From the Departments of Pharmacology and
§ Physiology, University of Tennessee, The Health Science
Center, Memphis, Tennessee 38163
Received for publication, October 9, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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Mammalian Sprouty proteins have been shown to
inhibit the proliferation and migration of cells in response to growth
factors and serum. In this communication, using HeLa cells, we have
examined the possibility that human Sprouty 2 (hSPRY2) mediates its
anti-migratory actions by modulating the activity or intracellular
localization of protein-tyrosine phosphatases. In HeLa cells,
overexpression of hSPRY2 resulted in an increase in protein-tyrosine
phosphatase (PTP1B) amount and activity in the soluble (100,000 × g) fraction of cells without an increase in total amount of
cellular PTP1B. This increase in the soluble form of PTP1B was
accompanied by a decrease in the amount of the enzyme in the
particulate fraction. The amounts of PTP-PEST or PTP1D in the soluble
fractions were not altered. Consistent with an increase in soluble
PTP1B amount and activity, the tyrosine phosphorylation of cellular
proteins and p130Cas was decreased in hSPRY2-expressing
cells. In control cells, overexpression of wild-type (WT) PTP1B, but
not its C215S catalytically inactive mutant mimicked the actions of
hSPRY2 on tyrosine phosphorylation of cellular proteins and migration.
On the other hand, in hSPRY2-expressing cells, the C215S mutant, but
not WT PTP1B, increased tyrosine phosphorylation of cellular proteins
and attenuated the anti-migratory actions of hSPRY2. Interestingly,
neither WT nor C215S mutant forms of PTP1B modulated the anti-mitogenic
actions of hSPRY2. Therefore, we conclude that an increase in soluble
PTP1B activity contributes to the anti-migratory, but not
anti-mitogenic, actions of hSPRY2.
Sprouty (SPRY)1 was
originally described as a protein that regulated fibroblast growth
factor signaling in Drosophila and inhibited tracheal
branching (1). Subsequently, SPRY was reported as an inhibitor of
signaling events initiated by the epidermal growth factor (EGF) and
fibroblast growth factor receptor tyrosine kinases (2) in
Drosophila and shown to modulate wing vein formation. To
date, four isoforms of mammalian SPRY proteins have been cloned (1,
3-5). These proteins have a highly conserved C terminus but their N
terminus is variable. Like the Drosophila SPRY proteins, a
decrease in mouse SPRY2 expression resulted in increased lung branching
morphogenesis (3). These findings demonstrated that SPRY proteins have
conserved function to modulate respiratory morphogenesis. The ability
of mouse SPRY4 to inhibit angiogenesis (6) also demonstrates that the
SPRY proteins plays a profound role in regulating tubular morphogenesis.
At the cellular level, we and others have shown that SPRY1 (7), SPRY2
(8), and SPRY4 (6) inhibit migration and proliferation of cells in
response to serum and growth factors. However, the mechanisms involved
in SPRY-elicited inhibition of cellular proliferation and migration
remain to be completely elucidated. SPRY1 and SPRY2 have been shown to
decrease Erk activation in response to fibroblast growth factor and
vascular endothelium-derived growth factor in vascular endothelial
cells (6, 7). However, even though EGF-stimulated cellular
proliferation was inhibited by SPRY proteins, the ability of EGF to
activate Erk activation was not affected (7). We have also observed
that although the activation of Erk by EGF is not altered by
overexpression of human SPRY2 (hSPRY2) in HeLa
cells,2 the proliferation and
migration of these cells in response to EGF is markedly attenuated by
hSPRY2 (8). Another level of regulation by SPRY proteins may involve
direct interactions with certain signaling molecules. For instance,
hSPRY2 has been shown to interact directly with c-Cbl, and this
interaction decreases internalization and down-regulation of the EGF
receptor (9).
Because the functions of receptor tyrosine kinases can be attenuated by
increase in protein-tyrosine phosphatase activities, we investigated
the possibility that hSPRY2 may alter the cellular activity of some
protein-tyrosine phosphatase(s) and thereby modulate the biological
actions of serum or growth factors. In this communication we
demonstrate that the amount of soluble protein-tyrosine phosphatase-1B (PTP1B) activity is increased in hSPRY2-expressing cells. This is
accompanied by a decrease in protein tyrosine phosphorylation. Expression of PTP1B mimicked the actions of hSPRY2 and inhibited serum-induced cell migration. More importantly, the inhibition of PTP1B
activity in hSPRY2-expressing cells by a dominant negative mutant of
PTP1B rescued cells from the anti-migratory actions of hSPRY2. These
data demonstrate that PTP1B plays an important role in mediating the
actions of hSPRY2 on cell migration.
