Syk, a Protein-tyrosine Kinase, Suppresses the Cell Motility
and Nuclear Factor
B-mediated Secretion of Urokinase Type
Plasminogen Activator by Inhibiting the Phosphatidylinositol
3'-Kinase Activity in Breast Cancer Cells*
Ganapati H.
Mahabeleshwar and
Gopal C.
Kundu
From the National Centre for Cell Science (NCCS), NCCS Complex,
Pune 411 007, India
Received for publication, August 30, 2002, and in revised form, November 7, 2002
 |
ABSTRACT |
Tumor growth and metastasis are
multifaceted processes that mainly involve cell adhesion, proteolytic
degradation of the extracellular matrix, and cell migration. Syk is a
member of a tyrosine kinase family that is expressed mostly in
hematopoietic cells. Syk is expressed in cell lines of epithelial
origin, but its function in these cells remains unknown. Here we report
that Syk is expressed in MCF-7 cells but not in MDA-MB-231 cells. The
overexpression of wild type Syk kinase but not kinase-negative Syk
suppressed cell motility and inhibited the activation of
phosphatidylinositol (PI) 3'-kinase in MDA-MB-231 cells. In contrast,
when Syk-specific antisense S-oligonucleotide but not the sense
S-oligonucleotide was transfected to MCF-7 cells the level of PI
3'-kinase activity as well as cell motility were increased. The
MDA-MB-231 cells transfected with wild type Syk cDNA followed by
treatment with piceatannol, a Syk inhibitor, enhanced cell motility and
PI 3'-kinase activity. Pervanadate, a phosphotyrosine phosphatase
inhibitor, induced PI 3'-kinase activity and stimulated the interaction
between the inhibitor of nuclear factor
B
(I
B
) and the
p85
domain of PI 3'-kinase through tyrosine phosphorylation of the
I
B
, which ultimately resulted in nuclear factor
B (NF
B)
activation. Pervanadate had no effect on the activation of Syk in these
cells. However, Syk suppressed the NF
B transcriptional activation
and interaction between I
B
and PI 3'-kinase by inhibiting the
tyrosine phosphorylation of I
B
. Syk, PI 3'-kinase inhibitors, and
NF
B inhibitory peptide inhibited urokinase type plasminogen
activator (uPA) secretion and cell motility in these cells. To our
knowledge, this is the first report that Syk suppresses the cell
motility and inhibits the PI 3'-kinase activity and uPA secretion by
blocking NF
B activity through tyrosine phosphorylation of I
B
.
These data further demonstrate a functional molecular link between
Syk-regulated PI 3'-kinase activity and NF
B-mediated uPA secretion,
and all of these ultimately control the motility of breast cancer cells.
 |
INTRODUCTION |
Cell migration and extracellular matrix invasion are two of the
major steps in embryonic development (1, 2), wound healing, and cancer
cell metastasis (3, 4). However, the exact molecular mechanisms that
regulate these processes are not well understood. Syk, a nonreceptor
protein-tyrosine kinase, is expressed widely in hematopoietic cells (5,
6). It has tandem amino-terminal SH21 domains and a
carboxyl-terminal kinase domain (7, 8). The SH2 domains bind
phosphorylated immunoreceptor tyrosine-based activation motifs and play
significant roles in signaling through immunoreceptors (9). ZAP-70 is a
cytoplasmic tyrosine kinase, and both Syk and ZAP-70 share the same
tandem SH2 domains at the amino terminus and play important roles in
coupling antigen and Fc receptors to downstream signaling events that
mediate diverse cellular responses including proliferation,
differentiation, and phagocytosis (9, 10). Fc receptors are members of
the family of membrane proteins, called immunoreceptors, and they are
expressed in all cells of the immune system. Both Syk and ZAP-70 are
regulated by
3 integrin-dependent cell
adhesion via phosphorylation-independent interaction with the
cytoplasmic domain of
3 integrin (11). The expression of
Syk has also been reported in cell lines of epithelial origin (12), but
its function in these cells is not well understood. Recently it has
been documented that Syk is commonly expressed in normal human breast
tissue, benign breast lesions, and low tumorigenic breast cancer cell
lines (13).
