Revealing Secrets of Cellular Phosphorylation Systems Using Biological Mass Spectrometry

所有生物体都含有蛋白质（例如酶），这些蛋白质进行必要的化学反应，使细胞生命得以存在。为了将蛋白质功能限制在细胞需要的时候，自然界已经开发出了能够以可逆方式改变蛋白质三维形状的分子系统，从而将蛋白质功能“打开”和“关闭”。蛋白质磷酸化是一种常见的化学修饰，被所有生物体用来改变蛋白质的形状和功能。介导磷酸分子的添加和去除的酶分别称为激酶和磷酸酶。由于可逆磷酸化的微妙平衡对于几乎所有细胞过程的正常运作都是必不可少的，因此所有激酶/磷酸酶的活性都受到严格控制。研究磷酸化系统的最强大的分析仪器之一是质谱仪。基于质谱的蛋白质组测序技术相当于基因组测序技术的蛋白质，因此有可能发现磷酸化系统的基本方面。因此，我们研究计划的长期目标是整合质谱，生物化学和细胞生物学技术，以更好地了解预测调节重要细胞过程的研究不足的激酶和磷酸酶的细胞作用。 我们建议利用先进的质谱技术对人类YVH 1（hYVH 1），一个很少研究的非典型双特异性磷酸酶（DUSP）亚家族成员的研究。也被称为DUSP 12，YVH 1直系同源物在动物和植物王国中广泛保守，我们实验室和其他人的最新研究提供了证据，证明hYVH 1是蛋白质合成，细胞存活和细胞DNA含量的调节剂。此外，我们最近报道了hYVH 1质谱研究的结果，发现hYVH 1与各种核糖核蛋白颗粒（RNP）和功能作为一种新的应力颗粒解体因子。这种最初的质谱分析也提供了初步证据，即一种被称为Src的充分表征的酪氨酸激酶可能控制hYVH 1的活性。我们已经证实，Src可以磷酸化人类细胞中的hYVH 1，并已测序一个新的磷酸酪氨酸位点使用质谱。我们现在有兴趣彻底表征Src介导的hYVH 1磷酸化的功能意义。具体来说，我们感兴趣的是确定这种磷酸化修饰如何改变hYVH 1的形状，以改变hYVH 1在细胞中的功能。在我们的建议中采用的方法将转移到其他鲜为人知的磷酸酶的研究，而研究结果将加深我们对hYVH 1在细胞调节中的作用的理解。

All organisms contain proteins (e.g. enzymes) that perform the necessary chemical reactions that allow cellular life to exist. In order to restrict protein function to when it is needed by the cell, nature has developed molecular systems that can change the three-dimensional shape of proteins in a reversible fashion, and thus turn protein function "on" and "off". Protein phosphorylation is a common chemical modification utilized by all living organisms to alter the shape and function of proteins. The enzymes that mediate the addition and removal of the phosphate molecules are called kinases and phosphatases respectively. Since a delicate balance of reversible phosphorylation is essential for nearly all cellular processes to function properly, the activities of all kinases/phosphatases are under tight control. One of the most powerful analytical instruments to study phosphorylation systems is a mass spectrometer. Mass spectrometry-based proteome sequencing techniques are the protein equivalent to genome sequencing techniques, and thus have the potential to discover fundamental aspects of phosphorylation systems. Accordingly, the long-term goal of our research program is to integrate mass spectrometry, biochemistry, and cell biology techniques to better understand the cellular role of poorly studied kinases and phosphatases predicted to regulate important cellular processes.    We propose to utilize advanced mass spectrometry techniques towards the study of human YVH1 (hYVH1), a scantily studied member of the atypical dual specificity phosphatase (DUSP) subfamily. Also known as DUSP12, YVH1 orthologues are widely conserved in the animal and plant kingdom, and recent studies from our laboratory and others have provided evidence that hYVH1 is a regulator of protein synthesis, cell survival, and cellular DNA content. Moreover, we recently reported the results of a hYVH1 mass spectrometry study that discovered hYVH1 associates with various ribonucleoprotein particles (RNP) and functions as a novel stress granule disassembly factor. This initial mass spectrometry analysis also provided preliminary evidence that a well characterized tyrosine kinase known as Src, may control the activities of hYVH1. We have confirmed that Src can phosphorylate hYVH1 in human cells and have sequenced a novel phospho-tyrosine site using mass spectrometry. We are now interested in thoroughly characterizing the functional significance of Src-mediated phosphorylation of hYVH1. Specifically, we are interested in determining how this phosphorylation modification changes the shape of hYVH1 in order to alter the function of hYVH1 in the cell. 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 of hYVH1 in cellular regulation.