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• W2112315450 abstract "The signaling pathways by which cell volume regulates ion transporters, e.g.Na+/H+ exchangers (NHEs), and affects cytoskeletal organization are poorly understood. We have previously shown that shrinkage induces tyrosine phosphorylation in CHO cells, predominantly in an 85-kDa band. To identify volume-sensitive kinases and their substrates, we investigated the effect of hypertonicity on members of the Src kinase family. Hyperosmolarity stimulated Fyn and inhibited Src. Fyn activation was also observed in nystatin-permeabilized cells, where shrinkage cannot induce intracellular alkalinization. In contrast, osmotic inhibition of Src was prevented by permeabilization or by inhibiting NHE-1. PP1, a selective Src family inhibitor, strongly reduced the hypertonicity-induced tyrosine phosphorylation. We identified one of the major targets of the osmotic stress-elicited phosphorylation as cortactin, an 85-kDa actin-binding protein and well known Src family substrate. Cortactin phosphorylation was triggered by shrinkage and not by changes in osmolarity or pHi and was abrogated by PP1. Hyperosmotic cortactin phosphorylation was reduced in Fyn-deficient fibroblasts but remained intact in Src-deficient fibroblasts. To address the potential role of the Src family in the osmotic regulation of NHEs, we used PP1. The drug affected neither the hyperosmotic stimulation of NHE-1 nor the inhibition of NHE-3. Thus, members of the Src family are volume-sensitive enzymes that may participate in the shrinkage-related reorganization of the cytoskeleton but are probably not responsible for the osmotic regulation of NHE. The signaling pathways by which cell volume regulates ion transporters, e.g.Na+/H+ exchangers (NHEs), and affects cytoskeletal organization are poorly understood. We have previously shown that shrinkage induces tyrosine phosphorylation in CHO cells, predominantly in an 85-kDa band. To identify volume-sensitive kinases and their substrates, we investigated the effect of hypertonicity on members of the Src kinase family. Hyperosmolarity stimulated Fyn and inhibited Src. Fyn activation was also observed in nystatin-permeabilized cells, where shrinkage cannot induce intracellular alkalinization. In contrast, osmotic inhibition of Src was prevented by permeabilization or by inhibiting NHE-1. PP1, a selective Src family inhibitor, strongly reduced the hypertonicity-induced tyrosine phosphorylation. We identified one of the major targets of the osmotic stress-elicited phosphorylation as cortactin, an 85-kDa actin-binding protein and well known Src family substrate. Cortactin phosphorylation was triggered by shrinkage and not by changes in osmolarity or pHi and was abrogated by PP1. Hyperosmotic cortactin phosphorylation was reduced in Fyn-deficient fibroblasts but remained intact in Src-deficient fibroblasts. To address the potential role of the Src family in the osmotic regulation of NHEs, we used PP1. The drug affected neither the hyperosmotic stimulation of NHE-1 nor the inhibition of NHE-3. Thus, members of the Src family are volume-sensitive enzymes that may participate in the shrinkage-related reorganization of the cytoskeleton but are probably not responsible for the osmotic regulation of NHE. Chinese hamster ovary 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein Na+/H+ exchanger N-methyl-d-glucammonium intracellular pH wild type Western blot immunoprecipitation The maintenance of normal cell volume is an essential homeostatic function. Most cells are equipped with a variety of volume-sensitive membrane transporters (e.g. isoforms of the Na+/H+ exchanger, Na+/K+/Cl− cotransporter, K+ and Cl− channels) that can effectively restore normal cell size after a perturbation caused either by exposure to an aniso-osmotic environment or by metabolic changes (for reviews, see Refs. 1Haussinger D. Biochem. J. 1996; 313: 697-710Crossref PubMed Scopus (497) Google Scholar and 2Lang F. Busch G.L. Ritter M. Volkl H. Waldegger S. Gulbins E. Haussinger D. Physiol. Rev. 1998; 78: 247-306Crossref PubMed Scopus (1582) Google Scholar). Alterations in the cell volume are also known to induce reorganization in the actin skeleton (3Foskett J.K. Spring K.R. Am. J. Physiol. 