The Role of Protein Phosphatases in Regulation of Drosophila S6 Kinase by Nutrient Signaling Pathways

The regulation of cellular growth is a highly complex, but fundamental, process and the inability of cells to initiate or repress cell growth in the presence of appropriate signals underlies many disease states including cancer and diabetes. Many studies have investigated the mechanisms by which cells activate growth; however, few have focused upon the means by which cells turn off growth in response to environmental signals. As many of the protein components of nutrient signaling pathways are proto-oncogenes, of equal importance is identifying and characterizing the components utilized to down-regulate growth, as mutations in these genes may also lead to genesis of disease. Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase implicated in the regulation of numerous processes, including cellular growth. Elucidating the role of PP2A in nutrient signaling pathways will aid in the understanding of the biochemical processes that eukaryotes employ to properly respond to their environment. I have cloned the Drosophila homolog of the Tap42/a4 gene (dTap), which retains 28% amino acid identity to the human a4 gene product. Utilizing a combination of RNA interference (RNAi) and nutrient starvation conditions, I have tested the sequestration model of dTap as a repressor of PP2A activity and shown that in S2 cells, dTap does not antagonize nutrient signaling pathways by repressing PP2A activity. Although dTap does not act as an inhibitor of PP2A activity towards dS6K, loss of dTap conferred rapamycin resistance to S2 cells and prolonged loss of dTap resulted in cell death. I have also utilized RNAi to screen the gene products of the PP2A-like family of phosphatases and Drosophila PP2A regulatory subunits for effects upon nutrient signaling pathways. I have shown that the PP2A catalytic subunit is required for the dephosphorylation of Drosophila S6 kinase (dS6K) during periods of inhibition of growth. The ablation of two other gene products, the PP2A-B56-2 regulatory subunit and the dPP4 catalytic subunits, results in hypophosphorylation of dS6K during amino acid starvation, suggesting that the activities of these two gene products are required for maintenance of nutrient signaling pathways and proper growth in Drosophila.