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.