1985; 248: C27-C36Crossref PubMed Google Scholar, 4Hallows K.R. Packman C.H. Knauf P.A. Am. J. Physiol. 1991; 261: C1154-C1161Crossref PubMed Google Scholar, 5Tilly B.C. Edixhoven N.J. Tertoolen L.G.J. Morii N. Saitoh Y. Narumiya S. de Jonge H.R. Mol. Biol. Cell. 1996; 7: 1419-1427Crossref PubMed Scopus (151) Google Scholar, 6Chowdhury S. Smith K. Gustin M.C. J. Cell Biol. 1992; 118: 561-571Crossref PubMed Scopus (196) Google Scholar) and to modulate gene transcription (7Handler J.S. Kwon H.M. Am. J. Physiol. 1993; 265: C1449-C1455Crossref PubMed Google Scholar, 8Waldegger S. Barth P. Raber G. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4440-4445Crossref PubMed Scopus (331) Google Scholar, 9Okazaki T. Ishikawa T. Nishimori S. Igarashi T. Hata K. Fujita T. J. Biol. Chem. 1997; 272: 32274-32279Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Moreover, the cellular hydration state is not only a subtly regulated parameter but is also an important regulatory signal (1Haussinger D. Biochem. J. 1996; 313: 697-710Crossref PubMed Scopus (497) Google Scholar). For example, hormones such as glucagon and insulin elicit volume changes which act as “second messengers” necessary for their cellular effects. Little is known about the volume-dependent signaling mechanisms and their relationship to the different effector systems such as the ion carriers or the cytoskeleton. Evidence has been accumulating that protein-tyrosine kinases may play a pivotal role in the signaling of both hypo- (5Tilly B.C. Edixhoven N.J. Tertoolen L.G.J. Morii N. Saitoh Y. Narumiya S. de Jonge H.R. Mol. Biol. Cell. 1996; 7: 1419-1427Crossref PubMed Scopus (151) Google Scholar, 10Tilly B.C. van den Berghe N. Tertoolen L.G. Edixhoven M.J. de Jonge H.R. J. Biol. Chem. 1993; 268: 19919-19922Abstract Full Text PDF PubMed Google Scholar, 11Wiese S. Schliess F. Haussinger D. Biochem. J. 1998; 379: 667-671Google Scholar) and hyperosmotic shock (12Szászi K. Buday L. Kapus A. J. Biol. Chem. 1997; 272: 16670-16678Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 13Krump E. Nikitas K. Grinstein S. J. Biol. Chem. 1997; 272: 17303-17311Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 14Junger W.G. Hoyt D.B. Davis R.E. Herdon-Remelius C. Namiki S. Junger H. Loomis W. Altman A. J. Clin. Invest. 1998; 101: 2768-2779Crossref PubMed Scopus (150) Google Scholar). Our recent studies using CHO1 cells (12Szászi K. Buday L. Kapus A. J. Biol. Chem. 1997; 272: 16670-16678Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) as well as that of Krump et al. on neutrophils (13Krump E. Nikitas K. Grinstein S. J. Biol. Chem. 1997; 272: 17303-17311Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) have shown that hypertonicity induces robust tyrosine phosphorylation in various protein bands. While the phosphorylation pattern differed in the two cell types studied, the trigger for the phosphorylation of most proteins was cell shrinkage and not an increase in osmolarity or in intracellular ion concentrations. In CHO cells, hypertonicity induced phosphorylation of proteins of ∼40, 85, and 110–130 kDa, with the most prominent response occurring in the ∼85-kDa band (p85). While the ∼40-kDa protein proved to be extracellular signal-regulated kinase-2, the identity of p85 and the other higher molecular weight proteins remains to be elucidated. Further, the kinase pathways responsible for these reactions are unknown. Some of the tonicity-sensitive proteins complexed with Src homology 2 (SH2) and SH3 domains, raising the possibility that the Src family of tyrosine kinases might be potential mediators of the osmotically induced phosphorylations. This notion is further strengthened by the finding that, in neutrophils, hypertonicity altered the activity of Fgr, Hck, and Lyn (13Krump E. Nikitas K. Grinstein S. J. Biol. Chem. 1997; 272: 17303-17311Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), members of the Src family expressed specifically in cells of hematopoietic origin. Furthermore, pharmacological data fostered the