Several cytokines, growth factors, and other agents control the
regulation of cell motility. Phosphatidylinositol 3'-kinase (PI
3'-kinase) also plays significant role in regulation of cell motility
(14). Two subunits are present among all of the classes of PI
3'-kinases. One is catalytic subunit p110 (
,
,
) or p110
, and the other is regulatory subunit p85 (
,
, p55
, and p101) (15). The regulatory subunit of PI 3'-kinase is responsible for B cell
development and proliferation (16), and the catalytic subunits are
critical for chemotactic activity (17).
The activation of NF
B is regulated by a number of
proinflammatory stimuli (18, 19). The NF
B family consists of
several members including p65, p50, RelB, and c-Rel molecules (18). The
activity of NF
B is also tightly controlled by its inhibitor, I
B
family of proteins (20). NF
B forms a complex with I
B
, and the
complex can be removed from the nucleus by exportin-mediated transport
to the cytoplasm. Recent report indicated that constitutively active PI
3'-kinase controls the activation of NF
B by the association of
tyrosine-phosphorylated I
B
with the regulatory subunit of PI
3'-kinase, p85
(21). The recent data also demonstrated that interleukin-1 stimulates PI 3'-kinase dependent phosphorylation and
transactivation of NF
B without nuclear translocation of NF
B, indicating an alternative pathway other than I
B
-mediated pathway (22). But the molecular mechanism by which Syk, a tyrosine kinase, regulates the PI 3'-kinase dependent activation of NF
B in breast cancer cells is not well defined.
Urokinase-type plasminogen activator (uPA) is a member of the serine
protease family which induces the conversion of plasminogen to plasmin
(23). Plasmin regulates cell invasion by degrading matrix proteins such
as fibronectin, type IV collagen, and laminin or indirectly by
activating matrix metalloproteinases and uPA (24-26). Previous reports
shown that uPA plays a significant role in tumor growth and metastasis.
The signaling pathway by which Syk controls uPA secretion through PI
3'-kinase-dependent activation of NF
B is not clearly understood.
In this study, we demonstrate that overexpression of wild type Syk in
MDA-MB-231 cells suppressed cell motility and reduced PI 3'-kinase
activity. Syk-specific antisense phosphorothioate oligonucleotide (ASSyk), when transfected to MCF-7 cells,
increased cell motility and up-regulated PI 3'-kinase activity.
Pervanadate (pV) stimulated PI 3'-kinase activity and induced
transactivation of NF
B through tyrosine phosphorylation of I
B
,
whereas Syk down-regulated the NF
B activity by inhibiting tyrosine
phosphorylation of I
B
in these cells. Syk, PI 3'-kinase
inhibitor, and NF
B inhibitors inhibited cell motility and uPA
secretion in these cells. Taken together, Syk suppressed cell motility,
uPA secretion, and PI 3'-kinase-mediated NF
B activation by
inhibiting the interaction between the p85
subunits of PI 3'-kinase
and tyrosine-phosphorylated I
B
.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The rabbit polyclonal anti-Syk and anti-I
B
and mouse monoclonal anti-PI 3'-kinase, p85
antibodies were obtained
from Santa Cruz Biotechnology. The rabbit polyclonal
anti-phospho-I
B
and mouse monoclonal anti-uPA antibodies were
purchased from Oncogene. Piceatannol, LY294002, and ST 638 (
-cyano-(3-ethoxy-4-hydroxy-5-phenylthiomethyl)cinnamide) were
purchased from Calbiochem. Wortmannin and TRITC-conjugated goat
anti-mouse IgG were obtained from Sigma. The PIwas from ICN. [
-32P]ATP was purchased from the Board of Radiation
and Isotope Technology (Hyderabad, India). The dual luciferase reporter
assay system was obtained from Promega. The rabbit anti-phosphotyrosine
antibody and LipofectAMINE Plus reagent were purchased from Invitrogen. The FITC-conjugated goat anti-rabbit IgG was from Pharmingen. Boyden
type cell migration chambers were obtained from Corning. All other
chemicals were analytical grade.
The pV was prepared by incubating 1 M sodium orthovanadate
with 33% H2O2 in phosphate-buffered saline (pH
7.4) at room temperature for 15 min. The pH of the solution was
neutralized with 1 N HCl, and excess
H2O2 was deactivated with catalase.
