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.