1 Department of Biology, Syracuse University, Syracuse, New York 13244; and 2 Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
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ABSTRACT |
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Indirect evidence has suggested that K-Cl cotransport in human and sheep erythrocytes is activated physiologically by a serine-threonine phosphatase. It is activated experimentally by H2O2 and by staurosporine, a kinase inhibitor. Activation by H2O2 and staurosporine is inhibited by serine-threonine phosphatase inhibitors, suggesting that the activators stimulate the phosphatase. The present study shows that sheep and human erythrocytes contain membrane-associated as well as cytosolic serine-threonine phosphatases, assayed from the dephosphorylation of 32P-labeled glycogen phosphorylase. In cells from both species, the relatively low sensitivity of the membrane enzyme to okadaic acid suggests it is type 1 protein phosphatase. The cytosolic phosphatase was much more sensitive to okadaic acid. Membrane-associated phosphatase was stimulated by both H2O2 and staurosporine. The results support earlier conclusions that the membrane-associated type 1 phosphatase identified here is regulated by phosphorylation and oxidation. The results are consistent with the phosphatase, or a portion of it, being responsible for activating K-Cl cotransport.
potassium-chloride cotransport; volume regulation; phosphorylation; oxidation
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INTRODUCTION |
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ACTIVATION OF THE MEMBRANE transporter K-Cl cotransport can lead to regulatory cell volume decrease by promoting water loss from cells. K-Cl cotransport is present in many cell types, including red blood cells of most mammals. In sheep and human red blood cells, K-Cl cotransport activity is very low at physiological volume but is activated by cell swelling (Ref. 7 and see Ref. 26 for a review). K-Cl cotransport can also be activated at physiological volume by various oxidizing agents (4, 26) and by the protein kinase inhibitor staurosporine (3). The signal transduction pathway that controls K-Cl cotransport following swelling or other activating treatments is not well understood. Okadaic acid and calyculin A, inhibitors of serine-threonine protein phosphatases types 1 and 2A (PP-1 and PP-2A) (20), inhibit swelling-stimulated, oxidant-stimulated, and staurosporine-stimulated K-Cl cotransport (3, 4, 22, 23, 33), suggesting that activation of cotransport is promoted by a dephosphorylation reaction. Activation by H2O2 and staurosporine appears to result from stimulation, direct or indirect, of a phosphatase (3, 4). There is good evidence indicating that the primary event following swelling is inhibition of the kinase, which allows increased dephosphorylation by a phosphatase (21). The evidence also indicates that the phosphatase is insensitive to cell volume changes (21).
The present work was undertaken in an attempt to demonstrate directly in red blood cells a phosphatase subject to inhibition by okadaic acid and stimulation by H2O2 and staurosporine. We found membrane-associated and cytosolic serine-threonine phosphatases in human and sheep erythrocytes and investigated several of their properties. Both cytosolic activity and membrane-associated phosphatase activity were inhibited by okadaic acid. The phosphatase associated with the membranes is inhibited by relatively high okadaic acid concentrations, suggesting it is PP-1 (13). We show that this phosphatase can be stimulated by pretreatment of intact cells with H2O2 or staurosporine, whereas the cytosolic phosphatase was unaffected. The results are consistent with the view that this membrane-associated phosphatase is the one that activates K-Cl cotransport.
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METHODS |
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Cells. Red blood cells from LK sheep were obtained as reported before (4) from sheep maintained at the Vinzant Family Farms (Borodino, NY). Human red blood cells were obtained at the University of Chile from three healthy adult volunteers after informed consent. Cells were washed by centrifugation in solutions described previously (3). For treatment of intact cells, cells were incubated for 30 min at 37°C, 10% hematocrit, with the desired agents. Control cells were incubated in parallel.
Preparation and treatment of membranes.