HA-hSPRY2-expressing Cell Lines--
Full-length
HA-tagged or red fluorescent protein-tagged hSpry2 were cloned from two
expressed sequence tag clones and stable clones of HeLa that express
HA-hSpry2 or hSpry2-red fluorescent protein were generated as reported
previously (8).
Construction of Recombinant Adenovirus Expressing Wild-type or
Mutant PTP1B--
Replication-deficient (E-1 deleted) recombinant type
5 adenovirus-expressing, HA-tagged wild-type, or C215S mutant PTP1B
were prepared using the commercial kit, Adeno-X
(Clontech, Palo Alto, CA). Plasmids containing
wild-type human PTP1B (pJ3H-PTP435) and catalytically inactive C215S
mutant PTP1B (pJ3H-PTP435-C215S) were kindly donated by Dr. Jonathan
Chernoff (Fox Chase Cancer Institute, Philadelphia, PA). These
PTP1B constructs containing an N-terminal hemagglutinin (HA) epitope
were used as templates to amplify PCR product expressing the
corresponding full-length HA-tagged PTP1B cDNAs (WT and mutant),
flanked by NotI and KpnI restriction sites. PCR
products were subcloned into the NotI/KpnI site
of the pShuttle vector provided in the Adeno-X kit
(Clontech). All sequences of PTP1B were verified by
DNA sequencing. An I-CeuI/PI-SceI restriction
fragment from pShuttle containing the cytomegalovirus-IE promoter/enhancer 5' to the PTP1B cDNA insert and the
polyadenylation signal was ligated into adenoviral DNA backbone that
was also restricted with I-CeuI and PI-SceI.
Following amplification and purification of recombinant viral DNA from
bacteria, adenovirus was generated by transfecting
PacI-linearized recombinant viral DNA into HEK 293 cells via
the use of the lipid transfection agent FuGENE 6 (Roche Diagnostics).
Cell Fractionation--
HeLa cells were lysed by incubation for
15 min on ice with a buffer containing the following: 20 mM
Tris-HCl, pH 7.5, 50 mM NaF, 2 mM sodium
orthovanadate, 20 mM p-nitrophenyl
phosphate, 2 mM EDTA, 2 mM EGTA, 5 mM benzamidine. Lysates were centrifuged for 60 min at
100,000 × g. The supernatants (soluble fraction) and
the pellet (particulate fraction) were then separated from each other.
SDS-PAGE and Western Blotting--
Cell lysates were mixed with
Laemmli sample buffer and proteins separated on polyacrylamide gels as
described by Laemmli (10). Proteins were transferred onto
nitrocellulose and incubated in 5% milk in PBS. The membranes were
incubated in primary antibody anti-HA (HA.11 from Covance Research
Products, Richmond, CA) or anti-PTP1B (BD Biosciences, San Diego, CA)
at 1:1000 dilution for 1 h, followed by secondary antibody (goat
anti-rabbit immunoglobulin G-horseradish peroxidase, 1:3000 dilution)
for 1 h at room temperature. Proteins were detected using an
enhanced chemiluminescence kit from Pierce.
Cell Migration Assays--
The modified Boyden chambers (Costar,
Corning, NY) were used for monitoring cell migration. Essentially,
overnight serum-starved cells were trypsinized and washed twice with
serum-free medium containing 0.5% BSA. Cells were plated on the upper
side of transwells (35,000 cells/well) in 100 µl of serum-free
medium, and 500 µl of the same medium was added to the lower chamber.
The cells were allowed to adhere for 1 h. Cells were treated with
virus in serum-free medium for 2 h. After 2 h of virus
treatment, serum was added (10%) to the lower chamber, and cells were
allowed to migrate overnight at 37 °C. At the end of the experiment
the transwell inserts were washed with PBS, fixed with
methanol/acetone, and stained with hematoxylin. Cells on the upper
chamber side of the membrane were removed with cotton swabs, and cells
that migrated through the membrane were counted.