concept that the hypertonicity-stimulated tyrosine phosphorylation might be causally connected to the osmotic regulation of Na+/H+ exchange. Specifically, the effect of hypertonicity on two osmotically sensitive, but oppositely regulated, isoforms of the Na+/H+ exchanger (15Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar, 16Soleimani M. Bookstein C. McAteer J.A. Hattabaugh Y.J. Bizal G.L. Musch M.W. Villereal M. Rao M.C. Howard R.L. Chang E.B. J. Biol. Chem. 1994; 269: 15613-15618Abstract Full Text PDF PubMed Google Scholar, 17Nath S.K. Hang C.Y. Levine S.A. Yun C.H. Montrose M.H. Donowitz M. Tse C.M. Am. J. Physiol. 1996; 270: G431-G441PubMed Google Scholar, 18Watts III, B.A. Good D.W. J. Biol. Chem. 1994; 269: 20250-20255Abstract Full Text PDF PubMed Google Scholar, 19Bianchini L. Kapus A. Lukacs G. Wasan S. Wakabayashi S. Pouyssegur J. Yu F.H. Orlowski J. Grinstein S. Am. J. Physiol. 1995; 269: C998-C1007Crossref PubMed Google Scholar) was abrogated by broad spectrum tyrosine kinase inhibitors; the hyperosmotic activation of Na+/H+ exchanger-1 (NHE-1) (in neutrophils) was inhibited by genistein (13Krump E. Nikitas K. Grinstein S. J. Biol. Chem. 1997; 272: 17303-17311Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), whereas the hyperosmotic inhibition of NHE-3 (in kidney cells) was prevented by genistein and herbimycin (20Good D.W. J. Biol. Chem. 1995; 270: 9883-9889Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Based on these observations, Krumpet al. (13Krump E. Nikitas K. Grinstein S. J. Biol. Chem. 1997; 272: 17303-17311Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) suggested that the hypertonic activation of NHE-1 in neutrophils may be due to the stimulation of certain Src kinases. To date, no information has been available about the osmotic responsiveness of the two most widely expressed, ubiquitous Src family members, p60src and p59fyn. In addition, none of the major osmosensitive phosphoproteins has been identified. The aim of this study was to gain further insight into the mechanisms of volume-dependent signaling by investigating the potential role of the Src family in the hypertonicity-induced tyrosine phosphorylation and the subsequent changes in ion transport. Specifically, we intended to investigate whether Src and Fyn can be regulated by a decrease in cell volume and whether any of the major phosphoproteins can be identified as a Src family substrate. We also wished to discern whether the Src family may play an essential role in the osmotic regulation of NHE-1 or NHE-3. Our results show that members of the Src family are regulated by cell shrinkage, and one of the major targets for volume-dependent tyrosine phosphorylation is the Src family substrate, actin cross-linking protein, cortactin. On the other hand, the osmotic regulation of NHE-1 and -3 does not appear to be mediated by Src-like kinases. Me2SO, nystatin (used from a stock solution of 400,000 units/ml in Me2SO, freshly prepared before each experiment), nigericin, monensin,N-methyl-d-glucammonium, and gluconic acid lactone were purchased from Sigma. Proteinase inhibitor mixture containing 0.8 mg/ml benzamidine HCl, 0.5 mg/ml aprotinin, 0.5 mg/ml leupeptin, 0.5 mg/ml pepstatin A, and 50 mmphenylmethylsulfonyl fluoride in pure ethanol was from PharMingen, Protein G-Sepharose beads from Amersham Pharmacia Biotech, 2‘,7‘-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)/acetoxymethylester and PP1 were from Calbiochem. Monoclonal anti-phosphotyrosine (4G10), anti-cortactin, anti-p60src, the Src assay kit including an Src family-specific substrate peptide, and the polyclonal anti-Fyn were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Monoclonal anti-Fyn was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-phospho-p38 was from New England Biolabs. Peroxidase-conjugated anti-mouse and anti-rabbit IgG, the Enhanced Chemiluminescence kit, and [γ-32P]ATP (3000 Ci/mmol) were from Amersham Pharmacia Biotech. Bicarbonate-free RPMI 1640 was buffered with 25 mm Hepes to pH 7.4 (osmolarity 290 ± 5 mosm). The Iso-Na medium consisted of 140 mmNaCl, 3 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 