Cell Culture--
The MDA-MB-231 and MCF-7 cells were purchased
from ATCC (Manassas, VA). Both MDA-MB-231 and MCF-7 cells were cultured
in Dulbecco's modified Eagle's medium. The medium was supplemented
with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 2 mM glutamine in a humidified atmosphere
of 5% CO2 and 95% air at 37 °C.
Western Blot Analysis--
To detect the level of Syk expression
in MCF-7 and MDA-MB-231 cells, the cells were lysed in lysis buffer
(1% Triton X-100 solution containing 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 2 mM EDTA). The cleared lysates were collected by
centrifugation at 12,000 × g for 15 min at 4 °C.
The protein concentration in the lysates was measured by Bio-Rad
protein assay. The lysates containing equal amounts of total proteins
were resolved by SDS-PAGE. The proteins were electrotransferred from
gel to nitrocellulose membrane. The membranes were incubated with
rabbit polyclonal anti-Syk antibody (1:200) and incubated further with anti-rabbit horseradish peroxidase-conjugated IgG (1:1,000). The membrane was washed and detected by the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences) according to the manufacturer's instructions. To check the tyrosine phosphorylation of
Syk, both MCF-7 and MDA-MB-231 cell lysates were immunoprecipitated individually with anti-Syk antibody. The immunoprecipitated samples were resolved by SDS-PAGE and detected by Western blot analysis using
rabbit anti-phosphotyrosine antibody as described above. These blots
were reprobed with anti-actin antibody as loading control.
DNA Transfection--
The wild type and kinase-negative Syk
(SykK
) cDNAs in an expression vector (pcDNA 3.1)
were a generous gift from Dr. Susette C. Mueller (Department of
Oncology, Georgetown University Medical School, Washington, D. C.).
The MDA-MB-231 cells were split 12 h prior to transfection in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
The cells were transiently transfected with Syk cDNA using
LipofectAMINE Plus according to the manufacturer's instructions.
Briefly, wild type or SykK
cDNA (8 µg) was mixed
with Plus reagent, and then cDNA reagent Plus was incubated with
LipofectAMINE. The LipofectAMINE Plus cDNA complex was added to the
cells and incubated further at 37 °C for 12 h. The control
cells received LipofectAMINE Plus alone. The cell viability was
detected by a trypan blue dye exclusion test. After incubation, the
medium was removed, and the cells were refed with fresh medium and
maintained for an additional 12 h. These transfected cells were
used for the detection of Syk and uPA expression by Western blot
analysis, NF
B activity by luciferase assay, and PI 3'-kinase
activity by kinase assay. These cells were also used for a migration
assay. In separate experiments, MCF-7 cells were transfected with
Syk-specific S-oligonucleotides according to the methods described
above. Human ASSyk (5'-TGC CGC TGC TGG CCA TGC TT-3') and SSyk (5'-AAG
CAT GGC CAG CAG CGG CA-3') with phosphorothioate linkages were
synthesized (Genomechanix). These oligonucleotides were purified by
column chromatography, and purity was checked by PAGE. These
oligonucleotide-transfected cells were used for the detection of Syk
and uPA expression by Western blot analysis, NF
B luciferase assay,
and PI 3'-kinase assay. These transfected cells were also used for a
cell migration assay. To check the dose-dependent response,
separate transfection experiments were performed with different doses
(0-10 µg) of ASSyk.
