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• W1511917665 abstract "Hypotonic volume expansion of skate erythrocytes rapidly stimulates the tyrosine phosphorylation of band 3, the membrane protein thought to mediate the osmotically sensitive taurine efflux. Skate erythrocytes possess numerous tyrosine kinases including p59fyn, p56lyn, pp60src, and p72syk, demonstrated by immune complex assays measuring autocatalytic kinase activity. Inclusion of the cytoplasmic domain of band 3 in this assay showed that only Syk and Lyn can directly phosphorylate the cytoplasmic domain of band 3. Upon cell volume expansion, Syk activity was increased as assessed by three different assays (immune complex assay measuring autophosphorylation, assay of the level of phosphotyrosine of the immunoprecipitated kinase, and assay of level of 32P in the kinase immunoprecipitated from cells prelabeled with32PO4 and then volume-expanded). The tyrosine kinase Lyn was also stimulated by volume expansion, most notably when analyzed by the latter two methods. Volume expansion stimulated a large increase in the ability of Syk to phosphorylate band 3 at times that coincide with the stimulation of taurine flux. The stilbene piceatannol inhibited Syk preferentially over Lyn and other tyrosine kinases and inhibited volume-stimulated taurine efflux in a concentration-dependent manner similar to that for the inhibition of Syk. Two major phosphorylation peaks were detected in tryptic digests of cdb3 separated by reverse phase HPLC. Edman degradation demonstrated a phosphotyrosine in a YXXL motif. In conclusion, p72syk appears to be a strong candidate as a pivotal signal-transducing step in the volume-activated taurine efflux in skate red cells. The level of band-3 phosphorylation may be regulated, in addition, by a protein-tyrosine phosphatase of the 1B variety. Hypotonic volume expansion of skate erythrocytes rapidly stimulates the tyrosine phosphorylation of band 3, the membrane protein thought to mediate the osmotically sensitive taurine efflux. Skate erythrocytes possess numerous tyrosine kinases including p59fyn, p56lyn, pp60src, and p72syk, demonstrated by immune complex assays measuring autocatalytic kinase activity. Inclusion of the cytoplasmic domain of band 3 in this assay showed that only Syk and Lyn can directly phosphorylate the cytoplasmic domain of band 3. Upon cell volume expansion, Syk activity was increased as assessed by three different assays (immune complex assay measuring autophosphorylation, assay of the level of phosphotyrosine of the immunoprecipitated kinase, and assay of level of 32P in the kinase immunoprecipitated from cells prelabeled with32PO4 and then volume-expanded). The tyrosine kinase Lyn was also stimulated by volume expansion, most notably when analyzed by the latter two methods. Volume expansion stimulated a large increase in the ability of Syk to phosphorylate band 3 at times that coincide with the stimulation of taurine flux. The stilbene piceatannol inhibited Syk preferentially over Lyn and other tyrosine kinases and inhibited volume-stimulated taurine efflux in a concentration-dependent manner similar to that for the inhibition of Syk. Two major phosphorylation peaks were detected in tryptic digests of cdb3 separated by reverse phase HPLC. Edman degradation demonstrated a phosphotyrosine in a YXXL motif. In conclusion, p72syk appears to be a strong candidate as a pivotal signal-transducing step in the volume-activated taurine efflux in skate red cells. The level of band-3 phosphorylation may be regulated, in addition, by a protein-tyrosine phosphatase of the 1B variety. elasmobranch incubation medium polyacrylamide gel electrophoresis 4-morpholineethanesulfonic acid high performance liquid chromatography protein-tyrosine phosphatase 1B Volume expansion stimulates the efflux of a variety of solutes from cells to accomplish a regulatory volume decrease because of the water that obligatorily follows the solutes. Among the most commonly utilized solutes to accomplish the volume decrease is the β-amino acid taurine, which can accumulate from 10–100 mm concentration in many cells (1Goldstein L. Davis-Amaral E.M. Musch M.W. Kidney Int. 1996; 49: 1690-1694Abstract Full Text PDF PubMed Scopus (30) Google Scholar). In erythrocytes of a number of species, the volume-stimulated taurine efflux appears to occur by a transport pathway that involves band 3 (2Goldstein L. Brill S.A. Am. J. Physiol. 