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First published online 30 September 2008
doi: 10.1242/jcs.035584

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Human kidney anion exchanger 1 localisation in MDCK cells is controlled by the phosphorylation status of two critical tyrosines

University of Bristol, Department of Biochemistry, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK

View larger version (37K): [in this window] [in a new window]   Fig. 1. Location of Y359 and Y904 and N-terminal truncation constructs on kAE1. Diagram of human kAE1 indicating the location of N-terminal tyrosine residue Y359, the locations of the N-terminal truncations and C-terminal tyrosine Y904. This illustration is adapted from a previous paper (Williamson and Toye, 2008).

View larger version (82K): [in this window] [in a new window]   Fig. 2. KAE1-Y359A is targeted to the plasma membrane in nonpolarised MDCKI cells and mistargeted to the apical membrane in polarised MDCKI cells. MDCKI cells stably expressing kAE1 or kAE1-Y359A were seeded on coverslips for nonpolarised cells (a,b) or polarised on filters (c-j). (a-c,g) kAE1 was detected using AE1 monoclonal antibody Bric170. (d,h) Rabbit anti-β-catenin antibody was used as a basolateral marker. The corresponding merged images for c and d, and g and h are shown in e and i, respectively. (f,j) An extracellular anti-AE1 antibody FITC-Bric6 was added to both the apical and basolateral surfaces of the intact cell monolayers. For the polarised images, the upper micrographs are focal planes taken parallel to the epithelium (X-Y) through the centre for kAE1 or the apical surface for kAE1-Y359A. The lower micrographs show focal planes perpendicular to the epithelium (X-Z) along the white line in the X-Y image. Normal kAE1 is at the plasma membrane in nonpolarised cells (a) and basolateral membrane in polarised cells using Bric170 (c) or FITC-Bric6 (f). The kAE1-Y359A protein is also at the plasma membrane in nonpolarised cells (b) but mistargeted to the apical membrane using Bric170 (g) or FITC-Bric6 (j). Scale bars: 30 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 3. The majority of N-terminal truncations of kAE1 are localised to the TGN. Nonpolarised MDCKI cells stably expressing Nt1-360AE1 (a), Nt1-338AE1 (b), Nt1-292AE1 (c), Nt1-269AE1 (d) or Nt1-155AE1 (e) or transiently expressing Nt1-338AE1(Y359A) (f), Nt1-244AE1 (g) and Nt1-214AE1 (h) were fixed and immunostained with Bric170 and a suitable secondary antibody. Only the Nt1-155AE1 truncation was correctly localised to the plasma membrane, whereas the other mutants were localised to the TGN. Merged image in k shows the overlap between Nt1-338AE1 detected with Bric170 (i) and TGN38 detected with a rabbit anti-TGN38 antibody (j). Mutation of Y359 to alanine did not alleviate the TGN trafficking of the Nt1-338AE1 protein when transiently expressed in MDCKI cells (compare f with b). Therefore inclusion of even small sections of the N-terminus to the Nt1-360AE1 protein appears to disrupt the steady state localisation of kAE1. Scale bar: 30 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 4. Pervanadate induces kAE1 phosphorylation on residues Y359 and Y904 and is sensitive to Src kinase family inhibitors. (A,B) Western blots of Bric170 immunoprecipitates from MDCKI or MDCKI-kAE1 cells treated with or without pervanadate for the indicated times. The membranes were sequentially probed with anti-Y904-P, anti-AE1Ct and anti-Y359-P, stripping after each antibody application. (A) kAE1 phosphorylation on Y359 and Y904 is detectable by the phosphospecific antibodies within 1 minute and the majority of the protein is phosphorylated within 5 minutes. The blot shown is representative of at least eight similar experiments. (B) MDCKI-kAE1 cells were preincubated with Src kinase inhibitors 25 µM PP1, 25 µM PP2, 25 µM PP3 (inactive analogue), 2 µM staurosporine (Stauro), 100 µM genistein (Gen), 5 µM herbimycin (Herb) and 25 µM SU6656 for 30 minutes before addition of 200 µM pervanadate for 5 minutes. kAE1 phosphorylation on residues Y359 and Y904 was inhibited by both PP1 and PP2, and to a lesser extent by staurosporine and SU6656. The broad-spectrum Src kinase inhibitors genistein and herbimycin did not inhibit kAE1 phosphorylation under the conditions tested. The blot shown is representative of at least three independent experiments for each inhibitor.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Sodium bicarbonate and hypertonicity or hyperosmolarity induce kAE1 phosphorylation. A range of treatments were added to MDCKI-kAE1 cells. Cells were then lysed and two immunoprecipitations conducted per 10 cm2 dish using Bric170. The immunoprecipitates were run on duplicate 8% SDS-PAGE gels and blots probed with either anti-Y359-P or anti-Y904-P, stripped and probed with anti-AE1Ct. A 5 minute pervanadate-positive control is included, loaded at half an immunoprecipitation equivalent to reduce the signal strength. (A) Effect of 1 hour exposure to either acidic pH 6.8 or increasing pH with either NaOH or NaHCO3. Only the pH 8.7 NaHCO3 medium induced kAE1 phosphorylation (on both Y359 and Y904) in MDCKI cells. (B) Effect of 1 hour exposure to various hypertonic media on kAE1 phosphorylation. Hypertonic solutions induce phosphorylation of kAE1 on Y359 but not Y904. NaHCO3 induced a consistently stronger response than hypertonicity alone and this was not inhibited by 1 mM DIDS (see supplementary material Fig. S6D for quantification). (C) Comparison of the effects of hypotonic medium (medium diluted 1:1 with water), cell swelling induced by 300 mM urea, 514 mM NaCl, 1 M sucrose or 1 M sorbitol. Hyperosmotic sucrose or sorbitol induced a greater phosphorylation of kAE1 than NaCl but this was still not as great as the effects of NaHCO3. The application of a hypotonic solution or cell swelling induced by urea had no effect on kAE1 phosphorylation. (D) Effect of incubation with 1 mM 8-bromo-cAMP, 60 µM forskolin, 5 µM ionomycin, 5 µM A23787, 1 µM thapsigargin, 10 µM PMA, either 200 µM H2O2 or 1 mM H2O2 and 100 µM ATP for 1 hour on MDCKI-kAE1 cells. No kAE1 phosphorylation was observed under the conditions tested using any of these reagents. Results representative of three separate experiments. (E) Effect of 1 hour exposure to vasopressin, angiotensin II or aldosterone. None of these hormones induced kAE1 phosphorylation under the conditions tested. Representative of at least two separate experiments. PV, pervanadate.

