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The Journal of Physiology logoLink to The Journal of Physiology
. 2018 Dec 21;597(6):1627–1642. doi: 10.1113/JP277024

Inhibition of non‐receptor tyrosine kinase Src induces phosphoserine 256‐independent aquaporin‐2 membrane accumulation

Pui W Cheung 1, Abby Terlouw 1, Sam Antoon Janssen 1, Dennis Brown 1, Richard Bouley 1,✉
PMCID: PMC6418769  PMID: 30488437

Abstract

Key points

  • Aquaporin‐2 (AQP2) is crucial for water homeostasis, and vasopressin (VP) induces AQP2 membrane trafficking by increasing intracellular cAMP, activating PKA and causing phosphorylation of AQP2 at serine 256, 264 and 269 residues and dephosphorylation of serine 261 residue on the AQP2 C‐terminus.

  • It is thought that serine 256 is the master regulator of AQP2 trafficking, and its phosphorylation has to precede the change of phosphorylation state of other serine residues.

  • We found that Src inhibition causes serine 256‐independent AQP2 membrane trafficking and induces phosphorylation of serine 269 independently of serine 256.

  • This targeted phosphorylation of serine 269 is important for Src inhibition‐induced AQP2 membrane accumulation; without serine 269, Src inhibition exerts no effect on AQP2 trafficking.

  • This result helps us better understand the independent pathways that can target different AQP2 residues, and design new strategies to induce or sustain AQP2 membrane expression when VP signalling is defective.

Abstract

Aquaporin‐2 (AQP2) is essential for water homeostasis. Upon stimulation by vasopressin, AQP2 is phosphorylated at serine 256 (S256), S264 and S269, and dephosphorylated at S261. It is thought that S256 is the master regulator of AQP2 trafficking and membrane accumulation, and that its phosphorylation has to precede phosphorylation of other serine residues. In this study, we found that VP reduces Src kinase phosphorylation: by suppressing Src using the inhibitor dasatinib and siRNA, we could increase AQP2 membrane accumulation in cultured AQP2‐expressing cells and in kidney collecting duct principal cells. Src inhibition increased exocytosis and inhibited clathrin‐mediated endocytosis of AQP2, but exerted its effect in a cAMP, PKA and S256 phosphorylation (pS256)‐independent manner. Despite the lack of S256 phosphorylation, dasatinib increased phosphorylation of S269, even in S256A mutant cells in which S256 phosphorylation cannot occur. To confirm the importance of pS269 in AQP2 re‐distribution, we expressed an AQP2 S269A mutant in LLC‐PK1 cells, and found that dasatinib no longer induced AQP2 membrane accumulation. In conclusion, Src inhibition causes phosphorylation of S269 independently of pS256, and induces AQP2 membrane accumulation by inhibiting clathrin‐mediated endocytosis and increasing exocytosis. We conclude that S269 can be phosphorylated without pS256, and pS269 alone is important for AQP2 apical membrane accumulation under some conditions. These data increase our understanding of the independent pathways that can phosphorylate different residues in the AQP2 C‐terminus, and suggest new strategies to target distinct AQP2 serine residues to induce membrane expression of this water channel when VP signalling is defective.

Keywords: non‐receptor kinase, water channel, aquaporin‐2, trafficking, phosphorylation

Key points

  • Aquaporin‐2 (AQP2) is crucial for water homeostasis, and vasopressin (VP) induces AQP2 membrane trafficking by increasing intracellular cAMP, activating PKA and causing phosphorylation of AQP2 at serine 256, 264 and 269 residues and dephosphorylation of serine 261 residue on the AQP2 C‐terminus.

  • It is thought that serine 256 is the master regulator of AQP2 trafficking, and its phosphorylation has to precede the change of phosphorylation state of other serine residues.

  • We found that Src inhibition causes serine 256‐independent AQP2 membrane trafficking and induces phosphorylation of serine 269 independently of serine 256.

  • This targeted phosphorylation of serine 269 is important for Src inhibition‐induced AQP2 membrane accumulation; without serine 269, Src inhibition exerts no effect on AQP2 trafficking.

  • This result helps us better understand the independent pathways that can target different AQP2 residues, and design new strategies to induce or sustain AQP2 membrane expression when VP signalling is defective.

Introduction

Aquaporin 2 (AQP2) is crucial for regulation of water balance by the kidney. Under basal conditions, cytosolic AQP2 constitutively traffics to the plasma membrane (Gustafson et al. 2000; Zhang et al. 2002; Lu et al. 2004), and once stimulated by VP, AQP2 phosphorylation is modified (Fushimi et al. 1997; Katsura et al. 1997), and the equilibrium between endocytosis and exocytosis changes to favour an increase of AQP2 membrane accumulation (Moeller et al. 2010, 2012; Kwon et al. 2013; Noda, 2014; Jung & Kwon, 2016). This increases water permeability in kidney collecting ducts, and effectively conserves water. Altered VP signalling and AQP2 trafficking occur in disease states such as nephrogenic diabetes insipidus, syndrome of inappropriate ADH production, cirrhosis and heart failure (Knoers & Deen, 2001; Morello & Bichet, 2001; Ranchin et al. 2010; Saigusa et al. 2012).

VP regulates AQP2 trafficking by phosphorylating three serine residues in the C‐terminus, namely serine 256 (S256), S264 and S269, and dephosphorylating S261 (Fenton et al. 2008; Moeller et al. 2010; Arthur et al. 2015; Cheung et al. 2017). S256 is believed to be the master regulator of AQP2 trafficking whose phosphorylation has to precede the change of phosphorylation states of other serine residues (Hoffert et al. 2006; Lu et al. 2008; Moeller et al. 2010). However, we have recently shown that S261 dephosphorylation occurs independently of S256 phosphorylation (Cheung et al. 2017), and more studies have suggested that the phosphorylation cascade of these serine residues is not sequential as previously thought (Hoffert et al. 2008). AQP2 membrane trafficking can also be facilitated by other receptor ligands, including angiotensin, prostaglandin, and calcitonin (Jensen et al. 2009, 2010; Li et al. 2009; Bouley et al. 2011), which affect the cAMP/PKA signalling pathway. In addition, we found that epidermal growth factor receptor (EGFR) inhibition can induce AQP2 phosphorylation in a similar pattern to VP, but independently of cAMP and PKA, suggesting a crosstalk between EGFR and VP signalling in AQP2 regulation (Cheung et al. 2016). One possible common denominator in this crosstalk is Src (pp60c‐SRC). In addition to its role in cell differentiation, proliferation and apoptosis, Src affects integrin receptor and ion channel activity (Wang et al. 2011; Bae et al. 2014; Stival et al. 2015), and plays an important role in clathrin‐coated pit endocytosis (Zimmerman et al. 2009; Cao et al. 2010; Reinecke & Caplan, 2014). Src was previously found in renal cortical collecting ducts, co‐localizing with AQP2 (Lin et al. 2004). Because of the proximity of Src and AQP2, and the endocytotic mechanism affected by Src and utilized by AQP2, we asked whether Src plays a regulatory role in AQP2 trafficking.

We report that Src inhibition stimulates AQP2 membrane accumulation independently of VP signalling and S256 phosphorylation (pS256). Inhibition of Src by dasatinib not only decreases AQP2 endocytosis, inhibiting clathrin‐mediated endocytosis, but also increases exocytosis in AQP2‐expressing cells. Finally, we found that S269 can be phosphorylated independently of pS256, and that pS269 is essential for dasatinib‐induced AQP membrane accumulation.

Methods

Ethical approval

All animal experiments were approved by the Massachusetts General Hospital Institutional Committee on Research Animal Care (Animal Protocol No. 2016N000040). Animals were inspected regularly by staff trained and experienced in small animal husbandry, with 24 h access to veterinary advice. Investigators were familiar with, and all experiments complied with, the ethical principles of The Journal of Physiology as outlined in Grundy (2015).

DNA constructs and cell culture

The AQP2 (S269A) and AQP2 (S256A, S269A) mutants were produced by site‐directed mutagenesis using the QuickChange site‐directed mutagenesis kit (Stratagene California, La Jolla, CA, USA) by point check mutation following the manufacturer's protocol. The cDNA constructs were confirmed by sequencing (MGH Sequencing Core). Both DNA constructs were stably expressed in LLC‐PK1 cells and selected as described for AQP2 wild‐type cDNA (Katsura et al. 1997; Lu et al. 2008). LLC‐PK1 cells stably expressing c‐myc‐tagged AQP2 (LLC‐AQP2), mutant cells including AQP2 S256A, AQP2 S269A, and AQP2 S256A‐S269A double mutant cells were all grown in Dulbecco's Modified Eagle's Medium (DMEM), FBS 10% and additional L‐glutamine. DAPI staining was used weekly to test for mycoplasma contamination.