1991; 260: R1014-R1020PubMed Google Scholar, 3Fievet B. Gabillat N. Borgese F. Motais R. EMBO J. 1995; 14: 5158-5169Crossref PubMed Scopus (97) Google Scholar). Oocyte expression of band 3 cloned from trout, but not mouse, results not only in a Cl− exchange activity, but also in a swelling-activated pathway for a number of solutes including taurine (4Fievet B. Perset F. Gabillat N. Guizouran H. Borgese F. Ripoche P. Motais R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10996-11001Crossref PubMed Scopus (31) Google Scholar). This latter finding strongly supports a role for band 3 as either the volume-activated transporter or a component of a volume-activated taurine transport pathway. Volume expansion causes skate band 3 to undergo structural changes and to modify its interaction with other red cells proteins. Band 3 exists in the membrane primarily as a dimer in human (5Jennings M.L. J. Membr. Biol. 1984; 80: 105-117Crossref PubMed Scopus (116) Google Scholar) and skate (6Musch M.W. Davis E.M. Goldstein L. J. Biol. Chem. 1994; 269: 19683-19686Abstract Full Text PDF PubMed Google Scholar) erythrocytes. However, in skate erythrocytes under volume-expanded conditions band 3 forms a tetramer (6Musch M.W. Davis E.M. Goldstein L. J. Biol. Chem. 1994; 269: 19683-19686Abstract Full Text PDF PubMed Google Scholar). The formation of this complex is thought to be related to the interaction of band 3 with cytoskeletal proteins in the cell as volume expansion stimulates a high affinity interaction of skate band 3 with ankyrin (7Musch M.W. Goldstein L. J. Biol. Chem. 1996; 271: 21221-21225Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). However, how this event relates to the formation of the tetramer and how this may mediate taurine efflux are as yet unknown. One biochemical change that occurs in skate band 3 in volume expansion is increased phosphorylation (8Musch M.W. Leffingwell T.R. Goldstein L. Am. J. Physiol. 1994; 266: R65-R74PubMed Google Scholar). This event occurs very rapidly, and band 3 phosphorylation, particularly that of tyrosine, has been documented in a large number of studies of human red cells (e.g. Refs. 9Tuy F.P.D. Henry J. Rosenfeld C. Kahn A. Nature. 1983; 305: 435-438Crossref PubMed Scopus (77) Google Scholar, 10Yannoukakos D. Meyer H.E. Vasseur C. Driancourt C. Wajeman H. Burseaux E. Biochim. Biophys. Acta. 1991; 1066: 70-76Crossref PubMed Scopus (42) Google Scholar, 11Harrison M.L. Isacsson C.C. Burg D.L. Geahlen R.L. Low P.S. J. Biol. Chem. 1994; 269: 955-959Abstract Full Text PDF PubMed Google Scholar) and recently has been shown to occur upon shrinkage of human erythrocytes (12Minetti G. Seppi C. Ciana A. Balduni C. Low P.S. Brovelli A. Biochem. J. 1998; 355: 305-311Crossref Scopus (40) Google Scholar). A number of tyrosine kinases have been detected in human erythrocytes, and of these, the kinase with the greatest ability to phosphorylate band 3 is p72syk (11Harrison M.L. Isacsson C.C. Burg D.L. Geahlen R.L. Low P.S. J. Biol. Chem. 1994; 269: 955-959Abstract Full Text PDF PubMed Google Scholar). In the present study we demonstrate that p72syk as well as p56lyn are activated by volume expansion of skate red blood cells. There is not a global stimulation of tyrosine kinases, because certain other tyrosine kinases investigated (pp60src and p59fyn) are not activated by volume expansion and do not readily phosphorylate band 3. Pervanadate treatment, which activates taurine efflux, also stimulates p72syk activity as well as its ability to tyrosine phosphorylate band 3. Thus, activation of p72syk by osmotic stress makes it an excellent candidate as a pivotal step in volume-stimulated taurine efflux through its phosphorylation of skate band 3. In addition, the level of band 3 phosphorylation may also be regulated by a protein-tyrosine phosphatase type 1B, which is closely associated with band 3. Little skates (Raja erinacea) were caught off Frenchman's Bay, ME or Woods Hole, MA and kept in running seawater. Blood was removed from a tail vessel into a heparinized syringe. Cells were pelleted (400 g for 2 min at room temperature), and the plasma and buffy coat were removed. Erythrocytes were resuspended in 5 volumes of isotonic (940 mosmol/liter) elasmobranch incubation medium (940 EIM)1 (composition: 300 mmol/liter NaCl, 5.2 mmol/liter KCl, 2.7 mmol/liter MgSO4, 5 mmol/liter CaCl2, 370 mmol/liter urea, 15 mmol/liter Tris, pH 7.4), washed twice, and resuspended at 50% hematocrit in 940 EIM. To volume-expand the cells, erythrocytes were diluted 1:10 into 460 EIM (NaCl was reduced to 100 mm and urea to 250 mm). At varying times, 1000 μl of incubation mixture (50 μl of cell equivalent) were removed, immediately pelleted for 10 s in a microcentrifuge, and the cell pellet was snap-frozen. When appropriate, cells were pretreated with pervanadate (prepared fresh for each experiment as described (13Musch M.W. Davis-Amaral E.M. Leibowitz K.L. Goldstein L. Am. J. Physiol. 