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• W2094567461 abstract "Phosphorylation of the epithelial Na+ channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the β and γ subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of β and γENaC, have been identified by an in vitro assay. One of these phosphorylates βThr-613 and γThr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of γThr-623 has also been demonstrated in vivo in channels expressed inXenopus oocytes, and mutating βThr-613 and γThr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions β613 or γ623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on βThr-613 and γThr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERK-mediated phosphorylation of βThr-613 and γThr-623 down-regulates the channel by facilitating its interaction with Nedd4. Phosphorylation of the epithelial Na+ channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the β and γ subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of β and γENaC, have been identified by an in vitro assay. One of these phosphorylates βThr-613 and γThr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of γThr-623 has also been demonstrated in vivo in channels expressed inXenopus oocytes, and mutating βThr-613 and γThr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions β613 or γ623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on βThr-613 and γThr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERK-mediated phosphorylation of βThr-613 and γThr-623 down-regulates the channel by facilitating its interaction with Nedd4. Active Na+ reabsorption in kidney collecting duct, distal colon, lung, and exocrine glands is mediated by an apical amiloride-blockable Na+ channel (1.Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1043) Google Scholar, 2.Horisberger J.D. Curr. Opin. Cell Biol. 1998; 10: 443-449Crossref PubMed Scopus (72) Google Scholar, 3.Palmer L.G. Garty H. Seldin D.W. Giebisch G. The Kidney, Physiology and Pathophysiology. 3rd Ed. Lippincott Williams and Wilkins, Philadelphia, PA2000: 251-276Google Scholar). The channel is composed of three homologous subunits, denoted α, β, and γENaC (Epithelial Na+Channel). 1The abbreviations used are: ENaCepithelial Na+ channelGSTglutathione S-transferaseERKextracellular regulated kinaseHAhemagglutininCHOChinese hamster ovaryMAPKmitogen-activated protein kinaseJNKc-Jun amino-terminal kinaseCK2casein kinase 2Its cell surface expression is determined by interactions of the C-tails of β and γ with the ubiquitin ligase Nedd4. The WW domains of Nedd4 bind to the proline-rich PY motifs on β and γENaC, leading to channel ubiquitination, internalization, and degradation (4.Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (601) Google Scholar, 5.Staub O. Dho S. Henry P.C. Correa J. Ishikawa T. Mcglade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (741) Google Scholar). A central role of ENaC in maintaining salt and water balance has been conclusively demonstrated by identifying genetic diseases associated with mutations in channel subunits, as well as by the phenotypic analysis of ENaC knockout mice (for review see Refs. 1.Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1043) Google Scholar, 2.Horisberger J.D. Curr. Opin. Cell Biol. 1998; 10: 443-449Crossref PubMed Scopus (72) Google Scholar, 3.Palmer L.G. Garty H. Seldin D.W. Giebisch G. The Kidney, Physiology and Pathophysiology. 3rd Ed. Lippincott Williams and Wilkins, Philadelphia, PA2000: 251-276Google Scholar). epithelial Na+ channel glutathione S-transferase extracellular regulated kinase hemagglutinin Chinese hamster ovary mitogen-activated protein kinase c-Jun amino-terminal kinase casein kinase 2 ENaC's activity is also controlled by a number of hormones such as the mineralocorticoid aldosterone, the anti-diuretic peptide vasopressin, and insulin (1.Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1043) Google Scholar, 3.Palmer L.G. Garty H. Seldin D.W. Giebisch G. The Kidney, Physiology and Pathophysiology. 