Signaling Mechanisms Regulating Pituitary Gene Expression

Pit-1/GHF-1 控制三种不同垂体细胞类型的个体发育：生长激素细胞、泌乳细胞和 促甲状腺素。由于所有三种垂体细胞类型都表达 Pit-1，但每种细胞都表达不同的垂体激素， GH、PRL 和 TSH β 的细胞特异性表达必须需要除 Pit-1 之外的因子。至 为了实现这一目标，Pit-1 建立了许多组合代码来控制特定细胞类型 这些激素标记基因的转录调控。这些代码可以涉及协同或 Pit-1 和其他转录因子之间的抑制蛋白-蛋白相互作用。此外，还有两个功能 Pit-1 中已鉴定出丝氨酸/苏氨酸磷酸化位点：丝氨酸 115 和苏氨酸 220。 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 细胞周期依赖性激酶。其中一个位点 T220 在同源域转录中高度保守 因素。不幸的是，Pit-1 中这些位点磷酸化的功能作用仍不清楚。突变 Pit-1 模仿苏氨酸 220、T220D 和 T220E 的磷酸化，降低了 Pit-1 的能力 Ras 和雌二醇刺激催乳素启动子。这种受损的信号传递能力与 T220D Pit-1 突变体无法作为单体与催乳素启动子中的位点结合。最后， 确定了 Pit-1 在垂体细胞增殖中的作用，以及 Pit-1 磷酸化与细胞的相关性 循环进程，表明 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 的磷酸化会引起结构变化，从而改变其对不同 DNA 的亲和力 结合位点和蛋白质伴侣。这些变化调节 Pit-1 响应特定信号的能力 介导生长和细胞特异性基因转录的途径。 总体目标：该提案的总体目标是确定生理效应和机制 其中，Pit-1 的磷酸化调节 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 磷酸化后的构象，4) 以及磷酸化对 Pit-1 整合细胞能力的影响 特定信号传导至垂体中的主要靶基因启动子。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.