Cell Migration Assay--
The migration assay was conducted
using a Transwell cell culture chamber according to the standard
procedure as described previously (27). Briefly, the Syk-specific
S-oligonucleotide-transfected MCF-7 cells or Syk cDNA-transfected
MDA-MB-231 cells were harvested with trypsin-EDTA and centrifuged at
800 × g for 10 min. The cell suspension (5 × 105 cells/well) was added to the upper chamber of the
prehydrated polycarbonate membrane filter. The lower chamber was filled
with fibroblast-conditioned medium, which acted as chemoattractant. In
a separate experiment, either wild type Syk cDNA-transfected MDA-MB-231 or nontransfected MCF-7 cells were incubated in the absence
or presence of 0-20 µM piceatannol, a Syk inhibitor, at 37 °C for 30 min and used for a migration assay. In other
experiments, both of these nontransfected cells were treated
individually with a PI 3'-kinase inhibitor (0-100 nM
wortmannin or 0-10 µM LY294002) at 37 °C for 3 h
or with 250 µM pV, a tyrosine phosphatase inhibitor, at
37 °C for 30 min or in combination and used for migration assay. To
check whether NF
B or uPA is involved in migration, both of these
nontransfected cell lines were treated individually with 100 µg/ml
SN-50, 100 µg/ml SN-50M, 10 µg/ml actinomycin-D, 50 µM curcumin, 5 ng/ml PMA, 10 µg/ml monoclonal uPA
antibody at 37 °C for 6 h. The transfected cells were also
treated with anti-uPA antibody and used for migration assay. After
treatment, these cells were incubated in a humidified incubator in 5%
CO2 and 95% air at 37 °C for 16 h. The nonmigrated
cells on the upper side of the filter were scraped, and the filter was
washed. The migrated cells in the reverse side of the filter were fixed
with methanol and stained with Giemsa. The migrated cells on the filter
were counted under an inverted microscope (Olympus). The experiments were repeated in triplicate. Preimmune IgG served as nonspecific control. These treated cells were also used for the detection of uPA by
Western blot analysis.
Immunoprecipitation and in Vitro Kinase Assay--
To examine
the autophosphorylation of Syk in MCF-7 and MDA-MB-231 cells, the cells
were immunoprecipitated with rabbit polyclonal anti-Syk antibody and
subjected to the the kinase assay. Briefly, the cells were lysed in
lysis buffer (1% Triton X-100 solution containing 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 2 mM EDTA), and the protein concentration in the cleared
lysates was measured by Bio-Rad protein assay. The samples containing equal amounts of total proteins were immunoprecipitated with rabbit polyclonal anti-Syk antibody according to the manufacturer's
instructions (Roche Molecular Biochemicals). The immunoprecipitated
samples were incubated with 2 µCi of [
-32P]ATP in
kinase assay buffer (50 mM Hepes buffer (pH 8.0) containing 10 mM Na3VO4, 50 mM
MgCl2, and 5 mM MnCl2) at 30 °C
for 10 min. The samples were resolved by SDS-PAGE, dried, and
autoradiographed. In separate experiments, the MCF-7 cells were treated
in the absence or presence of 250 µM pV for 0-60 min.
The cell lysates were immunoprecipitated with anti-Syk antibody, and a
Syk kinase assay was performed as described above.
To check the role of Syk in the regulation of PI 3'-kinase activity,
the MCF-7 cells were transfected with SSyk or ASSyk, the MDA-MB-231
cells were transfected with wild type or SykK
cDNA,
and the PI 3'-kinase activity was measured. Cells were lysed in lysis
buffer as described earlier and centrifuged at 12,000 × g for 15 min. The cleared lysates were collected, and the
protein concentration was measured by Bio-Rad protein assay. The PI
3'-kinase assay was performed with slight modification (28). Briefly,
cell lysates were immunoprecipitated with mouse monoclonal anti-p85
antibody, and the immunoprecipitated samples were incubated in kinase
assay buffer (25 mM Hepes (pH 7.4), 10 mM
MgCl2, and 1 mM EDTA) containing 0.25 mg/ml
phosphatidylinositol, 100 mM ATP, and 15 µCi of
[
-32P]ATP and incubated at 30 °C for 10 min. The
reaction was terminated by the addition of acidified
chloroform:methanol (2:1). Lipids were extracted according to the
procedure described previously (29) and separated on oxalate-treated
plastic TLC plates using a solvent system consisting of chloroform,
methanol, and 20% methylamine (65:35:10 v/v/v). The spots
corresponding to the position of radioactive phosphatidylinositol
phosphate (PIP) were visualized by autoradiography. In separate
experiments, wild type Syk cDNA-transfected MDA-MB-231 or
nontransfected MCF-7 cells were individually treated with 0-10 µM piceatannol, a Syk kinase inhibitor, at 37 °C for
30 min and used for the PI 3'-kinase assay. In other experiments,
nontransfected MDA-MB-231 or MCF-7 cells were also pretreated with 250 µM pV at room temperature for 0-30 min and used for the
PI 3'-kinase assay.