Red blood cells were lysed in 15 volumes of lysis buffer containing 10 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.4), 0.1% 2-mercaptoethanol, and 1 µM phenylmethylsulfonyl
fluoride. For human cells, lysis buffer also contained 10 µg/ml of
leupeptin, and, for sheep cells, the buffer also contained 25 µg/ml
each of leupeptin and aprotinin. Cell lysates were centrifuged (30,000 g for 20 min), and the membrane pellet
was washed by centrifugation in lysis buffer. The first supernatant was
used as the cytosolic fraction. The membrane and cytosolic fractions
were stored frozen (20°C) in lysis buffer until use, usually
within 1 wk.
Phosphatase activity.
Phosphatase activity was determined using
32P-labeled glycogen phosphorylase
as substrate as previously described (11). Briefly, 32P-labeled glycogen phosphorylase
a was prepared by reacting
[-32P]ATP with
purified glycogen phosphorylase b in the presence of purified
phosphorylase kinase. The reaction was allowed to go to completion, and
the excess [32P]ATP
was removed by precipitation of the phosphorylase with ammonium sulfate
and washing of the pellet. The ammonium sulfate was removed, and the
labeled phosphorylase a was stored until use at 4°C at a
concentration of 3 mg/ml. The phosphatase assay reaction media contained 16.7 mM Tris · HCl, 13.3 mM imidazole
(final pH of 7.4), 5 mM caffeine, 0.1 mM EDTA, 0.4% 2-mercaptoethanol,
0.67 mg/ml bovine serum albumin, and 1 mg/ml of the
32P-labeled substrate. Protein
concentrations in the red cell fractions to be assayed were 50-100
µg/ml for the membranes and 100-250 for the cytosol. Triton
X-100 was added to all samples before the phosphatase assay was
performed (final concentration, 0.5% vol/vol). The reaction mixture
was incubated for 15 min at 30°C. The fraction of substrate
dephosphorylated never exceeded 25%, and the reaction was linear with
time. Phosphatase activity is expressed as units per milligram protein,
in which 1 unit (U) equals 1 nmol of phosphate released per minute.
Concentrations giving half-maximal inhibition
(IC50) by okadaic acid were
estimated graphically. Protein concentrations were determined using the Lowry assay (28). The phosphatase assay measures serine-threonine phosphatase activity because the substrate used is specific for serine-threonine phosphatases.
Statistics. The results were analyzed for differences between means using Student's t-test when the data presented a single normal distribution or with a signed rank test when standardized skewness and kurtosis values were not within the range expected for data from a normal distribution (Statgraphics).
Materials.
Okadaic acid was obtained from LC Laboratories or from Sigma Chemical
and dissolved in either ethanol or dimethyl sulfoxide at 100 µM.
Calyculin was obtained from LC Laboratories and dissolved in ethanol at
100 µM. Both stock solutions were stored at 20°C. [
-32P]ATP was
obtained from Dupont New England Nuclear. Purified glycogen phosphorylase b and phosphorylase kinase were obtained from GIBCO BRL
(Gaithersburg, MD). All other chemicals were obtained from Sigma.
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RESULTS |
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Erythrocyte cytosolic and membrane-associated serine-threonine phosphatase activity. Human and sheep red cell cytosols and membranes contain significant phosphorylase phosphatase activity (Fig. 1, Table 1). In human and sheep red blood cells, the membrane-associated activity per milligram protein exceeds cytosolic activity. About 98% of the cell protein is in the cytosol, and the membrane fraction contains ~5% of the total phosphatase activity. Cytosolic phosphatase activity in sheep red blood cells is higher than in human red blood cells (0.8 vs. 0.3 U/mg protein). Membrane-associated phosphatase is also higher in sheep than in human red blood cells (3.1 vs. 0.9 U/mg protein). Phosphatase activity in membrane fractions obtained from sheep red blood cells ranged from 0.2 to 10.5 U/mg protein. We cannot explain the large range except to say that the few very high and low values (>8 and <0.5 U/mg) were all obtained early in the project. There was also a large range of values for membrane-associated phosphatase activities from human red blood cells (0.3 to 3.6 U/mg protein). After separation of the cytosolic and membrane-associated components by centrifugation at 30,000 g, 80-100% of the total lysate phosphatase activity was recovered.
