Revealing Secrets of Cellular Phosphorylation Systems Using Mass Spectrometry

蛋白质磷酸化是一种化学修饰，被所有生物用来改变蛋白质的功能，控制细胞通讯、生长、新陈代谢、分化和生存等过程。在蛋白质中添加和移除磷酸盐分子的酶分别称为激动酶和磷酸酶。由于可逆磷酸化的微妙平衡对细胞内稳态至关重要，所有激酶/磷酸酶的活动都受到密切关注。生物质谱学是研究磷酸化体系的最有力的分析工具之一。基于质谱学的方法可以快速鉴定含有激酶和磷酸酶的功能多蛋白复合体，并直接定位磷酸盐加成的位置，以便更好地研究蛋白质修饰的后果。因此，我们研究计划的长期目标是整合质谱学、生物化学和细胞生物学技术，以破译调控鲜为人知的激酶和磷酸酶的机制细节，预计这些酶和磷酸酶将调控基本的细胞过程。 然而，目前我们正将大部分注意力集中在一种知之甚少的磷酸酶YVH1(也称为DUSP12)上。人YVH1(HYVH1)是一种在人体组织中广泛表达的双特异性磷酸酶。YVH1基因广泛存在于动植物界，所有的基因产物除了其磷酸酶结构域外，还具有一个特征的锌结合结构域。值得注意的是，尽管YVH1是22年前发现的第一个真核数字信号处理器，但人们对其在人类细胞中的具体生物学功能知之甚少。 *最近在2014年收购了一种新的多路复用质谱学系统，该系统能够用于发现/靶向蛋白质组学、翻译后修饰图谱、结构蛋白质动力学和交互作用组分析等生物学应用，这从根本上改变了我们研究计划的分析深度。利用这个系统，我们最近利用经典的基于质谱学的亲和层析技术从人类细胞系中分离出了新的hYVH1结合蛋白。我们的初步发现表明，hYVH1在控制基因转化为功能蛋白的速度(信使核糖核酸调节)的细胞成分中是活跃的。我们现在有兴趣利用下一代亲和捕获方法和磁珠技术来彻底描绘和功能验证hYVH1互动组。后续实验将包括检查细胞如何控制hYVH1对这些大分子工作站的访问。我们建议中使用的方法将被转移到其他鲜为人知的磷酸酶的研究中，而这些发现将加深我们对DSP在细胞调节中所起作用的理解。

Protein phosphorylation is a chemical modification utilized by all living organisms to alter the function of proteins and controls processes such as cellular communication, growth, metabolism, differentiation, and survival. The enzymes that add and remove phosphate molecules to proteins are called kinases and phosphatases respectively.  Since a delicate balance of reversible phosphorylation is essential for cellular homeostasis, the actions of all kinases/phosphatases are under tight surveillance. One of the most powerful analytical tools to study phosphorylation systems is biological mass spectrometry. Mass spectrometry based methods can rapidly identify functional multiprotein complexes containing kinases and phosphatases as well as directly map the location of phosphate addition to better study the consequence of the protein modification. Accordingly, the long term goal of our research program is to integrate mass spectrometry, biochemistry, and cell biology techniques to decipher the mechanistic details regulating poorly understood kinases and phosphatases predicted to regulate fundamental cellular processes.      Currently we are directing much of our focus on a poorly understood phosphatase identified as YVH1 (also known as DUSP12). Human YVH1 (hYVH1) is a so called dual specificity phosphatase (DSP) widely expressed in human tissues. The YVH1 genes are found throughout the animal and plant kingdom and all gene products possess a characteristic zinc binding domain in addition to their phosphatase domain. Remarkably, although YVH1 was the first eukaryotic DSP identified over 22 years ago, relatively little is known about its specific biological function in human cells.      The recent acquisition in 2014 of a new multiplexed mass spectrometry system capable of biological applications such as discovery/targeted proteomics, mapping of post translational modifications, structural protein dynamics, and interactome analysis has fundamentally altered the depth of analysis for our research program. Utilizing this system, we have recently isolated novel hYVH1 associating proteins from human cell lines using classical mass spectrometry based affinity chromatography techniques. Our preliminary findings suggest that hYVH1 is active at cellular components that control the rate at which genes are transformed into functional proteins (mRNA regulation). We are now interested in employing next generation affinity capture methods and magnetic bead technology to thoroughly delineate and functionally validate the hYVH1 interactome. Follow-up experiments will include examining how the cell controls the access of hYVH1 to these macromolecular workstations.  The methods employed within our proposal will be transferrable to the study of other poorly understood phosphatases while the findings will deepen our understanding of the role DSPs play in cellular regulation.