The C Terminus of Sprouty Is Important for Modulation of Cellular
Migration and Proliferation*
Yinges
Yigzaw,
Laura
Cartin,
Sandra
Pierre,
Klaus
Scholich, and
Tarun B.
Patel
From the Department of Pharmacology and the Vascular Biology Center
of Excellence, The Health Science Center University of
Tennessee, Memphis, Tennessee 38163
Received for publication, January 5, 2001, and in revised form, March 9, 2001
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ABSTRACT |
The Drosophila Sprouty (SPRY) protein
has been shown to inhibit the actions of epidermal growth factor and
fibroblast growth factor. However, the role of mammalian SPRY proteins
has not been clearly elucidated. We postulated that human Sprouty2
(hSPRY2) is an inhibitor of cellular migration and proliferation.
Indeed, using stably transfected HeLa cells, which expressed
hemagglutinin (HA)-tagged hSPRY2 or hSPRY2 tagged at the C terminus
with red fluorescent protein, we demonstrated that hSPRY2 inhibits the migration of cells in response to serum, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor. Additionally, hSPRY2 also inhibited the growth of HeLa cells in response to serum. Previously, two C-terminal domains on hSPRY2, which
are necessary for its colocalization with microtubules (residues 123-177) or translocation to membrane ruffles (residues 178-194), have been identified (Lim, J., Wong, E. S., Ong, S. H.,
Yusoff, P., Low, B. C., and Guy, G. R. (2000) J. Biol. Chem. 275, 32837-32845). Therefore, using TAT-tagged
hSPRY2 and its mutants, we determined the role of these two C-terminal
domains in the inhibition of cell migration and proliferation. Our data
show that the deletion of either of these two regions in hSPRY2
abrogates its ability to modulate cell migration in response to
different growth factors and proliferation in response to serum.
Therefore, we conclude that hSPRY2 inhibits the actions of a number of
growth factors, and its C terminus, which is homologous among various
SPRY isoforms, is important in mediating its biological activity.
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INTRODUCTION |
In Drosophila, Sprouty has been demonstrated to be
important in regulating fibroblast growth factor
(FGF)1 and epidermal growth
factor (EGF)-mediated cellular actions. Thus, in Drosophila
Sprouty mutants, the fibroblast growth factor pathway is overactive,
and excessive branching is seen in Drosophila airways (1).
Kramer et al. (2) have shown antagonistic actions of Sprouty
on both the FGF and EGF signaling pathways in Drosophila, where loss of Sprouty results in supernumerary neurons and glia. In
addition, overexpression of Sprouty in wing veins and ovarian follicle
cells, tissues where EGF signaling is required for proper patterning,
results in phenotypes that resemble the loss-of-function phenotypes of
EGF receptors (3, 4). Sprouty has also been shown to interfere with
chick embryo development. Hence, infection of the prospective wing
territory with a retrovirus containing the Sprouty gene inhibits proper
limb growth and formation in the embryonic chick (5). Taken together,
these results suggest that Sprouty acts as an antagonist of both FGF
and EGF receptor signaling pathways.
To date, four isoforms of mammalian Sprouty protein have been described
(SPRY1-SPRY4) (1, 4-6). Among these, only partial clones of SPRY1 and
SPRY3 are available. The mouse as well as human SPRY2 (hSPRY2) and
mouse SPRY4 have been cloned (4, 6, 7), and both these proteins share
considerable sequence homology in the C terminus. However, their N
termini are different. By Northern analysis the mouse SPRY2 mRNA is
most abundant in brain, lung, and heart followed by kidney and skeletal
muscle (7). Despite their role in Drosophila and chick
development, relatively little is known about the biological actions of
the mammalian Sprouty proteins. Recently, one study (8) has
demonstrated that upon activation of cells by EGF, hSPRY2 is
translocated from the vicinity of microtubules to membrane ruffles.
Moreover, this translocation of hSPRY2 from microtubules to the
membrane ruffles is dependent upon certain portions of the C terminus.
