Identification of PP1c-PPP1R12A Substrates Using K-BIPS (Kinase-Catalyzed Biotinylation to Identify Phosphatase Substrates)

Protein phosphatase 1 regulatory subunit 12A (PPP1R12A) interacts with the catalytic subunit of protein phosphatase 1 (PP1c) to form one of the many protein phosphatase 1 complexes, PP1c-PPP1R12A (or myosin phosphatase). In addition to a well-documented role in muscle contraction, the PP1c-PPP1R12A complex is associated with cytoskeleton organization, cell migration, adhesion, cell division, and insulin signaling. Despite the variety of biological functions, only a few substrates of the PP1c-PPP1R12A complex are characterized, which limits a full understanding of PP1c-PPP1R12A activities. Here, the chemoproteomics method K-BIPS (Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates) was used to identify substrates of the PP1c-PPP1R12A complex in L6 skeletal muscle cells. K-BIPS enriched 136 candidate substrates with 14 high confidence hits. Two high confidence hits, AKT1 kinase and COPS4 signalosome proteins, were studied as novel PP1c-PPP1R12A substrates. Interestingly, both AKT1 and COPS4 were previously documented to regulate PP1c-PPP1R12A phosphatase activity. Therefore, the data suggest that PP1c-PPP1R12A influences its own phosphatase activity through substrate-dependent feedback mechanisms