Thymidine Incorporation--
Cells were plated in Dulbecco's
minimum essential medium containing 10% serum for 6 h at a
density of 7 × 104 cells/well in 24-well plates. Then
serum was withdrawn for an overnight period, and cells were treated
with adenoviral vectors for 2 h to express either EGFP, WT PTP1B,
or dominant negative (DN) PTP1B. The medium was exchanged with
Dulbecco's minimum essential medium containing 10% serum and cells
incubated for an overnight period. [3H]Thymidine, 1.5 µCi (PerkinElmer Life Sciences) was added to each well and
cells incubated for 4 h at 37 °C. At the end of this period,
plates were placed on ice, and cells were washed three times each with
ice-cold PBS, then with ice-cold 10% trichloroacetic acid, and
finally with ethanol/ether (2:1). The cells were dissolved with 0.1%
SDS in 0.1 N NaOH by incubating overnight at room
temperature. Aliquots were counted for 3H and protein
concentration determined using the bicinchoninic acid method (11)
(Micro BCA protein assay kit from Pierce).
PTP1B Activity Assays--
To monitor PTP1B activity, soluble
fractions (100 µg of protein) from control and hSPRY2-expressing
cells were used. The PTP1B was immunoprecipitated with anti-PTP1B
antibody, and the immunoprecipitates were washed three times with
ice-cold PBS and resuspended in 100 µl of buffer containing 20 mM HEPES, pH 7.5, 2.0 mM EDTA, 2.0 mM EGTA, 5 mM benzamidine, 20 µg/ml each of
soybean trypsin inhibitor, leupeptin, and aprotinin (12). Equal
aliquots of the immunoprecipitates were incubated in a reaction buffer
containing 40 mM MES, pH 6.5, and 20 mM
dithiothreitol in the presence and absence of 10 mM vanadate. The reactions were initiated by the addition of
32P-labeled RaytideTM. After incubation at room
temperature for the indicated times, the reactions were stopped by
addition of 1 ml of activated charcoal, and 500-µl aliquots of the
supernatant were counted for 32Pi released. The
vanadate-sensitive activity is presented as PTP1B activity.
RaytideTM Labeling--
RaytideTM (Oncogene, San Diego, CA) was
labeled according to the manufacturer's instructions. Briefly, 0.3 µg/µl Raytide was phosphorylated with 5 units of c-Src (Upstate
Biotechnology, Lake Placid, NY) in the presence of 0.3 mM
[ Human SPRY2 inhibits the biological actions of a number of growth
factors that exert their effects via activation of receptor tyrosine
kinases. Because the action of these kinases can be modulated by
protein-tyrosine phosphatases (PTPases), we hypothesized that hSPRY2
may alter the activity of PTPases. To address this hypothesis, we
utilized the previously described (8) HeLa cell lines that are
transfected to express hSPRY2. The Geneticin (G418)-resistant HeLa
cells that do not overexpress hSPRY2 protein were used as controls (8).
Initially, we monitored the amount of various protein-tyrosine
phosphatases in total cell lysates and in the soluble fractions of
hSPRY2-expressing cells. As shown in Fig. 1A, the soluble fraction of
hSPRY2-expressing cells contained more PTP1B as compared with controls.
However, the expression of PTP-PEST and PTP1D were not altered (Fig.
1A); the equal amounts of Erk in the same blots demonstrates
that the loading of proteins was the same. Interestingly, the amount of
PTP1B in the soluble fraction remained elevated irrespective of whether
the cells were grown in serum or serum-free medium for 1 day. Moreover,
the total amount of PTP1B in HeLa cells expressing hSPRY2 was not
altered (Fig. 1B). Consistent with this latter observation,
the increase in soluble PTP1B amount in hSPRY2-expressing cells was
accompanied by a decrease in the amount of PTP1B in the particulate
fraction (Fig. 1C). The increase in PTP1B in the soluble
fraction of cells was also confirmed with other clonal HeLa cell lines
expressing hSPRY2-red fluorescent protein (data not shown).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (1.1 µCi/nmol), 10 mM
MgCl2, in 50 mM HEPES, pH 7.5, 0.1 mM dithiothreitol, 0.015% Brij, and 0.1 mg/ml BSA buffer.
The reactions were incubated for 5 h at room temperature.
Thereafter, acetylated BSA was added to a final concentration of 7 mg/ml, and the reaction was stopped with ice-cold 20% trichloroacetic acid (final concentration). The BSA/RaytideTM co-precipitate was centrifuged (20,000 × g for 10 min) and washed five
times with 500 µl of 40% trichloroacetic acid until the background
in the blank reaction (reaction without RaytideTM) was diminished
significantly (
10% of that in reaction containing RaytideTM).