However, the functional role of hSPRY2, its colocalization with tubulin in microtubules, and its translocation remain to be described. Therefore, the purpose of this study was to determine the functional role of hSPRY2 in growth factor actions and determine whether the
translocation sequence on this protein alters its activity. Our data
show that in cells that are either transfected or transduced to express
hSPRY2, the migration in response to serum and a variety of growth
factors is attenuated. Additionally, serum-induced proliferation of
cells expressing hSPRY2 is also markedly inhibited. Moreover, our data
show that deletion of regions in the C terminus, which has been shown
to disrupt either the colocalization of hSPRY2 with tubulin or its
translocation to membrane ruffles, obliterates the biological actions
of the protein.
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MATERIALS AND METHODS |
Plasmid Constructs--
Full-length hSPRY2 was cloned from two
expressed sequence tag clones (accession numbers AA305092 and AA431912)
using the unique AflII site in their overlapping sequence.
The full-length clone was polymerase chain reaction (PCR)-amplified
with a forward primer containing KpnI and BamHI
sites (5-TATATATA GGT ACC GGA TCC GAG GCC AGA GCT CAG AGT GGC-3) and a reverse primer
containing a stop codon as well as XhoI and EcoRI
sites (5-ATATAT GAATTC CTCGAG
TTA CTA TGT TGG TTT TTC
AAA GTT CCT-3). This PCR product was digested with
KpnI/EcoRI and ligated to the
KpnI/EcoRI site of pRSET B. To generate a
construct to express hemagglutinin (HA)-hSPRY2, the full-length hSPRY2
cDNA from pRSET B was digested with
KpnI/EcoRI and subcloned into the
KpnI/EcoRI sites of HA-tag-containing mammalian
expression vector pHM6 (Roche Molecular Biochemicals).
To construct a TAT-HA-hSPRY2 expression plasmid, the hSPRY2 cDNA in
pRSET B was digested with KpnI/EcoRI and
subcloned into the KpnI/EcoRI site of pTAT-HA
bacterial expression vector (gift from Dr. Steven Dowdy, Washington
University School of Medicine, St. Louis, MO). Similarly, the
cDNAs encoding hSPRY2 deletion mutants (
123-177 and
178-194),
which were generated by PCR, were subcloned into the pTAT-HA expression
vector using KpnI and EcoRI restriction sites. To
express the C-terminal RFP-tagged hSPRY2, the full-length clone of
hSPRY2 was amplified by PCR with a forward primer containing a
XhoI site (5-TATATA CTCGAG ATG GAG GCC AGA
GCT-3) and a KpnI site containing reverse primer (5-ATATAT GG TAC CGT TGG TTT TTC AAA GT-3)
devoid of a stop codon. The PCR product was digested with
XhoI and KpnI and inserted into the XhoI/KpnI sites of the RFP expression mammalian
vector pDsRed1-N1 (CLONTECH). All hSPRY2 constructs
were sequenced fully for tag and sequence verification.
Selection of Stable Clones--
HeLa cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum without antibiotic for 2 days. The cells were transfected
with plasmid constructs encoding HA-hSPRY2, hSPRY2-RFP, RFP alone, or
vector alone using the FuGENE transfection reagent (Roche
Molecular Biochemicals). Twenty-four hours later, cells were grown in
media supplemented with the antibiotic Geneticin (G418, BioWhittaker)
at 200-800 µg/ml, and selection of clonal cell lines was initiated.
Individual G418-resistant clones were expanded and maintained in 800 µg/ml G418. Overexpression of HA-hSPRY2 was confirmed by Western
blotting and immunofluorescence. Similarly, overexpression of
hSPRY2-RFP was confirmed by fluorescence.
Expression and Purification of TAT-GFP and
TAT-HA-hSPRY2--
The TAT-HA tag encodes for an N-terminal
hexahistidyl leader followed by the 11-amino acid TAT protein
transduction domain (YGRKKRRQRRR) and an HA tag (YPYDVPDYA) (9, 10).
