Activation and Regulation of Bacterial Tyrosine Kinases

职务名称：细菌酪氨酸激酶的激活与调控三磷酸腺苷（ATP）的末端磷酸基团转移到特定蛋白质靶点上的特定酪氨酸残基对维持高等真核生物生命的所有过程具有深远的调节作用。事实上，有近百种不同的人类酶，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生命的真核域，并且实际上许多不同酪氨酸激酶的存在被认为是多细胞性的标志。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，在细菌生理学中发挥类似的重要作用。这些细菌酪氨酸激酶的最大家族，即BY激酶，在细菌界中是保守的，不包含真核酪氨酸激酶的序列特征，结构独特，并且通过不同但不完全理解的机制被激活和调节。该研究提出，使用实验和计算工具的综合应用，将从结构和机制方面破译BY-激酶功能的激活和调节。这项研究将提供多学科的培训，以广泛的学者在各个职业阶段，从高中生到博士后学员。这项工作将由纽约城市学院杰出的学术环境促进，该学院的学生团体反映了纽约市独特而多样的人口统计数据。这项研究的长期目标是了解原子细节中BY激酶的激活、活性和调节。为了实现这一目标，将确定BY激酶的催化亚基在实现有效的自磷酸化中所采用的机制以及它们与抵消PTP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状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的生化实验进行验证。拟议的研究将通过详细了解大自然利用的不同平台来执行这种具有深远生物学后果的简单化学反应，提供生命王国中蛋白质磷酸化的扩展视图。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。部署来自非致病性的、遗传上易处理的生物体的蛋白质将允许在细胞背景内对体外生物物理/生物化学见解进行稳健验证。该项目由生物科学理事会分子和细胞生物科学部的分子生物物理学小组支持。该奖项反映了NSF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.