【作者】 王斌；

【导师】 何正国；

【作者基本信息】 华中农业大学 ， 微生物学， 2010， 硕士

【摘要】 蛋白质磷酸化是细胞应对外界刺激最通常的反应机制,在细胞信号传导中发挥着重要的作用。然而,在古菌中,我们对磷酸化反应涉及的蛋白激酶以及相应的磷酸化底物知之甚少。在本论文研究中,我们从古菌Sulfolobus tokodaii str.7中第一次鉴定出一个典型的真核型丝／苏氨酸蛋白激酶(STPK) ST1565以及它的作用底物——-FHA结构域包含蛋白ST0829,并对磷酸化反应的条件以及二者保守结构域中的关键氨基酸位点进行了分析,主要取得了以下几个方面的结果：(1)通过细菌双杂交和Pull-down/western等试验证实了ST1565与ST0829能发生物理的相互作用。(2)蛋白激酶ST1565的自磷酸化与底物磷酸化过程均以Mn2+为最佳辅助因子,最适反应温度为45℃,最佳反应pH范围为5.5-7.5。(3)通过一系列定点突变对蛋白激酶功能结构域和FHA结构域的关键位点进行了分析和鉴别,发现多数保守氨基酸残基的突变使磷酸化反应的活性下降,其中T-329是激酶的主要活性位点,而T-326则是负调控位点。(4)ST0829的保守FHA结构域上一些氨基酸的替代突变使其丧失了与ST1565的物理相互作用,并且在进一步的ST0829的FHA结构域的空间模型构建中发现这些突变位点均位于蛋白质的边缘,可能组成潜在的互作结构域与蛋白激酶ST1565发生相互作用。本研究首次在第三域生命——古菌中发现了一个典型的类真核STPK能够相互作用并磷酸化它的FHA蛋白底物,这些结果不仅提高了我们对古菌激酶以及FHA蛋白结构和功能的认识,而且也增强了我们对整个三域生命中根本的磷酸化信号传导机制的理解。更多还原

【Abstract】 Protein phosphorylation is most commonly exploited by cells to allow appropriate responses to various environmental cues, which plays an important role in cell signaling. However, in the Archaea, little is known regarding which proteins are phosphorylated and which kinases are involved. In this study, we have identified, for the first time, a typical eukaryote-like Ser/Thr protein kinase (STPK) ST1565 and its protein partner, a forkhead-associated (FHA) domain-containing protein ST0829 from the archaeon Sulfolobus tokodaii str.7. We analyzed the reactive conditions of phosphorylation, and the critical amino acid residues in the conserved domain of both proteins, the major results were obtained as follows:(1) Using bacterial two-hybird assay and Pull-down/western experiments, the protein kinase ST1565 was shown to physically interact with the FHA domain protein ST0829. (2) Protein kinase ST1565 preferred Mn2+as its cofactor for auto-phosphorylation and for substrate-phosphorylation, at an optimal temperature 45℃and optimal of pH 5.5-7.5. (3) We analyzed and identified the critical amino acid residues in the conserved FHA and kinase domain sites through a series of mutation assays, and most mutional conservative amino acid residues lowered the activity of phosphorylation. Threonine-329 was suggested as a major activation site in the kinase. In contrast, Threonine-326 was a negative regulation site. (4) Several amino acid substitution mutants in the conserved FHA domain sites of ST0829 lost their physical interactions with ST1565, and the structure model for the FHA domain demonstrated that these mutation sites were located at the edge of the protein, and perhaps constituted the potential interaction domain with ST1565.This work presents pioneering work on the third domain of the Archaea, showing that a protein kinase interacts with and phosphorylates its FHA domain protein. These data provide critical information on the structural or functional characteristics of archaeal proteins and can accelerate the understanding of fundamental signaling mechanisms in all three domains of life forms.更多还原