The TAT-HA-hSPRY2 protein was expressed in BL21(DE3)pLys strain of
E. coli by induction with IPTG (0.4 mM) and
overnight growth at 37 °C. TAT fusion proteins were purified after
lysis of E. coli with 6 M guanidinium
hydrochloride. Cellular lysates were resolved by centrifugation, loaded
on a nickel-nitrilotriacetic acid column, washed successively with buffer (20 mM Tris HCl, pH 7.5) containing 8 M
urea, 4 M urea, and buffer alone. Proteins were eluted with
an imidazole gradient (40 mM-1 M) in the
same buffer containing 1 M NaCl. Fractions containing
TAT-HA-hSPRY2 were collected and pooled. This pool of TAT-HA-hSPRY2
protein was then desalted on a PD-10 column (Amersham Pharmacia
Biotech) into phosphate-buffered saline (PBS). After determining
protein concentration, TAT-HA-hSPRY2 was stored in 10% glycerol at
80 °C. The deletion mutants of TAT-HA-hSPRY2 (
123-177 and
178-194) were also expressed and purified using the same methodologies.
The plasmid pTAT-GFP was a gift from Dr. Steven Dowdy. TAT-GFP was
expressed and purified from E. coli by methods similar to
those described above for TAT-HA-hSPRY2. The peak fractions were
identified by absorbance at 280 nm, pooled, applied to a PD-10 column,
and eluted into PBS. Purified TAT-GFP was stored in 10% glycerol at
80 °C until use.
SDS-Polyacrylamide Gel Electrophoresis and Western Blotting with
Anti-HA Antibody--
Cell lysates were separated on polyacrylamide
gels as described by Laemmli (11). Proteins were transferred onto
nitrocellulose and incubated in 5% milk in PBS. The membrane was
incubated in primary antibody (anti-HA, HA.11 from Covance Research
Products, 1:400 dilution) for 1.5 h at 37 °C followed by
secondary antibody (goat anti-rabbit IgG-horseradish peroxidase, 1:5000
dilution) for 1 h at room temperature. Proteins were detected
using an enhanced chemiluminescence kit from Pierce.
Immunofluorescence--
Cells were grown on glass coverslips to
~75% confluence. The cells were then washed with PBS and
fixed by exposure to 4% paraformaldehyde in PBS for 10 min at room
temperature followed by 0.5% Triton X-100 in PBS for 15 min at room
temperature. After three washes with PBS, the coverslips were blocked
overnight with 1% bovine serum albumin in PBS. The coverslips were
exposed to a 1:1000 dilution of anti-HA antibody for 1 h at
37 °C followed by incubation with a 1:500 dilution of fluorescein
isothiocyanate-labeled goat anti-mouse secondary antibody (Jackson
Laboratories) for 1 h at 37 °C. Cells were then washed with PBS
containing 1% bovine serum albumin and mounted.
For fluorescence analysis of RFP, hSPRY2-RFP, fluorescein
isothiocyanate-labeled TAT-tagged proteins;Cy-3 conjugated anti-tubulin antibody (tubulin), and filamentous actin (Texas red
isothiocyanate-labeled phalloidin, Sigma), cells were rinsed in
Dulbecco's PBS (BioWhittaker), followed by fixation in 4%
paraformaldehyde at room temperature for 30 min. Cells were washed
twice with Dulbecco's PBS and mounted. Immunofluorescence was detected
using a Bio-Rad 1000 laser scanning confocal microscope or a Zeiss LSM
510 system.
Cell Migration Assays--
Migration of cells was measured using
a monolayer wounding protocol ("scratch wound" assay) in which
cells migrate from a confluent area to an area that has been
mechanically denuded of cells using a razor blade (12). Essentially,
confluent cells were treated with serum-free medium containing 0.1%
bovine serum albumin for 24 h before the start of the experiment.
After serum starvation, cells were rinsed with Dulbecco's modified
Eagle's medium and a scratch was made in the dish of cells using a
sterile, single-edged razor blade. The cells were then washed twice
with PBS and treated with the experimental medium. Sixteen h later, the
medium was removed, and the cells were rinsed twice with PBS and fixed
with 4% paraformaldehyde. The number of cells migrating was then
monitored by counting the number of cells in a field that had migrated
beyond the line of scratch. At least three fields per dish were
counted, and in each experiment and each condition, migration
was monitored in three different dishes.
