Dawn L. Duval - US grants

DESCRIPTION (taken from the application) Transcriptional activity is dependent not only on the binding of transcription factors to cis-acting elements, but also on the ability of those factors to recruit a complex hierarchy of proteins to stabilize the basal transcriptional machinery. Basal lactotroph specific expression of the rat prolactin (rPRL) promoter is dependent on the interaction of the proto-oncoprotein c-Ets-1, a member of the -Ets family of transcription factors, with the pituitary-specific transcription factor, Pit-1, at a composite Ets/Pit-1 DNA binding element in the rPRL promoter. While each of these factors alone is capable of activating rPRL gene transcription, the combination of these two transcription factors results in a marked synergistic response. Although the precise mechanism for this synergy remains unknown, it is clear that physical interaction of these two factors is required. Recent studies suggest that the activity of Pit-1 and Ets-1 may be determined by the formation of co-regulatory complexes. Thus, I hypothesize that the Pit-1/Ets-1 complex mediates the synergistic response of the rPRL promoter by recruiting specific transcription regulatory proteins. The goals of this study are to map the specific amino acids of Pit-1 and Ets-1 responsible for their physical and functional interaction, and to use liquid chromatography/mass spectrometry methods to identify the protein components that are recruited to the Ets-1/Pit-1 complex. Therefore, these studies are likely to provide critical insights into the molecular mechanisms underlying combinatorial factor interactions and how they mediate synergistic responses. Finally, these studies are important to my career development, as they will allow me to gain expertise in the cutting edge field of proteomics and protein structure-function relationships, which I can then utilize to establish my career as an independent investigator.

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[unreadable]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.