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First published online 12 February 2008
doi: 10.1242/jcs.025312

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Activation of the thiazide-sensitive Na⁺-Cl^– cotransporter by the WNK-regulated kinases SPAK and OSR1

¹ MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
² INSERM, U773, Centre de Recherche Biomédicale Bichat-Beaujon (CRB3), BP 416, F-75018, Paris, France
³ Université Paris 7 -Denis Diderot, site Bichat, Paris, F-75870, Paris, France

View larger version (41K): [in this window] [in a new window]   Fig. 1. Sequence alignment of the N-terminal region of SLC12 electroneutral cation-chloride-coupled cotransporters that is regulated by phosphorylation. Identical residues are highlighted in black and similar residues are in grey. Symbols indicate characterised phosphorylation sites.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Identification of residues on NCC that are phosphorylated by OSR1 in vitro. (A) E. coli-expressed NCC[1-100] was incubated with the indicated active (Act) and kinase-inactive (KI) forms of OSR1 or SPAK in the presence of Mg-[32P]ATP for 40 minutes. Phosphorylation of NCC[1-100] was determined following electrophoresis and subsequent autoradiography of the Colloidal-blue-stained bands corresponding to NCC[1-100]. Similar results were obtained in three separate experiments. KI-SPAK, kinase-inactive SPAK[D212A]; Act-SPAK, activated-SPAK[T233E]; KI-OSR1, kinase-inactive-OSR1[D164A]; Act-OSR1, activated OSR1[T185E]. (B) E. coli-expressed NCC[1-100] was incubated with active OSR1 in the presence of Mg-[32P]ATP for the indicated times and phosphorylation of NCC[1-100] was determined as in A. (C) The indicated forms of NCC[1-100] were phosphorylated with active OSR1 in the presence of Mg-[32P]ATP for 60 minutes. Phosphorylated NCC[1-100] was digested with trypsin and chromatographed on a C18 column. The peak fractions containing the major 32P-labelled peptides are labelled P1, P2, P3 and P4. (D) Summary of the mass spectrometry and solid-phase Edman sequencing data obtained following analysis of the indicated phosphopeptides. The deduced amino acid sequence of each peptide is shown and the phosphorylated residue is indicated by (p). `m' in the peptide sequence indicates methionine sulphoxide. (E) The indicated forms of NCC[1-100] were phosphorylated with kinase-inactive or active OSR1 or SPAK. Phosphorylation was analysed as in A as well as by quantification of 32P radioactivity associated with Colloidal-blue-stained bands corresponding to NCC[1-100]. Results of the latter are presented as a percentage of phosphorylation compared to wild-type (WT) NCC[1-100] by active OSR1/SPAK. Results of duplicate samples are shown and similar results were obtained in two separate experiments. AAA corresponds to a triple NCC[1-100] mutant in which Thr46, Thr55 and Thr60 are changed to Ala.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Characterisation of WNK1-SPAK/OSR1 activity and identification of in vivo phosphorylation sites on NCC. (A) HEK293 cells were treated with either basic (–) or hypotonic low-chloride (+) medium for 30 minutes. Endogenous WNK1 was immunoprecipitated and assayed employing kinase-inactive OSR1 as a substrate. Phosphorylation of OSR1 was determined after electrophoresis followed by autoradiography of the Colloidal-blue-stained bands corresponding to OSR1. The incorporation of 32P radioactivity was also quantified and results are presented as the mean activity ± s.d. for duplicate samples. WNK1 immunoprecipitates were also immunoblotted with the indicated antibodies. (B) As above, except that endogenous SPAK and OSR1 were immunoprecipitated employing an antibody that recognises both proteins. Immunoprecipitated SPAK/OSR1 was assayed employing the CATCHtide peptide substrate (Vitari et al., 2006). Results are presented as the mean activity ± s.d. for duplicate samples. Total cell extracts were also immunoblotted with the indicated antibodies. Results of duplicate samples are shown and similar results were obtained in two separate experiments for A and B. (C) HEK293 cells were transfected with a construct expressing human Flag-NCC. At 36 hours post-transfection, cells were stimulated as in A, and FLAG-NCC was immunoprecipitated and electrophoresed on a polyacrylamide gel. The Colloidal-blue-stained bands corresponding to FLAG-NCC were excised and digested with trypsin. Phosphopeptides were identified by combined liquid chromatography-mass spectrometry and tandem mass spectrometry analysis. The figure shows the signal intensity (cps, counts of ions per second detected) versus the m/z (amu, atomic mass units) for the phosphopeptides derived from NCC isolated from control (red) or hypotonic low-chloride (blue) cells.