5 mm glucose, 20 mm Hepes (pH 7.4). When required, Iso-Na was made hypertonic (Hyper-Na, 600 mosm) by the addition of 300 mm sucrose. The Iso- and Hyper-K media had the same composition, except NaCl was replaced by KCl. The permeabilization medium contained 140 mm KCl, 10 mm NaCl, 1 mm MgCl2, 1 mm EGTA, 0.193 mm CaCl2 (100 nm free Ca2+), 5 mm glucose, 10 mm Hepes (pH 7.2). To permeabilize the cells, this medium was supplemented with 400 units/ml nystatin and 60 mm sucrose. Sucrose was included to counterbalance the intracellular colloidosmotic pressure and thereby prevent swelling of the permeabilized cells, as reported earlier by us (12Szászi K. Buday L. Kapus A. J. Biol. Chem. 1997; 272: 16670-16678Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). To induce shrinkage, the nystatin-containing permeabilization buffer was supplemented with 350 mmsucrose. The Iso-NMG medium was composed of 140 mm NMG, 140 mm gluconic acid lactone, 3 mm NaCl, 1 mm MgCl2, 1 mm EGTA, 0.193 mm CaCl2 (100 nm free Ca2+), 5 mm glucose, 10 mm Hepes (pH 7.2). The osmolarity of the isotonic solutions was adjusted to 290 ± 5 mosm with the major salt. Osmolarity was checked with an Osmette osmometer. For most studies, we used a CHO cell line (AP-1) devoid of the endogenous NHE and stably transfected with the rat NHE-1 (referred to as NHE-1 cells) or NHE-3 (referred to as NHE-3 cells), as described previously (15Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar). These cells were grown in α-minimal essential medium, containing 25 mmNaHCO3 and supplemented with 10% fetal calf serum, and 1% antibiotic suspension (penicillin and streptomycin; Sigma) under a humidified atmosphere of air/CO2 (19:1) at 37 °C. To eliminate potential revertants and to maintain the high expression level of the NHE isoforms, cells were selected after every third passage for the Na+/H+exchange-dependent survival of an acute acid load (15Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar). WT, Fyn−/−, Src−/− fibroblasts were isolated from mouse embryos that were homozygous for disruption in Src or Fyngene and were immortalized with large T antigen (21Thomas S.M. Sorriano P. Imamoto A. Nature. 1995; 376: 267-271Crossref PubMed Scopus (304) Google Scholar). Cells were kindly provided by Sheila M. Thomas (Fred Hutchinson Cancer Center, Seattle, WA) and were maintained in Dulbecco's modified Eagle's medium. All other conditions and treatments were similar to those in CHO cells. Human neutrophils were prepared from healthy volunteers as described previously (22Suszták K. Mocsai A. Ligeti E. Kapus A. Biochem. J. 1997; 325: 501-510Crossref PubMed Scopus (33) Google Scholar) except that lysis of red blood cells was carried out using NH4Cl. Prior to use, cells (106/ml) were kept in Hepes-buffered RPMI medium at 37 °C. Confluent cultures were incubated for at least 3 h in serum- and HCO3−-free RPMI 1640 prior to experiments. Cells were preincubated in Iso-Na medium for 10 min and then subjected to various treatments as indicated. The medium was then aspirated, and the cells were vigorously scraped into ice-cold Triton-containing or modified radioimmune precipitation buffers, supplemented with 1 mm Na3VO4 and 20 μl/ml protease inhibitor mixture. The Triton lysis buffer contained 100 mm NaCl, 30 mm Hepes, 20 mm NaF, 1 mm EGTA, 1% Triton X-100, pH 7.5, and the radioimmune precipitation buffer was composed of 150 mm NaCl, 50 mm Tris-HCl, 1 mm NaF, 2 mmTris-EGTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, pH 7.4. Lysates containing equivalent amount of protein were either mixed with an equal amount of 2× Laemmli buffer (whole cell lysates) or were clarified by centrifugation at 12,000 × g for 10 min and further processed for immunoprecipitation. Extracts were precleared for 1 h using 40 μl of 50% suspension of Protein G-Sepharose beads and then incubated with the corresponding antibodies (see details in the figure legends to Figs. Figure 2, Figure 3, Figure 4, Figure 5, Figure 6) for 1 h. Immunocomplexes were captured using 40 μl of protein G-Sepharose, and the beads were washed four times with lysis buffer. Immunoprecipitated proteins