Cell treatment and immunocytochemistry

Cells plated onto glass coverslips were starved for at least 1 h in DMEM without FBS before treatment. Cells were treated for 30 min with dasatinib (Das, 10 nM; Selleckchem, Houston, TX, USA), bosutinib (Bos, 20 nM; Selleckchem), KX2‐391 (1 μM; Selleckchem) diluted in dimethyl sulfoxide (DMSO) or treated with methyl‐β‐cyclodextrin (MBCD, 50 mM; Sigma‐Aldrich, St Louis, MO, USA). Cells treated for 10 min with vasopressin (VP; 10 nM; Sigma Aldrich) were used as a positive control, and cells given the equivalent amount of water or DMSO served as negative controls. After treatment, c‐myc‐tagged AQP2 in LLC‐AQP2 cells was detected as previously described using c‐myc mouse monoclonal antibody (Ab), and donkey anti‐mouse IgG coupled to Alexa‐488 (15 μg/ml; Jackson ImmunoResearch, West Grove, PA, USA) (Bouley et al. 2000, 2005, 2011). Cells were then mounted with Vectashield/DAPI (Vector Laboratories, Burlingame, CA, USA) before images were acquired with a Nikon 90i microscope (Nikon Instruments Inc., Melville, NY, USA).

Short interfering RNA (SiRNA) transfection

LLC‐AQP2 cells and mutant AQP2 S256A cells were cultured as previously described above in the cell culture section to 50% confluency and transfected with 20 nM siRNA targeting Src (Invitrogen, Charlestown, MA, USA) 5ʹ‐GCAGAAGUAGAAGAUCCCAA‐3ʹ (Src siRNA1) or 5ʹ‐AAGGGUCUUGCACUAGAGG‐3ʹ (Src siRNA2) or control CT scrambled siRNA 5ʹ‐GCCAAGCGAGUAUUGUAUUCACAAA‐3ʹ for 48 h using Lipofectamine RNAiMAX according to the manufacturer's protocol. We have used either siRNA1 or 2 separately for our siRNA experiments, and both produced similar results. To avoid confusion, we only showed results from siRNA1. After transfection, cells were lysed for RNA extraction or Western blot analysis by methods described below, or fixed and stained for immunocytochemistry as described above.

RNA extraction and quantitative reverse transcriptase PCR

Total RNA was isolated using Qiashredder and RNeasy Mini Kit (Qiagen, Germantown, MD, USA). First‐strand cDNA was synthesized from 1 μg RNA using the High‐Capacity RNA‐to‐cDNA Kit (Applied Biosystems, Beverly, MA, USA). PCR reactions were performed using the PowerUp SYBR Green Master Mix on the QuantStudio3 Real‐Time PCR system (Applied Biosystems) using primers 5ʹ‐GGCCTGAGGATGGGTTAGAGA‐3ʹ and 5ʹ‐CAACAGCCCCACTGAGCAA‐3ʹ (Eurofins Genomics LLC, Louisville, KY, USA). Transcript levels were normalized to GAPDH as an internal control.

Kidney tissue preparation and immunohistochemistry

Adult male Sprague‐Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were housed individually and maintained in a temperature‐controlled room regulated on a 12 h light/dark cycle with free access to water and food. Rats were fed with normal chow (Pro‐Lab Isopro PMH 3000; LabDiet, St Louis, MO, USA) and acclimated 7 days in cages before any procedure. Kidneys were harvested from these rats under terminal anaesthesia with 2% isofluorane inhalation. The kidneys were perfused with PBS (37°C) for 1 min, and cut into thin slices (0.5 mm) using a Stadie‐Riggs microtome (Thomas Scientific, Swedesboro, NJ, USA). Before treatment, all kidney slices were equilibrated 30 min in CO2‐saturated Hanks’ balanced salt solution (HBSS: NaCl 110 mM, KCl 5 mM, MgSO4 1.2 mM, CaCl2 1.8 mM, NaOAc 4 mM, C6H7NaO7 1 mM, D‐glucose 6 mM, L‐alanine 6 mM, NaH2PO4 1 mM, Na2HPO4 3 mM and NaHCO3 25 mM) (Bouley et al. 2000, 2011). Drugs were added directly into the HBSS to obtain their final concentration. After treatment, slices were fixed in 4% paraformaldehyde‐lysine‐periodate fixative (PLP) overnight (Bouley et al. 2005, 2011; Cheung et al. 2016). After 5 washes in PBS, tissues were cryoprotected in PBS, 30% sucrose overnight before being embedded in Tissue‐Tek O.C.T. compound 4583 (Sakura Finetek, Torrance, CA, USA). Cryosections (5 μm) were cut using a Leica CM3050S microtome (Buffalo Grove, IL, USA) and placed onto positively charged slides (Denville Scientific Inc, Holliston, MA, USA). For immunostaining, sections were rehydrated in PBS for 20 min and then permeabilized with 0.1% Triton X‐100 for 10 min. After washing 3 times, non‐specific binding was blocked with 1% BSA in PBS for 30 min, and the tissues were incubated with goat anti‐AQP2 (C17; 0.2 μg/ml; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C. Sections were then incubated with secondary Ab, donkey anti‐goat IgG conjugated to Alexa‐488 (15 μg/ml) for 1 h at room temperature (RT). Tissue slices were washed 3 times then incubated with anti‐clathrin Ab (2.5 μg/ml) for 2 h. After incubation and further washes, the slices were incubated for 1 h with donkey anti‐mouse IgG conjugated to Alexa 568 (2 μg/ml) at RT. After three washes in PBS, sections were mounted in Vectashield/DAPI mounting medium. Anti‐Src Ab (0.7 μg/ml, 36D10; Cell Signaling Technology, Danvers, MA, USA) was used to study Src distribution in rat kidneys using a similar staining protocol as for the anti‐clathrin antibody. Here, the Src antibody was detected with donkey anti‐rabbit IgG conjugated to Alexa 647 (3.8 μg/ml) at RT. Furthermore, the Src antibody specificity was tested by peptide displacement: immunostaining was performed in the absence or the presence of the commercially available Src blocking peptide (1 μg/ml; Cell Signaling Technology) for 1 h at room temperature (RT). Both sets of images were taken under the same exposure conditions, and red pseudo‐color was applied to the far‐red channel. Images were acquired using a Nikon A1R confocal laser‐scanning microscope and/or a Zeiss LSM 800 with Airyscan (Peabody, MA, USA).

Quantification of AQP2 and clathrin membrane staining

We used Volocity software (PerkinElmer, Waltham, MA, USA) for quantification of AQP2 at the cell membrane. We first determined a region of interest corresponding to the membrane, cytoplasm, and nucleus of each cell. The fluorescence in the AQP2 channel was evaluated in each predetermined region of interest and corrected for non‐specific labelling by subtraction of the background fluorescence observed in the nucleus. The mean fluorescence intensity of the plasma membrane area was determined using 30 cells from five different images of each sample taken.

In kidney tissue slices, we quantified the fluorescence intensity of AQP2 or clathrin membrane accumulation in the apical pole of principal cells. Lines were drawn through perpendicularly sectioned cells from the lumen to the basal side of each cell. The peak fluorescence intensity along this line was measured and corrected by subtracting the average nucleus fluorescence pixel intensity of each cell. More than 30 lines (one per cell) were analysed for each tissue. The curve is the average of 3 independent experiments (mean ± SEM).

