Activation of the Na-K-Cl Cotransporter NKCC1 Detected with a Phospho-specific Antibody
2002; Elsevier BV; Volume: 277; Issue: 40 Linguagem: Inglês
10.1074/jbc.m206294200
ISSN1083-351X
AutoresAndreas W. Flemmer, Ignacio Gíménez, Brian F.X. Dowd, Rachel Darman, Biff Forbush,
Tópico(s)Ion channel regulation and function
ResumoThe Na-K-Cl cotransporter NKCC1 is activated by phosphorylation of a regulatory domain in its N terminus. In the accompanying paper (Darman, R. B., and Forbush, B. (2002)J. Biol. Chem. 277, 37542–37550), we identify three phosphothreonines important in this process. Using a phospho-specific antibody (anti-phospho-NKCC1 antibody R5) raised against a diphosphopeptide containing Thr212 and Thr217of human NKCC, we were readily able to monitor the cotransporter activation state. In 32P phosphorylation experiments with rectal gland tubules, we show that the R5 antibody signal is proportional to the amount of 32P incorporated into NKCC1; and in experiments with NKCC1-transfected HEK-293 cells, we demonstrate that R5-detected phosphorylation directly mirrors functional activation. Immunofluorescence analysis of shark rectal gland shows activation-dependent R5 antibody staining along the basolateral membrane. In perfused rat parotid glands, isoproterenol induced staining of both acinar and ductal cells along the basolateral membrane. Isoproterenol also induced basolateral staining of the epithelial cells in rat trachea, whereas basal cells in the subepithelial tissue displayed heavy, non-polarized staining of the cell membrane. In rat colon, agonist stimulation induced staining along the basolateral membrane of crypt cells. These data provide direct evidence of NKCC1 regulation in these tissues, and they further link phosphorylation of NKCC1 with its activation in transfected cells and native tissue. The high conservation of the regulatory threonine residues among NKCC1, NKCC2, and NCC family members, together with the fact that tissues from divergent vertebrate species exhibit similar R5-binding profiles, lends further support to the role of this regulatory locus in vivo. The Na-K-Cl cotransporter NKCC1 is activated by phosphorylation of a regulatory domain in its N terminus. In the accompanying paper (Darman, R. B., and Forbush, B. (2002)J. Biol. Chem. 277, 37542–37550), we identify three phosphothreonines important in this process. Using a phospho-specific antibody (anti-phospho-NKCC1 antibody R5) raised against a diphosphopeptide containing Thr212 and Thr217of human NKCC, we were readily able to monitor the cotransporter activation state. In 32P phosphorylation experiments with rectal gland tubules, we show that the R5 antibody signal is proportional to the amount of 32P incorporated into NKCC1; and in experiments with NKCC1-transfected HEK-293 cells, we demonstrate that R5-detected phosphorylation directly mirrors functional activation. Immunofluorescence analysis of shark rectal gland shows activation-dependent R5 antibody staining along the basolateral membrane. In perfused rat parotid glands, isoproterenol induced staining of both acinar and ductal cells along the basolateral membrane. Isoproterenol also induced basolateral staining of the epithelial cells in rat trachea, whereas basal cells in the subepithelial tissue displayed heavy, non-polarized staining of the cell membrane. In rat colon, agonist stimulation induced staining along the basolateral membrane of crypt cells. These data provide direct evidence of NKCC1 regulation in these tissues, and they further link phosphorylation of NKCC1 with its activation in transfected cells and native tissue. The high conservation of the regulatory threonine residues among NKCC1, NKCC2, and NCC family members, together with the fact that tissues from divergent vertebrate species exhibit similar R5-binding profiles, lends further support to the role of this regulatory locus in vivo. shark NKCC1 human NKCC1 enzyme-linked immunosorbent assay phosphate-buffered saline N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine NKCC1, the secretory or housekeeping isoform of the Na-K-Cl cotransporter, is expressed in most cell types, aiding in the regulation of cell volume. In polarized cells of secretory epithelia, NKCC1 is heavily expressed along the basolateral membrane, activated in response to secretagogues, and paramount for the transepithelial secretion of Cl− and water (2Haas M. Forbush III, B. Annu. Rev. Physiol. 