were diluted with Laemmli sample buffer, boiled for 5 min, and subjected to electrophoresis on 10% SDS-polyacrylamide gels. The separated proteins were transferred to nitrocellulose using a Bio-Rad Mini Protean II apparatus. To check the effectiveness of transfer and similarity of protein amount, lanes were visualized by staining with Ponceau S. Blots were blocked in Tris-buffered saline containing 5% bovine serum albumin and then incubated with the primary antibody. The binding of the antibody was visualized by peroxidase-coupled secondary anti-mouse or rabbit antibody (1:3000 dilution) using the enhanced chemiluminescence method.Figure 5Hypertonicity activates p59fyn. Cells were treated with iso- or hypertonic solutions for 10 min or for the indicated times and lysed. Fyn was immunoprecipitated from the extracts with a monoclonal antibody, and its activity was determined essentially as described for Src under Fig. 4. A, the Cdc2 peptide was used as substrate (n = 4, reflecting the Fyn activity of cell lysates containing 270 μg of protein), whereas in B, C, and E, enolase was applied (for B andE, n = 5). D, Fyn immunoprecipitates obtained from iso- and hypertonic samples were subjected to electrophoresis, blotted onto nitrocellulose, and probed with anti-phosphotyrosine (top). The same blot was stripped and reprobed with anti-Fyn (bottom). E, cells were permeabilized as detailed in the legend to Fig. 4 and were treated either with the permeabilization buffer ensuring isovolemia (I) or with this buffer supplemented with 300 mmextra sucrose, inducing shrinkage (S). After 10 min, cells were lysed and processed for the Fyn activity determination.View Large Image Figure ViewerDownload (PPT)Figure 4The involvement of NHE-1-mediated intracellular alkalinization in the inhibition of Src kinase. A, NHE-1 cells were pretreated with Iso-Na for 10 min, and (where indicated) the medium was supplemented with 15 μmof the NHE inhibitor HOE 694 for the last minute of the preincubation. Subsequently, the medium was exchanged to Iso- or Hyper-Na, with or without the drug, and after 2.5 min the cells were lysed with Triton buffer. In vitro kinase assays and the quantification of enolase phosphorylation (n = 3) were carried out as in Fig. 3. B, after a 10-min pretreatment with Iso-Na, the medium was replaced with Iso-Na without any ionophore (Control) or with Iso-Na supplemented with either 10 μg/ml monensin (Alkalinization (mon)) or 10 μg/ml nigericin (Acidification (nig)). The cells were lysed after 2.5 min, and the lysates were processed for the Src assay (n = 3). C, after a short pretreatment in Iso-Na, cells were briefly washed with the permeabilization medium and then were permeabilized under isovolemic conditions using the same solution supplemented with 400 units/ml nystatin and 60 mm sucrose. After 7 min, the medium was aspirated and replaced either by the same medium (Isovolemic) or by the permeabilization buffer containing an extra 300 mm sucrose (Shrunken). 10 min later, the cells were lysed, and the samples were processed for the immunocomplex Src kinase assays (n = 3).View Large Image Figure ViewerDownload (PPT)Figure 3The effect of hyperosmolarity on the activity of p60src kinase. NHE-1 cells were treated with isotonic solutions or challenged with hypertonicity for 10 min (A and B) or for the indicated times (C). Thereafter, cells were lysed with radioimmune precipitation (A) or Triton buffer (B andC) and subjected to immunoprecipitation using a monoclonal anti-Src antibody. Immunocomplex kinase assays were performed using [γ-32P]ATP and either a peptide fragment of Cdc2 (A) or enolase (B and C) as substrate (see “Experimental Procedures” for details). The peptide was separated by phosphocellulose filters, and the incorporated activity was measured by scintillation counting (n = 4, reflecting the Src activity of cell lysates containing 120 μg of protein). When enolase was used, the samples were subjected to SDS-polyacrylamide gel electrophoresis followed by radiography, and phosphorylation was quantified by a PhosphorImager. No radioactive bands were detected if the primary antibody or enolase was