The final RaytideTM/BSA pellet was washed once with acetone (500 µl)
and dissolved in 200 mM Tris-HCl, pH 8.0. Aliquots of the
32P-labeled RaytideTM were stored at
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
hSPRY2 increases the amount of PTP1B in
soluble fractions of Cells. Control and hSPRY2-expressing HeLa
cells were grown in 10% fetal bovine serum or serum-starved for
24 h. The cells were then harvested and soluble and particulate
fractions derived as described under "Materials and Methods."
A, soluble fractions (30 µg of protein each) of the cell
lysates were immunoblotted for PTP-PEST, PTP1B, and PTP1D using the
commercially available antibodies (BD Biosciences). The same blot was
also reprobed with anti-Erk1/2 antibody to verify efficiency of
transfer of protein to the nitrocellulose membrane. B, cells
were grown as described for A, and Western analysis was
performed in total cell lysate for PTP1B content. C, the
particulate and soluble fractions were isolated from control and
hSPRY2-expressing cells and analyzed for PTP1B content. The same blot
was reprobed with anti-Erk1/2 antibody to verify efficiency of transfer
of protein to the nitrocellulose membrane. For each panel, a
representative of at least three similar experiments is shown.
To determine whether the soluble PTP1B was active, we
immunoprecipitated PTP1B from the soluble fractions of control and
hSPRY2-expressing cells and monitored its ability to dephosphorylate
32P-labeled RaytideTM. As shown in Fig.
2, PTP1B activity in immunoprecipitates from the soluble fractions of hSPRY2-expressing cells was greater than
that in controls. These data (Fig. 2) demonstrate that the soluble
PTP1B in the hSPRY2-expressing cells is active. The increase in PTP1B
activity in the soluble fraction (Fig. 2) is consistent with the
observation that the amount of PTP1B in the soluble fraction of
hSPRY2-expressing cells is higher than that in controls (Fig. 1,
A and C). Although hSPRY2 expression increased
PTP1B protein in the soluble fraction by 5-fold (Fig. 1, A
and C), the PTP1B activity in immunoprecipitates was
increased by ~2-fold. This may be related to the fact that during the
immunoprecipitation procedure, despite our efforts to minimize
inactivation of PTP1B, oxidation of the critical cysteine
(Cys-215) in the catalytic site of PTP1B occurred and decreased
activity. It has been demonstrated that the oxidation of this cysteine
residue (Cys-215) to cysteine sulfenic acid decreases PTP1B activity
(13). Despite this discrepancy, the data in Fig. 2 clearly show that
the activity of PTP1B in immunoprecipitates of soluble fractions from
hSPRY2-expressing cells is greater than that in controls.
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An increase in PTP1B activity in the soluble fraction would suggest
that tyrosine phosphorylation of cellular proteins may also be altered.
Therefore, we compared lysates of hSPRY2-overexpressing cells with
control cells to monitor the tyrosine phosphorylation of proteins. As
shown in Fig. 3A, tyrosine
phosphorylation of several, but not all, proteins in cells expressing
hSPRY2 was decreased as compared with control cells. The same
observation was made in another clone of hSPRY2-expressing cells. The
most significant decrease in tyrosine phosphorylation was observed on
proteins of molecular mass of 130 and 66 kDa. Since one of the
substrates of PTP1B is known to be p130Cas (14), we
examined whether tyrosine phosphorylation of this protein was decreased
in cells overexpressing hSPRY2. As shown in Fig. 3B,
compared with control, tyrosine phosphorylation of the
immunoprecipitated p130Cas was decreased in
hSPRY2-overexpressing cells. Note that the amount of
p130Cas in the immunoprecipitates was the same (Fig.
3B). Taken together with the data in Fig. 2, the findings in
Fig. 3 are consistent with the notion that increase in PTPase activity
in hSPRY2-expressing cells would decrease tyrosine phosphorylation of
proteins. Moreover, since p130Cas is a substrate for PTP1B
(14), the decrease in p130Cas phosphorylation corroborates
the findings that soluble PTP1B is increased in SPRY2-expressing
cells.