Western blotting analysis

The Western blotting procedure was described in detail previously (Cheung et al. 2016, 2017). In brief, LLC‐AQP2 and AQP2 (S256A) cells were starved in DMEM without serum for 1 h. After treatment, cells were washed with cold PBS and lysed in cold RIPA buffer (Boston Bioproducts, Ashland, MA, USA) supplemented with a protease inhibitor cocktail (Complete Mini, Roche Diagnostics, Indianapolis, IN, USA), EDTA (5 mM) and phosphatase inhibitors NaF (1 mM), and sodium orthovanadate (1 mM). The protein concentration was determined by BCA (ThermoFisher Scientific, Waltham, MA, USA). 20 μg of solubilized protein was loaded in each well of NuPage 4–12% Bis‐Tris gels (ThermoFisher Scientific), and transferred onto Immun‐blot PVDF membranes (Bio‐Rad, Hercules, CA, USA). Membranes were blocked with PBS‐Tween‐20 0.1% (PBS‐T) containing 5% non‐fat milk for 1 h at RT. Primary Abs were incubated overnight at 4°C. Primary Abs (all raised in rabbit) were anti‐AQP2 (tAQP2; 1:1000; Alomone Labs, Jerusalem, Israel), anti‐phospho‐AQP2 (pSer269; 1:200; Cell Signaling Technology), anti‐phospho‐AQP2 (pSer261; 1:1000; Symansis, Temecula, CA, USA), anti‐phospho‐AQP2 (pSer256; 1:1000; Abcam, Cambridge, MA, USA), anti‐Src (1:2000) and anti‐phospho‐Src (pTyr416; 1:1000; Cell Signaling Technology).

To analyse the effect of Src inhibition on CREB or ERK phosphorylation, the primary Abs used (all raised in rabbit) were anti‐phospho‐CREB (1:1000) and anti‐CREB (1:2000) (Santa Cruz Biotechnology), anti‐phospho‐ERK (1:2000) and anti‐ERK (1:2000) (Cell Signaling Technology).

After incubation with primary antibodies, the membranes were washed 5 times with PBS‐T 5 min each before secondary anti‐rabbit IgG Ab conjugated to horseradish peroxidase (Jackson ImmunoResearch) was applied (0.16 μg/ml). Membranes were incubated in chemiluminescence Western Lightning ECL (Amersham, Pittsburgh, PA, USA) and were exposed to Hyblot ES film (Denville Scientific). Then, membranes were stripped with Western blot stripping buffer (ThermoFisher Scientific) for at least 15 min and re‐incubated with different Abs as listed previously. Band intensities in the different experimental conditions were quantified using Image J (NIH, Bethesda, MD, USA). Band intensity of the phospho‐serine Ab was corrected according to the band intensity of total AQP2 on the same membrane.

Fluorescence exocytosis and endocytosis assay

The exocytosis and endocytosis assays were described in detail in previous studies (Bouley et al. 2006; Nunes et al. 2008; Cheung et al. 2016). In brief, exocytosis was quantified in LLC‐PK1 cells stably expressing both c‐myc‐tagged AQP2 and the soluble yellow fluorescent protein (ssYFP) (LLC‐AQP2‐ssYFP cells) or ssYFP alone (LLC‐ssYFP cells). The cells were starved 1 h in HBBS (Hank's buffered saline solution; Invitrogen) supplemented with 20 mM HEPES and 2 g/l glucose, then drugs were added. At the end of each treatment, 150 μl of medium was transferred to a black half‐area 96‐well plate, and analysed using a multimode plate reader (model DTX880, Beckman‐Coulter, Fullerton, CA, USA). The fluorescent values represent five independent experiments performed in triplicate. Each fluorescence value is reported as a ratio of each background‐ and zero‐subtracted value.

The endocytosis assay was performed using the following protocol: after 1 h starving in serum‐free DMEM, cells were treated with drugs. Dialysed Texas Red dextran was added to a final concentration of 1.5 mg/ml 10 min before the end of the treatment. Cells were then washed with PBS then lysed in 150 μl of cold RIPA buffer and protease inhibitor cocktail. Fluorescence signals of the supernatants (100 μl) were read on a DTX880 Multimode plate reader. Endocytosis was also studied using a rhodamine‐tagged transferrin ligand (Rho‐Tf; Invitrogen). LLC‐AQP2 cells were incubated with drugs for 30 min, and Rho‐Tf was added to cells to reach the final concentration of 1 mg/ml for 15 min after incubation. Cells were then washed with cold PBS twice before fixing in 4% PFA. Images of cells were taken using a Nikon 90i microscope, and analysed by Volocity.

Cyclic AMP activity assay

The cAMP levels in cells treated with drugs were analysed using the BioTrack EIA system (GE Healthcare Life Sciences, Piscataway, NJ, USA) (Bouley et al. 2000). Here, cells were starved 2 h before drug administration and VP (10 nM) was used as positive control. Results were measured using the DTX880 Multimode plate reader. The assay was performed three times independently, and each treatment was done in triplicate (mean ± SEM).

Data analysis

Images of immunocytochemistry were analysed using Volocity. Western blotting results were quantified with Image J. Data are expressed as means ± SEM or means ± SD, and statistical analyses were performed as appropriate using the one‐way or two‐way ANOVA Tukey test. Differences were considered to be significant at P values < 0.05.

Results

Src kinase co‐localizes with AQP2 in kidney collecting ducts

Src kinase is expressed in the rat collecting duct, but its location differs along the tubule (Fig. 1). In the cortex, Src (Fig. 1, red) is most strongly expressed at the apical pole of intercalated cells, which also show basolateral staining. In principal cells, Src staining is also present in both the apical and basolateral membranes, whereas AQP2 (Fig. 1, green) is more strongly expressed at the apical pole. In the outer medulla, Src is equally expressed in the apical and basolateral poles, and co‐localizes with AQP2 mainly at the apical pole of principal cells. In the inner medulla, AQP2 and Src are co‐localized both apically and basolaterally, but Src staining is much stronger in the basolateral pole of principal cells. The insets in the left column of Fig. 1 show the lack of specific Src staining in the presence of blocking peptide, the peptide sequence used to develop the primary anti‐Src antibody. The insets in the middle and right columns show the expected AQP2 (green) staining in tissues double‐stained after application of the Src blocking peptide. The partial co‐localization and cell expression pattern of Src and AQP2 in the medullary collecting duct suggests a possible role of Src in AQP2 trafficking.

Figure 1.

Figure 1

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Src is differentially expressed in the cortex and medulla, and co‐localizes with AQP2 in the medulla

Immunocytochemistry shows Src (red, left column) and AQP2 (green, middle column) in cortex (upper row), outer medulla (middle row) and inner medulla (bottom row). The right column shows merged panels. In the cortex, Src is most strongly expressed apically in intercalated cells (which also have basolateral staining), as well as in both the apical and basolateral membranes of principal cells. In the inner and outer medulla, Src and AQP2 are co‐localized in principal cells, mainly at the apical pole in the inner stripe, but both apically and basolaterally in the inner medulla. The insets in the left column show the lack of specific Src staining in the presence of blocking peptide. The insets in the middle and right columns show the expected AQP2 (green) staining in tissues double‐stained after application of the Src blocking peptide. These images are representative of staining performed in three different animals. Bar is 10 μm.

VP reduces Src phosphorylation, and inhibiting Src alone induces membrane accumulation of AQP2

AQP2 shifted from the cytoplasm to the plasma membrane after VP treatment in LLC‐PK1 cells stably transfected with c‐myc‐tagged AQP2 (LLC‐AQP2) as previously described (Fig. 2 A, CT and VP) (Katsura et al. 1997; Bouley et al. 2000; Gustafson et al. 2000). A similar pattern was observed when cells were treated for 30 min with Src inhibitors dasatinib (Das), bosutinib (Bos) and KX2‐391. Quantification shows significant increases of AQP2 membrane expression with VP and all Src antagonists. We also tested the effect of Das in rat kidney slices (Fig. 2 B) and observed an increase of apical membrane accumulation of AQP2. Moreover, we found that VP and Das both decrease Src phosphorylation significantly (Fig. 2 C), suggesting that Src inhibition may also be a mechanism utilized by VP to exert its effect on AQP2. To further confirm the specific role of Src on AQP2 trafficking, we next transfected our cell models with Src siRNA to down‐regulate the expression of Src.

Figure 2.