View larger version (70K): [in this window] [in a new window]   Fig. 6. Pervanadate-induced tyrosine phosphorylation influences basolateral kAE1 localisation. MDCKI-cells stably expressing kAE1 (A,B,C,D) or four apically targeted kAE1 mutants kAE1Y359A (E), Nt1-360AE1 (F), kAE1(Y904A,Y907A) (H) or kAE1R901Stop (G) polarised on filters. 200 µM pervanadate (PV) was added to the cells in B-C, E-H or 514 mM NaHCO3 added to D for the times indicated. kAE1 was detected using AE1 monoclonal antibody Bric170 and a suitable secondary antibody. The upper micrographs are focal planes taken parallel to the epithelium (X-Y) near the centre for kAE1 or at the apical membrane for the kAE1 mutants. The lower micrographs show focal planes perpendicular to the epithelium (X-Z) along the white line in the X-Y image. Normal kAE1 is at the basolateral membrane in untreated control cells (A) and those treated with pervanadate for 15 minutes (B). kAE1 localisation was intracellular after 30 minutes of pervanadate treatment in C. (D) NaHCO3 had no effect on kAE1 localisation. (E-H) Pervanadate treatment for 30 minutes did not alter the apical localisation of kAE1 mutants that lack either Y359 or Y904. Scale bar: 30 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 7. Antibody uptake assay shows that phosphorylated kAE1 rapidly internalises from the plasma membrane in nonpolarised cells. MDCKI-kAE1, MDCKI-kAE1Y359A or MDCKI-Nt1-360AE1 cells were seeded onto coverslips and an antibody-uptake assay using FITC-Bric6 conducted under various conditions. (A-H) Pervanadate induces kAE1 internalisation. The majority of the kAE1 FITC-Bric6 is at the cell surface before internalisation (A,B). (C,D) FITC-Bric6 internalisation at 37°C for 30 minutes in the absence and presence of pervanadate (PV). (E-H) FITC-Bric6 is susceptible to acid wash in control cells (compare E and F) but pervanadate-treated cells are resistant to this process (compare G and H), consistent with the majority of the kAE1 having been internalised. (I-K) Hypertonicity inhibits kAE1 internalisation. (I) There is a lack of FITC-Bric6 internalisation following 1 hour exposure to 514 mM NaHCO3. (J,K) In the presence of 514 mM NaCl or 1 M sucrose, pervanadate-induced FITC-Bric6 internalisation is inhibited. (L-T) kAE1 mutants that lack Y359 have a rapid internalisation of FITC-Bric6 and this is inhibited by Src kinase inhibitor PP2. (L) FITC-Bric6 binding to kAE1Y359A cells before internalisation is allowed to commence. (M,N) kAE1Y359A uptake in the absence or presence of pervanadate. This demonstrates that Y359A rapidly internalises and there is no obvious difference between antibody uptake in the presence or absence of pervanadate. (O-T) Comparison of kAE1Y359A (O and P), Nt1-360AE1 (Q and R) or kAE1 (S and T) treated with either PP3 or PP2 as indicated. Scale bar: 30 µm.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Scheme to explain the role of Y359 and Y904 in regulating kAE1 localisation. kAE1 has two tyrosine residues, one in the N-terminus and one in the C-terminus, that are critical for basolateral localisation. We have shown that both of these tyrosines Y359 and Y904 can be phosphorylated. We propose that when Y359 and Y904 are phosphorylated at the plasma membrane, this marks the protein for internalisation by endocytosis. If Y359 is phosphorylated we hypothesise that this recruits a phosphatase (in this case SHP-2 via SH2 domain interaction), which then dephosphorylates Y904, blocking phosphorylation specific internalisation or revealing the basolateral targeting motif for recycling kAE1 back to the plasma membrane.

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