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Next, we reasoned that if hSPRY2 was attenuating the migratory and/or
proliferative actions of serum or growth factors by increasing PTP1B
activity, then antagonizing the activity of PTP1B in hSPRY2-expressing
cells should (i) reverse the decrease in tyrosine phosphorylation of
proteins and (ii) rescue the cells from the anti-migratory and/or
anti-proliferative actions of hSPRY2. To address this possibility,
using adenoviral vectors, we expressed either the WT or catalytically
inactive mutant (C215S) of PTP1B in control and hSPRY2-overexpressing
cells. Adenovirus that would express EGFP was used as a control. The
C215S mutant of PTP1B is catalytically inactive but can bind the
substrates and thereby protects substrate dephosphorylation by
endogenous (wild-type) PTP1B (14-16). Thus, the C215S mutant functions
as a DN PTP1B and is referred to as such from here on. Initially, we
investigated the effects of expressing either the WT or DN PTP1B on
tyrosine phosphorylation of cellular proteins in control and hSPRY2-
overexpressing cells. As shown in Fig.
4A, the expression of WT PTP1B
decreased tyrosine phosphorylation of cellular proteins in control
cells and cells expressing hSPRY2. Note that as shown before (Fig. 3), in cells infected with virus encoding EGFP, the tyrosine
phosphorylation of proteins in the hSPRY2-expressing cells was lower
than that in control. In control cells, DN PTP1B did not significantly
affect tyrosine phosphorylation of cellular proteins, but the tyrosine phosphorylation of proteins in hSPRY2-overexpressing cells was increased by DN PTP1B. It should be noted that the adenovirus-mediated expression of WT and DN PTP1B in control and hSPRY2 containing cells
was the same and ~10-fold higher than the levels of endogenous PTP1B
(Fig. 4B). Interestingly, analysis of the soluble fraction of cell lysates for the presence of adenoviral-induced PTP1B showed that in hSPRY2-expressing cells, the amounts of both WT and DN PTP1B
were greater than that observed in control cells (Fig. 4C). These latter data demonstrate that while overexpression of PTP1B permits the presence of soluble enzyme to be detected in control cells,
the overexpressed PTP1B is also subject to changes in cellular localization (i.e. increase in soluble fraction) by
hSPRY2.
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To determine whether the DN PTP1B would rescue the cells from the
anti-migratory actions of hSPRY2, the experiment depicted in Fig.
5A was performed. Essentially,
cells were infected with adenovirus for 2 h to express EGFP or WT
PTP1B or DN PTP1B. The migration of these cells in response to serum
was then monitored as described under "Materials and Methods." That
the expression of WT and DN PTP1B was similar in these cells was
confirmed at the end of the migration protocol as shown in Fig.
4B. In control cells, the expression of WT PTP1B decreased
migration of both control and hSPRY2-expressing cells (Fig.
5A). DN PTP1B did not appreciably alter the migration of
control cells, indicating that in these cells the endogenous PTP1B does
not significantly contribute to cell migration. On the other hand, in
hSPRY2-expressing cells, DN PTP1B markedly attenuated the ability of
hSPRY2 to inhibit cell migration. The ability of WT PTP1B to inhibit
migration and the ability of DN PTP1B to rescue cells from the
anti-migratory activity of hSPRY2 clearly show a role for PTP1B in
mediating the biological actions of hSPRY2. Since we and others have
shown that hSPRY2 also inhibits proliferation of cells (6-8), we
investigated whether the expression of PTP1B would protect cells
against the anti-mitogenic actions of hSPRY2. As shown in Fig.
5B, neither the WT nor the DN PTP1B altered thymidine
incorporation in control and hSPRY2-overexpressing cells.