3rd Ed. Lippincott Williams and Wilkins, Philadelphia, PA2000: 251-276Google Scholar). Previous studies have suggested the involvement of protein phosphorylation in these mechanisms. The serine/threonine kinase sgk (serum andglucocorticoid-dependent kinase) is induced by aldosterone and can activate the channel upon co-expression in Xenopus oocytes (6.Chen S.Y. Bhargava A. Mastroberardino L. Meijer O.C. Wang J. Buse P. Firestone G.L. Verrey F. Pearce D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2514-2519Crossref PubMed Scopus (643) Google Scholar, 7.Naray-Fejes-Toth A. Canessa C.M. Cleaveland E.S. Aldrich G. Fejes-Toth G. J. Biol. Chem. 1999; 274: 16973-16978Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 8.Shigaev A. Asher C. Latter H. Garty H. Reuveny E. Am. J. Physiol. 2000; 278: F613-F619Crossref PubMed Google Scholar). This response was recently found to involve phosphorylation of Nedd4-2 by sgk (9.Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Pratt J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (583) Google Scholar, 10.Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). The response of A6 cells to both aldosterone and insulin requires activation of phosphoinositide 3-kinase (11.Blazer-Yost B.L. Punescu T.G. Helman S.I. Lee K.D. Vlahos C.J. Am. J. Physiol. 1999; 277: C531-C536Crossref PubMed Google Scholar, 12.Record R.D. Froelich L.L. Vlahos C.J. Blazer-Yost B.L. Am. J. Physiol. 1998; 274: E611-E617PubMed Google Scholar). In addition, aldosterone and insulin, as well as intracellular signaling components such as protein kinases C and A, increase the in vivophosphorylation of the carboxyl termini of both β and γENaC (13.Shimkets R.A. Lifton R. Canessa C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3301-3305Crossref PubMed Scopus (162) Google Scholar). We have recently demonstrated phosphorylation of the carboxyl termini of ENaC subunits, expressed as glutathione S-transferase (GST) fusion proteins by crude cytosolic fractions (14.Chigaev A. Lu G. Shi H.-K. Asher C. Xu R. Latter H. Seger R. Garty H. Reuveny E. Am. J. Physiol. 2001; 280: F1030-F1036Crossref PubMed Google Scholar). The current study characterizes conserved residues phosphorylated in the carboxyl termini of ENaC subunits, explores their physiological role, and identifies the kinase involved. The data indicate that an extracellular regulated kinase (ERK)-dependent phosphorylation of βThr-613 and γThr-623 may be important in controlling interactions between the channel and Nedd4. The carboxyl termini of the rat β and γENaC (β 557–638 and γ 564–650) were subcloned downstream GST in the bacterial expression vector pGEX3X as described in a previous study (14.Chigaev A. Lu G. Shi H.-K. Asher C. Xu R. Latter H. Seger R. Garty H. Reuveny E. Am. J. Physiol. 2001; 280: F1030-F1036Crossref PubMed Google Scholar). cDNA constructs expressing fusion proteins between GST and the three WW domains of rat Nedd4 were kindly provided by D. Rotin (Hospital for Sick Children, Toronto, Canada) and are described in a previous study (5.Staub O. Dho S. Henry P.C. Correa J. Ishikawa T. Mcglade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (741) Google Scholar). GST fusion proteins were expressed in a protease-deficient Escherichia coli strain and purified on glutathione beads as detailed previously (14.Chigaev A. Lu G. Shi H.-K. Asher C. Xu R. Latter H. Seger R. Garty H. Reuveny E. Am. J. Physiol. 2001; 280: F1030-F1036Crossref PubMed Google Scholar). Functional expression in Xenopus oocytes was done using ENaC clones in pSPORT-1, obtained from B. C. Rossier (Institute of Pharmacology, University of Lausanne). Immunoprecipitation of phosphorylated ENaC was performed using a hemagglutinin A (HA)-tagged β and γ construct kindly provided by B. Schwappach (Zentrum für Molekulare Biologie, Universität Heidelberg). The HA epitope was introduced in the ecto domains of these subunits at a position shown before not to affect channel activity (15.Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (399) Google Scholar). Point mutations in the various constructs were introduced using a QuikChange site-directed mutagenesis kit and verified by sequencing. Cytosol was extracted from rat distal colon and fractionated by ion exchange chromatography using the following protocol. Rats (Wistar, 9–11 weeks old) were sacrificed by cervical dislocation. The distal colon was excised, cut open, and rinsed first in phosphate-buffered saline and then in buffer A composed of: 50 mm β-glycerophosphate, pH 7.3, 1.5 mm EGTA, 1.0 mm EDTA, 1.0 mm dithiothreitol, and 0.1 mm de-aerated sodium orthovanadate (16.Ahn N.G. Weiel J.E. Chan C.P. Krebs E.G. J. Biol. Chem. 1990; 265: 11487-11494Abstract Full Text PDF PubMed Google Scholar). The epithelial cells were scraped off the connective tissue using a glass slide and suspended in buffer A + a mixture of protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1.0 mmbenzamidine, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 2.0 μg/ml pepstatin-A). Cells were washed by centrifugation and then disrupted on ice by sonication (3 × 5 s). Cell homogenates were centrifuged for 30 min at 30,000 × g, and the supernatants were collected and further fractionated by ion exchange chromatography. 10 mg of protein in 50 ml of buffer A was loaded onto a MonoQ column (Amersham Biosciences, Inc.) at a rate of 1 ml/min. Bound proteins were eluted at the same rate by a linear NaCl gradient (0–30%) in buffer A. Ninety 1-ml fractions were collected, stored at 4 °C, and assayed for kinase activity within 24 h. Chinese hamster ovary (CHO) cells stably transfected with the human insulin receptor were kindly provided by Dr. Yehiel Zick (Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel). Cells were cultivated in a 1:1 mixture of Dulbecco's modified Eagle's medium and F12 supplemented with 10% fetal calf serum and 1% glutamine. After achieving 80% confluency, the medium was replaced by serum-free Dulbecco's modified Eagle's medium for a period of 24–36 h. Serum-depleted cells were incubated with 100 nm insulin for 5–120 min in the presence and absence of various inhibitors. They were washed in ice-cold phosphate-buffered saline, harvested, and suspended in buffer A + protease inhibitors. Cells were disrupted by 7-s sonication on ice. The homogenates were centrifuged for 60 min at 15,000 × g, and the supernatants were collected. Rat colon cytosol MonoQ fractions, whole CHO cytosol, and purified activated ERK2 were used in an in vitro phosphorylation assay of GST-ENaC fusion proteins. The fusion proteins were immobilized on glutathione-agarose beads and suspended in buffer A. Aliquots of 47 μl were mixed with 50-μl volumes of cytosolic fractions, whole cytosol (∼1 mg/ml protein), or purified ERK2 (1.5 ng/ml, specific activity of 0.45 mmol/min/mg), all in buffer A. Phosphorylation was initiated by the addition of 3 μl of ATP mixture composed of: 1.8 μl of 1 m MgCl2, 0.9 μl of 0.2 mm ATP, and 0.3 μl of [32P]ATP (10 mCi/ml, 3000 Ci/mmol). Suspensions were shaken for 30 min at 30 °C, pelleted, and washed several times in HB1B buffer (20 mmHEPES, pH 7.7, 50 mm NaCl, 0.1 mm EDTA, 25 mm MgCl2, and 0.05% Triton X-100). Pellets were suspended in Laemmli sample buffer, boiled for 5 min, resolved on 12% SDS-PAGE, and exposed to a PhosphorImager plate and/or x-ray film. Specific binding of cytosolic kinases to ENaC C-tail fusion proteins was determined by co-precipitation, as described previously (17.Rubinfeld H. Seger R. Current Protocols in Cell Biology. John Wiley & Sons, Inc, New York1998Google Scholar). In brief, fusion proteins immobilized on glutathione beads were incubated with either cytosolic fractions or purified kinases for 2 h. at 4 °C. Incubation was done in a binding buffer composed of 150 mm NaCl, 22 mm HEPES, pH 7.7, 2 mmMgCl2, 0.075% Triton X-100, 20 mmβ-glycerophosphate, 0.1 mm EDTA, 0.1 mmsodium orthovanadate, and protease inhibitors. The beads were sedimented and washed twice in HB1B buffer, a third wash in buffer A, and a final wash in a kinase assay buffer (20 mm HEPES, pH 7.7, 20 mm MgCl2, 20 mmβ-glycerophosphate, 2 mm dithiothreitol, and 0.1 mm