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

可逆性蛋白磷酸化是调节真核细胞大多数生理过程的主要机制，蛋白激酶和蛋白磷酸酶在细胞增殖、分化和许多其他关键事件的控制中起着关键作用。磷酸酶调控的一个共同主题是酶的定位决定其进入底物的机制。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被称为“靶向亚基”的调节蛋白的相互作用来介导的，从而产生一系列具有不同亚细胞作用的全酶复合物。这种独特的组合调节机制（三种PP1亚型加上与一组bbbb200调节亚基的互斥结合，形成具有高底物特异性的异源二聚体复合物）的进化，为开发基于靶向破坏单一复合物的高特异性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，但目前的列表仍然无法解释PP1已知发挥关键作用的大量调控途径，因此，继续鉴定和表征新的靶向亚基对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分析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定位到一系列功能多蛋白复合物中，这些复合物在核糖体生物发生，DNA损伤修复和染色体分离等多种过程中发挥作用。除了提供整个细胞功能PP1复合物的第一个全面图谱外，这也是第一个明确的迹象，表明同种异构体发挥着不同的和重叠的作用。同种异构体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是如何被动态调节以维持细胞稳态的也不清楚。初步结果表明，在转录水平和靶向亚基池之间PP1的重靶向两方面都有调控作用。需要添加到我们靶向PP1活性路线图的另一个关键信息层是鉴定其靶向分子复合物中的底物，为此我们开发了两种新技术，基于局部PP1水平扰动后的磷酸化蛋白质组学分析和催化受损的“底物陷阱”突变体。假设和研究目标：我的研究计划的长期目标是(i)定义PP1亚型特异性靶向的机制和意义，以及（ii）了解PP1全酶复合物的形成如何在细胞内协调。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稳态。具体的短期目标是：1 .绘制PP1催化亚基的动态再靶向图，并阐明潜在的机制。表征大规模复杂关联并直接鉴定磷酸酶底物III。确定PP1靶向亚基的异构体特异性结合的结构决定因素我相信，我们多方面和严格的方法，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上的进步。

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.