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DISCUSSION |
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PTP1B has been reported to be located in the endoplasmic reticulum by a hydrophobic C-terminal 35-amino acid anchoring region (17, 18). For activation of platelets by a variety of agonists that activate calcium-dependent neutral protease, calpain has been shown to cleave the protein above this anchoring region and render it soluble (19). The cleavage of PTP1B results in a 2-fold greater activity of the enzyme and is accompanied by changes in tyrosine phosphorylation of several proteins (19). On SDS-PAGE, the protease-cleaved PTP1B migrates as a 42-kDa species as compared with the full-length enzyme that migrates as a 50-kDa protein. In our experiments, the soluble form of PTP1B was not smaller in size as compared with the particulate enzyme, suggesting that the increase in soluble PTP1B in hSPRY2-expressing cells is not the result of cleavage of the C-terminal anchoring region. Moreover, although the presence of soluble PTP1B was difficult to detect in control cells, the overexpression of both WT and DN PTP1B was accompanied by the presence of PTP1B in the soluble fractions (Fig. 4B). As determined by their migration on SDS-PAGE, these ectopically expressed, soluble, PTP1B species were also not cleaved (Fig. 4B). Interestingly, the presence of full-length (50 kDa) PTP1B in the cytosolic fraction has also been observed in COS7 cells (20). Hence, it would appear that mechanisms other than cleavage of the full-length PTP1B are responsible for changes in its distribution between the endoplasmic reticulum and cytosol. In this context, the ability of hSPRY2 to redistribute PTP1B in cells may provide yet another means of regulating the biological actions of PTP1B. The precise mechanisms by which PTP1B is rendered soluble by hSPRY2 remain to be elucidated and form the subject of future studies.
Consistent with the observation that the amount of PTP1B and activity in the soluble fraction are elevated, we observed that the phosphorylation of several cellular proteins, including p130Cas was also decreased in hSPRY2-overexpressing cells (Fig. 3). Since p130Cas is a critical component of the focal adhesion complex (21), and because its phosphorylation status modulates the migratory response of cells (22-24), the decrease in phosphorylation of p130Cas would contribute to the anti-migratory actions of hSPRY2. We are currently in the process of determining the identity of the other proteins (~66 kDa) whose phosphorylation is decreased.
Among the mechanisms that mediate the actions of SPRY proteins, studies have suggested that SPRY proteins alter the activity of the Ras-Erk pathway by decreasing the activation of Ras (6, 25) or Raf (26) in response to growth factors. However, to date no attention has been given to the role of protein-tyrosine phosphatases in modulating the actions of SPRY proteins. In this respect, this is the first report to show that hSPRY2 modulates the cellular localization and activity of PTP1B in the soluble (cytosolic) fraction of cells. Indeed, our findings that overexpression of WT PTP1B, but not the DN PTP1B, in control cells decreases migration demonstrates that PTP1B plays a profound role in regulating this process. The fact that the DN PTP1B attenuates the anti-migratory actions of hSPRY2 (Fig. 5A) further reinforces the role of PTP1B in the migration of cells. More importantly, the experiments with the DN PTP1B demonstrate that the anti-migratory actions of hSPRY2 are, in part, mediated by an increase in PTP1B activity. Since DN PTP1B only partially rescues cells from the inhibition of migration in hSPRY2-expressing cells, the data in Fig. 5A also suggest that in addition to PTP1B, other mechanisms may also contribute to the anti-migratory actions of hSPRY2 in response to serum. These additional mechanisms remain to be identified. Interestingly, although hSPRY2 inhibits cellular proliferation (6-8), the expression of DN PTP1B did not rescue cells from the anti-mitogenic actions of hSPRY2 (Fig. 5B). These results demonstrate the specificity of the role of PTP1B in the anti-migratory actions of hSPRY2 and also imply that hSPRY2 inhibits cellular proliferation by modulating other mechanisms.
In summary, we have demonstrated that hSPRY2 expression is associated
with an increase in PTP1B in the soluble fraction of cells. The
experiments with exogenously expressed PTP1B demonstrate that this
enzyme plays a profound role in regulating cell migration, but not
proliferation, and that the increase in PTP1B activity in
hSPRY2-expressing cells contributes to the anti-migratory actions of hSPRY2.
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ACKNOWLEDGEMENT |
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We thank Dr. Jonathan Chernoff, Fox Chase Cancer Institute, for providing the PTP1B cDNAs.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL48308 (to T. B. P.) and HL063886 (to A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Pharmacology, University of Tennessee, The Health Science Center, 874 Union Ave., Memphis, TN 38163. Tel.: 901-448-6006; Fax: 901-448-4828; E-mail: tpatel@physio1.utmem.edu.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M210359200
2 Y. Yigzaw and T. B. Patel, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: SPRY, Sprouty; hSPRY2 (arabic numeral following SPRY designates isoform type), human Sprouty 2; EGF, epidermal growth factor; MES, 2-(N-morpholino)ethanesulfonic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HA, hemagglutinin; PTP1B, protein-tyrosine phosphatase-1B; EGFP, enhanced green fluorescent protein; WT, wild-type; DN, dominant negative; Erk, extracellular signal-regulated kinase.
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