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Stimulation of membrane-associated phosphatase activity by H2O2. Two protocols were used to study the effect of H2O2: pretreatment of intact cells and treatment of isolated membranes. Because K-Cl cotransport activity is maximally stimulated in intact sheep red blood cells by pretreatment with 5 mM H2O2 (4), phosphatase activity was determined in preparations of membranes from sheep red blood cells treated in this manner. Phosphatase activities in membrane fractions obtained from H2O2-treated cells were stimulated by 26 ± 9% compared with preparations from untreated cells (Fig. 3), and the stimulation was statistically significant (P = 0.014, paired t-test, 2-tailed, n = 19). In contrast, the phosphatase activities in cytosolic fractions were unaffected by pretreatment of the cells with H2O2. In membrane fractions from human red blood cells treated with 5 mM H2O2, there was also a stimulation of membrane-associated phosphatase activity (results not shown). The stimulation was less than in sheep red cell membranes, 8 ± 2%, but was statistically significant (P = 0.006, n = 13, signed rank test).
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Stimulation by H2O2 of phosphatase activity in membranes isolated from untreated cells. We also tested if direct treatment of isolated membranes with H2O2 could stimulate membrane-associated phosphatase activity. Isolated red cell membrane preparations do not contain catalase or glutathione peroxidase, two cytosolic enzymes that scavenge H2O2 (8). Therefore, much lower concentrations of H2O2 were used in these experiments than with intact cells. H2O2 treatment (50-100 µM) did stimulate phosphatase activity in the isolated membrane suspension of sheep cells (Fig. 4). The stimulation, ~15%, was smaller than that observed after treatment of intact cells (26%) but was statistically significant (P = 0.03, paired t-test of the slope from 0 to 100 µM H2O2, n = 4). Similar results were obtained with human red cell membranes treated with H2O2, a stimulation of ~12% (Fig. 4), also statistically significant (P = 0.04 for the slope from 0 to 200 µM H2O2, n = 2). In contrast to preparations from sheep cells, the direct treatment of human red cell membranes with H2O2 gave a larger stimulation of membrane-associated phosphatase (12%) than did treatment of intact cells (8%).
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Stimulation of membrane-associated phosphatase activity by staurosporine. Stimulation of sheep red cell K-Cl cotransport activity by 5 µM staurosporine is abolished by subsequent treatment of the cells with calyculin, suggesting that staurosporine may stimulate a serine-threonine phosphatase involved in activation of K-Cl cotransport (4). Therefore, we examined phosphatase activity of cytosol and membrane fractions from cells pretreated with 5 µM staurosporine. In preparations from sheep red blood cells, staurosporine pretreatment stimulated phosphatase activity in the membrane fraction but not in the cytosol (Fig. 5), just like the effect of H2O2 pretreatment of sheep cells. The stimulation of membrane phosphatase by staurosporine was 41 ± 8%, which was statistically significant (P = 0.002, paired t-test, n = 13). Also shown in Fig. 5 is the effect of pretreatment of human red blood cells with 5 µM staurosporine. Phosphatase activity in membrane from these cells was enhanced by 16 ± 5%, also statistically significant (P = 0.03, paired t-test, n = 14).
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Inhibition of H2O2- and staurosporine-stimulated phosphatase by okadaic acid. We determined the effects of varying concentrations of okadaic acid on the phosphatase activity of membranes from sheep red blood cells pretreated with H2O2 or staurosporine. The results are shown in Fig. 6 along with phosphatase activity of membranes from untreated cells. The IC50 for inhibition of the phosphatase in control membranes was ~30 nM, in the range of the IC50 in Fig. 1B. IC50 values were similar or slightly lower for the activity stimulated by H2O2 and staurosporine than that for membranes from untreated cells. Therefore, these agents stimulate a phosphatase activity that is inhibited by okadaic acid with a sensitivity similar or identical to that for untreated preparations.