1998; 275: R1677-R1686Google Scholar)) for 20 min and included during hypotonic exposure. The presence of various tyrosine kinases was determined using specific antibodies and immunoprecipitation. Anti-p72syk (polyclonal), anti-p59fyn (polyclonal), anti-p56/53lyn (polyclonal), anti-pp60src (monoclonal GD11), anti p56lck (polyclonal, carboxyl terminus) were purchased from Upstate Biotechnology (Lake Placid, NY) and prebound to protein A-Sepharose (Amersham Pharmacia Biotech). Cells were lysed in 9 volumes (450 μl) of Nonidet P-40 lysis (IP) buffer (25 mm HEPES, pH 7.4, 225 mmNaCl, 1% v/v Nonidet P-40, 5 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 2 mmbenzamidine with 10 μg/ml each leupeptin, aprotinin, and pepstatin A). Lysates were solubilized for 10 min and cleared by centrifugation. Supernatants were incubated with the antibodies at 4 °C for 120 min, and beads with immune complexes (antibodies and attached kinases) were washed two times with 25 mm HEPES, pH 7.4, 150 mm NaCl, and 0.1% v/v Nonidet P-40 with protease inhibitors as above and then once with assay buffer (50 mmHEPES, pH 7.4, 10 mm MnCl2, with protease inhibitors). The activity of the kinases attached to the beads was performed at 37 °C for 30 min in the assay buffer (50 μl) with 5 mm p-nitrophenylphosphate and 1 μm[γ-32P]ATP in the presence or absence of 2 μg of the cytoplasmic domain of band 3. Reactions were stopped by the addition of 25 μl of 3× SDS-PAGE stop solution and heated to 65 °C to elute the kinases from the beads (as well as the antibodies used to immunoprecipitate them). Beads were pelleted by centrifugation, and the samples were analyzed on 10% SDS-PAGE, transferred to a polyvinyl difluoride membrane (Immobilon, Millipore, Medford, MA), and alkali-treated before radiography. To determine whether 30 min was in the linear activity range for each kinase, assays were allowed to proceed for up to 120 min. For each kinase, the assays were linear for nearly 60 min, and therefore, the 30-min assay was selected (data not shown). Volume-activated taurine flux is bidirectional and may be measured in either efflux or uptake direction (2Goldstein L. Brill S.A. Am. J. Physiol. 1991; 260: R1014-R1020PubMed Google Scholar). Because it is more practical to measure the flux at early times in the uptake direction, we used taurine uptakes to determine the time course of volume-activated taurine flux. Uptakes were measured as follows. Briefly, erythrocytes were washed and resuspended in 940 EIM. Taurine uptake was initiated by adding cells either into 940 or 460 lithium EIM (with sodium salts replaced by lithium salts so that sodium-dependent taurine uptake did not contribute to the uptake) containing 0.4 μCi/ml of [3H]taurine. At varying times, samples were removed and immediately spun to pellet cells. Cells were washed three times with the appropriate EIM to remove labeled taurine in the extracellular spaces. Perchloric acid (7% final concentration) was added to the cells, and after being kept on ice for 15 min, precipitated material was pelleted in a microcentrifuge, and an aliquot of the supernatant was counted for radioactivity. When appropriate, cells were treated with the tyrosine kinase inhibitor piceatannol (from a 10 mm stock in Me2SO) for 30 min before taurine efflux measurements (2Goldstein L. Brill S.A. Am. J. Physiol. 1991; 260: R1014-R1020PubMed Google Scholar). Piceatannol was also included in the buffer used to remove extracellular radioactivity as well as in the flux buffer to ensure a constant level of the inhibitor from the beginning to the end of the flux measurement period. The activities of the tyrosine kinases were also measured in vivo by two different assays. First, as most of the tyrosine kinases require phosphorylation of tyrosine to become active, the kinases were immunoprecipitated after solubilization, run on SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed using the antiphosphotyrosine antibody 4G10. Kinase activities were also assessed by analyzing cells previously incubated with (32PO4) for 8 h in phosphate-free 940 EIM with 5 mm added glucose. The cells were then washed and diluted to 10% hematocrit in 940 or 460 EIM, 1-ml samples were removed at varying times, and the cells were pelleted