Regulation of AQP2 localization by S256 and S261 phosphorylation and ubiquitination

Abstract

Vasopressin-induced water reabsorption coincides with phosphorylation of aquaporin-2 (AQP2) at S256 (pS256), dephosphorylation at S261, and its translocation to the apical membrane, whereas treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA) induces AQP2 ubiquitination at K270, its internalization, and lysosomal degradation. In this study we investigated the relationship between S256 and S261 phosphorylation in AQP2 and its ubiquitination and trafficking in MDCK cells. Forskolin stimulation associated with increased pS256 and decreased pS261 AQP2, indicating that MDCK cells are a good model. After forskolin stimulation, TPA-induced ubiquitination of AQP2 preceded phosphorylation of AQP2 at S261, which in the first instance occurred predominantly on ubiquitinated AQP2. Forskolin-induced changes in pS261 were also observed for AQP2-S256A and AQP2-S256D, which constitutively localize in vesicles and the apical membrane, respectively. Although pS261 varies with forskolin as with wild-type AQP2, AQP2-S256A is not increased in its ubiquitination. Our data reveal that pS261 occurred independently of AQP2 localization and suggest that pS261 follows ubiquitination and endocytosis and may stabilize AQP2 ubiquitination and intracellular localization. The absence of increased ubiquitination of AQP2-S256A indicates that its intracellular location is due to the lack of pS256. Furthermore, AQP2-S261A and AQP2-S261D localized to vesicles, which was due to their increased ubiquitination, because changing K270 into Arg in both mutants resulted in their localization in the apical membrane. Although still increased in its ubiquitination, AQP2-S256D-S261D localized in the apical membrane. AQP2-S256D-K270R-Ub, however, localized to intracellular vesicles. Although our localization of AQP2-S261A/D is different from that of others, these data indicate that constitutive S256 phosphorylation counterbalances S261D-induced ubiquitination and internalization or changes its structure to allow distribution to the apical membrane. The vesicular localization of AQP2-S256D-K270R-Ub, however, indicates that the dominant apical sorting of S256D can again be overruled by constitutive ubiquitination. These data indicate that the membrane localization of AQP2 is determined by the balance of the extents of phosphorylation and ubiquitination.

Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

“…Tamma et al found that the cAMP/PKA-dependent S261 phosphorylation also occurs in the AQP2-S256A mutant, which cannot be phosphorylated at S256, and that forskolin induces dephosphorylation of S261 in this mutant. 18 However, our data seem in contrast to studies by Hoffert and coworkers, who had observed that cAMPstimulated and PKA-dependent phosphorylation of S256 precedes and primes for phosphorylation of S269. 15 The reason for the discrepancy is unclear.…”

“…12 AVP mediates dephosphorylation of S261. 15,18 This is associated with decreased polyubiquitination and proteasomal degradation and an enhanced AQP2 abundance, which contributes to the increase in water reabsorption of the collecting duct in response to AVP. 19 Although several proteins controlling AQP2 trafficking were identified and the paths of AQP2 to and from the plasma membrane are defined in general terms, 1,17 the molecular details underlying AQP2 trafficking are unclear.…”

A Protein Kinase A–Independent Pathway Controlling Aquaporin 2 Trafficking as a Possible Cause for the Syndrome of Inappropriate Antidiuresis Associated with Polycystic Kidney Disease 1 Haploinsufficiency

“…As reported, 34 addition of the prostaglandin synthesis inhibitor indomethacin was needed to reduce basal cAMP and AQP2-pS256 levels 12,34 and was present in all treatments.…”

“…Although vasopressin increases S264 and S269 phosphorylation, it decreases S261 phosphorylation. [9][10][11][12] Regarding the potential kinases responsible for the phosphorylation of these sites, c-Jun N-terminal kinase, p38, and cyclin-dependent kinases (cdks) cdk1 and cdk5 can phosphorylate AQP2 peptides at S261 in vitro. 13,14 Here, in the attempt to investigate the potential involvement of cdks in AQP2 regulation, we discovered a new PKA-independent signal transduction pathway regulating AQP2 phosphorylation and localization.…”

Phosphorylation and ubiquitylation are opposing players in regulating endocytosis of the water channel Aquaporin-2

“…The C-terminal 15 amino acids of AQP2 contain a vasopressin-regulated poly-phosphorylated region with four phosphorylated serine residues at S256, S261, S264 and S269 (T in human) (Hoffert et al, 2006). These sites have differing roles for regulation of channel function and localization (see, for example, Fushimi et al, 1997;Hoffert et al, 2008;Kamsteeg et al, 2000;Moeller et al, 2011;Moeller et al, 2010;Nedvetsky et al, 2010;Tamma et al, 2011). It has been proposed that phosphorylation of AQP2 results in increased accumulation of AQP2 on the plasma membrane owing to reduced internalization (Lu et al, 2007;Moeller et al, 2010).…”