Mechanisms of phosphorylation signaling by phosphoprotein phosphatases

磷蛋白磷酸酶磷酸化信号传导机制

• 批准号: 9490395
• 负责人: Arminja Nadine Kettenbach
• 金额: $ 40.34万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-05 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9490395
• 关键词: Address; Biological Process; Biology; Cells; Cellular biology; Communities; Data; Databases; Disease; Equilibrium; Financial compensation; Holoenzymes; Human; In Vitro; Knowledge; Laboratories; Malignant Neoplasms; Mutation; Nature; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological Processes; Positioning Attribute; Protein Dephosphorylation; Protein Kinase; Protein phosphatase; Proteins; Proteomics; Reaction; Reagent; Regulation; Research; Resources; Signal Transduction; System; Therapeutic Intervention; Validation; Work; combinatorial; human disease; innovation; insight; interest; new therapeutic target; phosphoproteomics; preference; programs; protein phosphatase 6; reconstitution

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.

抽象的 激酶引起的蛋白质磷酸化及其相反的磷酸酶去磷酸化调节大多数 生物过程。在人体细胞中，超过四分之三的细胞蛋白被磷酸化。这 给定磷酸化位点的占据反映了激酶活性和 磷酸酶。为了了解蛋白质磷酸化的可逆性质，我们必须研究正向磷酸化 通过将激酶和磷酸酶连接到其共享的感兴趣底物上来进行逆反应。蛋白质 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 仅由七种磷蛋白磷酸酶（PPP）完成。虽然已经取得了巨大的进步 人们对激酶的细胞信号传导了解甚少，但对磷酸酶的了解却少之又少。在我的实验室进行研究 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，识别相反的激酶，并确定监管输入。我的实验室很独特 旨在通过结合定量蛋白质组学和 磷酸蛋白质组学方法通过在体外重建最小信号单元来进行细胞研究。 PPP形式 具有重叠亚基的多聚体全酶作为不同的实体发挥作用，这阻碍了机械化 研究全酶在细胞中的特定功能和调节。我的研究计划直接解决这个问题 通过研究小区系统级的 PPP 信号网络以及小区中的隔离组件来解决问题 体外。通过结合细胞内发现、体外验证和机制还原，我们将区分直接 从间接效应或细胞补偿机制中确定特定的贡献 全酶。在此应用中，我们将重点关注 PP6，这是一种亚基多样性有限的 PPP，但 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 了解全酶特异性磷酸酶功能、底物偏好和调节，以及 通过将磷酸酶和激酶生物学整合到功能网络中将它们连接起来。定义互惠 PPP – 蛋白激酶调节和共享底物的对抗将提供对生理学如何影响的见解 过程由可逆磷酸化控制。我们将继续开发和实施创新 蛋白质组学和细胞生物学的方法可以更好地解决这些和新出现的问题。我们设想这个 致力于成为磷酸化信号转导界的资源以及未来研究的框架 进入磷酸酶生物学。我们将分享我们的数据、试剂和实验方法，并生成 我们将识别的磷酸酶底物的易于访问的数据库。