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

蛋白磷酸酶1调节亚基12A（Protein phosphatase 1 regulatory subunit 12A, PPP1R12A）可与蛋白磷酸酶1催化亚基（PP1c）结合，形成众多蛋白磷酸酶1复合物之一——PP1c-PPP1R12A复合物（又称肌球蛋白磷酸酶）。除了在肌肉收缩中已被充分阐释的功能外，PP1c-PPP1R12A复合物还参与细胞骨架组织、细胞迁移、黏附、细胞分裂以及胰岛素信号通路。尽管该复合物具备多样的生物学功能，但目前仅鉴定出少量其底物，这限制了对PP1c-PPP1R12A活性的全面理解。本研究采用化学蛋白质组学方法K-BIPS（激酶催化生物素化鉴定磷酸酶底物法，Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates），在L6骨骼肌细胞中对PP1c-PPP1R12A复合物的底物进行了鉴定。通过K-BIPS技术，共富集得到136个候选底物，其中14个为高置信度命中靶点。选取AKT1激酶与COPS4信号小体蛋白这两个高置信度命中物作为新型PP1c-PPP1R12A底物展开研究。值得注意的是，此前已有研究证实AKT1与COPS4均可调控PP1c-PPP1R12A磷酸酶的活性。因此，本研究数据表明PP1c-PPP1R12A可通过底物依赖性反馈机制，对自身的磷酸酶活性进行调控。