Cell Cycle Dependent Association of EBP50 with Protein Phosphatase 2A in Endothelial Cells

Abstract

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in the endothelium. We report unusual nuclear localization of EBP50 in bovine pulmonary artery endothelial cells (BPAEC). Immunofluorescent staining and cellular fractionation demonstrated that EBP50 is present in the nuclear and perinuclear region in interphase cells. In the prophase of mitosis EBP50 redistributes to the cytoplasmic region in a phosphorylation dependent manner and during mitosis EBP50 co-localizes with protein phosphatase 2A (PP2A). Furthermore, in vitro wound healing of BPAEC expressing phospho-mimic mutant of EBP50 was accelerated indicating that EBP50 is involved in the regulation of the cell division. Cell cycle dependent specific interactions were detected between EBP50 and the subunits of PP2A (A, C, and Bα) with immunoprecipitation and pull-down experiments. The interaction of EBP50 with the Bα containing form of PP2A suggests that this holoenzyme of PP2A can be responsible for the dephosphorylation of EBP50 in cytokinesis. Moreover, the results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells.

A Molecular Switch for the Orientation of Epithelial Cell Polarization

“…The PP2A phosphatase complex binds and dephosphorylates NHERF1 (Boratkó et al, 2012; Chen et al, 2012). We examined the localization of the NHERF1-associating PP2A-B55α subunit (3×HA-PP2A-Bα).…”

“…Internalized Podxl transcytoses to the AMIS (Bryant et al, 2010), likely now in association with NHERF2 (Figure 7G, box 4). The Podxl- NHERF1-Ezrin complex reforms at the AMIS via corecruitment of phosphatase PP2A (Figure 7G, box 5), probably promoting dephosphorylation of Podxl-NHERF1 (Boratkó et al, 2012; Chen et al, 2012). The AMIS then progresses to an open lumen, surrounded by a monolayer of AP-BL polarized epithelial cells.…”

Temporal and Spatial Regulation of Ezrin-Radixin-Moesin-Binding Phosphoprotein-50-kDa (EBP50) during Embryo Implantation in Mouse Uterus

“…Up to now, many studies have focused on EBP50, which is widely distributed and is particularly rich in tissues with polarized epithelia with numerous ezrin-radirin-moesin (ERM) family members, such as the liver, kidney, small intestine and placenta [20]. In addition to acting as a scaffold protein linking transmembrane proteins to various cytoskeletal proteins, EBP50 is localized in the plasma membrane and the cytoplasm of epithelial cells [21]. Hence, it can be deduced that EBP50 may be expressed in the apical plasma membrane of polarized epithelial cells.…”

“…During decidualization, uterine stroma cells start to proliferate and differentiate into decidual cells, which also undergo extensive remodeling to control embryo invasion and accommodate the growing embryo. Boratko et al [21] had reported that EBP50 is present in the nuclear and perinuclear regions in interphase cells. However, EBP50 redistributes to the cytoplasmic region during the prophase of mitosis.…”

Dynamics of ezrin and EBP50 in regulating microvilli on the apical aspect of epithelial cells

“…When EBP50 is phosphorylated by either Cdc2 (cell division cycle 2) or PKC, it is unable to bind to PDZ1 and PDZ2 ligands simultaneously and is unable to form microvilli perhaps due to unidentified PDZ2 ligand(s) which preferentially bind to its phosphorylated form [53]. The phosphoregulation of EBP50 activity is also involved in regulating EBP50’s localization and activity in endothelial cells and other epithelial cell lines [57, 58]. …”