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.

摘要 据估计，所有真核细胞蛋白中有70%是由磷酸化调控的。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。而当 磷酸化的重要性是显而易见的，在调节关键字的机制中仍然存在知识空白 参与这一过程的蛋白质，特别是磷酸蛋白磷酸酶(PPP)。我们的长期目标是 了解在健康和疾病中控制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，这两个蛋白在细胞分裂中都发挥着重要作用 和癌症。我们的目标是定义PP1-和PP2A：B55基底物的机制 招聘，以获得系统生物学的理解，由这些指导的蛋白质组和磷酸盐 酵素。对于PP2A家族的酶，已经确定底物由它们的变量B-来招募。 亚单位。我们最近发现PP2A B56亚基通过一种新的方式与其底物特异性结合 鉴定了短线状基序(SLIM)，LpSPIxE。这导致了数十种特定于B56的新奇生物的发现 底物和第一个PP2A：B56特异性调节剂的开发。在这里，我们调查了PP2A：B55， 细胞中最丰富的PP2A全酶和负责去磷酸化CDK1的主要酶 启动有丝分裂退出的目标。与此一致的是，在有丝分裂进入时，PP2A：B55的活性受到抑制。这是 通过两种B55特异性抑制剂：FAM122A和ARPP19实现。从分子上定义这些抑制剂是如何 阻断PP2A：B55的活性并阐明B55底物通过B55特异性募集的分子基础 斯利姆，我们将确定两种全酶(四重复合体)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，这是我们成功解决的一个问题 克服困难。此外，我们还开发了一种独特的PP1调节器(PhosTAP)，我们展示了它可以 成功地充分定义了PP1相互作用组和磷酸组。由于其100%的特异性和 仅对PP1具有特殊的亲和力，这种新型的PP1 PhosTAP也可以被用来专门招募PP1来 它在细胞内的作用点，以类似于PROTAC用于靶向降解的方式。 总之，提议的目标将提供急需的分子数据，证明关键的购买力平价如何 全酶，特别是PP1和PP2A全酶，结合它们的底物，以及这些相互作用是如何 在细胞周期中进行调节。因为这些全酶在多种人类疾病中起着关键作用， 尤其是癌症，拟议的工作将特别地建立这些全酶，以及一般的PPP，作为 有效和特定的药物靶点。