sodium orthovanadate). Beads were suspended in 30-μl volumes of the above kinase assay buffer, and phosphorylation was initiated by the addition of 2 μm ATP plus 2 μCi of [γ-32P]ATP. 30-μl aliquots of rat colon MonoQ fractions or whole CHO-T cytosol were resolved on 10% SDS-PAGE, blotted onto nitrocellulose, and blocked with 5% low fat milk. Samples were probed with either anti-ERK (1:20,000) or anti-phospho ERK (1:5000). Both anti-ERK antibodies were obtained from Sigma-Aldrich Fine Chemicals. Blots were overlaid with horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad, 1:10,000), and binding was detected by enhanced chemiluminescence. Binding of the WW domains of Nedd4 to β and γ PY peptides was monitored by surface plasmon resonance in a BIAcore 2000 sensor, as described previously (18.Asher C. Chigaev A. Garty H. Biochem. Biophys. Res. Commun. 2001; 286: 1228-1231Crossref PubMed Scopus (23) Google Scholar). In brief, peptides serving as substrates were immobilized on the sensor chip (SA, BIAcore, Uppsala Sweden) through amino termini biotin residues. In each chip, the peptides to be compared were immobilized on two of the channels. Free biotin and γ peptide carrying the mutationY628A were immobilized on the other two channels and served as negative controls. The three WW domains of rat Nedd4 were expressed as GST fusion proteins and used as analytes. They were suspended in HBS buffer (10 mm HEPES, pH 7.4, 140 mm NaCl, 3.4 mm EDTA, and 0.005% P20) and injected at a flow rate of 20 μl/min. Binding was monitored simultaneously in all four channels for at least 4 min and terminated by the application of HBS buffer without analyte (dissociation phase). The chip was regenerated by a subsequent injection of 10 μl of HBS + 0.05% SDS and extensively washed in HBS, and then the next analyte was applied. Each observation was confirmed by at least three measurements in two different chips. The ENaC-mediated Na+ current was determined in the oocyte expression system as described before (8.Shigaev A. Asher C. Latter H. Garty H. Reuveny E. Am. J. Physiol. 2000; 278: F613-F619Crossref PubMed Google Scholar, 14.Chigaev A. Lu G. Shi H.-K. Asher C. Xu R. Latter H. Seger R. Garty H. Reuveny E. Am. J. Physiol. 2001; 280: F1030-F1036Crossref PubMed Google Scholar). In brief, stage V–VI oocytes were injected with cRNA mixtures containing 2.5 ng of each of the ENaC subunits. Oocytes were incubated at 17 °C in a medium that contained 96 mm NaCl and 10 μmamiloride. Electrophysiological measurements were performed 48–72 h later by means of a two-electrode voltage clamp technique. Channel activity was determined as the amiloride-sensitive current amplitudes monitored at −100 mV. Phosphorylation was examined in oocytes injected with cRNA mixtures in which either β or γ were HA-tagged. Groups of ∼40 ENaC-expressing oocytes were incubated for 4 h with 3.3 mCi of orthophosphate (32Pi). A mixture of orthovanadate (0.5 mm) and H2O2 (1 mm) was added in the last 60 min of the incubation to stimulate endogenous kinases. Another aliquot of ∼40 injected oocytes was metabolically labeled by a 2-day incubation with 100 μCi of [35S]methionine. Oocytes labeled with 32P or35S were washed and homogenized in buffer A + protease inhibitors, and membranes were isolated by centrifugation through a sucrose cushion. Membranes were solubilized in 1% Triton X-100 in buffer A and centrifuged for 5 min at 11,000 × g to remove insoluble material. Aliquots of ∼200 μl of detergent-soluble membrane protein extracts were incubated for 12–16 h at 4 °C with 2 μg of a mouse monoclonal anti-HA antibody (clone 12CA5, Roche Molecular Biochemicals) and then for another 2 h with protein A-Sepharose beads. The beads were sedimented, washed twice in buffer A + 0.1% Triton X-100, and a third time in buffer A + 0.5 mLiCl. Immunopellets were suspended in SDS sample buffer, resolved on 8% SDS-PAGE, and assayed for radioactivity. Cytosol extracted from rat distal colon has been used to identify protein kinases capable of phosphorylating ENaC subunits. The colonic tissue was