Phosphorylation of Serine 256 Is Required for cAMP-dependent Regulatory Exocytosis of the Aquaporin-2 Water Channel

Abstract

The aquaporin-2 (AQP2) vasopressin water channel is translocated to the apical membrane upon vasopressin stimulation. Phosphorylation of serine 256 of AQP2 by cAMP-dependent protein kinase has been shown, but its relation to vasopressin-regulated translocation has not been elucidated. To address this question, wild type (WT) AQP2 and a mutant with alanine in place of serine 256 of AQP2 (S256A) were expressed in LLC-PK1 cells by electroporation. Measurements by a stopped-flow lightscattering method revealed that the osmotic water permeability (P f ) of LLC-PK1 cells transfected with WT was 69.6 ؎ 6.5 m/s (24.8 ؎ 2.2 m/s for mock-transfected), and stimulation by 500 M 8-(4-chlorophenylthio)-cAMP increased the P f by 85 ؎ 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in the P f was only 8 ؎ 5%. After cAMP stimulation, the increase in surface expression of AQP2 determined by surface biotin labeling was 4 ؎ 10%, significantly less than that for WT (88 ؎ 5%). In addition, an in vivo [ 32 P]orthophosphate labeling assay demonstrated significant phosphorylation of WT AQP2 and only minimal phosphorylation of S256A AQP2 in LLC-PK1 cells. Our results indicated that serine 256 of AQP2 is necessary for regulatory exocytosis and that cAMP-responsive redistribution of AQP2 may be regulated by phosphorylation of AQP2.The urine concentration system in the kidney is regulated mainly by vasopressin, an antidiuretic hormone that increases the osmotic water permeability of the collecting duct cells, resulting in bulk reabsorption of free water (1-3). The cellular actions of vasopressin in the collecting duct cell have been partially resolved since the identification of the vasopressin water channel aquaporin-2 (AQP2) 1 (4, 5). Vasopressin has been shown to induce regulatory redistribution of AQP2 from endosomal compartments to the apical membrane (6 -8). These observations have directly proved the shuttle hypothesis that exo-and endocytosis of water channel-containing vesicles account for the regulatory effects of vasopressin on water permeability of the collecting duct cells (9, 10).Despite elucidation of the regulatory translocation of AQP2, the potential interactions between vasopressin-induced intracellular cAMP accumulation and AQP2 trafficking remain unknown. It has been shown that cAMP-dependent phosphorylation of AQP2 expressed in the Xenopus oocyte slightly increased osmotic water permeability without changing AQP2 surface expression (11), but the increase was far too small to account for the dramatic increase in water permeability of collecting duct cells. Many channels have been shown to be functionally regulated by their phosphorylation (12), but the direct regulation of AQP2 channel functions through phosphorylation is minimal at best and is still controversial (13). On the other hand, the physiological significance of regulatory effects on AQP2 trafficking has been emphasized. Regulatory delivery and sorting of membrane proteins have been observed in many cell types (14, 15). Many vesicle-associated...

Phosphorylation of Soybean Nodulin 26 on Serine 262 Enhances Water Permeability and Is Regulated Developmentally and by Osmotic Signals

“…Numerous MIP proteins are subject to phosphorylation, and the effects on activity are varied, ranging from the stimulation (e.g., the plant MIPs ␣ TIP [Maurel et al, 1995] and PM28A [Johansson et al, 1998]) to the inhibition of water transport (e.g., mammalian AQP4 [Han et al, 1998;Zelenina et al, 2002]) and alterations in water permeability by affecting membrane trafficking and subcellular localization (e.g., AQP4 [Madrid et al, 2001] and AQP2 [Nielsen et al, 1995;Fushimi et al, 1997]). The present study shows that phosphorylation of nodulin 26 enhances its water permeability both upon expression in Xenopus oocytes and in symbiosome membrane vesicles.…”

Dual Role of Signaling Pathways Leading to Ca2+and Cyclic AMP Elevation in Host Cell Invasion byTrypanosoma cruzi

“…Although the exact mechanism by which cAMP regulates lysosome exocytosis is not known, it is possible that cAMP-dependent protein kinase A mediates phosphorylation of vesicle membrane components involved in docking and fusion events (10) or that the effects observed are due to phosphorylationdependent disassembly of cortical cytoskeleton components (8,12,27) or modulation of microtubule-dependent vesicular transport (9,13,33). We showed previously that membranepermeant analogs of cAMP enhance T. cruzi entry into NRK fibroblasts and that the infective trypomastigotes are able to stimulate cAMP production in host cells (23,32).…”

Overexpression of AQP5, a putative oncogene, promotes cell growth and transformation

“…However, stable cells expressing the Δ148-263 construct showed no significant difference in colony formation as compared to MOCK. From these two experiments and based on prior reports [3,4,14], we have hypothesized that this difference in proliferative ability between the Δ162-263 cells and the Δ148-263 cells was due to absence of the PKA substrate sequence in the Δ148-263 construct. Several reports have demonstrated that AQP5 expression is regulated by cAMP through a PKA pathway.…”

“…To further elucidate this observation, we have constructed two deletion mutants [3,4] based on the membrane topology of hAQP5 (Fig. 1).…”

“…The N-terminal and C-terminal halves are sequence-related, and loop B and E each contain the Asn-Pro-Ala (NPA) motif seen in the other aquaporins [3]. However, the cytoplasmic loop D contains a cAMP-protein kinase phosphorylation consensus site (Ser-Arg-Arg-Thr-Ser) which is seen only in AQP2 [3][4][5]. In normal tissues, AQP5 is expressed strongly in the salivary glands and the eye but expression is also seen in the lacrimal gland, trachea, and lung [3].…”