omitted from the reaction (not shown). Data are expressed as percentage change, compared with the isotonic activity (100%). Where error bars are indicated, the results are mean ± S.E. for 3–6 independent determinations. The amount of Src in the immunoprecipitates (B) and Triton X-100-insoluble (Tx) fractions (D) obtained from iso- and hypertonically treated cells were determined by immunoblotting with anti-Src. In four repeated experiments, no significant difference was observed between the iso- and hypertonic samples.View Large Image Figure ViewerDownload (PPT)Figure 2Cell shrinkage induces tyrosine phosphorylation of cortactin. A and B, after preincubation of cells expressing NHE-1 (A) or NHE-3 (B) for 10 min in Iso-Na, the medium was changed for either Iso-Na or Hyper-Na (HYP) for 10 min. Where indicated, 10 μm PP1 was present throughout the experiment (HYP + PP1). Cells were then lysed with radioimmune precipitation buffer, and cortactin was immunoprecipitated from extracts containing equal amounts of protein (280 μg) as described under “Experimental Procedures.” Immunoprecipitated molecules were separated, blotted onto nitrocellulose, and probed with anti-phosphotyrosine. To test the effectiveness of the immunoprecipitation, the same blots were stripped and reprobed with anti-cortactin. C, NHE-1 cells were preincubated in Iso-Na, and then the medium was changed to either Iso-Na or Iso-NMG or Iso-NMG supplemented with 400 units/ml nystatin, as indicated. After 10 min, cells were lysed, and cortactin was precipitated and analyzed as above.View Large Image Figure ViewerDownload (PPT) Quantification of the bands was performed using a Bio-Rad GS-690 imaging densitometer, and evaluation of data was carried out with the Molecular Analyst computer program (12Szászi K. Buday L. Kapus A. J. Biol. Chem. 1997; 272: 16670-16678Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The activity of Src and Fyn was determined by immunocomplex kinase assays (23Yamaji Y. Tsuganezawa H. Moe O.V. Alpern J.R. Am. J. Physiol. 1997; 272: C886-C893Crossref PubMed Google Scholar, 24Cheng H. Nishio H. Hatase O. Ralph S. Wang J.H. J. Biol. Chem. 1992; 267: 9248-9256Abstract Full Text PDF PubMed Google Scholar). Cell lysates obtained from iso- or hypertonically treated cells and containing equal amounts of protein (280–500 μg) were subjected to immunoprecipitation (see above), and the precipitates were washed with kinase buffer (20 mm Hepes, 10 mmMnCl2, 0.25 mm Na3VO4,pH 7.1). Kinase activity was measured as the phosphorylation of either the Src family-specific substrate peptide Cdc2-(6–20) or enolase. In the former case, the Upstate Biotechnology Src kinase assay kit was used according to the manufacturer's instructions. Briefly, the immunocomplexes were incubated with 20 μl of reaction buffer (100 mm Tris-HCl, pH 7.2, 125 mm MgCl225 mm MnCl2, 2 mm EGTA, 0.25 mm Na3VO4, 2 mmdithiothreitol), 10 μl of substrate peptide (0.6 mm stock in H2O), and the reaction was initiated by the addition of 10 μl of manganese/ATP mixture (0.5 mm ATP, 75 mm MnCl2 in reaction buffer) containing 10 μCi [γ-32P]ATP/sample. After 10 min at 30 °C, 20 μl of trichloroacetic acid was added, and 25 μl of the mixture was layered on P81 phosphocellulose squares. After extensive washing with 0.85% phosphoric acid, radioactivity bound to the filters was determined by scintillation counting. Nonspecific binding of radioactivity to the filters was determined in each experiment by measuring the activity of samples to which 10 μl of H2O was added instead of the peptide. The low activity measured in these samples was subtracted as background. Experiments were repeated at least three times, and the results were normalized to the amount of protein content of the initial cell lysate. When enolase was used, the immunocomplexes were incubated with 8 μl of kinase buffer, 12 μl of acid-denatured enolase (1.75 μg of enolase/sample), and 10 μl of ATP mixture (kinase buffer supplemented with 3 μm K-ATP and 10 μCi of [γ-32P]ATP/sample). After 5 or 10 min at 30 °C for Src and Fyn, respectively, the reaction was terminated by the addition of 10 μl of 4× Laemmli buffer, and