Cell Proliferation Assays--
Stable clones expressing either
HA-hSPRY2 or hSPRY2-RFP were plated in triplicate (100,000 cells/60-mm
dish) and grown in Dulbecco's modified Eagle's medium containing 10%
serum for 24, 48, and 72 h. Cells were then trypsinized and
counted using a hemocytometer as described by Lu and Serrero (13).
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RESULTS AND DISCUSSION |
Initially, we transfected HeLa cells with plasmid encoding either
HA-hSPRY2 or hSPRY2-RFP and selected several clones. Several G418-resistant clones were isolated. Fig.
1A shows a Western blot of
several stable cell lines which either expressed HA-hSPRY 2 (clones 3 and 9) or were G418-resistant but did not express HA-hSPRY2 (clones 5 and 8). Fluorescence microscopy was also used to confirm the presence
of HA-hSPRY2 by immunocytochemistry (Fig. 1B). Similarly, as
assessed by fluorescence microscopy, several clones expressing hSPRY2-RFP were also isolated. An example is shown in Fig.
1B with hSPRY-2RFP. Compared with control cells
expressing RFP alone, hSPRY2-RFP was exclusively located in the
cytoplasm (Fig. 1B); RFP was distributed in both the nuclei
and the cytoplasm. This would suggest that the hSPRY2 is responsible
for the differential localization of RFP and hSPRY2-RFP (Fig.
1B). This contention is supported by the similar
localization of HA-hSPRY2 (Fig. 1B). Lim et al.
(8) have demonstrated that in COS-1 cells, hSPRY2 is localized in
proximity to the microtubules, resulting in similar staining. Thus, the
localization of the tagged hSPRY2 proteins in our cells is similar to
that reported previously in Lim et al. (8). Moreover, our
data (Fig. 1B) demonstrate that neither the N terminus (HA)
tag nor the C terminus (RFP) tag interferes with the location of hSPRY2
in cells.

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Fig. 1.
Identification of HeLa clones overexpressing
HA-hSPRY2 or hSPRY2-RFP and their intracellular localization. HeLa
cells were transfected to express either HA-hSPRY2 or hSPRY2-RFP as
described under "Materials and Methods." Panel A, clonal
lines transfected with empty vector or vector-encoding HA-hSPRY2 were
screened by Western analyses with anti-HA antibody for the presence of
HA-hSPRY2. Panel B, RFP alone (control), hSPRY2-RFP-, and
HA-hSPRY2-expressing clonal cell lines were examined for the presence
of the proteins by fluorescence. For HA-hSPRY2, controls consisted of
cells which were G418-resistant but did not express the protein.
HA-hSPRY2 was detected by immunofluorescence as described under
"Materials and Methods." Note that the intracellular localization
of hSPRY2-RFP is different from RFP (control) but similar to
HA-hSPRY2.
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Because the Drosophila SPRY has been shown to be important
for tracheal formation and branching by opposing the actions of EGF and
FGF (2), we postulated that hSPRY2 would inhibit growth factor actions
and disrupt the migration of HeLa cells. For this purpose, we utilized
the scratch wound assay. As shown in Fig. 2A, control cells, which were
G418-resistant but did not express hSPRY2 (e.g.
HA-control, Fig. 1A), did not migrate in
serum-free medium but demonstrated increased migration when exposed to
either 10% serum or 100 nM EGF. Similar results (not
shown) were obtained with a second clone, which does not express
HA-hSPRY2 (Fig. 1A). In contrast to these findings, however,
when cells expressing HA-hSPRY2 were exposed to either serum or EGF, no
migration was observed (Fig. 2A). Results similar to those
shown for HA-hSPRY2 (Fig. 2A) were also observed with
another clone, which also expresses HA-hSPRY2 (not shown). In
additional migration assays, the ability of hSPRY2 to modulate the
migration of HeLa cells in response to a variety of growth factors was
tested. As shown in Fig. 2B, in control G418-resistant cells
not expressing HA-hSPRY2, serum, EGF, PDGF, and FGF markedly enhanced
migration of HeLa cells. However, in HA-hSPRY2-expressing cells,
migration in response to serum, EGF, PDGF, and FGF was inhibited (Fig.