To check the role of pV on the tyrosine phosphorylation of I
B
and
subsequent interaction between I
B
and PI 3'-kinase, both MCF-7
and MDA-MB-231 cells were treated individually with 250 µM pV tyrosine phosphatase inhibitor for 0-30 min, and
the total proteins in the lysates were measured by Bio-Rad protein assay. Half of the lysates were immunoprecipitated with
nonphosphorylated anti-I
B
antibody. The immunoprecipitated
samples were resolved by SDS-PAGE, and the level of
tyrosine-phosphorylated I
B
was detected by Western blot analysis
using anti-phosphotyrosine antibody. The remaining half of the lysates
was resolved by SDS-PAGE, and the serine-phosphorylated I
B
was
detected by Western blot analysis using anti-phospho-I
B
(serine-specific) antibody. Similarly, both MCF-7 and MDA-MB-231 cells
were treated individually in the absence or presence of 250 µM pV alone or with 100 µM ST 638, a
tyrosine kinase inhibitor, along with 250 µM pV at
37 °C for 0-30 min. The cells were lysed, and the lysates were
immunoprecipitated with anti-PI 3'-kinase, p85
antibody. The
immunoprecipitated samples were resolved by SDS-PAGE, and the tyrosine
phosphorylation of I
B
in lysates was detected by Western blot
analysis using rabbit anti-phosphotyrosine antibody. The blots were
reprobed with anti-actin antibody as loading control.
To check the effect of Syk on tyrosine phosphorylation of I
B
and
subsequent interaction between I
B
and PI 3'-kinase, the Syk-specific S-oligonucleotide-transfected MCF-7 or Syk
cDNA-transfected MDA-MB-231 cells were treated individually with
250 µM pV for 15 min, and the cells were lysed in lysis
buffer. Total proteins in the lysates were measured by Bio-Rad protein
assay. The cell lysates containing equal amounts of total proteins were
immunoprecipitated with anti-p85
antibody. The samples were resolved
by SDS-PAGE and analyzed by Western blot analysis using
anti-phosphotyrosine antibody. The blots were reprobed with anti-actin antibody.
Immunofluorescence Study--
Both MDA-MB-231 and MCF-7 cells
were grown in monolayer on glass slides and then treated individually
in the absence or presence of 250 µM pV at room
temperature for a period of 0-30 min. The cells were fixed with
paraformaldehyde for 10 min, blocked with 5% bovine serum albumin for
30 min, and washed with phosphate-buffered saline (pH 7.4). The fixed
cells were incubated with a mixture of mouse monoclonal anti-PI
3'-kinase, p85
(1:10 dilution), and rabbit polyclonal anti-I
B
antibodies (1:20 dilution) at room temperature for 2 h. The cells
were washed with phosphate-buffered saline (pH 7.4) and incubated with
a mixture of FITC-conjugated anti-rabbit IgG and TRITC-conjugated
anti-mouse IgG. The cells were washed, mounted with coverslips, and
analyzed under confocal microscopy (Zeiss).
NF
B Luciferase Reporter Gene Assay--
The semiconfluent
MCF-7 cells grown in 24-well plates were transiently transfected with
Syk-specific S-oligonucleotides and a luciferase reporter construct
(pNF
B-Luc) containing five tandem repeats of the NFkB binding site
(a generous gift from Dr. Rainer de Martin, University of Vienna,
Vienna, Austria) using LipofectAMINE Plus reagent for 12 h.
Similarly, the MDA-MB-231 cells were transfected with wild type or
SykK
cDNA and pNF
B-Luc under the same conditions
described above. The transfection efficiency was normalized by
cotransfecting the cells with pRL vector (Promega) containing a
full-length Renilla luciferase gene under the control of a
constitutive promoter. In separate experiments, MCF-7 cells transfected
with pNF
B-Luc or MDA-MB-231 cells transfected with wild type Syk and
pNF
B-Luc were treated with 0-10 µM piceatannol. In
other experiments, both MCF-7 and MDA-MB-231 cells were transfected
individually with pNF
B-Luc and treated with either 100 µM pV alone for 30 min, pV with 1-100 nM
wortmannin or 1-10 µM LY294002 for 3 h. Cells were
harvested in passive lysis buffer (Promega). The luciferase activities
were measured by luminometer (Lab Systems) using the dual luciferase
assay system according to the manufacturer's instructions (Promega).
