Mechanisms of Phosphorylation Signaling by Phosphoprotein Phosphatases

磷蛋白磷酸酶的磷酸化信号传导机制

• 批准号: 10625973
• 负责人: Arminja Nadine Kettenbach
• 金额: $ 44.28万
• 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 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625973
• 关键词: Biochemical; Biological; Catalytic Domain; Cell division; Cell physiology; Cells; Communities; Databases; Disease; Ensure; Enzymes; Equilibrium; Family; Foundations; Goals; Holoenzymes; In Vitro; Knowledge; Maps; Measurement; Mediating; Mitosis; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Protein Dephosphorylation; Protein phosphatase; Proteins; Proteome; Regulation; Research; Role; Signal Transduction; Site; Specificity; Substrate Specificity; Therapeutic Intervention; War; combinatorial; human disease; inhibitor; new therapeutic target; programs; reconstitution; recruit; user-friendly

Abstract Reversible protein phosphorylation is a major regulatory mechanism that controls most cellular processes, including mitosis. A ‘tug of war’ between kinases and phosphatases controls protein activity and function. The precise coordination of both activities is essential for the accurate execution of cell division. Despite substantial progress in deciphering kinase-mediated phosphorylation and its functional consequences, much less is known about phosphatases and how dephosphorylation is regulated. The long-term goal of our research program is to uncover the mechanisms that coordinate and integrate PPPs, kinases, and their shared substrates in the signaling networks that control cell division. The majority of cellular dephosphorylation is carried out by the 7 catalytic phosphatase subunits of the phosphoprotein phosphatase (PPP) family. Despite the apparent simplicity suggested by the small number of catalytic PPP enzymes, complexity and specificity arise through the formation of holoenzymes. Each holoenzyme functions as a distinct entity. The non-catalytic subunits modulate the activity of the catalytic subunits, enabling substrate specificity, dictating subcellular localization, and ensuring appropriate regulation. Combinatorially, there are several hundred functional PPP holoenzymes. There are key gaps in knowledge and challenges in studying PPP function and mechanisms of dephosphorylation. Although hundreds of thousands of phosphorylated sites on proteins have been identified, only ~450 have been matched to be dephosphorylated by a specific PPP holoenzyme. This is in part due to the lack of holoenzyme-specific inhibitors and the complexity of PPP holoenzymes. To overcome these obstacles and fill these gaps in knowledge, we will (i) identify holoenzyme-specific substrates; (ii) dissect mechanisms of substrate recruitment and site-specific dephosphorylation; and (iii) determine regulatory inputs governing specific PPP holoenzymes. We have developed new cell biological and biochemical approaches to investigate PPP signaling. We plan to map the PPP substrate space comprehensively, providing a foundation for studying PPPs for our lab and the signaling community by sharing our findings broadly and openly via a user-friendly database that we will maintain. The mechanisms that PPPs use to recruit substrates and catalyze site-specific dephosphorylation are still emerging. We will continue to dissect these mechanisms to predict and systematically dissect PPP-substrate relationships in cells. Finally, determining regulatory inputs that govern PPP holoenzyme formation, activity, and function is needed to understand their role in regulating mitosis. We combine quantitative measurements of the proteome and phosphoproteome with the reconstitution of minimal signaling units in vitro and in cells to precisely determine phosphatase function and regulation in cell division.

摘要 可逆的蛋白质磷酸化是控制大多数细胞过程的主要调节机制， 包括有丝分裂。蛋白激酶和磷酸酶之间的“拉锯战”控制着蛋白质的活性和功能。这个 这两种活动的精确协调对于准确地进行细胞分裂是至关重要的。尽管有大量的 破译激酶介导的磷酸化及其功能影响的进展，知之甚少 关于磷酸酶以及去磷酸化是如何被调控的。我们研究计划的长期目标是 揭示协调和整合PPP、激酶及其共享底物的机制 控制细胞分裂的信令网络。 大多数细胞去磷酸化是由7个催化磷酸酶亚基执行的。 磷酸蛋白磷酸酶(PPP)家族。尽管为数不多的 催化PPP酶的复杂性和特异性是通过全酶的形成而产生的。每个人 全酶作为一个不同的实体发挥作用。非催化亚基调节催化剂的活性。 亚基，使底物专一性，决定亚细胞定位，并确保适当的调节。 总体而言，有数百种具有功能的PPP全酶。 在研究PPP的功能和机制方面存在着关键的知识差距和挑战 去磷酸化。尽管已经确定了数十万个蛋白质上的磷酸化位点， 只有~450个匹配被特定的PPP全酶去磷酸化。这在一定程度上是由于 缺乏全酶特异性抑制剂和PPP全酶的复杂性。 为了克服这些障碍和填补这些知识的空白，我们将(I)确定全酶特异性 底物；(Ii)底物募集和位点特异性去磷酸化的机制；以及(Iii) 确定管理特定PPP全酶的调节输入。我们已经开发出新的细胞生物学和 研究PPP信号的生化方法。我们计划绘制PPP基板空间图 通过共享，为我们的实验室和信令界研究PPP提供了全面的基础 我们的调查结果通过一个用户友好的数据库广泛和公开地进行，我们将维护该数据库。PPP的作用机制 用来招募底物和催化位点特异性去磷酸化的方法仍在出现。我们将继续 分析这些机制以预测和系统地分析细胞中的PPP-底物关系。最后， 需要确定控制PPP全酶形成、活性和功能的调节输入 了解它们在调节有丝分裂中的作用。我们结合了蛋白质组的定量测量和 用磷蛋白组体外重组的最小信号单位和在细胞内精确测定 磷酸酶在细胞分裂中的作用和调节。

项目成果

Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division.

• DOI: 10.1016/j.cub.2017.11.034
• 发表时间: 2018-01-08
• 期刊: Current biology : CB
• 作者: Lee ME;Rusin SF;Jenkins N;Kettenbach AN;Moseley JB
• 通讯作者: Moseley JB

The Phospho-Code Determining Circadian Feedback Loop Closure and Output in Neurospora.

• DOI: 10.1016/j.molcel.2019.03.003
• 发表时间: 2019-05
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Bin Wang;A. Kettenbach;Xiaoying Zhou;J. Loros;J. Dunlap

Effects of carboxyl-terminal methylation on holoenzyme function of the PP2A subfamily.

• DOI: 10.1042/bst20200177
• 发表时间: 2020-10-30
• 期刊: Biochemical Society transactions
• 影响因子: 3.9
• 作者: Nasa I;Kettenbach AN
• 通讯作者: Kettenbach AN

Coupling of Cdc20 inhibition and activation by BubR1.

• DOI: 10.1083/jcb.202012081
• 发表时间: 2021-05-03
• 期刊: The Journal of cell biology
• 作者: Hein JB;Garvanska DH;Nasa I;Kettenbach AN;Nilsson J
• 通讯作者: Nilsson J

A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination.

• DOI: 10.1038/s41467-021-26079-0
• 发表时间: 2021-09-30
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Ambjørn SM;Duxin JP;Hertz EPT;Nasa I;Duro J;Kruse T;Lopez-Mendez B;Rymarczyk B;Cressey LE;van Overeem Hansen T;Kettenbach AN;Oestergaard VH;Lisby M;Nilsson J
• 通讯作者: Nilsson J