Activation and Regulation of Bacterial Tyrosine Kinases

标题：细菌酪氨酸激酶的激活和调控末端磷酸基团从三磷酸腺苷（ATP）转移到特定蛋白靶点上的特定酪氨酸残基，对高等真核生物维持生命的所有过程具有深远的调控作用。事实上，有近100种不同的人类酶，被称为酪氨酸激酶，促进这种转移。酪氨酸激酶活性的失调与多种人类疾病有关，包括几种癌症，使这些酶成为治疗干预的理想靶点。酪氨酸磷酸化驱动的信号转导被认为局限于真核生物的生命领域，事实上，许多不同的酪氨酸激酶的存在被认为是多细胞的标志。然而，最近的发现提供了明确的证据，表明细菌细胞也编码许多独特的酪氨酸激酶，这些酪氨酸激酶在细菌生理中起着类似的重要作用。这些细菌酪氨酸激酶中最大的家族，by激酶，在整个细菌王国中都是保守的，不包含真核酪氨酸激酶的序列特征，结构独特，通过不同的，但尚不完全了解的机制被激活和调节。本研究拟采用实验与计算相结合的方法，从结构和机制上解读by -激酶功能的激活与调控。本研究将为不同职业阶段的广泛学者提供多学科的培训，从高中生到博士后学员。纽约城市学院优秀的学术环境将为这项工作提供便利，该学院的学生群体反映了纽约市独特而多样化的人口结构。这项研究的长期目标是在原子细节上了解by激酶的激活、活性和调控。为了实现这一目标，将确定by激酶的催化亚基实现有效自磷酸化的机制，以及它们与抵消PTPs相互作用以逆转这种共价修饰的性质。拟议的研究将利用溶液态核磁共振和高分辨率质谱技术，这些技术由全原子计算研究提供信息，并通过体外和细胞内生化实验验证。拟议的研究将通过详细了解自然界利用的不同平台来进行这种具有深远生物学后果的简单化学反应，为生命王国中的蛋白质磷酸化提供一个扩展的视角。原型BY激酶大肠杆菌Wzc及其同源蛋白酪氨酸磷酸酶Wzb将用于这些研究。从非致病性、遗传易感的生物体中部署蛋白质，将允许在细胞背景下对体外生物物理/生化见解进行强有力的验证。该项目得到了生物科学理事会分子和细胞生物科学部分子生物物理组的支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Title: Activation and Regulation of Bacterial Tyrosine KinasesThe transfer of the terminal phosphate group from adenosine triphosphate (ATP) to specific tyrosine residues on defined protein targets has profound regulatory effects on all the processes that sustain life in higher eukaryotes. Indeed, there are nearly a hundred distinct human enzymes, known as tyrosine kinases, that facilitate this transfer. Dysregulation of tyrosine kinase activity has been implicated in a variety of human diseases including several cancers making these enzymes ideal targets for therapeutic intervention. Tyrosine phosphorylation driven signal transduction was considered to be restricted to the eukaryotic domain of life and indeed the presence of many distinct tyrosine kinases was believed to be the hallmark of multicellularity. However, recent discoveries have provided clear evidence that bacterial cells also encode numerous unique tyrosine kinases that play a similarly important role in bacterial physiology. The largest family of these bacterial tyrosine kinases, the BY-kinases, are conserved across the bacterial kingdom, contain none of the sequence signatures characteristic of eukaryotic tyrosine kinases, are structurally unique, and are activated and regulated through distinct, yet incompletely understood, mechanisms. The research proposed, using an integrated application of experimental and computational tools will decipher in terms of structure and mechanism, the activation and regulation of BY-kinase function. This research will provide multi-disciplinary training to a broad range of scholars at various career stages ranging from high-school students to postdoctoral trainees. The work will be facilitated by the outstanding intellectual environment at the City College of New York enabled by a student body that reflects the unique and diverse demographics of New York City.The long-term goal of the research is to understand the activation, activity and regulation of BY-kinases in atomic detail. Towards that goal, the mechanism/s employed by the catalytic subunit of BY-kinases in achieving efficient auto-phosphorylation and the nature of their interaction with counteracting PTPs to reverse this covalent modification, will be determined. The proposed research will utilize solution-state nuclear magnetic resonance and high-resolution mass-spectrometric techniques informed by all-atom computational studies and validated by biochemical experiments in vitro and in cell. The proposed studies will provide an expanded view of protein phosphorylation in life’s kingdoms through detailed insight into a divergent platform utilized by nature to perform this simple chemistry that has profound biological consequences. The archetypal BY kinase, Escherichia coli Wzc, and its cognate protein tyrosine phosphatase, Wzb will be utilized in these studies. Deploying proteins from a non-pathogenic, genetically tractable organism will allow the robust validation of the in vitro biophysical/biochemical insights within the cellular context. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.