2000; 62: 515-534Crossref PubMed Scopus (319) Google Scholar). NKCC1-mediated Cl− secretion has been well documented in rat parotid gland (3Tanimura A. Kurihara K. Reshkin S.J. Turner R.J. J. Biol. Chem. 1995; 270: 25252-25258Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 4Evans R.L. Turner R.J. J. Physiol. (Lond.). 1997; 499: 351-359Crossref Scopus (57) Google Scholar), shark rectal gland (5Forbush III, B. Haas M. Lytle C. Am. J. Physiol. 1992; 262: C1000-C1008Crossref PubMed Google Scholar), rat colon (6Payne J.A., Xu, J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), and dog trachea (7Haas M. Johnson L.G. Boucher R.C. Am. J. Physiol. 1990; 259: C557-C569Crossref PubMed Google Scholar). At least in shark rectal gland, the evidence is consistent with an indirect activation of NKCC1 upon agonist stimulation: secretagogues cause a drop in intracellular [Cl−] and volume via protein kinase A-mediated phosphorylation of apical chloride channels; in turn, low intracellular [Cl−] and low cell volume provide activation stimuli for the currently unidentified NKCC1 kinase(s) (2Haas M. Forbush III, B. Annu. Rev. Physiol. 2000; 62: 515-534Crossref PubMed Scopus (319) Google Scholar, 8Lytle C. Forbush III, B. Am. J. Physiol. 1996; 270: C437-C448Crossref PubMed Google Scholar). Our laboratory (9Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar) and others (10Kurihara K. Moore-Hoon M.L. Saitoh M. Turner R.J. Am. J. Physiol. 1999; 277: C1184-C1193Crossref PubMed Google Scholar) have linked the phosphorylation of the intracellular N-terminal domain with NKCC1 activation. In the accompanying paper, Darman and Forbush (1Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) describe the phosphorylation of three residues in this regulatory domain, of which Thr184 and Thr189 are necessary for maximal sNKCC11 activation. Thr189 in particular is demonstrated as being essential for sNKCC1 up-regulation. The three phosphoacceptor sites reside in a 30-amino acid region of the N terminus that exhibits 80% homology between sodium-coupled cation chloride cotransporter isoforms and 97% homology within NKCC1 proteins in species ranging from shark to human (shark Thr184/Thr189 correspond to human Thr212/Thr217). This region of the N terminus also contains a protein phosphatase-1-binding site (RVXF), which is conserved across divergent species and thought to mediate regulated dephosphorylation (11Darman R.B. Flemmer A. Forbush B. J. Biol. Chem. 2001; 276: 34359-34362Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). To date, most studies of NKCC regulation have been conducted in isolated tissue preparations, cell cultures, or cell-free systems utilizing [3H]benzmetanide binding,32P incorporation, or isotopic uptake. Although these studies have provided a wealth of information at the cellular and molecular levels, little is known about the phosphorylation state of NKCC in native tissue. To address this issue, we have developed a phospho-specific polyclonal antibody (anti-phospho-NKCC1 antibody R5) raised against the in vitro phosphorylated peptide corresponding to the regulatory domain. In this report, we demonstrate R5 specificity and sensitivity in recognizing the phosphorylated conserved threonines. We determine a positive correlation between NKCC1 activation and Thr184/Thr189 phosphorylation in sNKCC1-transfected HEK-293 cells using the R5 antibody. We also investigate in vivo NKCC1 activity and address the universality of this regulatory domain by examining R5 immunohistographs of shark rectal gland and several rat secretory tissues. These data provide a critical link between molecular regulation studies and the activation profile of NKCC1 in vivo. A 16-amino acid peptide, Tyrh208–Argh223-Lys (YYLRT*FGHNT*MDAVPRK) with an additional C-terminal lysine residue for coupling, was synthesized by the Keck Peptide Synthesis Facility at Yale University; the regulatory phosphothreonines Thr212 and Thr217 (in human NKCC1) were incorporated directly during synthesis. A rabbit antibody (anti-phospho-NKCC1 antibody R5; Pocono Rabbit Farms, Canadensis, PA) was raised against this peptide coupled to maleimidobenzoic acid-N-hydroxysuccinimide-activated keyhole limpet hemocyanin (Sigma) using standard procedures. The anti-phospho-NKCC1 antibody will be referred to as R5 for the remainder of the paper. For immunofluorescence studies, R5 was subjected to positive and negative affinity purification using the phosphorylated and non-phosphorylated kindred peptides coupled toN-hydroxysuccinimide-activated Sepharose beads (Amersham Biosciences) following the manufacturer's instructions. The specificity of R5 antisera for the immunizing peptide was measured using an ELISA format. As illustrated in Fig.1 (upper panel), R5 serum displayed >20-fold selectivity for the diphosphopeptide compared with the non-phosphorylated peptide. This high degree of selectivity enables a sensitive analysis of the phosphorylation state of the conserved threonines in situ and also Western blot analysis; for most purposes, the antibody can be effectively employed as diluted serum. Positive and negative affinity purification of the antibody further increased the phospho-specificity to >100-fold (Fig. 1, lower panel); the purified antibody was used for immunofluorescence analyses. Unnoticed until after the initial batch of R5 antibody had been produced, the second aspartate in this peptide was found to have been modified to an aspartimide during synthetic procedures; the unmodified peptide was subsequently synthesized for analytic and purification procedures. Fig. 1 shows that the R5 antibody had a slightly higher affinity for the modified peptide compared with the unmodified peptide. We speculate that the modification may have increased the immunogenicity of the Tyrh208–Argh223-Lys peptide. In any case, this antigen was quite successful; the second of two rabbits (R4, not discussed further here) also produced a high titer phospho-specific serum, with greater phospho-specificity, but less NKCC specificity, compared with R5. Several other antibodies were used to detect NKCC1 independent of its phosphorylation state. The T4 monoclonal antibody (12Lytle C., Xu, J.C. Biemesderfer D. Forbush III, B. Am. J. Physiol. 1995; 269: C1496-C1505Crossref PubMed Google Scholar) and the N1c polyclonal antibody (gift of Chris Lytle, University of California, Riverside, CA) (13McDaniel N. Lytle C. Am. J. Physiol. 1999; 276: G1273-G1278PubMed Google Scholar) both recognize the C terminus of human NKCC1 in a wide range of species. J3, J4, and J7 are anti-NKCC1 monoclonal antibodies that are highly specific for the shark cotransporter (14Lytle C., Xu, J.C. Biemesderfer D. Haas M. Forbush III, B. J. Biol. Chem. 1992; 267: 25428-25437Abstract Full Text PDF PubMed Google Scholar); these were used to detect sNKCC1 in ex vivo perfused fixed gland sections, isolated shark rectal gland tubules, and sNKCC1-transfected HEK-293 cells. For determination of antibody affinity and phospho-specificity by ELISA, phosphorylated and non-phosphorylated peptides were covalently coupled toN-oxysuccinimide binding plates (DNA-Bind, Costar Corp.). Plates were blocked with 20 mm ethanolamine, pH 8.2; washed twice with wash buffer (PBS, 1% bovine serum albumin, and 0.1% Triton X-100); and blocked with 7% milk in PBS in 0.1% Triton X-100. After washing once with wash buffer and sequential incubations with serially diluted sera and horseradish peroxidase-conjugated anti-rabbit IgG antibody, the optical density was measured with a spectrophotometer using o-phenylenediamine as a substrate. For Western blotting, samples were subjected to Tricine/SDS gel electrophoresis (7.5 or 10%) and transferred to polyvinylidene difluoride membranes (Immobilon, Millipore Corp., Bedford, MA). Blots were sequentially probed with