2B). Likewise, in hSPRY2-RFP-expressing cells, but not in
control cells expressing RFP alone, migration in response to serum,
EGF, PDGF, and FGF was inhibited (Fig. 2C). These data
demonstrate that hSPRY2 opposes the migration-enhancing actions of a
number of growth factors in HeLa cells. Moreover, the data in Fig. 2
show that the N- and C-terminal tags in SPRY do not interfere with its
ability to inhibit migration in response to a variety of stimuli.

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Fig. 2.
hSPRY2 inhibits migration of HeLa cells in
response to serum and different growth factors. Panel A,
stably transfected HeLa cells which expressed HA-hSPRY2 or did not
express the protein (clone 8) were treated with and without serum
(10%) or EGF (100 nM) for 16 h in a "scratch wound
assay" as described under "Materials and Methods." The cells were
fixed and examined for migration past the scratch boundary
(dotted line). An example of several experiments is shown.
Panel B, control and HA-hSPRY2-expressing HeLa cells were
exposed to serum (10%), PDGF (10 ng/ml), FGF (150 ng/ml), and EGF (100 nM) after a scratch wound (see "Materials and
Methods"). Controls were as follows: serum-free, serum in the medium
was substituted by 0.1% bovine serum albumin; Veh-EGF, cells received
the vehicle for EGF (0.01 N HCl); Veh-FGF, cells received
the vehicle for FGF (10 mM Tris-HCl, pH 7.5); Veh-PDGF,
cells were treated with the vehicle for PDGF (10 mM acetic
acid). Sixteen h later the cells that had migrated past the boundary of
the scratch were counted. The means ± S.E. of six experiments are
presented. **, p < 0.001. Panel C, same as
B except that cells expressing RFP alone (control) and hSPRY2-RFP were
used. The means ± S.E. of six experiments are presented. **,
p < 0.001; Student's unpaired t
test.
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Because hSPRY2 inhibited migration in response to a number of mitogenic
growth factors and serum, we assessed the effects of hSPRY2 on
proliferation of HeLa cells in response to serum. In HA-hSPRY2- (Fig.
3A) and hSPRY2-RFP-expressing
cells (Fig. 3B), the ability of serum to induce growth was
markedly attenuated. Therefore, as observed with the cell migration
assay, the N- and C-terminal tags do not alter the ability of hSPRY2 to
inhibit cell proliferation.

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Fig. 3.
HA-hSPRY2 and hSPRY2-RFP inhibit the
proliferation of HeLa cells in response to serum. Panel A,
HeLa cell lines which either expressed HA-hSPRY2 or did not express
this protein were plated at a density of 105 cells per
60-mm dish and treated with 10% serum. Cell proliferation was
monitored by counting the cells at 24, 48, and 72 h after plating
the cells. The means ± S.E. from six experiments are presented.
Panel B, same as A except that cells expressing hSPRY2-RFP
or RFP alone (control) were used. The means ± S.E. from six
experiments are presented. **, p < 0.001; Student's
unpaired t test.
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Lim et al. (8) have shown that in COS-1 cells growth factors
such as EGF translocate hSPRY2 from the proximity of microtubules to
membrane ruffles. Moreover, these authors demonstrated that the
deletion of amino acids 178-194 in hSPRY2 abolishes this translocation without changing the colocalization of hSPRY2 in the proximity of
microtubules (8). On the other hand, deletion of amino acids 123-177 selectively disrupts the colocalization of hSPRY2 with tubulin without
altering its translocation in response to growth factor (8). Therefore,
using the deletion mutants described by Lim et al. (8) we
investigated whether the regions of hSPRY2 that are important for
colocalization with tubulin and translocation are important for the
functional effects of hSPRY2 on cell migration and proliferation. To
facilitate these studies, we employed the approach of transducing
proteins into cells. It has been demonstrated that the addition of an
11-amino acid sequence from the TAT protein of human immunodeficiency
virus in the N terminus of proteins permits their transport across cell
membranes, and cells readily take up the protein of interest (9, 10,
14, 15). By this method, more than 50 proteins ranging in size from
15kDa to 115kDa have been shown to be successfully transduced into
cells (14, 15). There are several advantages to this approach. First,
transduction is achieved in nearly 100% of the cells ((15) and data
not shown). Therefore, one does not have to select clonal lines and
assay these. Second, the proteins enter the cell very rapidly (9, 10,
14, 15). Thus, 10 min after the cells were exposed to TAT-HA-hSPRY2,
the protein was detected inside the cells by Western analysis (Fig.
