Therefore , to determine which isoforms of 14-3-3 may interact with AQP2, HEK293T cells (do not express AQP2) co-transfected with various HA-14-3-3 constructs and AQP2 were stimulated with forskolin (to mimic dDAVP-induced increases in cAMP) followed by immunoprecipitation (Fig. in mpkCCD14cells, co-IP with phosphorylation deficient forms of AQP2 expressed in HEK293 cells, or surface plasmon resonance studies determined that the AQP2/14-3-3 interaction was modulated by phosphorylation of AQP2 at various sites in its carboxyl terminus, with Ser-256 phosphorylation critical for GLUFOSFAMIDE the interactions. shRNA-mediated knockdown of 14-3-3 in mpkCCD14cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life, and reduced AQP2 levels. In contrast, knockdown of 14-3-3 resulted in increased AQP2 half-life and increased AQP2 levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3 and -. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation, and degradation. Keywords: aquaporin, intracellular trafficking, kidney, phosphorylation, post-translational modification (PTM), ubiquitin, water channel, Vasopressin == Introduction == The 14-3-3 family are highly conserved proteins expressed in eukaryotic cells that play well established roles in interacting and modulating the function of target proteins at numerous levels (13). Seven 14-3-3 isoforms have been identified in mammalian cells (encoded by 7 different genes):,,,,,, and. Each are functional as homo- or heterodimers, displaying high affinity binding via selective interaction motifs in their target proteins (13). Although 14-3-3 protein interactions are often dependent on the phosphorylation status of their target protein, with binding motifs commonly containing phosphorylated serine or threonine residues (1, 4, 5), binding motifs containing non-phosphorylated residues have also been identified (6). Once docked, 14-3-3 dimers have been proposed to regulate the function of their target proteins in various manners, including: 1) acting as an adaptor for modulating interaction between two target proteins that each bind to a 14-3-3 monomer; 2) mediating conformational changes to modulate target protein activity; and 3) competing with other interaction proteins to control post-translational modification and/or localization of the target protein (1, 7). Due to essential roles of 14-3-3 proteins in modulating signal transduction pathways and protein trafficking (2, 8), dysfunction of 14-3-3 proteins are linked to diseases including cancer (8, 9), metabolic disorders, and altered cell proliferation (10). In the kidney, 14-3-3 proteins are expressed throughout the renal tubule (e. g. Refs. 1113), including the principal cells of the kidney collecting duct (14). The primary GLUFOSFAMIDE functions of these cells are to regulate NaCl (via the epithelial sodium channel, ENaC)2and water (via the water channel aquaporin-2, AQP2) transport from the preurine back to the blood and help maintain body NaCl and water homeostasis. A role of 14-3-3 to modulate ENaC function and NaCl transport is well established (e. g. Refs. 1518). The 14-3-3 isoforms and constitutes a heterodimer that interacts with a phosphorylated form of the E3 ligase Nedd4-2, blocking the interaction of Nedd4-2 with ENaC, reducing ENaC ubiquitylation and thereby increasing apical ENaC density and sodium transport. Furthermore, the steroid hormone aldosterone, which increases ENaC function, also increases abundance of 14-3-3 and -; suggesting that 14-3-3 isoform abundance can be selectively modulated by various hormones. In respect to water transport, although 14-3-3 and – are associated with intracellular AQP2 vesicles in the inner medullary collecting duct (19), a role for 14-3-3 proteins in modulating AQP2 function is not established. AQP2 functions as a tetrameric protein with the carboxyl termini located inside the cell (20). This tail domain is highly phosphorylated, with the levels of phosphorylation at Ser-256, Ser-261, Ser-264, and Ser-269 (Thr in human) residues being modulated by the hormone arginine vasopressin (AVP) (21, 22), acting via the vasopressin type 2 receptor (V2R). These phosphorylation sites play alternative Rabbit Polyclonal to ENDOGL1 roles in the subcellular distribution and function of AQP2, e. g. the apical plasma membrane accumulation of AQP2 in response to AVP treatment is modulated by Ser-256 and Ser-269 phosphorylation (2328). Although the underlying mechanisms for AQP2 regulation via phosphorylation are not completely clear, phosphorylation-dependent protein interactions appear to play a crucial role (29). In addition , the carboxyl-terminal tail of AQP2 is further modified by ubiquitylation (30). A complex interplay between AQP2 phosphorylation and ubiquitylation is responsible for modulating the abundance of AQP2 on the plasma membrane (23). In this study, we tested the hypothesis that AQP2 function is regulated by interaction with 14-3-3 proteins and that these interactions are modulated by AVP. In mouse kidney and a collecting duct cell line (mpkCCD14), 14-3-3 isoforms were identified at the mRNA and protein levels, several of which were modified in abundance by AVP. These 14-3-3 isoforms had alternative subcellular distributions in mouse kidney collecting duct cells. Biochemical GLUFOSFAMIDE studies identified an AVP-regulated and phosphorylation-dependent.