Dynamic targeting of protein phosphatase 1 (PP1) activity in vivo

体内蛋白磷酸酶 1 (PP1) 活性的动态靶向

• 批准号: RGPIN-2014-04077
• 负责人: TrinkleMulcahy, Laura
• 金额: $ 2.55万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566281
• 关键词: Dynamic; targeting; protein; phosphatase; PP1

Reversible protein phosphorylation is the major general mechanism regulating most physiological processes in eukaryotic cells, with protein kinases and protein phosphatases playing key roles in the control of cell proliferation, differentiation and a host of other critical events. A common theme in phosphatase regulation is a mechanism whereby localization of the enzyme determines its access to substrates. In the case of the ubiquitous serine/threonine protein phosphatase 1 (PP1), this is mediated by interaction of the catalytic subunit with a panel of regulatory proteins termed “targeting subunits” to generate a range of holoenzyme complexes with distinct subcellular roles. The evolution of this unique combinatorial regulatory mechanism (three PP1 isoforms plus mutually exclusive binding to a panel of >200 regulatory subunits to form heterodimer complexes with high substrate specificity) provides the opportunity to develop highly specific PP1 inhibitors, based on targeted disruption of single complexes. Although biochemical, bioinformatic, and proteomic approaches have identified a wide range of targeting subunits, the current list still cannot account for the large number of regulatory pathways in which PP1 is known to play a critical role and the continuing identification and characterization of novel targeting subunits is thus critical to our understanding of targeted phosphatase activity. Using a unique and powerful combination of live cell imaging with cell fractionation and quantitative interactome profiling we have mapped cell compartment- and isoform-specific targeting of PP1 to a range of functional multiprotein complexes, with roles in diverse processes including ribosome biogenesis, DNA damage repair and chromosome segregation. Along with providing the first comprehensive map of functional PP1 complexes throughout the cell, this is also the first clear indication that the isoforms play both distinct and overlapping roles. The mechanism and functional significance of isoform-specific targeting subunit binding remains unclear, as does the means by which levels and targeting of PP1 catalytic subunits are dynamically regulated to maintain cellular homeostasis. Preliminary results indicate regulation both at the level of transcription and by re-targeting of PP1 between pools of targeting subunits. Another key layer of information that needs to be added to our road map of targeted PP1 activity is the identification of its substrates within the molecular complexes to which it is targeted and we have developed two novel techniques to that end, based on phosphoproteomic analysis following perturbation of local PP1 levels and a catalytically compromised "substrate trap" mutant. Hypotheses and Research Objectives: The long-term objectives of my research program are to (i) define both the mechanism and significance of isoform-specific PP1 targeting and (ii) understand how the formation of PP1 holoenzyme complexes is coordinated within the cell. My hypothesis is that the targeting of PP1 catalytic subunits within the mammalian cell is a dynamic process that can be modulated as required to maintain cellular homeostasis. Specific short-term objectives are: I. Map the dynamic re-targeting of PP1 catalytic subunits and elucidate the underlying mechanism(s) II. Characterize large-scale complex associations and directly identify phosphatase substrates III. Identify the structural determinants of isoform-specific binding of PP1 targeting subunits I am confident that our multi-faceted and rigorous approach, combined with our dedication to technology development, will allow us to continue to make conceptual advances in the field of phosphatase research.

可逆蛋白质磷酸化是真核细胞中大多数物理过程的主要通用机制，蛋白激酶和蛋白质磷酸酶在控制细胞增殖，分化和许多其他关键事件中起着关键作用。磷酸酶调节中的一个共同主题是一种机制，酶的定位决定了其对底物的访问。在普遍存在的丝氨酸/苏氨酸蛋白磷酸酶1（PP1）的情况下，这是通过催化亚基与称为“靶向亚基”的调节蛋白的相互作用来介导的，以产生一系列的全酶复合物与独特的亚c骨角色。这种独特的组合调节机制的演变（三种PP1同工型以及与200个调节亚基的小组相互排斥的结合，形成具有高底物特异性的异二聚体复合物）为开发高度特定的PP1抑制剂提供了机会，尽管基于生物化学，蛋白质构成的范围广泛，但具有生物化学，蛋白质的范围广泛，构成了质量范围的范围。因此，已知PP1发挥关键作用，并且新型靶向亚基的持续鉴定和表征对我们对靶向磷酸酶活性的理解至关重要。使用活细胞成像与细胞分馏和定量相互作用组谱分析的独特而强大的组合，我们将PP1的细胞室和同工型特异性靶向映射到一系列功能性多蛋白质复合物中，并在包括核糖体生物学，DNA损伤修复和染色体组的不同过程中作用。除了在整个单元格中提供第一个综合功能PP1复合物的综合图，这也是第一个清晰的迹象表明，同工型既扮演着不同的角色又扮演重叠的角色。同工型特异性靶向亚基结合的机制和功能意义尚不清楚，PP1催化亚基的水平和靶向的平均值也是动态调节以维持细胞稳态的。初步结果表明在靶向亚基池之间的转录水平和通过重新定位PP1的调节。需要添加到目标PP1活性的路线图中的另一个关键信息层是鉴定其靶向的分子复合物中的底物，并且基于磷酸蛋白质组织的局部PP1水平和催化含量累积的“ cataly cymationally cymationally cyprational cyprationallate cyprationaltiqual蛋白质组学分析，我们已经为此开发了两种新型技术。假设和研究目标：我的研究计划的长期目标是（i）定义同工型特异性PP1靶向的机制和意义，以及（ii）了解PP1全酶复合物的形成如何在细胞内配合。我的假设是，哺乳动物细胞内PP1催化亚基的靶向是一个动态过程，可以根据需要调节维持细胞稳态的过程。特定的短期目标是：I。绘制PP1催化亚基的动态重新定位，并阐明基本机制II。表征大型复合物相关并直接识别磷酸酶底物III。确定PP1靶向亚基的同工型特异性结合的结构决定者，我相信我们的多方面和严格的方法，加上对技术开发的奉献，将使我们能够继续在磷酸酶研究领域的概念进步。