View larger version (56K): [in this window] [in a new window]   Fig. 4. NCC phosphorylation-site characterisation employing phosphospecific antibodies. (A) HEK293 cells were transfected with wild-type (WT) human FLAG-NCC or the indicated mutant forms of NCC. At 36 hours post-transfection, cells were treated with either basic (–) or hypotonic low-chloride (+) medium for 30 minutes and lysed. Total cell extracts were immunoblotted with NCC-phosphospecific and total antibodies. Similar results were obtained in two separate experiments. AAAA corresponds to a quadruple NCC[1-100] mutant in which Thr46, Thr55, Thr60 and Ser91 are changed to Ala. (B) Wild-type or indicated mutants of NCC[1-100] were phosphorylated with active (Act) or kinase-inactive (KI) mutants of OSR1 or SPAK and phosphorylation analysed by immunoblot analysis. Results of duplicate samples are shown. AAA corresponds to a triple NCC[1-100] mutant in which Thr46, Thr55 and Thr60 are changed to Ala; the kinase-inactive and activated mutants of SPAK/OSR1 employed are defined in the legend to Fig. 2A. (C) HEK293 cells were transfected with wild-type NCC. At 36 hours post-transfection, cells were treated with either basic or hypotonic low-chloride medium for the specified durations and lysed. The phosphorylation of WNK1 at Ser382 and total levels of WNK1 protein were analysed after its immunoprecipitation. Phosphorylation and total levels of SPAK/OSR1 and NCC were analysed in total cell extracts.

View larger version (67K): [in this window] [in a new window]   Fig. 5. SPAK/OSR1 interaction with NCC mediates NCC phosphorylation. (A) HEK293 cells were transfected with constructs encoding wild-type (WT) or indicated mutants of human FLAG-NCC. At 36 hours post-transfection, cells were lysed, and NCC was immunoprecipitated and subjected to immunoblot analysis with the indicated antibodies. Similar results were obtained in two separate experiments. (B) As above, except that cells were treated with either basic (–) or hypotonic low-chloride (+) medium for 30 minutes and lysed. Cell lysates were subjected to immunoblot analysis with the indicated antibodies.

View larger version (58K): [in this window] [in a new window]   Fig. 6. Endogenous NCC phosphorylation in the mpkDCT cell line. (A) Total cell extracts derived from mpkDCT, HEK293, mouse embryonic fibroblast (MEF) or embryonic stem (ES) cells were immunoblotted with a total NCC antibody from Chemicon (AB3553). Moesin levels were monitored as a loading control. (B,C) mpkDCT cells were treated with either basic (–) or hypotonic low-chloride (+) medium for 30 minutes and lysed. The activity and phosphorylation state of endogenous WNK1 and SPAK/OSR1 were analysed as described in the legend to Fig. 3A,B. Results of duplicate samples are shown and similar results were obtained in two separate experiments. (D) Phosphorylation of endogenous NCC in cell extracts from basic (–) and hypotonic low-chloride (+)-treated mpkDCT cells was determined by immunoblot analysis with the indicated total and phospho-NCC antibodies. Results of duplicate samples are shown and similar results were obtained in two separate experiments.

View larger version (53K): [in this window] [in a new window]   Fig. 7. Mutation of Thr60 of NCC prevents thiazide-sensitive hypotonic low-chloride-induced 22Na uptake in HEK293 cells. HEK293 cells were transfected with pCMV5 empty vector or constructs encoding the indicated wild-type (WT) or mutant forms of human NCC. At 36 hours post-transfection, 22Na uptake was assessed in control basic or hypotonic low-chloride-treated cells in the absence or presence of 0.1 mM metolazone in the uptake medium, as described in the Materials and Methods. For each transfection construct used, metolazone-sensitive 22Na uptake (i.e. metolazone-insensitive counts subtracted) is plotted for both basic and hypotonic low-chloride conditions. The results are presented as the mean 22Na uptake ± s.d. for triplicate samples. Similar results were obtained in three separate experiments. Expression and phosphorylation of NCC proteins were monitored in parallel experiments following immunoblot analysis using the indicated antibodies.

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