and snap-frozen. The samples were thawed on ice in IP buffer, and kinases were immunoprecipitated. The 32P-labeled kinases were eluted from the antibodies with Laemmli stop solution, run on SDS-PAGE, and autoradiographed after the gels were dried. Cells were incubated with (32PO4) for 8 h as above and washed, and their volume was expanded for 5 min. Time courses were not performed because of the amount of material required for the two conditions isotonic and hypotonic. The entire band 3 or the cytoplasmic domain (obtained by mild trypsinization, 50 ng/ml ghosts) were immunoprecipitated as described previously (13Musch M.W. Davis-Amaral E.M. Leibowitz K.L. Goldstein L. Am. J. Physiol. 1998; 275: R1677-R1686Google Scholar), run on SDS-PAGE, and electroeluted from the gel as described by Hunkapillar et al. (14Hunkapillar M.W. Luja E. Ostander F. Hood L.E. Methods Enzymol. 1983; 91: 227-236Crossref PubMed Scopus (684) Google Scholar). The samples were dried, resuspended in water, and treated with 10 μg/ml trypsin at 30 °C for 30 min, and the peptides were separated by reverse phase HPLC using a Waters C18 column (0.8 × 25 cm). The peptides were eluted at 0.5 ml/min using a solvent system of buffer A (0.05% trifluoroacetic acid in water) with a 90-min linear gradient to solvent B (0.05% trifluoroacetic acid with 75% acetonitrile). Fractions (0.5 min) were counted for32P by Cerenkov counting, and fractions with the greatest difference between isotonic and hypotonic conditions were analyzed for peptide sequence. The samples were dried, dissolved in 25% aqueous acetonitrile, and applied to Sequelon AA disks and dried overnight. Ten μl of fresh 0.1 m MES, pH 5, 10% acetonitrile, and 10 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/ml were applied and allowed to react for 30 min. Sequencing used an Applied Biosystems 470 gas phase sequencer connected on line to a phenylthiohydantoin analyzer. Following Edman degradation, a portion of the resulting derivatized amino acids was collected and counted for32P. Band 3 was immunoprecipitated from cells under isotonic and hypotonic conditions by the method previously described (8Musch M.W. Leffingwell T.R. Goldstein L. Am. J. Physiol. 1994; 266: R65-R74PubMed Google Scholar). For detection of co-immunoprecipitated PTP1B, Western blots of the immunoprecipitated proteins were incubated with anti-PTP1B polyclonal antiserum. Blots were washed and then developed using an enhanced chemiluminescence system. Erythrocytes from numerous species have been demonstrated to have high levels of protein-tyrosine kinase activity, and recently a number of receptor and nonreceptor tyrosine kinases have been identified in red cells (9Tuy F.P.D. Henry J. Rosenfeld C. Kahn A. Nature. 1983; 305: 435-438Crossref PubMed Scopus (77) Google Scholar, 11Harrison M.L. Isacsson C.C. Burg D.L. Geahlen R.L. Low P.S. J. Biol. Chem. 1994; 269: 955-959Abstract Full Text PDF PubMed Google Scholar). We measured the activities of tyrosine kinases in extracts of skate erythrocytes. Four tyrosine kinases found in human erythrocytes (Fyn, Lyn, Src, and Syk) and one tyrosine kinase not found in erythrocytes (lck found primarily in T-lymphocytes) were measured, and their ability to phosphorylate band 3 was determined by immunocomplex kinase assay (Fig. 1). Although it is difficult to compare activities of one kinase to another because of potential differences in sensitivity to assay conditions, the most active tyrosine kinase under the conditions measured was p72syk. Smaller activities of pp60src, p56/53lyn, and p59fyn were found, and no activity of p56lck could be detected (not shown). When the cytoplasmic domain of band 3 was included in the reactions, all the kinases demonstrated some ability to tyrosine phosphorylate band 3 upon very long exposures of the autoradiographs, but most signal was detected from p72sykfollowed by p56/53lyn (at least 5–10 times less active at phosphorylating band 3 than p72syk). Because cell volume expansion is known to increase phosphorylation of band 3 in skate erythrocytes (8Musch M.W. Leffingwell T.R. Goldstein L. Am. J. Physiol. 