Signaling Mechanisms Regulating Pituitary Gene Expression

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Pit-1/GHF-1 governs the ontogeny of three distinct pituitary cell types, somatotrophs, lactotrophs, and thyrotrophs. Since all three pituitary cell types express Pit-1, but each expresses a distinct pituitary hormone, factors in addition to Pit-1 must be required for cell-specific expression of GH, PRL, and TSH beta. To accomplish this, Pit-1 establishes a number of combinatorial codes that govern the cell-type specific transcriptional regulation of these hormonal marker genes. These codes can involve both synergistic or inhibitory protein-protein interactions between Pit-1 and other transcription factors. In addition, two functional serine/threonine phosphorylation sites have been identified in Pit-1: Serine 115 and Threonine 220. Both in vivo and in vitro studies have shown that these sites can be targeted by Protein Kinases A and C, as well as cell cycle dependent kinases. One of these sites, T220, is highly conserved among homeodomain transcription factors. Unfortunately, the functional role of phosphorylation of these sites in Pit-1 remains unclear. Mutations of Pit-1 which mimic the phosphorylation of threonine 220, T220D and T220E, reduced the ability of Pit-1 to target Ras and estradiol stimulation to the prolactin promoter. This impaired signaling capability correlated with an inability of the T220D Pit-1 mutant to bind to sites in the prolactin promoter as a monomer. Finally, the established role of Pit-1 in pituitary cell proliferation, as well as the correlation of Pit-1 phosphorylation with cell cycle progression, suggests that the phosphorylation of Pit-1 may serve as a regulatory switch between cell proliferation and differentiation in the Pit-1 lineages. Unifying hypothesis: Phosphorylation of Pit-1 induces structural changes that alter its affinity for distinct DNA binding sites and protein partners. These changes regulate Pit-1¿s ability to respond to specific signaling pathways that mediate growth and cell-specific gene transcription. Overall Goal: The overall goal of this proposal is to determine the physiological effects of, and mechanisms by which, phosphorylation of Pit-1 regulates transcription and proliferation of cells in the Pit-1 lineage. We will use in vitro cell models to determine 1) the effect of Pit-1 phosphorylation on cell cycle progression, 2) the cellular and developmental conditions which induce phosphorylation of Pit-1, 3) changes in Pit-1 structural conformations upon phosphorylation, 4) and the effect of phosphorylation on the ability of Pit-1 to integrate cell specific signaling to its primary target gene promoters in the pituitary. These results will be correlated with changes in cell growth and gene expression in vivo.