Protein Phosphatase 1 Holoenzyme Formation - ABSTRACT Phosphorylation is one of the most ubiquitous, reversible posttranslational modifications in cells. The enzymes responsible for controlling the phosphorylation state of the cell are kinases, which catalyze the transfer of the γ-phosphate moiety of ATP to substrates, and phosphatases, which catalyze the reverse hydrolysis reaction, the removal of the phosphate moiety from phosphorylated substrates. Thus, phosphatases dynamically reverse the effects of kinases. Because phosphorylation is critical for all biological processes from cell growth to differentiation to development, the location and duration of the reciprocal actions of kinases and phosphatases must be exquisitely regulated both temporally and spatially within the cell. Consequently, when this tight regulation is disrupted, dysregulation of phosphorylation signaling ensues and the consequence is most often disease. Deletion of either one of two PP1 regulators—SDS22 (PPP1R7) or Inhibitor-3 (I3; PPP1R11 or Ypi1 in yeast)—is lethal in yeast (essential genes), highlighting their biological significance. However, since their discovery, different biological roles have been assigned to SDS22 and I3, including roles in mitosis (SDS22), E3 ligase functionality (I3), PP1 biogenesis, among others. Thus, while it is clear that SDS22 and I3 are essential PP1 regulators, their true biological function(s) and especially their mechanism(s) of action are still unknown. This has hindered progress in understanding their roles in PP1 biology. In cells, these proteins form both heterodimeric (SDS22:PP1 and I3:PP1) and a heterotrimeric (SDS22:I3:PP1; SIP) PP1 complex. The structure and function(s) of the individual dimeric complexes, if and how the structure and function(s) of the trimeric complex differs from those of the dimeric complexes and the role(s) of each complex in PP1 holoenzyme formation are major questions in the field. Further, additional data suggest that dissociation of the SIP complex requires the AAA+ ATPase p37/p97. However, the molecular details of SIP complex dissociation have also remained elusive. The presented research project uses a powerful integrated approach that combines structural biology with biochemical and cell biology experiments to obtain novel insights into the molecular mechanisms used by these regulators to control PP1 activity and direct PP1 holoenzyme assembly. Because PP1 holoenzymes have critical roles in human diseases, the proposed work will provide novel strategies for selectively inhibiting PP1 activity by targeting the PP1 holoenzyme formation and subunit exchange, which is essential for understanding how distinct PPPs contribute to disease.