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DISCUSSION |
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We found serine-threonine protein phosphatase activity in human and sheep red blood cells, both in the cytosol and associated with the membranes. The conclusion that the phosphatases are serine-threonine phosphatases is based on the substrate used in the assays and on their sensitivity to okadaic acid (Fig. 1, Table 1); the membrane-associated activity from sheep red blood cells is also inhibited by calyculin A (Fig. 2). These two inhibitors are specific for the serine-threonine phosphatases PP-1 and PP-2A. On the basis of their very different sensitivities to okadaic acid, we conclude that the cytosolic phosphatase is mainly PP-2A and the membrane-associated phosphatase is mainly PP-1. The IC50 values for the membrane-associated phosphatase in cells from the two species were 27 and 16 nM (Table 1), in the range of IC50 values for inhibition of PP-1 from various sources by okadaic acid (12-75 nM; Refs. 10 and 12). The comparison of sensitivities to calyculin A and okadaic acid for the membrane-associated phosphatase from sheep red blood cells (Fig. 2) was consistent with at least a large fraction of the membrane-associated activity being PP-1, since this phosphatase activity was considerably less sensitive to okadaic acid than to calyculin (10).
The IC50 values for cytosolic phosphatase in cells from the two species were 0.23 and 0.03 nM, in or near the range of IC50 for inhibition of PP-2A by okadaic acid (0.1-2 nM; Refs. 13 and 20). The locations in the cells of the two types of phosphatases are consistent with earlier reports: PP-1 is generally bound to particles or membranes, whereas PP-2A is usually cytosolic (29).
There have been previous reports of at least two distinct serine-threonine phosphatases in whole red cell lysates (24, 35). Using a variety of substrates, Kiener et al. (24) isolated a type 1 phosphatase from human red blood cells and Usui et al. (35) described a type 2A phosphatase, also in human red blood cells. Neither of these reports included studies of isolated red cell membranes or experiments using okadaic acid. Studies using phosphorylated casein as a substrate did not detect serine-threonine phosphatases associated with human red cell membranes (6). The present report is the first demonstration of serine-threonine phosphatase associated with red cell membranes.
The results of this study show that H2O2 and staurosporine, agents that stimulate K-Cl cotransport in sheep red blood cells (3, 4), also stimulate membrane-associated serine-threonine phosphatase in red blood cells from humans and sheep. H2O2 stimulates membrane-associated phosphatase activity when intact cells are pretreated with 5 mM H2O2 (Fig. 3) or isolated membranes are treated with 50 µM H2O2 (Fig. 4). In sheep red cell membranes, phosphatase activity stimulated by H2O2 is almost entirely inhibited by okadaic acid (Fig. 6). The IC50 for inhibition of the H2O2-stimulated phosphatase activities, 10-15 nM (Fig. 6), is in the range of IC50 values for inhibition of PP-1 by okadaic acid (10, 12).
The mechanism by which the mild oxidant H2O2 stimulates phosphorylase phosphatase activity is unknown. Stimulation can occur in the absence of cytosolic components (Fig. 4); therefore, a direct effect of H2O2 on the phosphatase is likely. The activities of the catalytic subunits of types 1 and 2A phosphatases are regulated by differential association with regulatory and targeting subunits (5, 29). A possible mechanism for stimulation by H2O2 of the phosphatase is oxidation of sulfhydryl groups involved in maintaining the structure of the holoenzyme. Oxidation could lead to the release of inhibitory subunits known to be associated with phosphatases (29). For a recent discussion of the mechanism of regulation of serine-threonine phosphatases, see Barford (1).
