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
¿-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.