Mechanisms of phosphorylation signaling by phosphoprotein phosphatases

摘要 激酶引起的蛋白质磷酸化和磷酸酶引起的蛋白匡威去磷酸化作用， 生物过程。在人类细胞中，超过四分之三的细胞蛋白质被磷酸化。的 给定磷酸化位点的占有率反映了激酶活性和 磷酸酶为了理解蛋白质磷酸化的可逆性，我们必须研究其正向作用。 以及通过将激酶和磷酸酶连接到它们共同感兴趣的底物上来进行逆反应。蛋白 磷酸化是由500多种蛋白激酶催化的，然而，大多数蛋白去磷酸化是 仅由七种磷蛋白磷酸酶（PPPs）进行。虽然在这方面取得了巨大的进展， 通过激酶破译细胞信号，对磷酸酶的了解要少得多。我实验室的研究 通过构建磷酸酶底物， 关系，识别对立的激酶，并确定监管投入。我的实验室 通过结合定量蛋白质组学和 磷酸化蛋白质组学方法在细胞中进行体外最小信号单位的重建。购买力平价表 多聚体全酶具有重叠的亚基，作为不同的实体发挥功能，这阻碍了 研究全酶在细胞中的特殊功能和调节。我的研究计划直接解决了这个问题 通过研究PPP信令网络的系统范围内的细胞和隔离组件， 体外通过结合细胞内发现和体外验证和机制还原，我们将区分直接 间接效应或细胞补偿机制，并确定特定的 全酶在本申请中，我们将重点关注PP6，这是一种亚基多样性有限的PPP， 准确地反映了PPP全酶的组合性质。总的来说，这些发现将进一步促进我们的 了解全酶特异性磷酸酶功能，底物偏好和调节， 将磷酸酶和激酶生物学整合到功能网络中。定义互惠PPP - 蛋白激酶的调节和共同底物的反对将提供洞察如何生理 过程由可逆磷酸化控制。我们将继续开发和实施创新的 蛋白质组学和细胞生物学的方法，以更好地解决这些问题和新出现的问题。我们设想 致力于成为磷酸化信号社区的资源，以及未来研究的框架 磷酸酶生物学我们将分享我们的数据，试剂和实验方法，并产生一个 容易获得的数据库的磷酸酶底物，我们将确定。

ABSTRACT Protein phosphorylation by kinases with its converse dephosphorylation by phosphatases regulates most biological processes. In human cells, more than three-quarters of cellular proteins are phosphorylated. The occupancy of a given phosphorylation site reflects the balance between the activities of kinases and phosphatases. To understand the reversible nature of protein phosphorylation, we must investigate the forward and reverse reaction by connecting kinases and phosphatases on their shared substrates of interest. Protein phosphorylation is catalyzed by more than 500 protein kinases, however, most protein dephosphorylation is carried out by only seven phosphoprotein phosphatases (PPPs). While there has been tremendous progress in deciphering cellular signaling by kinases, much less is known about phosphatases. Research in my laboratory is focused on integrating phosphatases into cellular signaling networks by establishing phosphatase-substrate relationships, identifying opposing kinases, and determining regulatory inputs. My laboratory is uniquely positioned to address these outstanding challenges by combining quantitative proteomics and phosphoproteomics approaches in cells with reconstitution of minimal signaling units in vitro. PPP form multimeric holoenzymes with overlapping subunits that function as distinct entities, which hampers mechanistic studies of holoenzyme specific functions and regulation in cells. My research program directly addresses this problem by studying PPP signaling networks on a system-wide level in cells and with isolated components in vitro. By combining in-cell discovery and in vitro validation and mechanistic reduction, we will distinguish direct from indirect effects or cellular compensation mechanisms and determine the contribution of specific holoenzymes. In this application we will focus on PP6, a PPP with limited subunit diversity that nonetheless accurately reflects the combinatorial nature of PPP holoenzymes. Collectively, these findings will further our understanding of holoenzyme specific phosphatase function, substrate preferences, and regulation, and connect phosphatase and kinase biology by integrating them into functional networks. Defining reciprocal PPP – protein kinase regulation and opposition of shared substrates will provide insights into how physiological processes are controlled by reversible phosphorylation. We will continue to develop and implement innovative approaches in proteomics and cell biology to better address these and emerging questions. We envision this work to be a resource for the phosphorylation signaling community, as well as a framework for future research into phosphatase biology. We will share our data, reagents, and experimental approaches and generate an easily accessible database for the phosphatase substrates we will identify.