Serine-threonine phosphatase activity of the membrane fraction was also stimulated in cells pretreated with staurosporine (Fig. 5). In contrast to the result with H2O2, incubation of isolated membranes with staurosporine did not result in stimulation of phosphatase activity. Because pretreatment of intact cells is necessary for the stimulation by staurosporine, the effect is indirect and may be exerted on a cytosolic agent regulating the phosphatase. The results support an earlier conclusion that staurosporine stimulates phosphatase activity indirectly by inhibition of a kinase that in turn inhibits the phosphatase (3). The addition of diluted cytosol to isolated membranes did not reconstitute phosphatase stimulation by staurosporine, suggesting that the kinase or other target of staurosporine is too dilute or that the response requires cellular integrity. Similar to the phosphatase activity stimulated by H2O2, the staurosporine-stimulated activity is inhibited by okadaic acid with an IC50 consistent with the stimulated phosphatase being PP-1 (Fig. 6). The stimulation of a membrane-associated phosphatase activity when intact cells are treated with staurosporine indicates the physiological relevance of a staurosporine-sensitive kinase in the regulation of a serine-threonine phosphatase. There are other recent reports on regulation of both PP-1 and PP-2A by phosphorylation in intact cells (2, 9, 17).
In a recent related study, evidence was presented that suggested that the Src family tyrosine kinases Fgr and Hck inhibit the phosphatase that activates K-Cl cotransport in mouse red blood cells (15). These observations are consistent with our proposed mechanism for the stimulation of cotransport by staurosporine: inhibition by staurosporine of a tyrosine kinase that inhibits the phosphatase (3).
The parallels between stimulation by H2O2 and staurosporine of calyculin-sensitive K-Cl cotransport (3, 4) and of membrane-associated phosphatase activity (this study) support the view that the membrane-associated phosphatase studied in this work is the one that regulates K-Cl cotransport. There were earlier proposals that the phosphatase involved in regulation of K-Cl cotransport is a membrane-associated enzyme (4). The sensitivity of K-Cl cotransport to okadaic acid suggests that it is PP-1 (33), as does the sensitivity of the membrane-associated enzyme to okadaic acid (present study). Other evidence supporting this conclusion comes from a study in which PP-1 incorporated into resealed ghosts made from calyculin-treated red blood cells reactivated K-Cl cotransport (25).
The stimulation of cotransport by both H2O2 and staurosporine was severalfold, whereas the stimulation of membrane-associated phosphatase by these agents was 25% or less. The explanation for this is not clear, and there are a number of possibilities. The simplest explanation is that there is more than one phosphatase associated with the membrane, as suggested by the okadaic sensitivity curve, and one of these phosphatases is involved in regulation of K-Cl cotransport and is activated by H2O2 and staurosporine. On the other hand, it is possible that all of the membrane-associated phosphatase dephosphorylates K-Cl cotransporters and some other explanation accounts for the discrepancy noted.
Several enzymes produce H2O2 as a normal by-product of their activity (14), stressing the importance of oxidative processes in cellular functions. Moreover, evidence from plant and animal cells suggests that H2O2 may act as a second messenger (19, 34). For example, in mouse fibroblasts, H2O2 rapidly induces the binding of iron regulatory protein to iron-responsive elements. This induction is blocked by okadaic acid, suggesting the presence of a protein phosphatase in a transduction pathway induced by oxidative stress (31). Furthermore, recent data suggest a role for reactive oxygen species in apoptosis (16, 30, 36) and in cellular aging (27, 36). Our data on stimulation of K-Cl cotransport by treatment with H2O2, which leads to cell shrinkage, is consistent with reports that oxidizing agents can cause red cell dehydration (18). Because aging red blood cells lose water (32), oxidants may participate in cell aging and initiate apoptosis in cells in general by stimulating a membrane-associated phosphatase that in turn activates K-Cl cotransport and the associated water loss.
In summary, this study demonstrates a membrane-associated serine-threonine phosphatase, largely PP-1, in sheep and human red blood cells. This phosphatase is stimulated by H2O2 and staurosporine, two agents that activate K-Cl cotransport in sheep red blood cells. Therefore, the membrane-associated phosphatase activity, or a portion of it, may be the phosphatase that regulates cotransport.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33640, Fondecyt 1960879 and 1940339 from Conicyt, Chile, and Fundaciùn Andes.
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FOOTNOTES |
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Address for reprint requests: I. Bize, Laboratory Medicine, The Children's Hospital, 300 Longwood Ave., Bader 760, Boston, MA 02115.
Received 16 June 1997; accepted in final form 31 October 1997.
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