1994; 266: R65-R74PubMed Google Scholar), the activities of three of the nonreceptor tyrosine kinases were measured under cell volume expansion conditions for 10 min. Only p72sykdemonstrated an increase in activity after volume expansion when assessed using autophosphorylation as the measure of activation (Fig.2). Utilizing this assay protocol, neither p56/53lyn nor pp60src increased in activity after hypotonic exposure. p72syk was rapidly activated (within 2 min) after hypotonic stress and can be most readily observed when the cytoplasmic domain of band 3 is included in the kinase reaction (Fig.3).Figure 3Time course of activation of p72sykin volume-expanded skate erythrocytes. Cells were incubated for the times indicated under isotonic (ISO) or hypotonic (HYPO) conditions and then frozen. Cells were thawed, and p72syk activity was measured by an immune complex assay using [32P]ATP in the absence (−cdb3) or presence (+cdb3) of the cytoplasmic domain of band 3. The autoradiographs shown are representative of those from three different experiments.View Large Image Figure ViewerDownload (PPT) Because the immune complex assay conditions may alter the activity of kinases, we determined whether certain kinases were activated in vivo by two additional methods. The first was to investigate the level of phosphotyrosine under stimulated conditions, and the second was to determine the level of 32P of the kinases from [32P]phosphate-labeled cells. The first has the disadvantage that it only analyzes phosphotyrosine and if serine or threonine is phosphorylated, this would not be shown. However, analysis for phosphotyrosine is also very sensitive. The second has the advantage that all three potential phosphorylated amino acids could be observed, but it is quite difficult to label erythrocytes well with32PO4, and the signals are often quite low. Using both of these latter techniques, we observed stimulation of Syk as well as Lyn under volume-expanded conditions (Fig.4), whereas pp60src did not demonstrate any activation. Because band 3 has been implicated as an osmolyte channel or channel regulator involved in volume-activated taurine transport (1Goldstein L. Davis-Amaral E.M. Musch M.W. Kidney Int. 1996; 49: 1690-1694Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 3Fievet B. Gabillat N. Borgese F. Motais R. EMBO J. 1995; 14: 5158-5169Crossref PubMed Scopus (97) Google Scholar), we compared the time courses of p56lyn and p72syk activation with that for volume-activated taurine transport. Fig.5 shows that the time courses for activation of the two processes were roughly similar. All were activated by 2 min, peaked at about 5 min, and fell after that. However, there is not an exact quantitative correlation in the three time courses, because Syk and Lyn activities peaked a little later and fell somewhat faster than the taurine flux. The time courses for Syk and Lyn activation are also quite similar to what we found previously for the volume-activated phosphorylation of band 3 in hypotonically stressed intact skate erythrocytes (8Musch M.W. Leffingwell T.R. Goldstein L. Am. J. Physiol. 1994; 266: R65-R74PubMed Google Scholar).Figure 5Effect of volume expansion on taurine flux, p72syk and p56lyn activities. Taurine uptake was measured under isotonic and hypotonic conditions in lithium-containing incubation medium to prevent sodium-dependent uptake from obscuring the signal through the volume-sensitive pathway. Kinase activities presented are kinase activities from 32P-labeled cells. Data shown are means ±S.E. for four experiments. ■, Syk; ▴, Lyn; ●, taurine uptake.View Large Image Figure ViewerDownload (PPT) To determine which kinase(s) is (are) required in the activation of taurine efflux, we used the stilbene piceatannol, which has been described as a selective inhibitor of Syk (15Peters J.D. Furlong M.T. Asai D.J. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1996; 271: 4755-4762Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The selectivity of piceatannol was confirmed as shown in Fig.6. We used the immune complex assay to determine the selectivity because of the strong signal in this assay, allowing better quantitation of the effect of piceatannol on the kinases. It is an assumption that this selectivity carries over into the intact cell. When hypotonic-stimulated taurine efflux was measured after piceatannol treatment, the 50% inhibitory concentration agreed well with that of inhibition of Syk, supporting an important role for this kinase in activation of the taurine transporter. We previously found that pervanadate stimulated taurine efflux in both intact skate erythrocytes and in inside-out membrane vesicles prepared from these erythrocytes (13Musch M.W. Davis-Amaral E.M. Leibowitz K.L. Goldstein L. Am. J. Physiol. 1998; 275: R1677-R1686Google Scholar). Stimulation was observed in both control and volume-expanded cells. Because pervanadate is known to be an inhibitor of phosphotyrosine phosphatase (16Heffetz D. Buskin I. Dror R. Zick Y. J. Biol. Chem. 1990; 265: 2896-2902Abstract Full Text PDF PubMed Google Scholar, 17Zick Y. Sagi-Eiserberg R. Biochemistry. 