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INTRODUCTION |
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
-amino acid taurine, which can accumulate from 10-100 mM concentration in many cells (1). In erythrocytes of a
number of species, the volume-stimulated taurine efflux appears to
occur by a transport pathway that involves band 3 (2, 3). 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
(4). 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 (5) and skate (6)
erythrocytes. However, in skate erythrocytes under volume-expanded
conditions band 3 forms a tetramer (6). 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 (7). 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 (8). 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. 9-11) and recently has been shown to occur upon
shrinkage of human erythrocytes (12). 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 (11). 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.
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MATERIALS AND METHODS |
Isolation of Cells--
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 (13)) for 20 min and included during hypotonic exposure.
Detection of Tyrosine Kinases--
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 mM
NaCl, 1% v/v Nonidet P-40, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 mM
benzamidine 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 mM
HEPES, 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 [
-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).
Taurine Flux--
Volume-activated taurine flux is bidirectional
and may be measured in either efflux or uptake direction (2). 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 (2). 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.
Assessment of in Vivo Kinase Activity--
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.
Identification of Phosphotyrosines of Band 3--
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 (13), run on SDS-PAGE, and
electroeluted from the gel as described by Hunkapillar et al. (14). 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 for
32P 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 for
32P.
Detection of PTP1B--
Band 3 was immunoprecipitated from cells
under isotonic and hypotonic conditions by the method previously
described (8). 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.
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RESULTS |
Detection of Tyrosine Kinases and Abilities to Phosphorylate Band
3--
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 (9, 11). 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 p72syk
followed by p56/53lyn (at least 5-10 times less active at
phosphorylating band 3 than p72syk).

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Fig. 1.
Identification of nonreceptor tyrosine
kinases in skate erythrocytes. Fifty µl of packed cells were
lysed in Nonidet P-40 buffer, and kinases were immunoprecipitated using
specific antisera bound to protein A-Sepharose. Kinases were detected
through activation of autocatalytic activity by including
[ -32P]ATP in the reaction. Samples are analyzed on
10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane,
alkali-treated, and exposed to film. The image shown is an overnight
exposure representative of three experiments. + indicates
inclusion or omission of 2 µg of the purified cytoplasmic domain of
band 3 in the immune complex assay.
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Volume Expansion, p72syk Activity, and Taurine
Transport--
Because cell volume expansion is known to increase
phosphorylation of band 3 in skate erythrocytes (8), the activities of
three of the nonreceptor tyrosine kinases were measured under cell
volume expansion conditions for 10 min. Only p72syk
demonstrated 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).

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Fig. 2.
Effect of volume expansion on activity of
p72syk, pp60src, and p56/53lyn. Samples
were prepared from cells incubated under isotonic (ISO, 940 mosmol/liter) or hypotonic (HYPO, 460 mosmol/liter)
conditions for the times indicated. Kinase activity was measured as
autocatalytic activity only in this series of experiments.
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Fig. 3.
Time course of activation of p72syk
in 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.
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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 with
32PO4, 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 (1, 3), 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 (8).

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Fig. 4.
Time course of activation of p72syk,
p56lyn, and pp60src in volume-expanded erythrocytes as
assessed by immunoprecipitation from cells labeled for 8 h with
32PO4 or antiphosphotyrosine content by
Western blotting after immunoprecipitation.
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Fig. 5.
Effect 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.
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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 (15). 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.

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Fig. 6.
Effect of piceatannol on taurine efflux and
kinase activities. Taurine efflux was measured in different cells
from those used for inhibition of kinases activities. Kinase activities
were measured using the immune complex assay procedure and inclusion of
varying concentrations of piceatannol solely in the kinase assay.
Values shown are means ±S.E. for n = 3 for all data.
, p72syk; , flux inhibition; , p56lyn; ,
pp60src.
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Pervanadate and p72syk Activity--
We previously found
that pervanadate stimulated taurine efflux in both intact skate
erythrocytes and in inside-out membrane vesicles prepared from these
erythrocytes (13). Stimulation was observed in both control and
volume-expanded cells. Because pervanadate is known to be an inhibitor
of phosphotyrosine phosphatase (16, 17), 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 (13).

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Fig. 7.
Effect of pervanadate on p72syk
activity. Cells were treated for 10 min with 0.5 mM
pervanadate and then used exactly as in Fig. 3. Activity of
p72syk was measured in the absence ( cdb3) or
presence (+cdb3) of the cytoplasmic domain of band 3. The
autoradiograph shown is representative of those from three different
experiments. ISO, isotonic; HYPO,
hypotonic.
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Identification of Phosphotyrosines--
In preliminary
experiments, both the entire immunoprecipitated
32PO4-labeled band 3 molecule and
32PO4-labeled cdb3 (prepared by mild tryp-
sinization of band 3) were analyzed. The major
32PO4-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 showing
32PO4-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 (18) and corresponds to residues 75-90
(18). 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 (18). 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 (18).

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Fig. 8.
Determination of tyrosine phosphorylation
sites of cdb3. 32PO4-labeled cdb3 was
obtained by trypsin treatment (50 ng/ml) of ghosts from cells labeled
with 32PO4. After gel purification, cdb3 was
subjected to extensive proteolysis with trypsin (10 µg/ml), and the
peptides were separated by C18 reverse phase-HPLC. Peaks A and B from
hypotonic cells were selected for amino acid sequence using Edman
degradation. Tracings shown are representative of those of
three different experiments. ABS, absorbance.
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Fig. 9.
32P 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.
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Detection of a Type 1B Protein-tyrosine Phosphatase Associated with
Skate Band 3--
The enhancement of protein tyrosine phosphorylation
by p72syk in the presence of pervanadate suggests that the
level of phosphorylation of proteins phosphorylated by p72syk
is regulated by protein-tyrosine phosphatases. Because erythrocytes are
known to be a rich source of these phosphatases (20, 21), 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 (20). 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 p72syk
may rapidly phosphorylate band 3, it does not form a complex of
sufficient affinity to withstand the immunoprecipitation
conditions.

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Fig. 10.
Association of PTP1B with band 3. Band
3 was immunoprecipitated from cells incubated in isotonic and hypotonic
medium for the times indicated. Proteins were eluted from protein
A-Sepharose, analyzed on 10% SDS-PAGE, and transferred to a
polyvinylidene difluoride membrane. PTP1B was detected in the samples
using a polyclonal antisera and an enhanced chemiluminescence system.
The image shown is representative of those from two different
experiments. ISO, isotonic; HYPO,
hypotonic.
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DISCUSSION |
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
(13), 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. (19). 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
(1, 3). A very recent study using oocyte expression of trout band 3 has further reinforced its role in volume-activated solute fluxes (4). 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 (13). This evidence raises the possibility that activation of p72syk
may 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 (22), neutrophil signaling through adhesion
complexes (23), apoptosis stimulated by osmotic stress or ultraviolet
radiation in B cells (24), integrin cross-linking in myeloid cells
(25), and thrombin stimulation of platelet aggregation (26). 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. (27) 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 (28), 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.