selected, because it is relatively homogenous and has high Na+ channel abundance. Proteins were fractionated on a MonoQ column, and fractions were tested for their ability to phosphorylate fusion proteins having the carboxyl termini of γ ENaC. Fig. 1 depicts a typical assay demonstrating the existence of at least three protein kinase-enriched fractions phosphorylating GST-γ. The first peak of protein kinase activity was eluted around fractions 38–41 (∼0.07–0.09m NaCl) and is characterized in this study. A second peak, seen around fraction 59 (∼0.19 m NaCl), was found to phosphorylate residue γThr-630 (data not shown). Hence, it is likely to be the same kinase detected in a crude DE-52 fraction reported before (14.Chigaev A. Lu G. Shi H.-K. Asher C. Xu R. Latter H. Seger R. Garty H. Reuveny E. Am. J. Physiol. 2001; 280: F1030-F1036Crossref PubMed Google Scholar). A third, major peak was seen around fractions 74–77 (∼0.25–0.27 m NaCl) and will be described elsewhere. Initial experiments have used mutagenesis to identify ENaC residues phosphorylated by the kinase eluted around fraction 40. Phosphorylation of GST-γ appeared to be on Thr-623, and mutating this site to Ala residue completely inhibited incorporation of 32P into this fusion protein (Fig. 2A). Mutating γThr-630, another residue found to be phosphorylated by crude cytosol, had no effect. Cytosolic fractions 38–41 phosphorylated GST-β as well. In this case, however, mutating the β residue analogous to γThr-623 (βThr-613) into an Ala residue had only a minor effect on the incorporation of 32P into this fusion protein (not shown). This may be due to the phosphorylation of additional residues on the carboxyl tail of β. Phosphorylation of βThr-613 could be convincingly shown in subsequent experiments using purified kinase and cytosol from CHO cells. It was further demonstrated that the protein kinase eluted in fraction 40 tightly binds to the γ C-tail and can be co-precipitated with it. In this assay, GST-γ immobilized on glutathione beads was incubated with the above cytosolic fraction in the absence of ATP. The beads were washed several times and then incubated with [γ-32P]ATP with no added protein kinases. Substantial phosphorylation of the fusion protein can take place under these conditions only if some of the kinase is precipitated with its substrate. As shown in Fig. 2B, GST-γ could be effectively phosphorylated following such co-precipitation, and the phosphorylation occurred on Thr-623. Next, it was determined that the above phosphorylation takes place also in Xenopus oocytes expressing the three ENaC subunits. Accordingly, HA-tagged wild type and mutated subunits were translated in the oocyte system and metabolically labeled with either [35S]methionine or 32Pi. Orthovanadate and H2O2 were added during the incubation with 32P to achieve activation of endogenous protein kinases. Immunoprecipitation of HA-tagged β and γ demonstrated phosphorylation of both subunits (Fig. 3A). Mutating γThr-623 into an Ala residue significantly decreased incorporation of 32P into this subunit without affecting labeling by [35S]methionine. In three different experiments using ∼40 oocytes each, the mutation of Thr-623 lowered the32P/35S ratio of the immunoprecipitated subunit by 47 ± 15%. Thus, Thr-623 is one of the γ residues phosphorylated in the oocyte system. Phosphorylation of the β subunit on the other hand, was not significantly affected by mutating Thr-613 into Ala. Thus, this residue is either not phosphorylated in oocytes or its phosphorylation is masked by 32P incorporation into other residues. This may be similar to the phenomenon described above where mutating βThr-613 into Ala had a minor effect on the overall β phosphorylation by fractionated cytosol, even though it is clearly one of the phosphorylated residues (see subsequent experiments in CHO-T cells). Expressing the double mutant βT613A/γT623A in oocytes resulted in macroscopic Na+ currents that were 3.5-fold higher than that evoked by the wild type channel (Fig. 3B). Mutating the same residues into