primary and horseradish peroxidase-conjugated secondary antibodies. Chemiluminescent substrate was detected using a cooled CCD camera. Lines of stably transfected HEK-293 cells were handled as described in the accompanying paper (1Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Following appropriate preincubations, cells were solubilized by one of two procedures. (a) Cells were lysed either in 20 mm HEPES, pH 7.2 (see Fig. 2), or in phosphatase inhibitory buffer (300 mm NaCl, 60 mm NaF, 10 mm EDTA, 30 mm Na2HPO4, 30 mm sodium pyrophosphate, 40 mm HEPES, 0.2 mm NaVO4, and 0.5 μm calyculin A) (see Fig. 3), with both buffers containing 1% Triton X-100 and protease inhibitors (1Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Lysate was centrifuged to remove insoluble material and diluted into SDS sample buffer. (b) Alternatively, cells were lysed in 1% SDS and 1 mH3PO4 and subsequently diluted into sample buffer for Western blotting or dot blotting. For reasons that are yet unclear, we consistently saw a higher level of background R5 signal on dot blots (see Figs. 5 and 6) and gels (data not shown) of samples stopped with H3PO4/SDS stop medium compared with that seen when cells were harvested in 1% Triton X-100 (see Figs.3 and 4).Figure 3Recognition of Thr184 and Thr189 mutants by the R5 antibody. sNKCC1- and vector-transfected HEK-293 cells were preincubated in low Cl− hypotonic medium (+) or high K+ medium (−) for 50 min prior to lysis in Triton X-100 with protease and phosphatase inhibitors. After centrifugation and dilution into SDS sample buffer, identical volumes of solubilized protein representing 6 mm2 of tissue culture monolayer were loaded. Coomassie Blue staining of blots showed ∼20% variation in protein levels among these samples; to correct for this variation, individual lanes in all three panels have been digitally corrected accordingly. The sNKCC1 mutants are described in the accompanying paper (1Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) (PKA-1 denotes186GHNT → RRNT). Upper panel, R5 Western blot analysis of sNKCC1 phosphorylation site mutants and wild-type sNKCC1- and vector-transfected HEK-293 cells; middle panel, a parallel blot probed with the J4 antibody to detect total sNKCC1 expression; lower panel, Coomassie Blue stain of the blot inA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Time courses of phosphorylation of NKCC1 and of activation of NKCC-mediated 86Rb influx.Transfected HEK-293 cells were preincubated for 30 min in basic medium (135 mm NaCl, 5 mm RbCl, 1 mm CaCl2/MgCl2, 1 mmNa2HPO4/Na2SO4, and 15 mm NaHEPES, pH 7.4) (open symbols) or low Cl− hypotonic medium (closed symbols). A second preincubation, for the times plotted on the abscissa, was carried out in 0Na-0K-130Cl hypertonic medium (red dashed lines, ●, Ο), low Cl− hypotonic medium (black solid lines, ▵), low Cl−medium (purple dotted lines, ▿), hypertonic medium (blue solid lines, ○), or basic medium (green dashed lines, ▾). In alternate rows of the plate, a 2-min 86Rb influx was then carried out in regular flux medium (basic medium with ∼1 μCi/ml 86Rb and 10−4m ouabain) (upper panels); or the wells were sucked dry, rapidly solubilized in 70 μl of H3PO4/SDS, and dot-blotted with the R5 antibody (lower panels). Each point shows the value of the flux in a single well or, in the two curves for which error bars are shown, the mean and range in duplicate wells. Similar results were obtained in three other experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 686 Rb influx and phosphorylation in the T202E mutant. sNKCC1-, T202E-, and vector-transfected cells were preincubated for two sequential 30-min preincubation periods as follows: black bars, basic/basic medium; dark gray bars, basic/0Na-0K-130Cl hypertonic medium; light gray bars, low Cl− hypotonic/0Na-0K-130Cl hypertonic medium; white bars, basic/low Cl− hypotonic medium. Following preincubation, 86Rb influx was performed on one-fourth of the plate (upper panel, n = 6 on one plate); and the other wells were sucked dry, and H3PO4/SDS was added for dot blotting with the R5 antibody (lower panel, n = 18 on the same plate). A constant background value was subtracted from all dot-blot values; this represented 22, 50, and 54% of the maximum R5 dot signal in sNKCC1, T202E, and the vector, respectively. Data are plotted as means ± S.E. relative to the value with the last of the four preincubation protocols.