Role of DNA binding in the regulation and function of ribosomal S6 kinase 2

核糖体蛋白S6激酶(S6K)是丝氨酸蛋白激酶AGC家族中的一员，也包括PKA、PKB(Akt)、PKCs等。细胞模型和动物模型的生化和遗传学研究表明，S6K在调节细胞生长、大小和能量代谢中起主要作用。磷脂酰肌醇3-激酶(PI3K)和哺乳动物雷帕霉素靶标(MTOR)这两条主要的信号转导通路协调S6K的活性，以响应细胞外和细胞内的刺激，如生长因子、有丝分裂原、代谢产物和营养物质。在激活状态下，S6K转位到离散的细胞室/多酶复合体，在那里它们与多种底物相互作用并磷酸化，这些底物参与调节翻译、RNA加工、细胞骨架重排、细胞生长和生存。越来越多的证据表明，S6K信号与多种人类疾病有关，包括糖尿病、衰老和癌症。在哺乳动物细胞中，S6K有两种亚型，分别称为S6K1和S6K2。S6K的两种异构体都有核质穿梭的报道，但它们的核功能还不清楚。这项拨款申请是基于我们的新发现，这些发现暗示了S6K2在转录调控中的作用。我们提供的证据表明，S6K2在C端的自身抑制域上有一个AT-钩状DNA结合域，它介导了与DNA的特异性相互作用。我们还发现，S6K2的活性是由与DNA的直接相互作用以剂量依赖的方式诱导的。此外，对AT-Hook基序的突变研究揭示了它在调节S6K2促生存信号中的重要性。为了进一步推进这些原始发现，我们建议通过一系列生物物理、生化和细胞方法来研究S6K2-DNA相互作用的分子机制和S6K2激活DNA结合的替代模型。我们的工作还将集中在探索S6K2和转录因子之间的合作相互作用模式，以靶向一组特定基因的启动子和增强子区，这涉及到S6K2介导的细胞过程的调节。将利用哺乳动物细胞模型和裸鼠异种移植研究来研究S6K2 AT-Hook基序在转录、核糖体生物发生、细胞生存和生长调节中的作用。这项研究有望对S6K2的核功能及其在转录调控中的意义有更深入的了解。S6K2-DNA相互作用的独特特征不仅具有学术意义，而且可能为药物开发提供新的见解。

Ribosomal protein S6 kinase (S6K) is a member of the AGC family of Ser Thr kinases which also includes PKA, PKB (Akt), PKCs etc. Biochemical and genetic studies in cell-based and animal models have provided evidence that S6K is a principal player in the regulation of cell growth, size and energy metabolism. Two major signal transduction pathways, phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin (mTOR), coordinate the activity of S6Ks in response to extracellular and intracellular stimuli, such as growth factors, mitogens, metabolites and nutrients. In an activate state, S6Ks translocate to discrete cellular compartments/multienzyme complexes, where they interact with and phosphorylate diverse substrates implicated in the regulation of translation, RNA processing, cytoskeletal rearrangement, cell growth and survival. A growing body of evidence links S6K signalling to various human pathologies, including diabetes, ageing and cancer. In mammalian cells, there are two isoforms of S6K, termed S6K1 and S6K2. Nucleocytoplasmic shuttling has been reported for both isoforms of S6Ks, but their nuclear functions are not well understood. This grant application is based on our novel findings which imply the role of S6K2 in the regulation of transcription. We provide the evidence that S6K2 possesses an AT-hook DNA-binding domain at the C-terminal autoinhibitory domain, which mediates specific interaction with DNA. We also found that the activity of S6K2 is induced by direct interaction with DNA in a dose-dependent manner. Furthermore, mutational studies of the AT-hook motif revealed its importance in the regulation of S6K2 pro-survival signalling. To further advance these original findings, we propose to study molecular mechanisms of S6K2-DNA interaction and an alternative model of S6K2 activation upon DNA binding by employing a range of biophysical, biochemical and cellular approaches. Our efforts will be also focused on exploring a cooperative mode of interaction between S6K2 and transcription factors in targeting promoter and enhancer regions of a specific set of genes, implicated in the regulation of S6K2-mediated cellular processes. Mammalian cell models and xenograft studies in nude mice will be employed to study the role of the S6K2 AT-hook motif in the regulation of transcription, ribosome biogenesis, cell survival and growth. It is expected that the proposed study will give deeper insight into nuclear functions of S6K2 and its implication in the regulation of transcription. Unique features of the S6K2-DNA interaction are not only of academic interest, but may also provide novel insights applicable to drug development.