the samples were boiled and subjected to SDS-polyacrylamide gel electrophoresis. The gels were dried and used for direct quantification of radioactivity with a Molecular Dynamics PhosphorImager using ImageQuant software. The gels were also subjected to radiography with an intensifying screen. Each experiment was performed at least three times, and each time duplicates or triplicates were measured. Results are expressed as -fold increase compared with the controls. pHiwas measured fluorometrically using the indicator dye BCECF, essentially as described (15Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar). Confluent cultures of NHE-1 or NHE-3 cells grown on glass coverslips were loaded with 1 μmBCECF/acetoxymethylester for 10 min in Iso-Na medium. Ratio fluorometry was performed on small populations of cells (6–12 cells/measurement) using an illumination system from Photon Technologies, Inc., in the dual excitation (495 ± 10 nm/445 ± 10 nm) single emission (530 ± 30 nm) configuration. Excitation light was reflected to the cell by a 510-nm dichroic mirror, and emitted light was selected by a 520-nm longpass filter. Cells were visualized with a Nikon Diaphot TMD microscope and a Hoffman modulation contrast video system through a CCD video camera connected to a Panasonic monitor. Dye-loaded NHE-1 cells were preincubated with or without PP1, and then each coverslip was mounted to form the bottom of a thermostatted, perfusable Leydig chamber into which 0.5 ml of the same medium was added, and the basal fluorescence was recorded. The medium was then rapidly exchanged (by the addition of 10 × 0.5 ml in less than 15 s) to a hypertonic medium with or without the drug. To follow the osmotic effects on NHE-3 cells, these cells were first acidified by the ammonium prepulse technique. After 10 min of dye loading, in the presence or absence of PP1, cells were washed and then incubated in Iso- or Hyper-Na medium, supplemented with 20 mmNH4Cl, and supplemented, where indicated, with PP1 again. Thereafter, cells were washed with Iso- or Hyper-K medium and placed under the microscope in the same medium. Recovery of pHi was initiated with Iso- or Hyper-Na, again in the absence or presence of the Src inhibitor. After each measurement, fluorescence was calibrated in terms of pHi by sequential perfusion of the chamber using nigericin-containing Iso-K medium (or for hypertonic samples Iso-K supplemented with 150 mm extra KCl), at various pH values between 6 and 8 with 0.5 pH unit increments. Analysis of the data was carried out using Felix® software. In neutrophils, pHi was monitored in cell suspension (0.5 × 106/ml) using a Beckman fluorimeter (22Suszták K. Mocsai A. Ligeti E. Kapus A. Biochem. J. 1997; 325: 501-510Crossref PubMed Scopus (33) Google Scholar). Protein concentration was determined by the BCA assay (Pierce) using bovine serum albumin as a standard. Data are presented as representative immunoblots of at least three similar experiments or as mean ± S.E. of the number of experiments indicated (n). To assess whether Src kinases might play a role in the hypertonicity-induced tyrosine phosphorylation observed in CHO cells, we used PP1, a newly developed, selective pyrazolo pyrimidine-type inhibitor of this enzyme family (25Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brisette W.H. Weringer E.J. Pollok Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar,26Amoui M. Draber P. Draberova L. Eur. J. Immunol. 1997; 27: 1881-1886Crossref PubMed Scopus (77) Google Scholar). Fig. 1 A shows that osmotic shock evoked strong tyrosine phosphorylation of several proteins, the predominant response occurring in 80–85- and 110–130-kDa bands, as reported earlier by us (12Szászi K. Buday L. Kapus A. J. Biol. Chem. 1997; 272: 16670-16678Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). PP1 caused a concentration-dependent inhibition of the hypertonicity-triggered tyrosine phosphorylation in most bands. Half-maximal inhibition of p85 phosphorylation was obtained at ∼2 μm, whereas 10 μm completely abolished phosphotyrosine accumulation in this band (Fig. 1 B). This concentration dependence corr" @default.