4A). Similarly, as monitored
by fluorescence, fluorescein isothiocyanate-labeled TAT-HA-hSPRY2 and
its C-terminal deletion mutants were observed inside the cells after
exposing the cells to the protein for 30 min and up to 24 h (Fig.
4B). It should be noted that for the experiments in Fig. 4,
A and B, the cells were extensively washed to
remove the TAT-HA-hSPRY2 or its deletion mutants that may have adhered
to the outside of the cells. Moreover, the proteins were visualized by
confocal microscopy, and several "z" plane images demonstrated that
the fluorescent tagged proteins were located in the cytoplasm (data not
shown).

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Fig. 4.
Transduction of TAT-tagged hSPRY2 proteins in
HeLa cells. Panel A, HeLa cells were transduced with
TAT-HA-hSPRY2 for the times indicated. Cells were then washed
extensively, and cell lysates were subjected to Western analyses with
anti-HA antibody for the presence of TAT-HA-hSPRY2 protein. Panel
B, HeLa cells were exposed to fluorescein isothiocyanate-labeled
TAT-HA-hSPRY2 or its deletion mutants (1 µg of protein/ml) at
37 °C for the different times indicated. Cells were washed
extensively at the end of the incubation and fixed as described under
"Materials and Methods." The fluorescein isothiocyanate-labeled
hSPRY2 was detected by confocal fluorescence microscopy.
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Next we investigated whether the TAT-HA-hSPRY2, like its FLAG-tagged
counterpart (8), colocalized with microtubules and was translocated to
membrane ruffles in the presence of growth factors such as EGF. We
could not detect membrane ruffling in HeLa cells under any experimental
condition, and therefore, these experiments were performed in COS-7
cells. COS-7 cells are identical to the COS-1 cells that Lim et
al. (8) had used to characterize the cellular localization and
translocation of hSPRY2. As shown in Fig.
5A, TAT-HA-hSPRY2 was
colocalized with tubulin, a microtubule component. On the other hand,
the deletion mutant TAT-HA-hSPRY2
123-177 was not colocalized with
tubulin to the same extent as was the full-length TAT-HA-hSPRY2 (Fig.
5A). These results demonstrate that the TAT-tagged proteins
localized like their FLAG-tagged counterparts (8). Moreover, after
treatment with EGF for 10 min, the TAT-HA-hSPRY2, but not its deletion
mutant TAT-HA-hSPRY2
178-194, translocated to membrane ruffles (Fig.
5B) as described for the FLAG-tagged hSPRY2 (8). In these
latter experiments, translocation of filamentous actin to the membrane
ruffles in response to EGF was used as a positive control (8). This
control also permitted the colocalization of filamentous actin with
TAT-HA-hSPRY2 in membrane ruffles to be discerned.

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Fig. 5.
Intracellular localization of TAT-HA-hSPRY2
and its deletion mutants. COS-7 cells (104
cells/35-mm plate) were exposed to TAT-HA-hSPRY2 or its deletion
mutants (10 µg of protein/ml) for 1 h in serum-free medium.
Thereafter, the cells were washed with PBS and incubated in the
presence or absence of EGF (100 nM) for 10 min. The cells
were then fixed as described under "Materials and Methods" for
immunocytochemistry. Panel A, TAT-HA-hSPRY2 and
TAT-HA-hSPRY2 123-177 transduced COS-7 cells were exposed to
anti-tubulin (1:100 dilution) and anti-HA antibodies to visualize
tubulin and TAT-HA-hSPRY2 (and its mutant), respectively. The
localization of tubulin (red) and TAT-tagged hSPRY2 and its
123-177 mutant (green) are shown separately and
together. Panel B, COS-7 cells transduced with TAT-HA-hSPRY2
or its deletion mutant 178-194 were incubated in the presence
(lower two rows) and absence (top row) of EGF
(100 nM). After fixing (red) the cells were
exposed to Texas red isothiocyanate-labeled phalloidin, (1:100
dilution), (red) to visualize filamentous actin and anti-HA
antibody to visualize TAT-HA-tagged hSPRY2 and its deletion mutant
( 178-194) (green). Note: in the presence of EGF,
TAT-HA-hSPRY2 178-194 does not translocate to membrane ruffles.