Figure 2

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Inhibition of Src activity induces AQP2 membrane accumulation

A, under control conditions (CT), AQP2 is located throughout the cytoplasm of LLC‐AQP2 cells. In contrast, AQP2 is accumulated in the plasma membrane in the presence of VP. When treated with Src inhibitors, dasatinib (Das), bosutinib (Bos) or KX2‐391, AQP2 is also located mainly at the plasma membrane. Membrane AQP2 accumulation was quantified on at least 30 cells per treatment in each experiment. These images are representative of 4 different experiments (mean ± SD, n = 4, * P < 0.0001). Bar is 10 μm. B, immunocytochemistry on in situ kidney slices treated with Das showed an increase of AQP2 at the plasma membrane compared to tissues treated with buffer alone (CT). These images are representative of 4 different experiments. Bar is 20 μm. C, Western blot analysis of LLC‐AQP2 cell lysate treated with DMSO (CT), vasopressin (VP, 10 nM) or Das (10 nM) was performed in triplicate. Phospho‐Src (pSrc) was detected using a specific phospho Src‐antibody. After detection, membranes were stripped and re‐probed with total Src antibody (tSrc). Band intensities were quantified and corrected based on tSrc expression. The quantification is the average of 4 independent experiments (mean ± S.E.M, n = 4, * P < 0.0001).

Down‐regulation of Src kinase shifts AQP2 localization from the cytoplasm to the plasma membrane, and this process is S256 independent

We transfected our LLC‐AQP2 cells with Src siRNA for 48 h and found a greater than 50% reduction of Src mRNA (data not shown) and a significant decrease in Src protein expression (Fig. 3 A). We found that by down‐regulating Src, we induced AQP2 membrane expression, similar to the effect in cells treated with Src inhibitors. We furthered our study by transfecting our mutant cell line (LLC‐AQP2 S256A) in which serine 256 cannot be phosphorylated, and found that Src siRNA again caused a shift of AQP2 from the cytoplasm to the plasma membrane. This finding suggests that AQP2 membrane accumulation induced by Src suppression is S256 independent (Fig. 3 B). We continued our investigation by using specific phospho‐antibodies to study the phosphorylation states of other essential serine residues.

Figure 3.

Figure 3

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Down‐regulation of Src expression using siRNA shifts AQP2 from the cytoplasm to the plasma membrane

A, Western blot analysis (left) showed that Src expression in LLC‐AQP2 cells was decreased significantly when cells were transfected with Src siRNA (20 nM) for 48 h (right). B, immunocytochemistry showed that transfection with 20 nM of scrambled (CT) and Src siRNA did not induce significant morphological changes in our cells (left), and cells transfected with src SiRNA had a significant increase in AQP2 membrane expression compared to cells transfected with scrambled siRNA both in LLC‐AQP2 (upper right) and LLC‐AQP2 S256A mutant cells (lower right). The quantification for Western blot analysis is the average of 4 independent experiments (mean ± S.E.M, n = 4, * P = 0.005). Quantification of AQP2 was performed in at least 30 cells per experiment and 3 experiments were included (mean ± SD, n = 3, * P < 0.0001 for both LLC‐AQP2 and LLC‐AQP2 S256A cells). Bar is 10 μm.

Src inhibition shows a different AQP2 phosphorylation pattern from VP: it does not change S256 and S261 phosphorylation, but increases S269 phosphorylation

In contrast to VP, which induces S256 and S269 phosphorylation and S261 dephosphorylation, Das treatment of LLC‐AQP2 cells did not result in S256 phosphorylation or S261 dephosphorylation (Fig. 4 A and B). However, there was an increase in S269 phosphorylation similar to VP treatment (Fig. 4 C). In order to confirm these findings, we created stable mutant cell lines that have point mutations at the pertinent serine residues that would not allow phosphorylation of these residues.

Figure 4.

Figure 4

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Src inhibition increases AQP2 S269 phosphorylation

Western blot analysis of LLC‐AQP2 cell lysate treated with DMSO (CT), vasopressin (VP, 10 nM) or Das (1 nM or 10 nM) was performed in duplicate. Src inhibition with Das, in contrast to VP, does not increase AQP2 S256 phosphorylation (A), nor does it decrease S261 phosphorylation (B). However, it increases S269 phosphorylation significantly (C). AQP2 pS256, pS261 and pS269 were detected using specific phospho‐Ab. After detection with each specific phospho‐Ab, membranes were stripped and re‐probed with a different phospho‐Ab, and finally with total AQP2 Ab (tAQP2). Of note, panels A and B are from the same membrane and, thus, they share the same tAQP2 loading control. Band intensities were quantified and expressed relative to tAQP2 expression. The quantification is the average of 4 independent experiments. (mean ± S.E.M, n = 4, * P < 0.05. For pS269, P = 0.0029 for CT vs. VP, P = 0.0149 for CT vs. 1 nM Das and P = 0.0052 for CT vs. 10 nM Das).

S269 can be phosphorylated independently of S256, and S269 phosphorylation is important for Das‐induced AQP2 membrane accumulation

Src inhibition by Das treatment, similar to cells transfected with Src siRNA (Fig 3 B), induced AQP2 accumulation at the plasma membrane in LLC‐AQP2‐S256A cells (Fig. 5 A). This result is similar to cells incubated with MBCD (methyl‐β‐cyclodextrin, Fig 5 A, inset), a non‐specific endocytosis inhibitor (Lu et al. 2004; Cheung et al. 2016). On the other hand, consistent with previous findings (Lu et al. 2004; Arthur et al. 2015), VP did not cause AQP2 membrane accumulation. Western blot analysis of LLC‐AQP2 S256A cells shows a significant increase in phosphorylation of AQP2 S269 with VP and Das, despite the lack of S256 phosphorylation (Fig. 5 B). LLC‐AQP2 cell lysates were used in parallel as a positive control.

Figure 5.

Figure 5

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Effect of Src inhibition on AQP2 trafficking is independent of AQP2 S256 phosphorylation

A, under basal conditions, the AQP2 S256A mutant is located throughout the cytoplasm in LLC‐AQP2 S256A cells (CT). Das induces AQP2 membrane accumulation similar to cells treated with MBCD (inset), a non‐specific endocytosis blocker. In contrast, VP treatment does not induce AQP2 membrane accumulation in these cells. B, Western blot analysis of LLC‐AQP2 S256A cells treated with VP or Das was performed in triplicate using a specific pS269 AQP2 antibody; LLC‐AQP2 cells (WT) serve as controls. Quantification (see the panels on the right) shows the average of 4 experiments performed in triplicate (mean ± S.E.M, n = 4, * P < 0.05, P = 0.0375 for CT vs. VP and P = 0.0315 for CT vs. Das). C and D, LLC‐AQP2 S269A mutant (C) and LLC‐AQP2 double mutant cells (S256A, S269A; D) were treated with MBCD (insets), VP (10 nM) or Das (1 nM). MBCD caused membrane accumulation of AQP2 in both cell lines. VP caused membrane accumulation in S269A cells but not in S256A cells, and Das was without effect in both cell lines. Quantification of AQP2 was performed in at least 30 cells per experiment and 3 experiments were included (mean ± SD, n = 3, * P < 0.0001). Bar is 10 μm.

To further examine the role of S269 phosphorylation, we established a stable mutant cell line, LLC‐AQP2‐S269A, to evaluate whether S269 phosphorylation is essential for Das‐induced AQP2 membrane accumulation (Fig. 5 C). The S269A mutant accumulates at the membrane when cells are treated with VP or MBCD (Arthur et al. 2015). However, Das completely lost its effect: the distribution of AQP2 remained cytoplasmic, similar to controls. We repeated the experiment in another mutant cell line, in which two serine‐to‐alanine mutations were introduced into AQP2 (S256A, S269A) (Fig. 5 D). The cytoplasmic distribution pattern with VP and dasatinib was similar to untreated cells (CT). Neither VP nor Das induced AQP2 (S256A, S269A) membrane accumulation, but the AQP2 double mutant did accumulate on cell membranes upon MBCD treatment, suggesting that this AQP2 double mutant is still recycling at the plasma membrane. This is consistent with our previous findings in cells expressing mutations in multiple AQP2 phosphorylation sites (Arthur et al. 2015). After observing the effect of Src inhibition on AQP2 phosphorylation, we investigated whether the major pathways of AQP2 signalling were activated.

The effect of Src inhibition on AQP2 does not utilize the cAMP/PKA pathway

Using a cAMP ELISA assay, we found that Das does not cause an increase in intracellular cAMP level, in contrast to VP, our positive control, which increases cAMP more than 1000‐fold compared to CT and Das treated cells (Fig. 6 A). Moreover, Das did not result in phosphorylation of the PKA substrate cAMP response element binding protein (CREB), whereas CREB was phosphorylated significantly upon VP treatment (Fig. 6 B). These results suggest that Das does not use the cAMP/PKA pathway to induce AQP2 membrane accumulation.

