Administrative supplement for Ti2E microscope

Ti2E显微镜行政补充

• 批准号: 10386283
• 负责人: Arminja Nadine Kettenbach
• 金额: $ 9.41万
• 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/10386283
• 关键词: Administrative Supplement; 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; Microscope; Mitosis; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Protein Dephosphorylation; Protein phosphatase; Proteins; Proteome; Regulation; Research; Role; Signal Transduction; Site; Specificity; Substrate Specificity; Therapeutic Intervention; War; combinatorial; human disease; inhibitor/antagonist; new therapeutic target; phosphatase-1 kinase; 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。激酶和磷酸酶之间的“拔河”控制蛋白质的活性和功能。的 两种活动的精确协调对于细胞分裂的精确执行是必不可少的。尽管作出了重大的 在破译激酶介导的磷酸化及其功能后果的进展，知之甚少 关于磷酸酶和去磷酸化是如何调节的。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， 控制细胞分裂的信号网络。 大多数细胞去磷酸化是由7个催化磷酸酶亚基进行的。 磷蛋白磷酸酶（PPP）家族。尽管少数人提出的明显简单， 催化PPP酶，复杂性和特异性通过全酶的形成而产生。每个 全酶作为一种独特的实体发挥作用。非催化亚基调节催化亚基的活性。 亚基，使底物特异性，支配亚细胞定位，并确保适当的调节。 组合起来，有几百种功能性PPP全酶。 在研究公私伙伴关系的功能和机制方面存在着主要的知识差距和挑战， 去磷酸化虽然蛋白质上有成千上万的磷酸化位点已经被鉴定出来， 只有约450个被特定的PPP全酶去磷酸化。这部分是由于 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，填补这些知识空白，我们将（i）确定全酶特异性 （ii）剖析底物募集和位点特异性去磷酸化的机制;和（iii） 确定特定PPP全酶的调控输入。我们开发了新的细胞生物学， 生物化学方法来研究PPP信号。我们计划绘制PPP基底空间 全面地，通过共享，为我们的实验室和信号社区提供了研究PPP的基础 我们将通过一个用户友好的数据库，广泛和公开地公布我们的调查结果。公私伙伴关系 用于募集底物和催化位点特异性去磷酸化的用途仍在出现。我们将继续 剖析这些机制来预测和系统地剖析细胞中PPP-底物的关系。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。我们将蛋白质组的联合收割机定量测量和 磷酸化蛋白质组与重建的最小信号单位在体外和细胞中，以精确地确定 磷酸酶在细胞分裂中的功能和调节。