Function of Initiation Factor Kinases in Regulating Dictyostelium Development

当营养缺乏时，变形虫盘基骨柱停止分裂并聚集成多细胞实体。多细胞发育之后是独特细胞类型的细胞分化。该奖项的PI将解决环境信号（特别是氨基酸剥夺）如何触发变形虫聚集的基本问题。PI先前发现，蛋白质GCN2是盘齿柱中氨基酸剥夺的传感器。盘基骨柱实际上有三种GCN2蛋白，称为Ifks（起始因子激酶），它们在蛋白质合成中很重要。GCN2有两个不同的结构域：一个是磷酸化eIF2 α亚基的蛋白激酶结构域，另一个是结合在氨基酸缺乏过程中积累的不带电trna的组氨酸- trna合成酶（HisRS）结构域。PI提出了一个模型，表明GCN2与不带电的tRNA结合，从而引起GCN2的构象改变。这种变化促进了eIF2结构域的磷酸化，从而调节了其他蛋白质的翻译。有了这个奖项，PI将通过以下方法来测试该模型：1)将GCN2蛋白的遗传分析扩展到IfkC， 2)创建eIF-2alpha的突变形式，以及3)使用蛋白质组学方法寻找Ifks的新底物。除了发现GCN2蛋白在调节盘基骨柱发育中的功能外，本研究的发现还可能揭示GCN2蛋白和其他eIF2激酶在包括人类在内的其他生物发育中的功能。过去，PIs实验室的大部分工作都是由本科生独立完成的。拟议的实验将继续这样做，同时还将开展适当和广泛的指导活动，以协助培训下一代科学家。

When starved of nutrients, Dictyostelium amoebae stop dividing and aggregate into multicellular entities. Multicellular development is followed by the cellular differentiation of unique cell types. The PI of this award will address the fundamental question of how environmental signals (amino acid deprivation in particular) trigger the amoeba to aggregate. The PI previously found that the protein GCN2 is a sensor of amino acid deprivation in Dictyostelium. Dictyostelium actually has three GCN2 proteins, termed Ifks (initiation factor kinases) that are important in the synthesis of proteins. GCN2 has two distinct domains: a protein kinase domain that phosphorylates the alpha subunit of eIF2, and a histidyl-tRNA synthetase (HisRS) domain that binds uncharged tRNAs that accumulate during amino acid starvation. The PI has proposed a model suggesting that GCN2 binds to uncharged tRNA, which elicits a conformation change in GCN2. This change facilitates phosphorylation of the eIF2 domain and, thus, regulates translation of other proteins. With this award, the PI will test this model by 1) extending their genetic analyses of GCN2 proteins to IfkC, 2) creating mutant forms of eIF-2alpha, and 3) searching for new substrates of the Ifks using a proteomics approach. In addition to discovering how GCN2 proteins function in regulating Dictyostelium development, the findings from this study may reveal the functions of GCN2 proteins and other eIF2 kinases in the development of other organisms, including humans. Much of the past work in the PIs laboratory was accomplished by undergraduates performing independent and honors research. This will continue for the proposed experiments, along with appropriate and extensive mentoring activities to assist in training the next generation of scientists.