Figure 6.

Figure 6

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Src inhibition does not increase intracellular cAMP, PKA activity or MAPK/ERK phosphorylation

A, cAMP ELISA assay shows no increase of intracellular cAMP level in LLC‐AQP2 cells treated with Das, in contrast to the large increase seen with VP. This is an average of 3 independent experiments performed in triplicate (n = 3, * P < 0.0001). B, Western blot analysis using a specific phospho‐CREB (a substrate of PKA) Ab does not show any increase of PKA activity in cells treated with Das, again in contrast to the large effect of VP (n = 3, * P < 0.0001). C, Western blot analysis shows that EGF increases phosphorylation of ERK significantly but Das has no effect. The Western blots are representative of 5 independent experiments. The band density quantification on the right shows an average of 5 independent experiments performed in duplicate (means ± S.E.M, n = 5, * P = 0.0001).

Since MAPK/ERK is the main downstream effector associated with Src signalling (Moon et al. 2002; Johnson, 2008), we examined the effect of Das on ERK phosphorylation in our LLC‐AQP2 cells. Das treatment caused a slight but not statistically significant increase in pERK in Das treatment, in contrast to EGF, our positive control (Fig. 6 C).

Das treatment of LLC‐AQP2 cells increases exocytosis and reduces endocytosis

To explain how AQP2 accumulates at the plasma membrane, we studied endocytosis and exocytosis of AQP2 when Src is inhibited. Using soluble secreted yellow fluorescent protein (ssYFP) as a surrogate marker of AQP2 exocytosis in LLC‐AQP2 cells (Fig. 7 A) (Nunes et al. 2008), we found that both VP and Das significantly increased exocytosis of ssYFP by at least 50% compared to control (CT). In cells without AQP2, VP caused a reduced but still significant increase in exocytosis, but Das had no effect. This result suggests that the presence of AQP2 in vesicles is necessary to reveal the effect of the Src inhibition, and the effect of Das may be specific to AQP2‐containing vesicles. We also examined the effect of Das on endocytosis and found a significant 20% reduction of endocytosis of Texas Red‐tagged dextran in LLC‐AQP2 cells when they were treated with dasatinib (Fig. 7 B). MBCD, our positive control, a non‐specific endocytosis inhibitor, also decreased endocytosis significantly. A similar result was found when we studied the internalization of rhodamine‐tagged transferrin in LLC‐AQP2 cells (Fig. 7 C). Under control conditions, transferrin is internalized and distributed throughout the cytoplasm. In contrast, in cells treated with Das or MBCD, we found an accumulation of transferrin at the membrane. This result suggests that Das inhibits clathrin‐mediated endocytosis (since transferrin is internalized by this mechanism). Previously, we showed that the internalization of AQP2 is clathrin‐coat pit dependent (Sun et al. 2002), and we further confirmed this by staining kidney tissues with clathrin and AQP2 antibodies. We quantified an increase in clathrin at the apical membrane of principal cells treated with Das, co‐localizing with AQP2 (Fig. 7 D), suggesting a block in clathrin‐mediated internalization of AQP2. In summary, Das inhibits clathrin‐mediated endocytosis of AQP2 and increases exocytosis, and as a result, increases AQP2 membrane accumulation.

Figure 7.

Figure 7

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Src inhibition increases exocytosis and decreases endocytosis

A, Das increases the exocytosis of soluble secreted yellow fluorescent protein (ssYFP) in LLC‐AQP2 cells but not in LLC‐PK1 cells that do not express AQP2. These results are an average of 4 independent experiments performed in triplicate (n = 4, * P < 0.05. For LLC‐AQP2 cells, P = 0.0163 for CT vs. VP, P = 0.0293 for CT vs. 1 nM Das, and P = 0.0447 for CT vs. 10 nM Das. For LLC‐PK1 cells, P = 0.0002). B, Das and MBCD (used as a positive control) reduced the endocytosis of Texas Red‐tagged dextran in LLC‐AQP2 cells. This result is an average of 3 independent experiments performed in triplicate (n = 3, * P = 0.0052 and ** P = 0.0002). C, rhodamine‐tagged transferrin accumulates at the plasma membrane of LLC‐AQP2 cells treated for 30 min with Das and MBCD, in contrast to control cells (CT), where transferrin is diffusely localized in the cytoplasm after endocytosis. (mean ± S.E.M, n = 3, * P < 0.0001. Bar is 10 μm. D, immunohistochemistry using anti‐AQP2 and anti‐clathrin antibodies shows an increase of clathrin (orange) and AQP2 (green) in the apical pole of principal cells upon treatment with Das when compared to control. The quantification is the average of 3 independent experiments (mean ± S.E.M, n = 3, * P = 0.0133. Bar is 20 μm.

Discussion

Our study suggests a role of the non‐receptor tyrosine kinase, Src in AQP2 trafficking and phosphorylation. Src inhibition results in an accumulation of plasma membrane AQP2 by increasing exocytosis and decreasing clathrin‐mediated endocytosis, in a cAMP/PKA signalling pathway‐independent manner. Importantly, Src inhibition phosphorylates S269 independently of S256, and its effect on AQP2 membrane accumulation is S269 phosphorylation dependent. Src kinases are a family of non‐receptor tyrosine kinases including nine members that are expressed at various levels in different tissues (Schartl & Barnekow, 1984). Utilizing three inhibitors, dasatinib, bosutinib and KX2‐391, we conclude that only Src, but not other family members, or other pathways (such as Abl or c‐kit which can be affected by dasatinib but not by KX2‐391), is involved in AQP2 trafficking (Lombardo et al. 2004; Berger et al. 2005; Fallah‐Tafti et al. 2011). We further confirmed the role of Src using siRNA. Our study shows a high expression of Src in kidney collecting ducts (CD), and a differential Src expression along the CD that, in the medulla, strongly coincides with AQP2 localization in the principal cells. While cortical collecting ducts play a significant role in urinary concentration, we have previously found differences in signalling pathway regulation between cortex and medulla (Bouley et al. 2000, 2005). Our finding with Src is perhaps one more example of non‐homogenous AQP2 regulation in different collecting duct regions. It is possible that this allows the kidney to fine‐tune water balance more selectively. Although the effect of Src on AQP2 may be more predominant in the medulla, inhibition of Src with FDA‐approved Src inhibitors such as dasatinib can cause syndrome of inappropriate secretion of anti‐diuretic hormone (SIADH) (Hill et al. 2016), a state of excessive water reabsorption secondary to AQP2 up‐regulation and overexpression. In addition, fluid retention and hyponatraemia, an electrolyte imbalance caused by dysregulated retention of water by the kidney, is frequently reported as a common adverse effect of Src inhibitors in clinical trials (Brave et al. 2008; Gold et al. 2014; Schuetze et al. 2017). These clinical findings may be related to the effect of Src inhibition on AQP2 that we have uncovered here.

Our study shows that Src kinase affects AQP2 trafficking and induces differential phosphorylation of AQP2; inhibition of Src results in accumulation of AQP2 at the plasma membrane, and this process involves phosphorylation of S269, but not S256. This unexpected result suggests that phosphorylation of AQP2 residues can occur independently, and that S256 phosphorylation is not a pre‐requisite for S269 phosphorylation. S269 phosphorylation is thought to be important for optimal AQP2 membrane retention (Hoffert et al. 2008; Moeller et al. 2009), and as AQP2 recycles constitutively from the cytoplasm to the membrane, a dasatinib‐induced increase in pS269 may, therefore, play an important role in the observed increase in AQP2 membrane accumulation. However, in our S256A mutant cells (Fig. 5 B), we also saw a significant increase of S269 phosphorylation in VP‐treated cells, even though VP does not induce AQP2 membrane accumulation in these cells. This discrepancy between the phenotypic effect of VP and dasatinib on S256A cells suggests that dasatinib is utilizing additional mechanisms that allow AQP2 to accumulate at the membrane. Our data also imply that these mechanisms are dependent on S269 phosphorylation because dasatinib does not induce AQP2 membrane accumulation in S269A AQP2 cells. The kinase involved in S269 phosphorylation remains elusive, but prior in vitro studies have shown that PKA does not phosphorylate S269 (Hoffert et al. 2008; Cao et al. 2010), and homology sequence analysis predicts no Src phosphorylation motif on AQP2 (Brown et al. 2008). Of note, when Src was inhibited in our LLC‐AQP2 cells, we detected a slight but not significant increase in MAPK/ERK phosphorylation. Whether this is important for S269 phosphorylation or AQP2 trafficking is unclear, as VP treatment induces variable and inconsistent results in the MAPK/ERK phosphorylation pattern across different cell types (Pisitkun et al. 2008; Rinschen et al. 2010; Cheung et al. 2017).

