Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary.
Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary
J Biol Chem. 2019 Dec 27;294(52):20196-20206. doi: 10.1074/jbc.RA119.007980. Epub 2019 Nov 21.
Endothelial cells have key functions in endothelial barrier integrity and in responses to angiogenic signals that promote cell proliferation, cell migration, cytoskeletal reorganization, and formation of new blood vessels. These functions highly depend on protein-protein interactions in cell-cell junction and cell attachment complexes and on interactions with cytoskeletal proteins. Protein phosphatase 2A (PP2A) dephosphorylates several target proteins involved in cytoskeletal dynamics and cell adhesion. Our goal was to find new interacting and substrate proteins of the PP2A-B55α holoenzyme in bovine pulmonary endothelial cells. Using LC-MS/MS analysis, we identified flotillin-1 as a protein that binds recombinant GSH -transferase-tagged PP2A-B55α. Immunoprecipitation experiments, proximity ligation assays, and immunofluorescent staining confirmed the interaction between these two endogenous proteins in endothelial cells. Originally, flotillins were described as regulatory proteins for axon regeneration, but they appear to function in many cellular processes, such as membrane receptor signaling, endocytosis, and cell adhesion. Ser is a known PKC-targeted site in flotillin-1. Utilizing phosphomutants of flotillin-1 and the NanoBiT luciferase assay, we show here that phosphorylation/dephosphorylation of Ser in flotillin-1 significantly affects its interaction with PP2A-B55α and that PP2A-B55α dephosphorylates phospho-Ser Spreading, attachment, migration, and tube formation rates of S315A variant-overexpressing cells were faster than those of nontransfected or S315D-transfected cells. These results indicate that the PP2A-flotillin-1 interaction identified here affects major physiological activities of pulmonary endothelial cells.
内皮细胞在维持内皮屏障完整性和对促进细胞增殖、细胞迁移、细胞骨架重排和新血管形成的血管生成信号的反应中具有关键功能。这些功能高度依赖于细胞-细胞连接和细胞附着复合物中的蛋白质-蛋白质相互作用，以及与细胞骨架蛋白的相互作用。蛋白磷酸酶 2A（PP2A）去磷酸化参与细胞骨架动力学和细胞黏附的几个靶蛋白。我们的目标是在牛肺内皮细胞中寻找新的 PP2A-B55α 全酶的相互作用和底物蛋白。使用 LC-MS/MS 分析，我们鉴定出 flotillin-1 是与重组 GSH-转移酶标记的 PP2A-B55α 结合的蛋白。免疫沉淀实验、临近连接分析和免疫荧光染色证实了这两种内源性蛋白质在内皮细胞中的相互作用。最初， flotillin 被描述为轴突再生的调节蛋白，但它们似乎在许多细胞过程中发挥作用，如膜受体信号转导、内吞作用和细胞黏附。丝氨酸是 flotillin-1 中已知的 PKC 靶向位点。利用 flotillin-1 的磷酸突变体和 NanoBiT 荧光素酶测定，我们在这里表明， flotillin-1 中丝氨酸的磷酸化/去磷酸化显著影响其与 PP2A-B55α 的相互作用，并且 PP2A-B55α 去磷酸化了丝氨酸。S315A 变异体过表达细胞的扩展、附着、迁移和管状形成速度比非转染或 S315D 转染细胞更快。这些结果表明，这里鉴定的 PP2A-flotillin-1 相互作用影响肺内皮细胞的主要生理活动。