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Having shown that the TAT-tagged hSPRY2 and its deletion mutants were
behaving like their FLAG-tagged counterparts, we investigated whether
these proteins alter migration and proliferation of HeLa cells. As
demonstrated in Fig. 6A, in
cells exposed to TAT-HA-hSPRY2, the migration in response to serum,
EGF, PDGF, and FGF was markedly diminished compared with control cells,
which were exposed to TAT-tagged GFP. These data demonstrate that the
TAT-HA-hSPRY2 was inhibiting cells in the same manner as that observed
with transfected HA-hSPRY2 (c.f. Figs. 2B and
6A). Additionally, because the TAT-GFP-transduced cells did
not alter migration (c.f. controls in Figs. 2B
and 6A), our data show that the effects of TAT-HA-hSPRY2 on
cell migration cannot be attributed to the TAT tag. Most interestingly, our data demonstrated that when cells were transduced with either hSPRY2
123-177 or hSPRY2
178-194 the inhibitory effect of the protein on cell migration in response to growth factors and serum was
obliterated. Because residues 123-177 and 178-194 in hSPRY2 are
necessary for colocalization with microtubules and translocation to
membrane ruffles, respectively (8), it would follow that the disruption
of either of these functions obliterates the activity of SPRY as an
inhibitor of cell migration in response to growth factors and
serum.

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Fig. 6.
Deletion of two C-terminal domains within
hSPRY2 abolishes its ability to inhibit cellular migration and
proliferation. Panel A, HeLa cells were grown close to
confluency and serum-starved for 24 h. Thereafter, a scratch was
made in the dishes, and cells were grown in medium containing 10 µg/ml TAT-HA-hSPRY2 or one of its deletion mutants. Controls were
grown in the presence of 10 µg/ml TAT-GFP protein. Cells in this
medium were treated with serum (10%), EGF (100 nM), FGF
(150 ng/ml), or PDGF (10 ng/ml). Controls for these conditions were
0.1% bovine serum albumin (serum control) and vehicles for the growth
factors (see legend to Fig. 2). The migration of cells was measured as
described in the legend to Fig. 2. The means ± S.E. of three
experiments are presented. Panel B, HeLa cells were plated
as described in the legend to Fig. 3 and exposed to serum (10%) either
in the presence of 10 µg/ml of one of the TAT-tagged proteins,
i.e. TAT-GFP (control) or TAT-HA-hSPRY2 or
TAT-HA-hSPRY2 123-177 or TAT-HA-hSPRY2 178-194. The medium
containing the TAT-tagged proteins was changed every 24 h. Cells
were counted 72 h after initiating the experiment. The means ± S.E. of three experiments are shown. **, p < 0.001;
Student's unpaired t test.
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To test the effects of the deletion mutants of hSPRY2 on cell
proliferation, the experiment depicted in Fig. 6B was
performed. Similar to our findings with the migration assay, in cells
exposed to TAT-HA-hSPRY2 proliferation in response to serum was
attenuated. This is similar to the findings with cells overexpressing
HA-hSPRY2 and hSPRY2-RFP (Fig. 3). However, in cells transduced with
hSPRY2
123-177 and hSPRY2
178-194, serum-induced cell growth was
not altered. Therefore, as discussed above for cell migration, these
latter data also demonstrate that the C-terminal regions encompassed by
amino acids 123-177 and 178-194 in hSPRY2 are necessary to observe inhibition of cell growth.
Although SPRY has been shown to be important in modulating the actions
of growth factors in the Drosophila and chicken, the role of
SPRY2 in mammalian systems has remained unknown. More elusive has been
the determination of the mechanism(s) and signaling pathways involved
in the actions of SPRY. Therefore, the identification of hSPRY2 as a
modulator of migration and proliferation is a starting point in the
elucidation of the mechanisms underlying the actions of this protein.