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Correlation between R5 antibody signal and32P incorporation. Shark rectal gland tubules were preloaded with 32P for 40 min and then incubated in shark Ringer's solution with forskolin (10 μm) and calyculin A (0.5 μm) or with the addition of 580 mmsucrose for the indicated times prior to solubilization in 1% Triton X-100 with phosphatase and protease inhibitors. A, phosphorimage of whole cell lysate subjected to SDS gel electrophoresis and transferred to polyvinylidene difluoride membrane; B, phosphorimage of NKCC1 immunopurified with the J4 antibody;C, blot of whole cell lysate from A probed with the R5 antibody (Western blot); D–F, comparison of NKCC132P incorporation and R5 binding by quantitation of phosphorimages and Western blots. Closed symbols, activation in hypertonic media; open symbols, forskolin and calyculin A activation. D shows data from the images in A andC (analysis of whole cell lysates). E andF show combined data from three experiments (not including the one presented in D) in which the samples were analyzed together. Different symbols indicate different experiments.E shows the results from the analysis of whole cell lysates. F shows the results from the analysis of J4 immunoprecipitates.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Shark rectal gland tubule isolation and 32P incorporation were conducted as previously described (1Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 15Lytle C. Forbush III, B. Am. J. Physiol. 1992; 262: C1009-C1017Crossref PubMed Google Scholar). At specific time points following appropriate incubations, 200-μl aliquots of tubules were pelleted; resuspended in 100 μl of phosphatase inhibitory buffer containing 3% Triton X-100, 1 μm calyculin A, and a mixture of protease inhibitors; snap-frozen in liquid nitrogen; and stored at −80 °C. For NKCC1 immunoprecipitation, samples were thawed and centrifuged, and J4 antibody-Sepharose beads (15Lytle C. Forbush III, B. Am. J. Physiol. 1992; 262: C1009-C1017Crossref PubMed Google Scholar) were added to the supernatant. After an overnight incubation at 4 °C with subsequent washing, samples were subjected to SDS gel electrophoresis with Western blotting as described above. 32P incorporation into proteins was analyzed on gels and blots using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were anesthetized via intraperitoneal injection of sodium pentobarbital (50 mg/kg of body weight). Each animal was kept warm using a heated plate, and body temperature was monitored with a rectal thermometer. In most experiments, a polyvinyl catheter was introduced into the appropriate artery (common carotid for the parotid gland, thoracic for the trachea, and mesenteric for the colon), and the animals were infused with prewarmed Krebs-Henseleit bicarbonate solution (140 mmNa+, 5.4 mm K+, 1.2 mmMg2+, 1.2 mm Ca2+, 124 mm Cl−, 21 mmHCO 3−, 2.4 mmHPO 42−, 0.6 mmH2PO 4−, pH 7.4; 300 mosmol/liter adjusted with mannitol). Agonists were added to the perfusate to stimulate cotransporter activity as explained in the figure legends for each particular tissue. For Western blot analysis of parotid tissue, the gland was removed and snap-frozen in liquid nitrogen. Frozen, pestle-ground tissue was placed in boiling SDS sample buffer. For organ fixation, periodate/lysine/paraformaldehyde fixative was perfused through the same catheter for 5–15 min (16McClean W. Nakane P.F. J. Histochem. Cytochem. 