1990; 29: 10240-10245Crossref PubMed Scopus (92) Google Scholar), we tested the effect of the inhibitor on the autophosphorylation of p72syk and the phosphorylation of the cytoplasmic domain of band 3 (cdb3) by p72syk. As seen in Fig. 7, pervanadate enhanced the level of protein tyrosine phosphorylation produced by p72syk both in the presence and absence of cdb3 at all time points examined under both isotonic and hypotonic conditions. The enhancement of the level of protein tyrosine phosphorylation by pervanadate is quite similar to the stimulation of taurine efflux by this agent previously observed in intact erythrocytes and membrane vesicles from skate red blood cells (13Musch M.W. Davis-Amaral E.M. Leibowitz K.L. Goldstein L. Am. J. Physiol. 1998; 275: R1677-R1686Google Scholar). In preliminary experiments, both the entire immunoprecipitated32PO4-labeled band 3 molecule and32PO4-labeled cdb3 (prepared by mild tryp- sinization of band 3) were analyzed. The major32PO4-labeled HPLC peaks (which increased during hypotonic stimulation) were always observed in both analyses. Because the separations with the cdb3 provided less background and better resolution of the HPLC peaks, these were selected for amino acid analysis, and a representative tracing is presented in Fig.8 showing32PO4-labeling as well as absorbance at 215 nm. The two largest peaks were analyzed by Edman degradation. For the peak that eluted later (peak B on Fig. 8), 16 amino acids were determined. The amino acid sequence determined was GDAQAYVELNELMGNS, which is the same as for cloned trout band 3 (18Hubner S. Michel F. Rudloff V. Appelhans H. Biochem. J. 1992; 285: 17-23Crossref PubMed Scopus (54) Google Scholar) and corresponds to residues 75–90 (18Hubner S. Michel F. Rudloff V. Appelhans H. Biochem. J. 1992; 285: 17-23Crossref PubMed Scopus (54) Google Scholar). 32P analysis of the eluate from the Edman analysis is presented in Fig. 9, which demonstrates that the Tyr (Y) at position 6 was found to contain 32P, which increased significantly during volume expansion. The serine at position 16 contained 32P, which did not change during volume expansion. For peak A, we were only able to obtain 5 amino acids from the amino terminus of this tryptic peptide with the sequence HEEDS: residues 44–48 of the trout band 3 (18Hubner S. Michel F. Rudloff V. Appelhans H. Biochem. J. 1992; 285: 17-23Crossref PubMed Scopus (54) Google Scholar). If the tryptic fragment we have does correspond to the fragment in the trout, a tyrosine is anticipated in another 16 amino acids. The tyrosine we identified as well as the one we believe may be there in peak A are both in YXXL sequences. This sequence is often required for the activation of a number of tyrosine kinases as is part of an ITAM motif (18Hubner S. Michel F. Rudloff V. Appelhans H. Biochem. J. 1992; 285: 17-23Crossref PubMed Scopus (54) Google Scholar).Figure 932 P content of eluate of Edman analysis of peak B of Fig. 8. A portion of the post-column-derivatized material was collected, and the 32P content was counted. Data presented is counts/5 min and is the mean ±S.E. for 3 analyses. The difference observed at the tyrosine (Y) at position 6 is significantly different, p < 0.05.View Large Image Figure ViewerDownload (PPT) The enhancement of protein tyrosine phosphorylation by p72syk in the presence of pervanadate suggests that the level of phosphorylation of proteins phosphorylated by p72sykis regulated by protein-tyrosine phosphatases. Because erythrocytes are known to be a rich source of these phosphatases (20Clari G. Brunati A.M. Moret V. Biochem. Biophys. Res. Commun. 1987; 142: 587-594Crossref PubMed Scopus (26) Google Scholar, 21Zipser Y. Kosower N.S. Biochem. J. 1996; 314: 881-887Crossref PubMed Scopus (53) Google Scholar), we assayed extracts of skate erythrocytes for protein-tyrosine phosphatases. Among the protein-tyrosine phosphatases, a type 1B has been reported to be closely associated with band 3 in human red blood cells (20Clari G. Brunati A.M. Moret V. Biochem. Biophys. Res. Commun. 