Src is also known to be a multifunctional protein that affects protein exocytosis and endocytosis. For example, Src affects the release of neurotransmitter (Ely et al. 1994; Ohnishi et al. 2001), recycling of receptors (Auciello et al. 2013; Vistein & Puthenveedu, 2014), uptake of transferrin (Cao et al. 2010), and internalization of growth factors and integrins (Belleudi et al. 2011; Wang et al. 2011). In addition, Src is also implicated in vesicular trafficking. The role of Src may be direct: it is recruited to and continuously associated with vesicles generated at membrane ruffles up to their fusion with late endosomes and lysosomes (Kasahara et al. 2007). The effect of Src can also be indirect: Src signalling activates caveolin‐1 and induces albumin transcytosis in vascular epithelial cells (Minshall et al. 2000). Here, we showed that Src inhibition affects both AQP2 exocytosis and endocytosis simultaneously. These synergistic effects combine to increase AQP2 membrane accumulation. Moreover, our data suggest that Src kinases are present and potentially active under basal conditions. When Src activity is blocked, AQP2 accumulates at the membrane, implying that Src plays a role in maintaining the balance of AQP2 constitutive recycling so that membrane accumulation does not normally occur under baseline conditions. It is perhaps not surprising that Src should play a role in AQP2 constitutive recycling, as Src has been previously implicated in the constitutive internalization of G‐protein coupled receptors or channels (Chao et al. 2005; Lee et al. 2013). In our study, we were not able to measure a significant change in actin depolymerization (data not shown), which is believed to be an important step in AQP2 membrane accumulation (Noda et al. 2008; Yui et al. 2012), and the mechanism by which Src inhibition increases exocytosis remains elusive. Inhibition of Src also blocks clathrin‐mediated endocytosis and this contributes to AQP2 accumulation at the cell surface. It is possible that Src inhibition blocks the fission of the clathrin‐coated pits from the plasma membrane, as Src has been shown to phosphorylate the GTPase dynamin 2, the protein responsible for membrane scission at the neck of clathrin‐coated pits (Wang et al. 2011). Our results showing membrane accumulation of transferrin with Src inhibition support the role of Src phosphorylation of dynamin in the constitutive recycling of transferrin (Cao et al. 2010). Indeed, we have previously shown that inhibition of dynamin activity results in constitutive membrane accumulation of AQP2 (Zhang et al. 2002; Lu et al. 2004).

In summary, our data highlight the role of the non‐receptor tyrosine kinase Src in AQP2 water channel trafficking. Src plays a significant role in the constitutive recycling of AQP2 by decreasing the efficiency of AQP2 internalization, leading to AQP2 plasma membrane accumulation. The S269 AQP2 phosphorylation that occurs after Src inhibition by dasatinib is a critical and necessary step in this process, but this phosphorylation event does not depend on prior S256 phosphorylation. Because of its differential expression in renal tubules, and its function in the regulation of other receptors and channels (Sterling et al. 2003; Liang et al. 2006; Thebault et al. 2009; Li et al. 2015), Src is poised to play a role in the fine‐tuning of water and electrolyte homeostasis by the kidney.

Additional information

Competing interests

None declared.

Author contributions

P.W.C, D.B. and R.B. conceived and designed the paper; A.T. and S.A.J. contributed ideas and data; all authors analysed data and refined both text and intellectual content. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was supported by National Institutes of Health (NIH) grant DK096586 (D.B.). P.W.C. was supported by a Ben J. Lipps Research Fellowship Award from the American Society of Nephrology, by the National Fellow‐to‐Faculty Transition Award from the American Heart Association, and by an NIH K‐award 1K08DK115901‐01. A.T. was supported by funding from The Harvard Summer Research Program in Kidney Medicine (HSRPKM) grant R25DK101398. S.A.J. was supported by an internship stipend from Radboud University Medical Center, The Netherlands. The Nikon A1R and the Zeiss LSM 800 with Airyscan confocals in the Program in Membrane Biology (PMB) Microscopy Core were purchased using NIH Shared Instrumentation Grants 1S10RR031563‐01 and 1S10OD021577‐01 (D.B.), respectively. Additional support for the Program in Membrane Biology Microscopy Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (DK043351).

Biography

Pui Cheung is an Instructor in Medicine at Harvard Medical School and a physician in the Division of Nephrology at Massachusetts General Hospital. Her research interest is in renal water handling, focusing on modulating aquaporin‐2 water channel phosphorylation and trafficking in physiological and diseased states. She showed that epidermal growth factor (EGF) antagonizes the effect of vasopressin on aquaporin‐2 trafficking, and her current effort is to dissect the complex EGF pathways that control water homeostasis. Her goal is to uncover novel treatments for water balance such as the syndrome of inappropriate ADH production, heart failure and cirrhosis.

graphic file with name TJP-597-1627-g001.gif

Edited by: Kim Barrett & Robert Fenton

D. Brown and R. Bouley made equal contributions to this work.

This is an Editor's Choice article from the 15 March 2019 issue.

Linked articles: This article is highlighted in a Perspectives article by Moeller. To read this article, visit https://doi.org/10.1113/JP277502.