• W2112315450 created "2016-06-24" @default.
• W2112315450 creator A5004819829 @default.
• W2112315450 creator A5033704842 @default.
• W2112315450 creator A5034774105 @default.
• W2112315450 creator A5055198525 @default.
• W2112315450 creator A5086761442 @default.
• W2112315450 date "1999-03-01" @default.
• W2112315450 modified "2023-10-13" @default.
• W2112315450 title "Cell Shrinkage Regulates Src Kinases and Induces Tyrosine Phosphorylation of Cortactin, Independent of the Osmotic Regulation of Na+/H+ Exchangers" @default.
• W2112315450 cites W1486289829 @default.
• W2112315450 cites W1493020321 @default.
• W2112315450 cites W1542805579 @default.
• W2112315450 cites W1548272930 @default.
• W2112315450 cites W1580762299 @default.
• W2112315450 cites W1584026320 @default.
• W2112315450 cites W1594394126 @default.
• W2112315450 cites W1602747859 @default.
• W2112315450 cites W1607594871 @default.
• W2112315450 cites W1858501591 @default.
• W2112315450 cites W1964350811 @default.
• W2112315450 cites W1965860054 @default.
• W2112315450 cites W1972811230 @default.
• W2112315450 cites W1981707775 @default.
• W2112315450 cites W1983620623 @default.
• W2112315450 cites W1987872719 @default.
• W2112315450 cites W1997986332 @default.
• W2112315450 cites W1999673185 @default.
• W2112315450 cites W2001306989 @default.
• W2112315450 cites W2001453486 @default.
• W2112315450 cites W2003962192 @default.
• W2112315450 cites W2005313327 @default.
• W2112315450 cites W2009582035 @default.
• W2112315450 cites W2014286995 @default.
• W2112315450 cites W2022064841 @default.
• W2112315450 cites W2024630630 @default.
• W2112315450 cites W2025800972 @default.
• W2112315450 cites W2030570585 @default.
• W2112315450 cites W2031983592 @default.
• W2112315450 cites W2036431526 @default.
• W2112315450 cites W2054956895 @default.
• W2112315450 cites W2056426987 @default.
• W2112315450 cites W2056596114 @default.
• W2112315450 cites W2061229454 @default.
• W2112315450 cites W2063224946 @default.
• W2112315450 cites W2064540749 @default.
• W2112315450 cites W2067437104 @default.
• W2112315450 cites W2070651101 @default.
• W2112315450 cites W2072373148 @default.
• W2112315450 cites W2076482516 @default.
• W2112315450 cites W2077080151 @default.
• W2112315450 cites W2082710015 @default.
• W2112315450 cites W2085635715 @default.
• W2112315450 cites W2090634323 @default.
• W2112315450 cites W2090895449 @default.
• W2112315450 cites W2096679179 @default.
• W2112315450 cites W2096873347 @default.
• W2112315450 cites W2119261959 @default.
• W2112315450 cites W2126107464 @default.
• W2112315450 cites W2150125913 @default.
• W2112315450 cites W2164384086 @default.
• W2112315450 cites W2167956058 @default.
• W2112315450 cites W2186358919 @default.
• W2112315450 cites W2224021986 @default.
• W2112315450 cites W2276541799 @default.
• W2112315450 cites W2286893858 @default.
• W2112315450 cites W2310097628 @default.
• W2112315450 cites W2324328780 @default.
• W2112315450 cites W2342744565 @default.
• W2112315450 cites W25348686 @default.
• W2112315450 cites W342550691 @default.
• W2112315450 cites W5386695 @default.
• W2112315450 cites W78644067 @default.
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