) Protein Phosphatase 2A (PP2A) is an important regulator of signal transduction pathways, a target of viral oncoproteins, and a potentially useful target for therapeutic drug design. However, PP2A's broad substrate specificity in vitro has hampered attempts to understand its normal regulation and functionally relevant substrates in vivo. We identified a PR55/B regulatory subunit of Protein Phosphatase 2A (SUR-6 PP2A-B) as a positive modulator of Ras signaling in C. elegans, and our genetic data suggest that SUR-6 acts in a common process with KSR and directs PP2A to a specific Ras pathway substrate. Two mutations in SUR-6 PP2A-B preferentially affect Ras-mediated signaling, with minimal effects on other SUR-6- or PP2A-regulated processes. We will use C. elegans vulva development as a genetic model system to elucidate how PP2A-B subunits regulate PP2A catalytic activity and/or substrate specificity, and how PP2A regulates the Ras/Raf/MEK/ERK signaling cascade. To test the absolute requirements for SUR-6 PP2A-B during Ras-mediated vulval induction, we will isolate sur-6 null alleles and use a mosaic analysis strategy to determine their effects on vulval induction. To determine how SUR-6 PP2A-B influences PP2A catalytic activity, we will use RNA-mediated interference, PP2A-C mutations, PP2A-C transgenes and SV4O small t antigen expression to test how altering PP2A catalytic activity influences vulval induction. We will also use RNA-mediated interference to test the requirements for other PP2A-B regulatory subunits that might direct PP2A to alternative Ras pathway substrates, and test which domains of SUR-6 are important for binding to PP2A-A. To test if candidate proteins are likely substrates of PP2A, we will test if specific phospho-acceptor site mutants can bypass the requirement for SUR-6 during vulva induction. Finally, to identify other regulators and targets of PP2A activity during vulval induction, we will conduct a yeast two-hybrid screen for proteins that bind to SUR-6 PP2A-B, and conduct genetic modifier screens for mutations with properties similar to those of sur-6 mutations. Our studies will provide important insights into the normal in vivo regulation of PP2A and its targets, and how PP2A activity might be manipulated for therapeutic effects.
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