It has been shown previously that Drosophila SPRY associates
with the Grb2 homologues Drk and Gab1 and that the Ras pathway below
the activation of Ras may be the locus of SPRY action (3, 4). However,
whether the mitogen-activated protein kinase pathway in mammalian cells
is the target of hSPRY2 is a question that remains to be resolved.
Interestingly, among the various isoforms of SPRY, the N-terminal
domains are different, but the C terminus is similar. Because the
C-terminal domains that are conserved are important in the actions of
hSPRY2 as an inhibitor of cell migration and proliferation, it would
appear that these functions may be displayed by all isoforms of SPRY in
different species. Indeed, lack of this inhibitory action in SPRY
mutants of Drosophila would explain the excessive branching of trachea (1).
We used HeLa cells in our studies because we were interested in
determining whether hSPRY2 inhibits the migration and/or
proliferation of transformed carcinoma cells. Because the migration of
HeLa cells may be an index of their metastatic capacity, it would be tempting to speculate that hSPRY2 may decrease the metastatic potential
of cancer cells. Likewise, one would predict that in cells that
proliferate and migrate very rapidly, the amount of hSPRY2 protein may
be decreased. Presently, there are no antibodies available that can
detect the endogenously expressed hSPRY2 in HeLa or other cell types.
However, using reverse transcription-PCR methodology, we did detect
endogenous hSPRY2 mRNA in HeLa cells (data not shown). Whether the
amount of this transcript and its protein are regulated by specific
growth factors or other stimuli remains to be determined.
In conclusion, our data demonstrate that hSPRY2 is an inhibitor of cell
migration in response to a number of growth factors and also exerts
anti-proliferative actions. These actions of hSPRY2 require the domains
in the molecule that have been shown previously to be necessary for
colocalization with tubulin in microtubules or translocation of hSPRY2
to the ruffles. The deletion of either of these latter domains of
hSPRY2 obliterates its ability to inhibit migration and cell
proliferation. Because hSPRY2 inhibits cell migration in response to
several growth factors it appears to be a general inhibitor of receptor
tyrosine kinases. Moreover, because the conserved C-terminal domains
are important for SPRY function and because these regions are
conserved in the different isoforms, our data suggest that all SPRY
isoforms may inhibit migration and proliferation of cells.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Steven Dowdy for providing
the pTAT-HA plasmid and the pTAT-GFP construct. We also thank
Dr. Mark Krasnow, Stanford University School of
Medicine, Stanford, CA, for his help in verifying the published
nucleotide and protein sequences of mouse and human SPRY2. Finally, we
extend our gratitude to Dr. Trevor Sweatman for help with the graphics.
 |
Note Added in Proof |
After acceptance of this article for
publication, two other groups have reported that Spry4 (Lee, S. H.,
Schloss, D. J., Jarvis, L., Krasnow, M. A., and Swain, J. L. (2001)
J. Biol. Chem. 276, 4128-4133) and SPRY-1 and -2 (Impagnatiello, M. A., Weitzer, S., Gannon, G., Compagni, A., Cotten,
M., and Christofori, G. (2001) J. Cell Biol. 152, 1087-1098) inhibit endothelial cell proliferation, migration, and
differentiation. Thus the inhibition of cellular migration and
proliferation by SPRY proteins may be a general phenomenon.
 |
FOOTNOTES |
*
This work was supported by Grants HL48308 and HL07641 from
the National Institutes of Health and a postdoctoral fellowship from
the American Heart Association, Southeast Affiliate (to Y. Y.).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, 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, March 13, 2001, DOI 10.1074/jbc.M100123200
 |
ABBREVIATIONS |
The abbreviations used are:
FGF, fibroblast growth
factor;
SPRY, Sprouty;
hSPRY, human Sprouty;
RFP, red fluorescent
protein;
EGF, epidermal growth factor;
PDGF, platelet-derived growth
factor;
IPTG, isopropyl
-D-thiogalactopyranoside;
HA, hemagglutinin;
GFP, green fluorescent protein;
PBS, phosphate-buffered
saline;
PCR, polymerase chain reaction.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.