Department of Cellular and Molecular Medicine, University of Ottawa, Faculty of Medicine, Ottawa, Canada; Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Canada.
Department of Cellular and Molecular Medicine, University of Ottawa, Faculty of Medicine, Ottawa, Canada.
J Biol Chem. 2023 Feb;299(2):102893. doi: 10.1016/j.jbc.2023.102893. Epub 2023 Jan 10.
The subcellular localization, activity , and substrate specificity of the serine/threonine protein phosphatase 1 catalytic subunit (PP1) is mediated through its dynamic association with regulatory subunits in holoenzyme complexes. While some functional overlap is observed for the three human PP1 isoforms, they also show distinct targeting based on relative preferences for specific regulatory subunits. A well-known example is the preferential association of MYPT1 with PP1β in the myosin phosphatase complex. In smooth muscle, MYPT1/PP1β counteracts the muscle contraction induced by phosphorylation of the light chains of myosin by the myosin light chain kinase. This phosphatase complex is also found in nonmuscle cells, where it is targeted to both myosin and nonmyosin substrates and contributes to regulation of the balance of cytoskeletal structure and motility during cell migration and division. Although it remains unclear how MYPT1/PP1β traffics between microtubule- and actin-associated substrates, our identification of the microtubule- and actin-binding protein SPECC1L in both the PP1β and MYPT1 interactomes suggests that it is the missing link. Our validation of their association using coimmunoprecipitation and proximity biotinylation assays, together with the strong overlap that we observed for the SPECC1L and MYPT1 interactomes, confirmed that they exist in a stable complex in the cell. We further showed that SPECC1L binds MYPT1 directly and that it can impact the balance of the distribution of the MYPT1/PP1β complex between the microtubule and filamentous actin networks.
丝氨酸/苏氨酸蛋白磷酸酶 1 催化亚基（PP1）的亚细胞定位、活性和底物特异性是通过其与全酶复合物中的调节亚基的动态结合来介导的。虽然三种人类 PP1 同工酶存在一些功能重叠，但它们也表现出基于对特定调节亚基相对偏好的独特靶向性。一个众所周知的例子是 MYPT1 与肌球蛋白磷酸酶复合物中的 PP1β 的优先结合。在平滑肌中，MYPT1/PP1β 抵消了肌球蛋白轻链激酶对肌球蛋白轻链磷酸化引起的肌肉收缩。该磷酸酶复合物也存在于非肌肉细胞中，在非肌肉细胞中，它靶向肌球蛋白和非肌球蛋白底物，并有助于调节细胞迁移和分裂过程中细胞骨架结构和运动的平衡。尽管 MYPT1/PP1β 如何在微管和肌动蛋白相关底物之间运输仍不清楚，但我们在 PP1β 和 MYPT1 相互作用组中鉴定出微管和肌动蛋白结合蛋白 SPECC1L，表明它是缺失的环节。我们使用共免疫沉淀和邻近生物素化测定验证了它们的关联，并且我们观察到 SPECC1L 和 MYPT1 相互作用组之间存在很强的重叠，这证实了它们在细胞中存在稳定的复合物。我们进一步表明 SPECC1L 直接结合 MYPT1，并且它可以影响 MYPT1/PP1β 复合物在微管和丝状肌动蛋白网络之间分布的平衡。
