Ser‐256 phosphorylation dynamics of aquaporin 2 during maturation from the endoplasmic reticulum to the vesicular compartment in renal cells

Procino, Giuseppe; Carmosino, Monica; Marin, Oriano; Brunati, Anna Maria; Contri, Antonella; Pinna, Lorenzo A.; Mannucci, Roberta; Nielsen, Søren; Kwon, Tae‐Hwan; Svelto, María; Valenti, Giovanna

doi:10.1096/fj.02-0870fje

Cited by 85 publications

(71 citation statements)

References 42 publications

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“…It is located within the Golgi and has been suggested to phosphorylate AQP2 in this compartment ( Figure 5). 44 Other candidates are casein kinase II and protein kinase Ca, which phosphorylates S256 in vitro. 15,45 In IMCD cells, 4AD does not affect the phosphorylations at S261 and S269 (Figures 3 and 7), suggesting that the responsible kinases (e.g., p38 mitogen-activated protein kinases or Jun kinase 19 ) are insensitive to 4AD or that they catalyze the phosphorylations independently from V-ATPase activity.…”

Section: Discussionmentioning

confidence: 99%

Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

Bogum

Faust

Zühlke

et al. 2013

Journal of the American Society of Nephrology

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In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H + -ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKAdependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.

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Section: Discussionmentioning

confidence: 99%

Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

Bogum

Faust

Zühlke

et al. 2013

Journal of the American Society of Nephrology

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“…This might reflect cAMP-independent activation of AQP2 in AC6 Ϫ/Ϫ mice, which may involve V 2 R activation of phosphoinositide-specific phospholipase C 7 and PKA-independent AQP2 phosphorylation at S256. 36,37 These mechanisms may also contribute to intact AQP2 regulation in renal cortex of AC6 Ϫ/Ϫ mice. A prevalent dysfunctional missense single nucleotide polymorphism of AC6 with a reduced ability to form cAMP has been described with a frequency of approximately 3% in a white population.…”

Section: Discussionmentioning

confidence: 99%

Adenylate Cyclase 6 Determines cAMP Formation and Aquaporin-2 Phosphorylation and Trafficking in Inner Medulla

Rieg

Tang²,

Murray³

et al. 2010

Journal of the American Society of Nephrology

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Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V 2 receptor (V 2 R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V 2 R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus. The anti-diuretic hormone arginine-vasopressin (AVP) is the primary regulator of water reabsorption in the renal collecting duct (CD) and is critically involved in the regulation of water balance and maintenance of plasma osmolality. 1 AVP acts on the CD through the G s protein-coupled vasopressin V 2 receptor (V 2 R) to stimulate adenylyl cyclase (AC) and thus the synthesis of cAMP. 2 cAMP activates protein kinase A (PKA), which phosphorylates the water channel aquaporin-2 (AQP2) in its COOH-terminal tail on serine residue 256 (S256), thereby resulting in apical plasma membrane accumulation of AQP2. [3][4][5][6] In addition, cAMP-independent activation of AQP2 has been reported, which may involve V 2 R action of phosphoinositide-specific phospholipase C. 7 Other non-PKA-targeted AQP2 phosphorylation sites include S261, S264, and S269. 8 Phosphorylation of AQP2 at S264 and S269 requires prior phosphorylation of S256. 9 Immunohistochemistry showed that pS269-AQP2 localizes exclusively in the apical plasma

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“…However, because the S256A mutation also inhibits hsc70 binding without causing membrane accumulation of AQP2, other factors must also be involved. Interestingly, it has been reported that impaired constitutive phosphorylation of Ser-256 (S256A) in the Golgi causes AQP2 routing to lysosomes rather than to AQP2-containing vesicles designated for exocytosis (55). Heat shock protein 70 (hsp70) has been well documented to be involved in endoplasmic reticulum-associated degradation (32,56), and facilitates the degradation of cystic fibrosis transmembrane conductance regulator in yeast (41)).…”

Section: Discussionmentioning

confidence: 99%

Heat Shock Protein 70 Interacts with Aquaporin-2 and Regulates Its Trafficking

Sun²,

Matsuzaki

et al. 2007

Journal of Biological Chemistry

114

115

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The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodaminetransferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.Aquaporin 2 is expressed in principal cells of the kidney collecting duct and mediates water absorption and urinary concentration in response to vasopressin (VP). 4 Upon stimulation by VP, AQP2 accumulates on the plasma membrane of collecting duct principal cells (1-4). Activation of both cAMP-dependent, VP/forskolin-stimulated and cAMP-independent, cGMP-responsive signaling pathways stimulates membrane accumulation of AQP2 (5). Recent evidence also indicates that cAMP-and cGMP-independent membrane accumulation of AQP2 can also occur in the absence of hormonal stimulation (6 -8), such as by blocking endocytosis (6, 8) and by disrupting the actin cytoskeleton (9, 10).Although the complicated pathways of intracellular AQP2 trafficking remain to be completely elucidated, even less is known about the direct interaction of AQP2 or AQP2-containing vesicles with a host of accessory factors that must be involved in these processes. Understanding these interactions is a critical step in dissecting the processes of exocytosis, endocytosis, intracellular translocation, as well as degradation and shedding of AQP2 that all contribute to the final physiological response to VP in vivo. Many of the cytosolic proteins known to be involved in vesicular trafficking in general have also been impli...

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Ser‐256 phosphorylation dynamics of aquaporin 2 during maturation from the endoplasmic reticulum to the vesicular compartment in renal cells

Cited by 85 publications

References 42 publications

Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

Adenylate Cyclase 6 Determines cAMP Formation and Aquaporin-2 Phosphorylation and Trafficking in Inner Medulla

Heat Shock Protein 70 Interacts with Aquaporin-2 and Regulates Its Trafficking

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