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 估计所有真核细胞蛋白的70％受磷酸化调节。严格的时间和 空间控制对于此过程的保真度至关重要，因为脱轨的信号级联反应导致疾病。尽管 磷酸化的重要性是明显的，知识差距仍然存在于调节密钥的机制中 参与此过程的蛋白质，尤其是磷酸蛋白磷酸酶（PPP）。我们的长期目标是 了解控制健康和疾病中PPP活性的结构和功能机制。在这里，我们 侧重于蛋白质磷酸酶1（PP1）和PP2A的功能，这两者在细胞分裂中具有重要作用 和癌症。我们的目标旨在定义PP1-和PP2A的机制：基于B55的底物 招募以获得系统生物学的理解，对这些蛋白质组和磷酸盐 酶。对于PP2A酶家族，确定底物是由其可变b-募集的 亚基。我们最近表明，PP2A B56亚基通过新的 鉴定出短的线性基序（Slim），lpspixe。这导致发现了数十种新颖的B56特异性 第一个PP2A的底物和开发：B56特异性调节剂。在这里，我们调查PP2A：B55， 细胞中最丰富的PP2A全酶和负责去磷酸化CDK1的主要酶 启动有丝分裂出口的目标。与此一致，在有丝分裂进入时，pp2a：B55活性受到抑制。这是 由两个B55特异性抑制剂实现：FAM122A和ARPP19。分子定义这些抑制剂如何 块PP2A：B55活性并通过B55特异性的B55底物募集的分子基础 Slim，我们将确定两个Holoenzyme（四倍复合物）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表达，这是我们成功的问题 克服。此外，我们已经开发了独特的PP1调节器（PHOSTAP），我们表明可以是 成功利用以充分定义PP1相互作用组和磷酸化组。由于其100％的特异性和 这种新颖的PP1 PhoStap也可以利用仅对PP1的特殊亲和力，以专门招募PP1 它在细胞内的作用点，类似于Protac用于靶向降解的方式。 共同的目标将提供急需的分子数据，以证明如何关键PPP 全酶，尤其是pp1和pp2a全酶，结合其底物以及这些相互作用如何 在细胞周期中调节。因为这些全酶在多种人类疾病中具有关键作用，所以 尤其是癌症，拟议的工作将专门建立这些全酶，而PPP通常是 有效和特定的药物靶标。