Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

Abstract

Vasopressin triggers the phosphorylation and apical plasma membrane accumulation of aquaporin 2 (AQP2), and it plays an essential role in urine concentration. Vasopressin, acting through protein kinase A, phosphorylates AQP2. However, the phosphorylation state of AQP2 could also be affected by the action of protein phosphatases (PPs). Rat inner medullas (IM) were incubated with calyculin (PP1 and PP2A inhibitor, 50 nM) or tacrolimus (PP2B inhibitor, 100 nM). Calyculin did not affect total AQP2 protein abundance (by Western blot) but did significantly increase the abundances of pS256-AQP2 and pS264-AQP2. It did not change pS261-AQP2 or pS269-AQP2. Calyculin significantly enhanced the membrane accumulation (by biotinylation) of total AQP2, pS256-AQP2, and pS264-AQP2. Likewise, immunohistochemistry showed an increase in the apical plasma membrane association of pS256-AQP2 and pS264-AQP2 in calyculin-treated rat IM. Tacrolimus also did not change total AQP2 abundance but significantly increased the abundances of pS261-AQP2 and pS264-AQP2. In contrast to calyculin, tacrolimus did not change the amount of total AQP2 in the plasma membrane (by biotinylation and immunohistochemistry). Tacrolimus did increase the expression of pS264-AQP2 in the apical plasma membrane (by immunohistochemistry). In conclusion, PP1/PP2A regulates the phosphorylation and apical plasma membrane accumulation of AQP2 differently than PP2B. Serine-264 of AQP2 is a phosphorylation site that is regulated by both PP1/PP2A and PP2B. This dual regulatory pathway may suggest a previously unappreciated role for multiple phosphatases in the regulation of urine concentration.

Aquaporins in Renal Diseases

“…AQP2 is one of the most important channel proteins involved in regulating urine concentration, and is located at the apical membrane of principal cells in the collecting duct [14]. The water reabsorption function of AQP2 is mainly regulated by arginine vasopressin (AVP) via increasing intracellular production of cyclic adenosine monophosphatase (cAMP) and further phosphorylation of AQP2 at Ser256 and Ser269 to stimulate the intracellular trafficking of AQP2 to the plasma membrane [15,16,17] (Figure 2). Besides AVP, researchers have demonstrated that activation of bile acid receptor TGR5 and hydrogen sulfide (H 2 S) stimulates the expression of AQP2 via cAMP-protein kinase A (PKA) signaling pathway and attenuates the defection of urinary concentration in mice [18,19].…”

Molecular mechanisms regulating aquaporin-2 in kidney collecting duct

“…In addition to the involvement of PP2A in AQP2 regulation, protein phosphatase 2B (PP2B), one of components of AKAPs-containing multiple complex has also been proposed to dephosphorylate endosome-bounded AQP2 (44). A recent study demonstrated that multiple phosphatases are involved in the subcellular localization of phosphorylated AQP2, and particularly PP1/PP2A plays a role in the AQP2 phosphorylation and AQP2 expression in the apical plasma membrane (116). Comprehensive understanding of the effects of multiple kinases under different conditions, such as activity and specificity against each phosphorylation site of AQP2 and the interaction with other proteins, could provide information for better understanding of the phosphorylationmediated AQP2 trafficking.…”

Aldosterone Decreases Vasopressin-Stimulated Water Reabsorption in Rat Inner Medullary Collecting Ducts

“…Aldosterone increases calcineurin activity in rat cortical collecting ducts through an MR-dependent but transcription-independent mechanism [11]. Our published data show that inhibiting calcineurin alters the phosphorylation and the activity of the urea transporter UT-A1 in the IMCD [12,13].…”

“…Previous studies show aldosterone increases urine production and decreases apical AQP2 expression in rats with diabetes insipidus, suggesting that aldosterone may decrease vasopressinstimulated osmotic water permeability [13,22]. There are some sex-specific differences in blood pressure control and water homeostasis (reviewed in [29]).…”