Mechanisms of phosphorylation signaling by phosphoprotein phosphatases

摘要 蛋白激酶的磷酸化和磷酸酶的反向去磷酸化是调节最大的 生物过程。在人类细胞中，超过四分之三的细胞蛋白是磷酸化的。这个 一个给定的磷酸化位点的占有率反映了激酶活性和磷酸化活性之间的平衡 磷酸酶。为了了解蛋白质磷酸化的可逆性，我们必须研究正向 并通过连接它们共同的底物上的激酶和磷酸酶来逆转反应。蛋白 磷酸化由500多种蛋白激酶催化，然而，大多数蛋白质去磷酸化是 仅由七种磷酸蛋白磷酸酶(PPP)执行。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。我的实验室里的研究 专注于通过建立磷酸酶底物将磷酸酶整合到细胞信号网络中 关系，识别对立的激酶，并确定调节输入。我的实验室独一无二 通过将定量蛋白质组学和 磷蛋白质组学方法在细胞中的最小信号单位的重建在体外。PPP表单 具有重叠亚基的多聚体全酶，其功能是不同的实体，这阻碍了机械性 全酶在细胞中的特定功能和调节的研究。我的研究计划直接解决了这个问题 通过在全系统级别的小区中研究PPP信令网络以及在 体外培养。通过结合细胞内发现和体外验证以及机械还原，我们将区分直接 从间接效应或细胞补偿机制来确定具体的贡献 全酶。在本应用程序中，我们将重点介绍PP6，这是一种具有有限的子单元多样性的PPP 准确地反映了PPP全酶的组合性质。总的来说，这些发现将进一步推动我们的 了解全酶特异性磷酸酶的功能、底物偏好和调节，以及 通过将磷酸酶和激酶生物整合到功能网络中，将它们联系起来。定义互惠PPP -蛋白激酶的调节和共享底物的反对将提供对如何生理的洞察 过程是由可逆的磷酸化控制的。我们将继续发展和实施创新 蛋白质组学和细胞生物学的方法，以更好地解决这些和新出现的问题。我们预见到了这一点 努力成为磷酸化信号转导社区的资源，以及未来研究的框架 变成了磷酸酶生物学。我们将共享我们的数据、试剂和实验方法，并生成 易于访问的磷酸酶底物数据库，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.