The regulation of phosphoprotein phosphatases in the nucleus

细胞核中磷蛋白磷酸酶的调节

• 批准号: 10656696
• 负责人: Rebecca Page
• 金额: $ 36.13万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656696
• 关键词: Affinity; Anaphase; Binding; Binding Proteins; Biochemical; Biological; Biology; Biophysics; CDC2 gene; Calcineurin; Cell Cycle; Cell Nucleus; Cell division; Cell physiology; Cells; Cellular biology; Characteristics; Chemicals; Chimera organism; Code; Complex; Coupled; Cryoelectron Microscopy; Data; Development; Disease; Drug Targeting; Enzymes; Escherichia coli; Family; Genes; Goals; Health; Histidine; Holoenzymes; Human Genome; Insecta; Knowledge; Lead; Malignant Neoplasms; Methods; Mitosis; Mitotic; Molecular; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Post-Translational Protein Processing; Process; Protein Dephosphorylation; Protein Engineering; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Protein-Serine-Threonine Kinases; Proteins; Proteome; Regulation; Research; Research Personnel; Role; Serine; Signal Transduction; Site; Specificity; Structure; System; Systems Biology; Threonine; Tyrosine; Work; design; glycogen metabolism; human disease; inhibitor; insight; novel; phosphoproteomics; premature; prevent; protein function; rational design; recruit; tool

ABSTRACT An estimated 70% of all eukaryotic cellular proteins are regulated by phosphorylation. Strict temporal and spatial control are essential for the fidelity of this process, as derailed signaling cascades lead to disease. While the importance of phosphorylation is clear, knowledge gaps remain in the mechanisms that regulate key proteins involved in this process, especially phosphoprotein phosphatases (PPP). Our long-term goal is to understand the structural and functional mechanisms that control PPP activity in health and disease. Here, we focus on the function of protein phosphatase 1 (PP1) and PP2A, both of which have major roles in cell division and cancer. Our aims are designed to define the mechanisms of PP1- and PP2A:B55-based substrate recruitment to obtain a systems biology understanding of the proteomes and phosphatomes directed by these enzymes. For the PP2A family of enzymes, it is established that substrates are recruited by their variable B- subunits. We recently showed that the PP2A B56 subunit binds specifically to its substrates via a newly identified short linear motif (SLiM), LpSPIxE. This has led to the discovery of scores of novel B56-specific substrates and the development of the first PP2A:B56-specific regulator. Here, we investigate PP2A:B55, the most abundant PP2A holoenzyme in cells and the primary enzyme responsible for dephosphorylating CDK1 targets to initiate mitotic exit. Consistent with this, at mitotic entry, PP2A:B55 activity is inhibited. This is achieved by two B55-specific inhibitors: FAM122A and ARPP19. To molecularly define how these inhibitors block PP2A:B55 activity and to elucidate the molecular basis of B55 substrate recruitment via a B55-specific SLiM, we will determine both holoenzyme (quadruple complexes) structures. This is technically challenging, as these PPPs cannot be functionally expressed in E. coli or insect cells, a problem we have successfully overcome. Furthermore, we have developed a unique PP1 regulator (PhosTAP), which we show can be successfully leveraged to fully define the PP1 interactome and phosphatome. Due to its 100% specificity and exceptional affinity for only PP1, this novel PP1 PhosTAP can also be leveraged to specifically recruit PP1 to its point of action within the cell, in a manner similar to that used by PROTACs for targeted degradation. Together, the proposed aims will provide the much-needed molecular data that demonstrate how key PPP holoenzymes, especially PP1 and PP2A holoenzymes, bind their substrates and how these interactions are regulated during the cell cycle. Because these holoenzymes have critical roles in multiple human diseases, especially cancer, the proposed work will establish these holoenzymes specifically, and PPPs generally, as potent and specific drug targets.

抽象的 据估计，所有真核细胞蛋白质中有 70% 受到磷酸化的调节。严格的时间和 空间控制对于这一过程的保真度至关重要，因为脱轨的信号级联会导致疾病。尽管 磷酸化的重要性是显而易见的，但调节关键机制的知识差距仍然存在 参与该过程的蛋白质，尤其是磷蛋白磷酸酶（PPP）。我们的长期目标是 了解控制健康和疾病领域 PPP 活动的结构和功能机制。在这里，我们 重点关注蛋白磷酸酶 1 (PP1) 和 PP2A 的功能，两者在细胞分裂中发挥重要作用 和癌症。我们的目标是定义基于 PP1 和 PP2A:B55 的底物的机制 招募以获得对这些指导下的蛋白质组和磷酸组的系统生物学理解 酶。对于 PP2A 酶家族，已确定底物是通过其变量 B- 招募的 亚单位。我们最近表明，PP2A B56 亚基通过新的特异性结合其底物 鉴定出短线性基序（SLiM），LpSPIxE。这导致了数十种新的 B56 特异性的发现 底物和第一个 PP2A:B56 特异性调节剂的开发。在这里，我们研究 PP2A:B55， 细胞中最丰富的 PP2A 全酶，也是负责 CDK1 去磷酸化的主要酶 目标启动有丝分裂退出。与此一致的是，在有丝分裂进入时，PP2A:B55 活性受到抑制。这是 这是通过两种 B55 特异性抑制剂实现的：FAM122A 和 ARPP19。从分子上定义这些抑制剂如何 阻断 PP2A:B55 活性并通过 B55 特异性阐明 B55 底物招募的分子基础 SLiM，我们将确定两种全酶（四联复合物）结构。这在技术上具有挑战性，因为 这些 PPP 不能在大肠杆菌或昆虫细胞中功能性表达，我们成功解决了这个问题 克服。此外，我们还开发了一种独特的 PP1 调节剂 (PhosTAP)，我们证明它可以 成功地充分定义了 PP1 相互作用组和磷酸化组。由于其 100% 的特异性和 这种新型 PP1 PhosTAP 仅对 PP1 具有特殊的亲和力，也可用于专门招募 PP1 它在细胞内的作用点，其方式类似于 PROTAC 用于定向降解的方式。 总之，拟议的目标将提供急需的分子数据，以证明 PPP 的关键作用 全酶，尤其是 PP1 和 PP2A 全酶，结合其底物以及这些相互作用是如何进行的 在细胞周期中受到调节。因为这些全酶在多种人类疾病中发挥着关键作用， 特别是癌症，拟议的工作将具体建立这些全酶，以及一般的 PPP，如 有效且特定的药物靶点。