1987; 142: 587-594Crossref PubMed Scopus (26) Google Scholar). To determine whether a similar association occurs in skate red cells, skate band 3 was immunoprecipitated, and protein-tyrosine phosphatases were detected by probing Western blots of co-immunoprecipitated proteins. As shown in Fig. 10, a protein reacting with anti-1B tyrosine phosphatase and consistent with the molecular weight of a PTP1B is associated with band 3. The distribution of this protein does not vary with hypotonic exposure. When three separate experiments were analyzed by densitometry, no significant difference at any time point was observed. When we tried to probe the same samples for p72syk, we were unable to detect p72syk in the immunoprecipitates. Thus although p72sykmay rapidly phosphorylate band 3, it does not form a complex of sufficient affinity to withstand the immunoprecipitation conditions. Although band 3 is known to be a prime target for tyrosine phosphorylation by p72syk, we now demonstrate the tyrosine phosphorylation of band 3 under physiologically relevant conditions, namely volume expansion. In the present studies, we have presented data showing the activation of p72syk during hypotonic expansion of skate red blood cells. Other nonreceptor tyrosine kinases, which are present in the erythrocytes, including pp60src and Fyn, are not activated. The activation of the kinase p56lyn was not observed using the immune complex assay but was seen when assessed by analyzing activation in the intact cell. It may be that the artificial conditions used in the immune complex assay may alter the activity of the kinase and/or remove regulatory proteins. The results from the intact cell demonstrate Lyn activation and could be considered the more appropriate measure of activity. The pivotal role of Syk was demonstrated using the selective inhibitor piceatannol, which preferentially inhibits Syk more than Lyn and inhibits pp60src only poorly. One cannot state that Lyn activation is not involved in the stimulation of taurine efflux, but Syk activation is a required step in this process. Activation of Lyn and Syk may both be required to stimulate taurine efflux maximally. Inclusion of the phosphatase inhibitor pervanadate, which has the physiologic effect in skate erythrocytes of potentiating a volume-induced taurine efflux during the regulatory volume decrease (13Musch M.W. Davis-Amaral E.M. Leibowitz K.L. Goldstein L. Am. J. Physiol. 1998; 275: R1677-R1686Google Scholar), also increases the tyrosine phosphorylation of band 3 by p72syk. Because many of the tyrosine kinases are activated by tyrosine phosphorylation, inhibition of the phosphatases that inactivate these kinases may leave them in an active state. The effect of pervanadate was only pursued for Syk, as the latter appears to be required for the stimulation of taurine efflux. Lyn activity may be stimulated in pervanadate-treated cells, but we did not test this possibility. The tyrosine phosphorylation of band 3 occurs, at least in part and likely predominantly, on the cdb3. We have identified one tyrosine in a YXXL sequence in skate band 3 that is phosphorylated under volume-expanded conditions, and a sequence from another tryptic peptide indicates that this may also have a phosphotyrosine in a YXXL sequence. This belief is based on the amino acid sequence of trout band 3, which has been cloned. The two peptides we have analyzed are consistent with tryptic predictions from this sequence. It is interesting that in the trout sequence, these two YXXL motifs, are separated by 13 amino acids. Certain tyrosine kinases require phosphorylation of one or both Y in two YXXL sequences 4–7 amino acids apart. (19Weiss A. Cell. 1993; 73: 209-212Abstract Full Text PDF PubMed Scopus (476) Google Scholar). This is termed an ITAM-motif, and it is possible that these two YXXL are essential in the activation of volume-expanded activation of band 3. The phosphorylation may act to bring kinases or other associated proteins to band 3, and this may regulate band 3 interaction with additional regulatory proteins including ankyrin or band 4.1. Precise kinetic comparisons between the kinase activations and taurine flux should not be made. There are technical limitations on how rapidly taurine flux may be measured so that a very rapid phase, within 2 min, cannot be measured. Thus one cannot state that there are activation steps between kinase activation and taurine flux simply based on the data presented. Also, quantitative comparisons should not be made because one of the major purposes of kinases is amplification of a signal. Therefore, a small activation of Syk may lead to a large activation of taurine flux. One may be able to stimulate a greater percentage of the cell Syk without further additional effect on taurine flux; however, this is only speculative. p72syk may play an important role in the volume-activated taurine efflux of the skate