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

可逆性蛋白磷酸化是真核细胞中调节大多数生理过程的主要一般机制，其中蛋白激酶和蛋白磷酸酶在控制细胞增殖、分化和其他一系列关键事件中起着关键作用。磷酸酶调节的一个共同主题是一种机制，通过这种机制，酶的定位决定了它对底物的访问。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一组称为靶向亚基的调节蛋白质相互作用来产生一系列具有不同亚细胞作用的全酶复合体来调节的。这种独特的组合调控机制(三种PP1亚型加上相互排斥地与一组&gt；200调节亚基结合形成具有高底物专一性的异二聚体复合体)的进化为基于单个复合体的靶向干扰开发高度特异性的PP1抑制剂提供了机会。尽管生化、生物信息学和蛋白质组学方法已经确定了广泛的靶向亚基，但目前的清单仍然不能解释PP1在其中发挥关键作用的大量调控途径，因此继续鉴定和鉴定新的靶向亚基对于我们理解靶向磷酸酶活性至关重要。利用活细胞成像与细胞分离和定量相互作用组谱的独特而强大的组合，我们已经将PP1的细胞室和异构体特异性靶向映射到一系列功能多蛋白复合体，这些复合体在不同的过程中发挥作用，包括核糖体生物发生、DNA损伤修复和染色体分离。除了提供第一张细胞内功能PP1复合体的全面图谱外，这也是第一个明确的迹象，表明异构体既发挥着不同的作用，又发挥着重叠的作用。目前尚不清楚异构体特异性靶向亚基结合的机制和功能意义，也不清楚PP1催化亚基的水平和靶向是如何动态调节以维持细胞内稳态的。初步结果表明，PP1在转录水平和靶向亚基池之间通过重新靶向PP1进行调控。需要添加到我们的靶向PP1活性路线图中的另一个关键信息层是在其靶向的分子复合体中识别其底物，我们为此开发了两项新技术，这两项技术是基于对局部PP1水平的扰动和一个催化受损的“底物陷阱”突变体进行的磷酸蛋白质组分析。假设和研究目标：我的研究计划的长期目标是(I)确定异构体特异性PP1靶向的机制和意义，以及(Ii)了解PP1全酶复合体的形成是如何在细胞内协调的。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，可以根据需要进行调节，以维持细胞内的稳态。具体的短期目标是：I.绘制PP1催化亚基的动态重靶向图并阐明其潜在机制(S)II.表征大规模的复杂关联并直接鉴定磷酸酶底物III.确定靶向亚基的PP1异构体特异性结合的结构决定因素我相信，我们多方面的、严格的方法，加上我们对技术开发的奉献，将使我们能够继续在磷酸酶研究领域取得概念性进展。

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.