Understanding how serine/threonine phosphatase reactions are controlled by regulatory subunits is a fundamental question in cell biology. For example, the ubiquitous protein phosphatase 1 catalytic subunit (PP1c) conducts most dephosphorylation events in eukaryotic cells, but its substrate specificity and spatiotemporal precision are thought to be achieved through interactions with regulatory subunits such as the ankyrin repeat, SH3 domain, proline-rich-region-containing proteins (ASPPs). ASPPs are associated with cardiocutaneous diseases, but if and how they tune PP1c in these contexts remains unknown. We found that loss of the sole C. elegans ASPP homolog, APE-1, suppresses a distinct pathology in worms called ‘jowls’—providing an unrivaled in vivo assay for ASPP activity. Using this assay, we found that APE-1 binds phosphatase catalytic subunits via its ankyrin repeats and SH3 domain and localizes to epithelial junctions through its uncharacterized amino terminus. Yet, simply localizing the phosphatase to these junctions is not sufficient for activity. A second, but unclear, regulatory function via the ankyrin repeats is required and conserved. Prior models for PP1c regulation include specifying cellular localization or altering the PP1c catalytic site to favor a subset of targets. However, our recent biochemical and in vivo data suggest ASPPs regulate PP1c via a novel mechanism—multimerization of the catalytic subunit. In this project, I aim to evaluate the ASPPs as a phosphatase amplifier at intercellular contacts by combining our in vivo assay for ASPP activity with biochemical and genetic analyses. Aim 1: Evaluate how ASPPs multimerize phosphatase catalytic subunits. Rationale: Quantitative blots, fluorescent size exclusion chromatography, mass photometry, and in vivo oligomerization suggest ASPPs multimerize PP1c. Approach: Quantify ASPP-regulated PP1c multimerization via single-molecule TIRF microscopy. Use extra-oligomerization domains in vivo to see what ratio of PP1c to ASPP is required for activity. Outcome: A novel mechanism of PP1c regulation via multimerization.

Aim 2: Define the intercellular contacts to which ASPPs localize PP1c. Rationale: Recent in vivo data suggest APE-1 localizes to puncta at epithelial junctions via a conserved N-terminal coiled-coil. Approach: Use in vivo fluorescence to identify the region of vertebrate ASPPs sufficient for localization to junctions. Identify binding partners and potential targets at junctions via proteomic analysis. Outcome: ASPP/PP1c complex binding partners at intercellular junctions.

Aim 3: Identify the target(s) of the ASPP/PP1c complex. Rationale: Our preliminary proteomics data suggest several candidate targets for ASPP-directed dephosphorylation. Approach: Use RNAi to quickly screen candidates in our in vivo assay. Use CRISPR-Cas9 to create phospho-dead and phospho-mimetic mutations at known phosphorylation sites within promising candidates (3B). Outcome: A clear target for the ASPP/PP1c complex.