Activation and Regulation of Bacterial Tyrosine Kinases

细菌酪氨酸激酶的激活和调节在高等真核生物维持生命的所有过程中，末端磷酸基团从三磷酸腺苷(ATP)转移到特定蛋白靶标上的特定酪氨酸残基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，促进了这种转移。酪氨酸激酶活性的失调与包括几种癌症在内的多种人类疾病有关，这使得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，表明细菌细胞还编码许多独特的酪氨酸激酶，这些酪氨酸激酶在细菌生理学中发挥着同样重要的作用。这些细菌酪氨酸激酶中最大的家族，即副激酶，在整个细菌界中都是保守的，不包含真核细胞酪氨酸激酶的序列特征，在结构上是独特的，并通过不同的但尚未完全了解的机制来激活和调节。这项研究提出，利用实验和计算工具的综合应用，将从结构和机制方面破译BK功能的激活和调节。这项研究将为不同职业阶段的学者提供多学科培训，范围从高中生到博士后实习生。这项工作将得到纽约市立大学杰出的智力环境的推动，学生群体反映了纽约市独特和多样化的人口统计结构。这项研究的长期目标是了解原子细节的激活、活性和副激酶的调节。为了实现这一目标，将确定副激酶催化亚单位在实现有效的自动磷酸化方面所采用的机制/S，以及它们与中和PTPs相互作用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学技术，以全原子计算研究为基础，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自然界用来执行这一具有深远生物后果的简单化学的不同平台，提供对生命王国中蛋白质磷酸化的扩展视角。在这些研究中，将使用由激酶组成的原型大肠杆菌WZC及其同源蛋白酪氨酸磷酸酶WZB。部署来自非致病的、遗传上易处理的生物体的蛋白质将允许在细胞范围内对体外生物物理/生化洞察力进行强有力的验证。这个项目得到了生物科学局分子和细胞生物科学部分子生物物理学分部的支持。这个奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.