In conclusion, we have determined that an AVP-regulated and phosphorylation-dependent interaction occurs between AQP2 and 14-3-3 and -. These interactions play a divergent role in modulating AQP2 trafficking, phosphorylation, ubiquitylation, and degradation. Our studies confirm that AQP2 function is highly dependent on phosphorylation-dependent protein interactions with its carboxyl-terminal domain and furthermore, provide additional evidence that various isoforms of 14-3-3 play divergent roles in the cell. FIGURE 14. Stable knockdown of 14-3-3 and -in mpkCCD 14 cells alters AQP2 membrane targeting, phosphorylation, and ubiquitylation. The three mpkCCD 14 cell lines under control (C), acute dDAVP stimulated (V), or acute dDAVP stimulated followed by dDAVP washout (V ϩ WO) conditions were assessed for AQP2 membrane abundance via biotinylation, alongside analysis of AQP2 phosphorylation levels and ubiquitylation status. A, representative immunoblotting **data** of AQP2, pS269-AQP2, ubiquitin, total 14-3-3, 14-3-3, and 14-3-3 levels in the various fractions. B and C, summary quantification **data** for biotinylated AQP2. D and E, summary quantification **data** for ubiquitylated AQP2. F and G, summary quantification **data** for pS269-AQP2. Each paired graph represents **data** normalized and analyzed in two different ways to facilitate comparisons between the experimental conditions within an individual cell line, or between individual cells lines for each experimental condition. * indicates p Ͻ 0.05 relative to the control condition/cell line, or from the group as indicated. . Proposed models for 14-3-3 regulation of AQP2. A, upon AVP stimulation AQP2 undergoes phosphorylation at Ser-256 that facilitates its trafficking to the plasma membrane. At the membrane AQP2 undergoes Ser-269 phosphorylation. The individual or combined effect of the two AQP2 phosphorylation events mediates an interaction with 14-3-3. The AQP2⅐14-3-3 complex prevents or limits binding of an unknown E3 ligase or an adapter protein to AQP2 thus reducing AQP2 ubiquitylation, internalization, and subsequent degradation. B, upon AVP stimulation AQP2 undergoes phosphorylation at Ser-256 (and possibly Ser-264) that facilitates its trafficking to the plasma membrane. At the membrane, AQP2 undergoes Ser-269 phosphorylation. The individual or combined effect of the three AQP2 phosphorylation events mediates an interaction with 14-3-3, which recruits a protein phosphatase to AQP2 resulting in dephosphorylation and internalization.