References

  1. Arthur J, Huang J, Nomura N, Jin WW, Li W, Cheng X, Brown D & Lu HJ (2015). Characterization of the putative phosphorylation sites of the AQP2 C terminus and their role in AQP2 trafficking in LLC‐PK1 cells. Am J Physiol Renal Physiol 309, F673–F679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auciello G, Cunningham DL, Tatar T, Heath JK & Rappoport JZ (2013). Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8. J Cell Sci 126, 613–624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bae SH, Kim DH, Shin SK, Choi JS & Park KS (2014). Src regulates membrane trafficking of the Kv3.1b channel. FEBS Lett 588, 86–91. [DOI] [PubMed] [Google Scholar]
  4. Belleudi F, Scrofani C, Torrisi MR & Mancini P (2011). Polarized endocytosis of the keratinocyte growth factor receptor in migrating cells: role of SRC‐signaling and cortactin. PLoS One 6, e29159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berger DM, Dutia M, Birnberg G, Powell D, Boschelli DH, Wang YD, Ravi M, Yaczko D, Golas J, Lucas J & Boschelli F (2005). 4‐Anilino‐7,8‐dialkoxybenzo[g]quinoline‐3‐carbonitriles as potent Src kinase inhibitors. J Med Chem 48, 5909–5920. [DOI] [PubMed] [Google Scholar]
  6. Bouley R, Breton S, Sun T, McLaughlin M, Nsumu NN, Lin HY, Ausiello DA & Brown D (2000). Nitric oxide and atrial natriuretic factor stimulate cGMP‐dependent membrane insertion of aquaporin 2 in renal epithelial cells. J Clin Invest 106, 1115–1126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bouley R, Hawthorn G, Russo LM, Lin HY, Ausiello DA & Brown D (2006). Aquaporin 2 (AQP2) and vasopressin type 2 receptor (V2R) endocytosis in kidney epithelial cells: AQP2 is located in ‘endocytosis‐resistant’ membrane domains after vasopressin treatment. Biol Cell 98, 215–232. [DOI] [PubMed] [Google Scholar]
  8. Bouley R, Lu HA, Nunes P, Da Silva N, McLaughlin M, Chen Y & Brown D (2011). Calcitonin has a vasopressin‐like effect on aquaporin‐2 trafficking and urinary concentration. J Am Soc Nephrol 22, 59–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bouley R, Pastor‐Soler N, Cohen O, McLaughlin M, Breton S & Brown D (2005). Stimulation of AQP2 membrane insertion in renal epithelial cells in vitro and in vivo by the cGMP phosphodiesterase inhibitor sildenafil citrate (Viagra). Am J Physiol Renal Physiol 288, F1103–F1112. [DOI] [PubMed] [Google Scholar]
  10. Brave M, Goodman V, Kaminskas E, Farrell A, Timmer W, Pope S, Harapanhalli R, Saber H, Morse D, Bullock J, Men A, Noory C, Ramchandani R, Kenna L, Booth B, Gobburu J, Jiang X, Sridhara R, Justice R & Pazdur R (2008). Sprycel for chronic myeloid leukemia and Philadelphia chromosome‐positive acute lymphoblastic leukemia resistant to or intolerant of imatinib mesylate. Clin Cancer Res 14, 352–359. [DOI] [PubMed] [Google Scholar]
  11. Brown D, Hasler U, Nunes P, Bouley R & Lu HA (2008). Phosphorylation events and the modulation of aquaporin 2 cell surface expression. Curr Opin Nephrol Hypertens 17, 491–498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cao H, Chen J, Krueger EW & McNiven MA (2010). SRC‐mediated phosphorylation of dynamin and cortactin regulates the “constitutive” endocytosis of transferrin. Mol Cell Biol 30, 781–792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chao C, Ives KL, Goluszko E, Kolokoltsov AA, Davey RA, Townsend CM Jr & Hellmich MR (2005). SRC regulates constitutive internalization and rapid resensitization of a cholecystokinin 2 receptor splice variant. J Biol Chem 280, 33368–33373. [DOI] [PubMed] [Google Scholar]
  14. Cheung PW, Nomura N, Nair AV, Pathomthongtaweechai N, Ueberdiek L, Lu HA, Brown D & Bouley R (2016). EGF receptor inhibition by erlotinib increases aquaporin 2‐mediated renal water reabsorption. J Am Soc Nephrol 27, 3105–3116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cheung PW, Ueberdiek L, Day J, Bouley R & Brown D (2017). Protein phosphatase 2C (PP2C) is responsible for VP‐induced dephosphorylation of AQP2 serine 261. Am J Physiol Renal Physiol, 313, F404–F413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ely CM, Tomiak WM, Allen CM, Thomas L, Thomas G & Parsons SJ (1994). pp60c‐src enhances the acetylcholine receptor‐dependent catecholamine release in vaccinia virus‐infected bovine adrenal chromaffin cells. J Neurochem 62, 923–933. [DOI] [PubMed] [Google Scholar]
  17. Fallah‐Tafti A, Foroumadi A, Tiwari R, Shirazi AN, Hangauer DG, Bu Y, Akbarzadeh T, Parang K & Shafiee A (2011). Thiazolyl N‐benzyl‐substituted acetamide derivatives: synthesis, Src kinase inhibitory and anticancer activities. Eur J Med Chem 46, 4853–4858. [DOI] [PubMed] [Google Scholar]
  18. Fenton RA, Moeller HB, Hoffert JD, Yu MJ, Nielsen S & Knepper MA (2008). Acute regulation of aquaporin‐2 phosphorylation at Ser‐264 by vasopressin. Proc Natl Acad Sci U S A 105, 3134–3139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fushimi K, Sasaki S & Marumo F (1997). Phosphorylation of serine 256 is required for cAMP‐dependent regulatory exocytosis of the aquaporin‐2 water channel. J Biol Chem 272, 14800–14804. [DOI] [PubMed] [Google Scholar]
  20. Gold KA, Lee JJ, Harun N, Tang X, Price J, Kawedia JD, Tran HT, Erasmus JJ, Blumenschein GR, William WN, Wistuba, II & Johnson FM (2014). A phase I/II study combining erlotinib and dasatinib for non‐small cell lung cancer. Oncologist 19, 1040–1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Grundy D ( 2015). Principles and standards for reporting animal experiments in The Journal of Physiology and Experimental Physiology . J Physiol 593, 2547–2549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gustafson CE, Katsura T, McKee M, Bouley R, Casanova JE & Brown D (2000). Recycling of AQP2 occurs through a temperature‐ and bafilomycin‐sensitive trans‐Golgi‐associated compartment. Am J Physiol Renal Physiol 278, F317–F326. [DOI] [PubMed] [Google Scholar]
  23. Hill J, Shields J & Passero V (2016). Tyrosine kinase inhibitor‐associated syndrome of inappropriate secretion of anti‐diuretic hormone. J Oncol Pharm Pract 22, 729–732. [DOI] [PubMed] [Google Scholar]
  24. Hoffert JD, Fenton RA, Moeller HB, Simons B, Tchapyjnikov D, McDill BW, Yu MJ, Pisitkun T, Chen F & Knepper MA (2008). Vasopressin‐stimulated increase in phosphorylation at Ser269 potentiates plasma membrane retention of aquaporin‐2. J Biol Chem 283, 24617–24627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hoffert JD, Pisitkun T, Wang G, Shen RF & Knepper MA (2006). Quantitative phosphoproteomics of vasopressin‐sensitive renal cells: regulation of aquaporin‐2 phosphorylation at two sites. Proc Natl Acad Sci U S A 103, 7159–7164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jensen AM, Bae EH, Fenton RA, Norregaard R, Nielsen S, Kim SW & Frokiaer J (2009). Angiotensin II regulates V2 receptor and pAQP2 during ureteral obstruction. Am J Physiol Renal Physiol 296, F127–F134. [DOI] [PubMed] [Google Scholar]
  27. Jensen AM, Bae EH, Norregaard R, Wang G, Nielsen S, Schweer H, Kim SW & Frokiaer J (2010). Cyclooxygenase 2 inhibition exacerbates AQP2 and pAQP2 downregulation independently of V2 receptor abundance in the postobstructed kidney. Am J Physiol Renal Physiol 298, F941–F950. [DOI] [PubMed] [Google Scholar]
  28. Johnson DE ( 2008). Src family kinases and the MEK/ERK pathway in the regulation of myeloid differentiation and myeloid leukemogenesis. Adv Enzyme Regul 48, 98–112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jung HJ & Kwon TH (2016). Molecular mechanisms regulating aquaporin‐2 in kidney collecting duct. Am J Physiol Renal Physiol 311, F1318–F1328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kasahara K, Nakayama Y, Sato I, Ikeda K, Hoshino M, Endo T & Yamaguchi N (2007). Role of Src‐family kinases in formation and trafficking of macropinosomes. J Cell Physiol 211, 220–232. [DOI] [PubMed] [Google Scholar]
  31. Katsura T, Gustafson CE, Ausiello DA & Brown D (1997). Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin‐2 in transfected LLC‐PK1 cells. Am J Physiol Renal Physiol 272, F817–F822. [PubMed] [Google Scholar]
  32. Knoers NV & Deen PM (2001). Molecular and cellular defects in nephrogenic diabetes insipidus. Pediatr Nephrol 16, 1146–1152. [DOI] [PubMed] [Google Scholar]
