PHAS-I is the first member of a newly discovered family of proteins that regulate the rate-limiting step in translation initiation and that appear to be key mediators of the effects of hormones and growth factors on protein synthesis. Recent evidence suggests that PHAS-I is involved in mitogenic control; and, in preliminary experiments overexpressing PHAS proteins in ras-transformed fibroblasts markedly decreased cell proliferation and promoted reversion to a normal phenotype. The major objective of this proposal is to investigate the regulation and function of this important family, PHAS-I & -II bind to eIF-4E, the mRNA cap binding protein, and inhibit translation in vitro and in cells. Phosphorylation of PHAS-I in response to insulin or growth factors causes dissociation of PHAS-I and eIF-4E, allowing eIF-4E to participate in translation initiation. Rapamycin or increasing cAMP promotes PHAS-I dephosphorylation and increases binding to eIF-4E. PHAS-II is highest in tissues where PHAS-I is lowest. Unlike PHAS-I, PHAS-II is readily phosphorylated by PKA, suggesting that regulation of the two proteins differs. We propose to investigate the signaling mechanisms involved in regulating the proteins. Their phosphorylation in response to agents that regulate association with eIF-4E will be characterized. Peptide mapping and site-directed mutagenesis will be used to identify important phosphorylation sites. Bicistronic mRNAs that allow assessment of cap- dependent protein synthesis will be used to investigate the effects of PHAS proteins on translation in vitro and in cells. Secondary structure will be introduced into the 5'-UTR to investigate the hypothesis that the inhibitory effect of PHAS proteins on translation is greatest with mRNAs that are most dependent of eIF-4E for initiation. Wildtype and phosphorylation-site mutants of PHAS proteins will be introduced into cells by transient expression, microinjection, and stable transfection to investigate the role of PHAS-I & -II in mediating the actions of insulin and growth factors on translation initiation, protein synthesis, and cell proliferation. The diversity of the family will be investigated by attempting to identify new PHAS proteins.
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