erythrocyte. Skate band 3, which is a substrate of p72syk, is thought to act as a channel or channel activator for the volume-activated taurine efflux in fish erythrocytes (1Goldstein L. Davis-Amaral E.M. Musch M.W. Kidney Int. 1996; 49: 1690-1694Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 3Fievet B. Gabillat N. Borgese F. Motais R. EMBO J. 1995; 14: 5158-5169Crossref PubMed Scopus (97) Google Scholar). A very recent study using oocyte expression of trout band 3 has further reinforced its role in volume-activated solute fluxes (4Fievet B. Perset F. Gabillat N. Guizouran H. Borgese F. Ripoche P. Motais R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10996-11001Crossref PubMed Scopus (31) Google Scholar). Not only may band 3 mediate Cl− exchange when expressed in oocytes, but when trout (but not mouse) band 3 is expressed, a channel is introduced that transports taurine, sorbitol, and urea. This transport pathway has very similar pharmacologic inhibitory properties to those we have previously described in skate red blood cells. This data strongly supports a role of band 3 in the transport of organic osmolytes under volume-expanded conditions. Band 3 has been shown to be rapidly phosphorylated during volume expansion of the skate erythrocyte. In the present study we have shown that the time courses for activation of the taurine flux measured in skate red blood cells and activation of p72syk activity measured in red blood cell extracts using the cytoplasmic domain of band 3 as a substrate are quite similar. Furthermore, pervanadate, which increases the level of phosphorylation of cdb3 brought about by volume-activated p72syk activity, also enhances the volume-activated efflux of taurine in both intact skate erythrocytes and membrane vesicles (13Musch M.W. Davis-Amaral E.M. Leibowitz K.L. Goldstein L. Am. J. Physiol. 1998; 275: R1677-R1686Google Scholar). This evidence raises the possibility that activation of p72sykmay be a crucial step in the volume recovery process. Nonreceptor tyrosine kinases are recognized as a major signal transduction mechanism used in a vast array of cells. Specifically with regard to Syk, this kinase has been implicated in such events as B cell antigen receptor signaling (22Richards J.D. Gold M.R. Hourihane S.L. DeFranco A.L. Matsuuchi L. J. Biol. Chem. 1996; 271: 6458-6466Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), neutrophil signaling through adhesion complexes (23Yan S.R. Huang M. Berton G. J. Immunol. 1997; 158: 1902-1910PubMed Google Scholar), apoptosis stimulated by osmotic stress or ultraviolet radiation in B cells (24Qin S. Minami Y. Kurosaki T. Yamamura H. J. Biol. Chem. 1997; 272: 17994-17999Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), integrin cross-linking in myeloid cells (25Gotoh A. Takahira H. Geahlen R.L. Broxmeyer H.E. Cell Growth Differ. 1997; 8: 721-729PubMed Google Scholar), and thrombin stimulation of platelet aggregation (26Wang X. Yanagi S. Yang C. Inatome R. Yamamura H. J. Biochem. (Tokyo). 1997; 121: 325-330Crossref PubMed Scopus (20) Google Scholar). In the case of the fish erythrocytes, an important step will be to define the signal between cell swelling and Syk activation. In addition to the SH2 or SH3 domains, other domains of Syk are critical as Latour et al. (27Latour S. Chow L.M.L. Veillette A. J. Biol. Chem. 1996; 271: 22782-22790Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) elucidate while making chimeras of Syk and ZAP-70. The SH2 as well as portions of the carboxyl-terminal domains both play a role in the regulation of kinase activity. Kinases may not only be important for their ability to phosphorylate, but they have been speculated to serve structural roles in addition. In the B-lymphocyte cell line DT-40, p72syk plays a central role in cell activation (28Wan Y. Bence K. Hata A. Kurosaki T. Veillette A. Huang X.Y. J. Biol. Chem. 1997; 272: 17209-17215Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), potentially through its interactions with Grb2/SOS (which may then activate the mitogen-activate protein kinase cascade). Therefore, p72syk, potentially through its SH2 domain and other domains, may bring together the requisite proteins for a functional response to occur and be part of a complex of cytosolic and membrane proteins involved in activating the efflux of taurine." @default.
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• W1511917665 title "Volume Expansion Stimulates p72 and p56 in Skate Erythrocytes" @default.
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