  33. Kwon TH, Frokiaer J & Nielsen S (2013). Regulation of aquaporin‐2 in the kidney: A molecular mechanism of body‐water homeostasis. Kidney Res Clin Pract 32, 96–102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lee KH, Ho WK & Lee SH (2013). Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase‐dependent tyrosine phosphorylation. Front Cell Neurosci 7, 14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Li JH, Chou CL, Li B, Gavrilova O, Eisner C, Schnermann J, Anderson SA, Deng CX, Knepper MA & Wess J (2009). A selective EP4 PGE2 receptor agonist alleviates disease in a new mouse model of X‐linked nephrogenic diabetes insipidus. J Clin Invest 119, 3115–3126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Li X, Yang B, Chen M, Klein JD, Sands JM & Chen G (2015). Activation of protein kinase C‐alpha and Src kinase increases urea transporter A1 alpha‐2,6 sialylation. J Am Soc Nephrol 26, 926–934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Liang M, Cai T, Tian J, Qu W & Xie ZJ (2006). Functional characterization of Src‐interacting Na/K‐ATPase using RNA interference assay. J Biol Chem 281, 19709–19719. [DOI] [PubMed] [Google Scholar]
  38. Lin DH, Sterling H, Yang B, Hebert SC, Giebisch G & Wang WH (2004). Protein tyrosine kinase is expressed and regulates ROMK1 location in the cortical collecting duct. Am J Physiol Renal Physiol 286, F881–F892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lombardo LJ, Lee FY, Chen P, Norris D, Barrish JC, Behnia K, Castaneda S, Cornelius LA, Das J, Doweyko AM, Fairchild C, Hunt JT, Inigo I, Johnston K, Kamath A, Kan D, Klei H, Marathe P, Pang S, Peterson R, Pitt S, Schieven GL, Schmidt RJ, Tokarski J, Wen ML, Wityak J & Borzilleri RM (2004). Discovery of N‐(2‐chloro‐6‐methyl‐phenyl)‐2‐(6‐(4‐(2‐hydroxyethyl)‐piperazin‐1‐yl)‐2‐methylpyrimidin‐4‐ylamino)thiazole‐5‐carboxamide (BMS‐354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. J Med Chem 47, 6658–6661. [DOI] [PubMed] [Google Scholar]
  40. Lu H, Sun TX, Bouley R, Blackburn K, McLaughlin M & Brown D (2004). Inhibition of endocytosis causes phosphorylation (S256)‐independent plasma membrane accumulation of AQP2. Am J Physiol Renal Physiol 286, F233–F243. [DOI] [PubMed] [Google Scholar]
  41. Lu HJ, Matsuzaki T, Bouley R, Hasler U, Qin QH & Brown D (2008). The phosphorylation state of serine 256 is dominant over that of serine 261 in the regulation of AQP2 trafficking in renal epithelial cells. Am J Physiol Renal Physiol 295, F290–F294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Minshall RD, Tiruppathi C, Vogel SM, Niles WD, Gilchrist A, Hamm HE & Malik AB (2000). Endothelial cell‐surface Gp60 activates vesicle formation and trafficking via Gi‐coupled Src kinase signaling pathway. J Cell Biol 150, 1057–1070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Moeller HB & Fenton RA (2012). Cell biology of vasopressin‐regulated aquaporin‐2 trafficking. Pflugers Arch 464, 133–144. [DOI] [PubMed] [Google Scholar]
  44. Moeller HB, Knepper MA & Fenton RA (2009). Serine 269 phosphorylated aquaporin‐2 is targeted to the apical membrane of collecting duct principal cells. Kidney Int 75, 295–303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Moeller HB, Praetorius J, Rutzler MR & Fenton RA (2010). Phosphorylation of aquaporin‐2 regulates its endocytosis and protein‐protein interactions. Proc Natl Acad Sci U S A 107, 424–429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Moon SK, Lee HY, Li JD, Nagura M, Kang SH, Chun YM, Linthicum FH, Ganz T, Andalibi A & Lim DJ (2002). Activation of a Src‐dependent Raf‐MEK1/2‐ERK signaling pathway is required for IL‐1alpha‐induced upregulation of beta‐defensin 2 in human middle ear epithelial cells. Biochim Biophys Acta 1590, 41–51. [DOI] [PubMed] [Google Scholar]
  47. Morello JP & Bichet DG (2001). Nephrogenic diabetes insipidus. Annu Rev Physiol 63, 607–630. [DOI] [PubMed] [Google Scholar]
  48. Noda Y ( 2014). Dynamic regulation and dysregulation of the water channel aquaporin‐2: a common cause of and promising therapeutic target for water balance disorders. Clin Exp Nephrol 18, 558–570. [DOI] [PubMed] [Google Scholar]
  49. Noda Y, Horikawa S, Kanda E, Yamashita M, Meng H, Eto K, Li Y, Kuwahara M, Hirai K, Pack C, Kinjo M, Okabe S & Sasaki S (2008). Reciprocal interaction with G‐actin and tropomyosin is essential for aquaporin‐2 trafficking. J Cell Biol 182, 587–601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Nunes P, Hasler U, McKee M, Lu HA, Bouley R & Brown D (2008). A fluorimetry‐based ssYFP secretion assay to monitor vasopressin‐induced exocytosis in LLC‐PK1 cells expressing aquaporin‐2. Am J Physiol Cell Physiol 295, C1476–C1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Ohnishi H, Yamamori S, Ono K, Aoyagi K, Kondo S & Takahashi M (2001). A src family tyrosine kinase inhibits neurotransmitter release from neuronal cells. Proc Natl Acad Sci U S A 98, 10930–10935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Pisitkun T, Jacob V, Schleicher SM, Chou CL, Yu MJ & Knepper MA (2008). Akt and ERK1/2 pathways are components of the vasopressin signaling network in rat native IMCD. Am J Physiol Renal Physiol 295, F1030–F1043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Ranchin B, Boury‐Jamot M, Blanchard G, Dubourg L, Hadj‐Aissa A, Morin D, Durroux T, Cochat P, Bricca G, Verbavatz JM & Geelen G (2010). Familial nephrogenic syndrome of inappropriate antidiuresis: dissociation between aquaporin‐2 and vasopressin excretion. J Clin Endocrinol Metab 95, E37–43. [DOI] [PubMed] [Google Scholar]
  54. Reinecke J & Caplan S (2014). Endocytosis and the Src family of non‐receptor tyrosine kinases. Biomol Concepts 5, 143–155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Rinschen MM, Yu MJ, Wang G, Boja ES, Hoffert JD, Pisitkun T & Knepper MA (2010). Quantitative phosphoproteomic analysis reveals vasopressin V2‐receptor‐dependent signaling pathways in renal collecting duct cells. Proc Natl Acad Sci U S A 107, 3882–3887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Saigusa T, Reichert R, Guare J, Siroky BJ, Gooz M, Steele S, Fenton RA, Bell PD & Kolb RJ (2012). Collecting duct cells that lack normal cilia have mislocalized vasopressin‐2 receptors. Am J Physiol Renal Physiol 302, F801–F808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Schartl M & Barnekow A (1984). Differential expression of the cellular src gene during vertebrate development. Dev Biol 105, 415–422. [DOI] [PubMed] [Google Scholar]
  58. Schuetze SM, Bolejack V, Choy E, Ganjoo KN, Staddon AP, Chow WA, Tawbi HA, Samuels BL, Patel SR, von Mehren M, D'Amato G, Leu KM, Loeb DM, Forscher CA, Milhem MM, Rushing DA, Lucas DR, Chugh R, Reinke DK & Baker LH (2017). Phase 2 study of dasatinib in patients with alveolar soft part sarcoma, chondrosarcoma, chordoma, epithelioid sarcoma, or solitary fibrous tumor. Cancer 123, 90–97. [DOI] [PubMed] [Google Scholar]
  59. Sterling H, Lin DH, Wei Y & Wang WH (2003). Tetanus toxin abolishes exocytosis of ROMK1 induced by inhibition of protein tyrosine kinase. Am J Physiol Renal Physiol 284, F510–F517. [DOI] [PubMed] [Google Scholar]
  60. Stival C, La Spina FA, Baro Graf C, Arcelay E, Arranz SE, Ferreira JJ, Le Grand S, Dzikunu VA, Santi CM, Visconti PE, Buffone MG & Krapf D (2015). Src kinase is the connecting player between protein kinase A (PKA) activation and hyperpolarization through SLO3 potassium channel regulation in mouse sperm. J Biol Chem 290, 18855–18864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Sun TX, Van Hoek A, Huang Y, Bouley R, McLaughlin M & Brown D (2002). Aquaporin‐2 localization in clathrin‐coated pits: inhibition of endocytosis by dominant‐negative dynamin. Am J Physiol Renal Physiol 282, F998–F1011. [DOI] [PubMed] [Google Scholar]
  62. Thebault S, Alexander RT, Tiel Groenestege WM, Hoenderop JG & Bindels RJ (2009). EGF increases TRPM6 activity and surface expression. J Am Soc Nephrol 20, 78–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Vistein R & Puthenveedu MA (2014). Src regulates sequence‐dependent beta‐2 adrenergic receptor recycling via cortactin phosphorylation. Traffic 15, 1195–1205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wang Y, Cao H, Chen J & McNiven MA (2011). A direct interaction between the large GTPase dynamin‐2 and FAK regulates focal adhesion dynamics in response to active Src. Mol Biol Cell 22, 1529–1538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Yui N, Lu HJ, Bouley R & Brown D (2012). AQP2 is necessary for vasopressin‐ and forskolin‐mediated filamentous actin depolymerization in renal epithelial cells. Biol Open 1, 101–108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhang Y, Chen R & Sun S (2002). The influence of Annexin32, a new Ca(2+)‐dependent phospholipid‐binding protein, on coagulation time and thrombosis. Sci China C Life Sci 45, 186–190. [DOI] [PubMed] [Google Scholar]
  67. Zimmerman B, Simaan M, Lee MH, Luttrell LM & Laporte SA (2009). c‐Src‐mediated phosphorylation of AP‐2 reveals a general mechanism for receptors internalizing through the clathrin pathway. Cell Signal 21, 103–110. [DOI] [PubMed] [Google Scholar]

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