Signaling Mechanisms Regulating Pituitary Gene Expression

PIT-1/GHF-1调控三种不同类型的垂体腺细胞的个体发育，生长激素细胞、催乳素细胞和 促甲状腺激素。由于三种类型的垂体细胞都表达Pit-1，但每一种都表达一种不同的垂体激素， GH、PRL和TSHβ的细胞特异性表达必须需要Pit-1以外的因子。至 为此，Pit-1建立了许多组合代码，这些组合代码管理特定的单元格类型 这些荷尔蒙标记基因的转录调控。这些代码可以涉及协同或 抑制Pit-1和其他转录因子之间的蛋白质-蛋白质相互作用。此外，还有两种功能 已在Pit-1中确定了丝氨酸/苏氨酸磷酸化位点：丝氨酸115和苏氨酸220。两个都在 体内和体外研究表明，这些位点可以被蛋白激酶A和C以及 细胞周期依赖的蛋白激酶。其中一个位点T220在同源结构域转录中高度保守 各种因素。不幸的是，这些位点的磷酸化在Pit-1中的功能作用仍不清楚。突变 模拟苏氨酸220、T220D和T220E的磷酸化的Pit-1降低了Pit-1的活性 靶向RAS和雌二醇对催乳素启动子的刺激。这种受损的信号传递能力与 T220D Pit-1突变体不能作为单体与催乳素启动子中的位点结合。最后， Pit-1在垂体细胞增殖中的作用及其与细胞的相关性 周期进程，提示Pit-1的磷酸化可能作为细胞之间的调节开关 Pit-1谱系的增殖和分化。 统一假设：Pit-1的磷酸化导致结构变化，改变其与不同DNA的亲和力 结合位点和蛋白质伙伴。这些变化调节了Pit-1？S对特定信号的反应能力 调节生长和细胞特异性基因转录的途径。 总体目标：本提案的总体目标是通过以下方式确定的生理影响和机制 其中，Pit-1的磷酸化调控Pit-1谱系中细胞的转录和增殖。 我们将使用体外细胞模型来确定1)Pit-1磷酸化对细胞周期进程的影响，2) 诱导Pit-1磷酸化的细胞和发育条件，3)Pit-1结构的变化 磷酸化的构象，4)磷酸化对Pit-1整合细胞能力的影响 在脑下垂体中向其主要靶基因启动子发出特异性信号。这些结果将与 体内细胞生长和基因表达的变化。

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.