glutamic acid had a smaller but nevertheless significant stimulatory effect. Thus, γThr-623 and/or βThr-613 have a functional role and their mutation largely activates the channel. However, because mutation to a neutral or negatively charged amino acid evoked the same response, it is not certain that this activation is due to the inability to phosphorylate threonine 613/623. γThr-623 and βThr-613 are well-conserved residues located immediately before the PY motifs (Fig. 2C). Their phosphorylation could affect the channel by altering its interaction with Nedd4. Assessment of such a mechanism has been done by determining association of the WW regions of Nedd4 with phosphorylated and non-phosphorylated PY peptides using BIAcore (18.Asher C. Chigaev A. Garty H. Biochem. Biophys. Res. Commun. 2001; 286: 1228-1231Crossref PubMed Scopus (23) Google Scholar). Accordingly, various β and γ peptides, listed in Table I, were synthesized and attached to streptavidin-coated sensor chips via an amino-terminal biotin moiety. The three WW domains of rat Nedd4, expressed as GST fusion proteins, were passed over the chip and binding was recorded as a change in refractive index. A typical sensogram-monitoring interactions of WW2 to γPY peptides is illustrated in Fig. 4A. Binding of the recombinant protein to the phosphorylated peptide (γ pThr-623) was 2- to 3-fold higher than that recorded for the non-phosphorylated analogue (γ). No significant binding was observed with a peptide carrying a point mutation that impairs Nedd4-ENaC interactions (γ Y628A), or to free biotin. The small rapid signal seen in these channels is probably due to bulk or nonspecific effects. Therefore, in all subsequent experiments the signal obtained in the biotin channel was subtracted from readings in other channels to obtain ENaC-specific signals. We have also tested a γ peptide with phospho-threonine at position 630, shown to be phosphorylated by another cytosolic fraction (14.Chigaev A. Lu G. Shi H.-K. Asher C. Xu R. Latter H. Seger R. Garty H. Reuveny E. Am. J. Physiol. 2001; 280: F1030-F1036Crossref PubMed Google Scholar). This peptide (γ pThr-630) appeared to bind WW2 at a rate that was close to that of the wild type peptide (Fig. 4B).Table IENaC peptides used to study WW bindingPeptide sequenceSpecificationsI P G T P P P N Y D S L R L QβPY peptideI P G pT P P P N Y D S L R L QβPY peptide with pThr-613V P G T P P P R Y N T L R L DγPY peptideV P G pT P P P R Y N T L R L DγPY peptide with pThr-623V P G T P P P R Y N pT L R L DγPY peptide with pThr-630V P G T P P P R A N T L R L DγPY peptide with Y628A Open table in a new tab The above protocol was repeated for different concentrations of all three WW domains using both β and γ peptides (Fig. 5). All three WW domains were found to bind better to PY peptides that have phospho-threonine at position β613 or γ623. The experiment of Fig. 5 also confirms a previous observation that WW2 and WW3 bind PY sequences much better than WW1 (19.Farr T.J. Coddington-Lawson S.J. Snyder P.M. McDonald F.J. Biochem. J. 2000; 345: 503-509Crossref PubMed Google Scholar, 20.Harvey K.F. Dinudom A. Komwatana P. Jolliffe C.N. Day M.L. Parasivam G. Cook D.I. Kumar S. J. Biol. Chem. 1999; 274: 12525-12530Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Additional competition experiments summarized in Fig. 6, further establishes the above findings. In this experiment the WW3 fusion protein was preincubated in solution with different non-biotinylated peptides, and then applied to the sensor chip. The competing soluble peptide largely inhibited association of WW3 with the immobilized peptide, and phospho-peptides were more effective competitors then the non-phosphorylated analogues. This result was observed irrespective of the immobilized substrate (β or γ) or analyte (WW2 or WW3, data not shown). It demonstrates that the phosphorylated peptides better associate with the WW fusion proteins also in solution; i.e. it rules out the possibility that their larger binding reflects differences in peptide immobilization or packing on