We propose to investigate, by molecular genetic approaches, the role of reversible protein phosphorylation in signal transduction and development. The phospho-protein phospho-protein phosphatase PP2A is the major ser/thre phosphatase in eukaryotes. The enzyme is assumed to play an important role in signal transduction either by constitutively dephosphorylating proteins that are phosphorylated in response to extracellular stimuli, or becoming activated directly in response to such stimulation. However, this conclusion is based on the ability of the enzyme to dephosphorylate many proteins in vitro, where its specificity is low, or on the action of inhibitors that affect many phosphatases. We propose to test the hypothesis that PP2A play an important role in vivo in the dephosphorylation of specific proteins which are activated by phosphorylation during signal transduction and development. The eukaryotic microorganism Dictyostelium discoideum, is particularly suitable for these studies, because of its sample genome, short, well- defined developmental cycle and ease of making mutants by reverse genetics. We had previously shown that extracellular cAMP, which activates cell surface receptors in Dictyostelium, induces genes by signal transduction. The proteins involved in the pathways are highly conserved. In preliminary studies we cloned the gene for the catalytic subunit PP2A (PP2Ac) and transfected it intro wild type cells. We discovered dominant negative mutations by the transfection technique, and constructed others by site specific mutagenesis. The mutants appear to be defective in development. We plan to confirm and extend these studies, to look for substrate proteins that may dephosphorylated by PP2A or proteins that may interact with the enzyme in response to signaling. We also plan to construct more okadaic acid resistant mutants by site specific mutagenesis of PP2Ac. We propose to exploit these mutants to determine precisely the points at which PP2A is involved in development or growth and the pathways that are activated. PP2A is presumed to dephosphorylate an important and signaling enzyme, protein kinase B (PKB) which is intensively studied. The mammalian PKB gene is a proto-oncogene and is also involved in the release from apoptosis. We have knocked out the Dictyostelium PKB gene and observed that it affects development. We propose to test the hypothesis that PP2A regulates the signal transduction activities of PKB by use of the dominant negative and okadaic acid mutants described above. The studies should define more clearly the interactions that involve PP2A in signaling in vivo. In addition the institution should benefit from the interactions that quality arises from this research with students and faculty and the technologies provided.
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