EBP50 binds ezrin through its C-terminal domain, and this interaction, as well as a functional PDZ1 domain, is required for microvillar assembly and maintenance ( Garbett et al., 2010). ![]() EBP50 has two postsynaptic density 95/discs large/zona occludens-1 (PDZ) domains, which mediate interactions with multiple PDZ ligands, including transporters and receptors in the plasma membrane ( Weinman et al., 1995 Short et al., 1998 Cao et al., 1999 Hernando et al., 2002) and the cytoplasmic protein EBP50 PDZ interactor of 64 kDa (EPI64 Reczek and Bretscher, 2001). We find that E3KARP’s function and dynamics are regulated during the cell cycle in a manner mechanistically different from those of EBP50.ĮBP50 was identified as a binding partner of activated ezrin ( Reczek et al., 1997) and as a factor necessary to confer cAMP regulation on NHE3 ( Weinman et al., 1993), hence the alternate names. In this study, we explore the biochemical properties of E3KARP and examine whether it is similarly regulated. Recent work has shown that EBP50’s function and dynamics are regulated by its ability to bind ligands ( Garbett and Bretscher, 2014). Very little is known about the regulation of scaffolding proteins despite their importance in the functional organization of plasma membrane domains. ERM proteins can bind directly to plasma membrane proteins and also associate with scaffolding proteins ezrin-binding phosphoprotein of 50 kDa (EBP50)/Na +-H + exchanger-3 regulatory factor 1 (NHERF1) or its paralogue, exchanger 3 kinase A regulatory protein (E3KARP)/Na +-H + exchanger-3 regulatory factor 2 (NHERF2 Fehon et al., 2010). The apical domain of epithelial cells is decorated by microvilli that contain a core of actin filaments linked to the plasma membrane in part by activated ezrin, a member of the ezrin/radixin/moesin (ERM) family. Polarized cells establish and maintain compositionally and morphologically distinct plasma membrane domains, the classic example being an epithelial cell, with its distinct apical and basolateral domains. Of interest, in both cases, the mechanisms regulating dynamics involve the tails, which are the most diverged region of the paralogues and probably evolved independently after a gene duplication event that occurred early in vertebrate evolution. Regulation of the dynamics of EBP50 is known to be dependent on its tail region but modulated by PDZ domain occupancy, which is not the case for E3KARP. A-Raf is found to be required for S303 phosphorylation in mitotic cells. Moreover, the S303D mutation enhances the in vivo dynamics of the E3KARP tail alone, whereas in vitro the interaction of E3KARP with active ezrin is unaffected by S303D, implicating another factor regulating dynamics in vivo. Whereas E3KARP can substitute for the function of the closely related scaffolding protein EBP50/NHERF1 in the formation of interphase microvilli, E3KARP S303D cannot. The exchange rate of E3KARP is greatly enhanced during mitosis due to phosphorylation at Ser-303 in its tail region. We examine the dynamics and function of the apical scaffolding protein E3KARP/NHERF2, which consists of two PDZ domains and a tail containing an ezrin-binding domain.
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