the chip.Figure 6Competition of phosphorylated and non-phosphorylated peptides. Aliquots of WW3 (0.6 μm) were preincubated with either diluent or different peptides (250 μg/ml). The mixtures were applied onto the sensor chip, and binding to phosphorylated β peptide was monitored. A, sequential recordings following the injections of: WW3, WW3 + β, WW3 + βp, WW3 + γ, WW3 + γp, and WW3. Injection of the analyte (first arrow) was followed by injection of HBS (second arrow). The chip was then regenerated with 10 μl of HSB + 0.05% SDS (third arrow) and finally equilibrated with HBS (fourth arrow). B, means ± S.E. of the maximal bindings recorded in three independent experiments. Data are presented as fraction of WW3 binding in the absence of competing peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although the above data clearly show effects of phosphorylation on the interactions between WW proteins and PY peptides, kinetic analysis of these effects was not trivial. As reported previously, the association could not be well fitted to simple 1:1 Langmuir binding and a considerable fraction of the response was attributed to a very slow and non-saturable component (18.Asher C. Chigaev A. Garty H. Biochem. Biophys. Res. Commun. 2001; 286: 1228-1231Crossref PubMed Scopus (23) Google Scholar). This phase was preceded by a faster saturable component that was particularly noticeable at high concentrations of the analytes. This phenomenon was independent of the flow rate (20–75 μl/min) and hence not likely to reflect mass transfer effects. Data could in principle be fitted to a sum of two exponents, but the fit was satisfactory only for the interaction of β and β pThr-613 with WW3. In this case, the faster event had the characteristics of a simple 1:1 binding and its apparent association rate constants (ks) showed the expected linear dependence on the analyte concentration (Fig. 7). The dissociation rate constants (kd) were concentration independent. The kinetics parameters extracted from this analysis, and the equilibrium constants calculated as ratios of the association and dissociation rate constants, are summarized in Table II. The phospho-β peptide was found to have ∼4-fold higher affinity toward WW3, due to an increased association rate constant. The second, slower phase could not be analyzed kinetically, but this component too was markedly affected by phosphorylation.Table IIKinetic parameters of the interaction between WW3 and β peptidesAnalytesubstratekakdKDm−1×s−1s−1μmWW3β pT6132.3 × 1040.026 ± 0.0101.1WW3β0.5 × 1040.021 ± 0.0034.2The association rate constants (ka) were calculated from slopes of the best-fitted lines from plots of ks versus the analyte concentration (Fig. 7, r<0.99). The dissociation rate constants (kd) are means ± S.D. of values obtained for different concentrations of the analyte. The equilibrium constants (KD) are ka/kd. Open table in a new tab The association rate constants (ka) were calculated from slopes of the best-fitted lines from plots of ks versus the analyte concentration (Fig. 7, r<0.99). The dissociation rate constants (kd) are means ± S.D. of values obtained for different concentrations of the analyte. The equilibrium constants (KD) are ka/kd. Next, we studied the identity of the cytosolic kinase eluted in fraction 40, which phosphorylates γThr-623. The presence of multiple proline residues near the phosphorylation site suggested involvement of a proline-directed kinase, e.g. a member of the MAPK family such as ERK, p38, or" @default.
• W2094567461 created "2016-06-24" @default.
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• W2094567461 creator A5047228848 @default.
• W2094567461 creator A5059428666 @default.
• W2094567461 creator A5065267260 @default.
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• W2094567461 date "2002-04-01" @default.
• W2094567461 modified "2023-10-15" @default.
• W2094567461 title "Interactions of β and γENaC with Nedd4 Can Be Facilitated by an ERK-mediated Phosphorylation" @default.
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