1973; 22: 1077-1083Crossref Scopus (3173) Google Scholar). Tissues were removed from the animal and further fixed for 2–4 h at 4 °C. To achieve best fixation results, the trachea was also filled with fixative at 20 cm of H2O. Shark rectal glands were isolated from the animals, and each gland was perfused through its unique artery and treated as described for rat tissues. Small sections of fixed tissues were cryoprotected with 50% polyvinylpyrrolidone in 2.3 msucrose overnight at 4 °C. Tissue sections were then mounted on aluminum nails and snap-frozen in liquid nitrogen. Semi-thin 0.5–1-μm sections were cut using a Reichert Ultracut E ultramicrotome fitted with a FC-4E cryoattachment. Sections were placed on coated slides (Superfrost Plus, Erie Scientific, Portsmouth, NH) and washed with PBS prior to antigen retrieval by incubating the tissues with 1% SDS for 5 min. After quenching with NH4Cl for 15 min, sections were blocked with 0.1% bovine serum albumin and 10% goat serum in PBS, incubated for 1–2 h with the primary antibody, washed, and incubated with the appropriate secondary antibody conjugated to fluorescein isothiocyanate or Alexa dyes (Molecular Probes, Inc.). Sections were washed three times with PBS and mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA), and micrographs were taken with a Zeiss Axiophot microscope on Kodak Tmax 100 film (Eastman Kodak Co.) or using an Olympus Fluoview confocal microscope. In the accompanying paper (1Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), Darman and Forbush identify three phosphoacceptor threonines in an activation domain in the N terminus of shark NKCC1. In particular, phosphorylation of Thr184 and Thr189 is highlighted as a key element in NKCC1 cotransport activation. Because this region is well conserved both across species and between isoforms, it is an excellent candidate as a universal regulatory motif for NKCC. Thus, we prepared a diphosphorylated peptide corresponding to this region of the human NKCC1 sequence (Tyrh208–Argh223-Lys) and raised a polyclonal antibody (R5) using the keyhole limpet hemocyanin-conjugated peptide as antigen. The specificity of R5 for phosphorylated NKCC is illustrated by the Western blot in Fig. 2. As shown here, R5 recognized a single protein of ∼180 kDa, corresponding to the Na-K-Cl cotransporter (see Fig. 3). HEK-293 cells transfected with shark or human NKCC showed a strong signal compared with native NKCC in HEK-293 cells transfected with vector alone. Cross-reactivity of the R5 antibody with other proteins was not detected in HEK-293 cells and is rarely seen in lysates of eukaryotic cells. To investigate the activation dependence of the R5 signal, we incubated NKCC1-transfected HEK-293 cells in low Cl− hypotonic medium (3 mm Cl− solution diluted 2-fold with water) or in high K+ medium (135 mm NaCl, 5 mm RbCl, 1 mmCaCl2/MgCl2, 1 mmNa2HPO4/Na2SO4, 15 mmNaHEPES, pH 7.4, and 10 mm K+) to activate or deactivate the transporter, respectively. As illustrated in Fig. 2, the antibody signal was severalfold greater in sNKCC1- and hNKCC1-transfected HEK-293 cells under activating conditions. As a further confirmation that the R5 signal is dependent upon phosphorylation of the cotransporter, the samples in this experiment were incubated at room temperature for 90 min following lysis of the cells to allow endogenous protein phosphatases to dephosphorylate NKCC1. As shown here, the R5 signal was almost completely eliminated by incubation in lysis buffer devoid of the protein phosphatase-1 inhibitor calyculin A. We examined various NKCC phosphorylation site mutants to determine whether R5 recognizes the monophosphorylated as well as the diphosphorylated regulatory domains. Fig.3 presents Western blots of transfected HEK-293 cells from such an experiment comparing vector alone, wild-type sNKCC1, and Thr184/Thr189 mutants under the activation and deactivation conditions described above. Total shark NKCC1 was indicated by J3 antibody binding (Fig. 3, middle panel); as shown here, the expression was similar for each of the constructs. For both Thr184 and Thr189 single mutants, the R5 signal was found to increase with activation (Fig. 3,upper panel), and although the level was clearly greater than in control HEK-293 cells, there was much less signal than for wild-type sNKCC1. The simplest explanation for this result is that the