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.
Highlights
Ezrin-radixin-moesin (ERM) binding phosphoprotein of 50 kD (EBP50) is a member of the Na+/H+ exchanger regulatory factor (NHERF) family which consists of four related PDZ domain containing scaffolding proteins termed as NHERF1/EBP50, NHERF2/ E3KARP, NHERF3/PDZK1, and NHERF4/IKEPP [1]
Immunofluorescent staining with tag specific antibody showed the same result as above, namely, the recombinant EBP50 was in the nuclear and perinuclear region in endothelial cells (EC) (Fig. 1A.m–o), it was present in the cytoplasm of the epithelial cells (Fig. 1A.p–r)
Based on studies of epithelial cells where EBP50 is localized to the plasma membrane and the cytoplasm, it is accepted that its primary function is to act as a scaffold protein linking transmembrane proteins to various cytoskeletal proteins
Summary
Ezrin-radixin-moesin (ERM) binding phosphoprotein of 50 kD (EBP50) is a member of the Na+/H+ exchanger regulatory factor (NHERF) family which consists of four related PDZ (postsynaptic density 95/discs-large/zona occludens-1) domain containing scaffolding proteins termed as NHERF1/EBP50, NHERF2/ E3KARP, NHERF3/PDZK1, and NHERF4/IKEPP [1]. For example, in microvillar assembly as part of the PDZK1/NHERF3-EBP50-ezrin complex was studied in details [5]. It seems that EBP50 binds ezrin in epithelial cells and there is an interdependence of EBP50 and ezrin for their apical localization [6,7]. Most of the interacting proteins bind to the first PDZ domain, only a few partners relate with the second PDZ, like betacatenin [9]. Protein-protein interactions between the members of the NHERF family [5,12] have been described, as well
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