR, PP2A and Rac will provide important insights into how different Ras signaling pathways are normally coordinated in vivo.