The regulation of phosphoprotein phosphatases in the nucleus

摘要 据估计，70%的真核细胞蛋白质受磷酸化调节。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 磷酸化的重要性是显而易见的，但在调节关键蛋白质的机制中仍然存在知识空白。 参与这一过程的蛋白质，特别是磷蛋白磷酸酶（PPP）。我们的长期目标是 了解控制PPP活动在健康和疾病的结构和功能机制。这里我们 蛋白磷酸酶1（PP1）和PP2A在细胞分裂中起重要作用 和癌症我们的目的是确定PP1和PP2A：B55基底物的作用机制 招募获得由这些指导的蛋白质组和磷酸化酶的系统生物学理解 内切酶对于酶的PP2A家族，已经确定底物是通过其可变B募集的。 亚单位。我们最近发现PP2A B56亚基通过一种新的 确定了短线性基序（SLiM），LpSPIxE。这导致了大量新的B56特异性的发现。 底物和第一个PP2A：B56特异性调节剂的开发。在这里，我们研究PP2A：B55， 细胞中最丰富的PP2A全酶和负责CDK1去磷酸化的主要酶 启动有丝分裂退出的靶点。与此一致，在有丝分裂进入时，PP2A：B55活性被抑制。这是 通过两种B55特异性抑制剂：FAM122A和ARPP19实现。为了从分子上定义这些抑制剂 阻断PP2A：B55活性，并通过B55特异性的 SLiM，我们将确定两个全酶（四重复合物）的结构。这在技术上具有挑战性，因为 这些PPP不能在E.大肠杆菌或昆虫细胞，我们已经成功地解决了这个问题， 克服此外，我们还开发了一种独特的PP1调节剂（PhosTAP），我们表明它可以 成功地充分利用了PP1相互作用组和磷酸酶组。由于其100%的特异性和 由于仅对PP1具有特殊的亲和力，这种新型PP1 PhosTAP也可用于特异性募集PP1， 其在细胞内的作用点，以类似于PROTAC用于靶向降解的方式。 总之，提出的目标将提供急需的分子数据，证明关键的PPP 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发生的。 在细胞周期中受到调节。因为这些全酶在多种人类疾病中起着关键作用， 特别是癌症，拟议的工作将建立这些全酶具体，和PPP一般， 有效和特异的药物靶点。

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.