R5 antibody, although having the greatest affinity for the diphosphorylated regulatory domain, also has significant although lower affinity for the monophosphorylated forms. The J3 antibody consistently recognized two bands in transfected cells (Fig. 3, middle panel), and we presume that this is due to different degrees of glycosylation, possibly reflecting different subcellular compartmentalization. Only the upper of these two bands was phosphorylated upon activation, as demonstrated by R5 recognition (the T189D mutant may exhibit a small amount of another lower band). The identity of the R5-positive band of lesser mobility in deactivated cells is a puzzle. Our interpretation is that this signal is from the endogenous HEK-293 cell cotransporter, based on two observations. (a) There was little or no J3 signal at this position (this R5 signal was from the region between the two strong J3 bands, better seen in superimposition of reblotting experiments (data not shown)); and (b) the upper J3 band exhibited only a very small upward mobility shift upon activation of the transporter. Surprisingly, and somewhat at odds with this interpretation, the native HEK-293 cell cotransporter appeared to undergo a mobility shift upon phosphorylation, despite the fact that the amount of HEK-293 cell R5 signal did not increase much upon activation (Figs. 2 and 3 and discussion below). To analyze the phosphorylation state of sNKCC1 residues Thr184/Thr189 in their native cellular environment, we examined 32P incorporation and R5 binding in isolated shark rectal gland tubules preloaded with32Pi (1Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Tubules were activated by incubation either in hypertonic media or in isotonic media containing forskolin (to activate apical Cl− channels and to lower intracellular [Cl−] (8Lytle C. Forbush III, B. Am. J. Physiol. 1996; 270: C437-C448Crossref PubMed Google Scholar)) and calyculin A. Under both conditions, NKCC1 displayed a time-dependent increase of32P incorporation in the 195-kDa band of NKCC1 as detected by 32P phosphorimaging of samples subjected to gel electrophoresis and transferred to polyvinylidene difluoride membranes (Fig. 4 A); immunoprecipitation with the J4 antibody highlighted 32P phosphorylation of NKCC1 (Fig. 4 B). Using the R5 antibody, NKCC1 phosphorylation was readily detected even in the crude lysates (Fig.4 C). Fig. 4 (D–F) illustrate the quantitative relationship between the R5 signal and 32P incorporation. It is clear from these data that R5 binding varies linearly with32P incorporation within our measurements, both in crude lysates (Fig. 4, D and E) and in immunoprecipitates (Fig. 4 F). Thus, Thr184 and Thr189 are indicators of overall sNKCC1 phosphorylation under these conditions, providing rational support for the use of R5 as a quantitative tool. To further establish the relationship between phosphorylation and NKCC1 activation, we compared the effects of several activation media on the R5 signal and 86Rb influx. Fig.5 illustrates the results of an experiment, similar to that of Fig. 8 of the accompanying paper (1Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), in which the time course of activation and deactivation was determined in various media. Following preincubation of cells in a single 96-well plate for each cell line, alternate rows were either lysed in H3PO4/SDS for dot-blot analysis (possible because only a single band was detected by R5; Fig. 1) or subjected to a 1-min 86Rb influx assay. It is readily seen in Fig. 5that function mirrors phosphorylation, i.e. activating and deactivating conditions had similar effects on both measurements (a small discrepancy between phosphorylation and flux for hNKCC1 cells in low Cl− hypotonic medium transferred to 0Na-0K-130Cl hypertonic medium was seen in two of four such experiments). Human and shark NKCC1 exhibited similar behavior, except that hNKCC1 was less activated by the hypertonic sucrose medium. We have used similar experiments to answer a question regarding the mec
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