Changes in luciferase activity with respect to the control were calculated.
 |
RESULTS |
Detection of Tyrosine-phosphorylated Syk Expression by Western Blot
Analysis and Autophosphorylation by in Vitro Kinase Assay--
The
expression of Syk was analyzed by SDS-PAGE followed by Western blot in
MCF-7 (Fig. 1A, lane
1) and MDA-MB-231 (lane 2) cells. To assess the
autophosphorylation activity of Syk, both of these cell lines were
lysed in lysis buffer, and the lysates were immunoprecipitated with
rabbit polyclonal anti-Syk antibody. The immunoprecipitated samples
were incubated with [
-32P]ATP in kinase assay buffer.
The samples were resolved by SDS-PAGE and autoradiographed. Fig.
1B shows the autophosphorylated Syk expression in MCF-7
cells (lane 1), but this was absent in MDA-MB-231 cells
(lane 2). To check whether the phosphorylation of Syk is tyrosine-specific, both of the cell lysates were immunoprecipitated with anti-Syk antibody and detected by Western blot analysis using rabbit anti-phosphotyrosine antibody. The MCF-7 (Fig. 1C,
lane 1) but not MDA-MB-231 (lane 2) cells
recognized tyrosine-phosphorylated Syk expression, suggesting that
tyrosine residue of Syk is involved in autophosphorylation. All of
these blots were reprobed with anti-actin antibody as loading control.
To check whether pV, a tyrosine phosphatase inhibitor, has any role in
the autophosphorylation activity of Syk, the MCF-7 cells were treated
with 250 µM pV for 0-60 min, and the cell lysates were
immunoprecipitated with anti-Syk antibody. The activity of Syk in the
immunoprecipitated samples was detected by kinase assay, and the
results showed that pV had no effect on Syk activity in these cells
(Fig. 1D, lanes 1-5).

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Fig. 1.
Expression and autophosphorylation of Syk in
breast cancer cells. A, equal amounts of total proteins
in cell lysates from MCF-7 and MDA-MB-231 cells were resolved by
SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-Syk
antibody. Lane 1, MCF-7 cells; lane 2, MDA-MB-231
cells. B, equal amounts of total proteins in both cell
lysates were immunoprecipitated with rabbit polyclonal anti-Syk
antibody, and the immunoprecipitates were incubated with 2 µCi of
[ -32p]ATP in kinase assay buffer as described under
"Experimental Procedures." The sample was resolved by SDS-PAGE and
autoradiographed. Lane 1, MCF-7 cells; lane 2,
MDA-MB-231 cells. C, cell lysates containing equal amounts
of total proteins were immunoprecipitated with anti-Syk antibody, and
the immunocomplex was resolved by SDS-PAGE and analyzed by Western blot
using rabbit anti-phosphotyrosine antibody. Lane 1, MCF-7
cells; lane 2, MDA-MB-231 cells. The arrow
indicates the Syk-specific band. As loading controls, all of these
blots were reprobed with goat polyclonal anti-actin antibody
(lower panels in A-C). D, effect of
pV on autophosphorylation activity of Syk in MCF-7 cells. The cells
were treated with 250 µM pV for 0-60 min, and the cell
lysates containing equal amounts of total proteins were
immunoprecipitated with anti-Syk antibody. The immunoprecipitates were
incubated with 2 µCi of [ -32p]ATP in kinase assay
buffer as described above. The sample was resolved by SDS-PAGE and
autoradiographed. Lane 1, control; lane 2, pV 5 min; lane 3, pV 15 min; lane 4, pV 30 min; and
lane 5, pV 60 min.
|
|
To control the expression of Syk and to check the status of
Syk-dependent downstream signaling events and cell
motility, the low invasive MCF-7 cells were transfected with
Syk-specific phosphorothioate-linked SSyk or ASSyk oligonucleotides in
the presence of LipofectAMINE Plus. The expression of Syk was detected
by Western blot analysis, and the data indicated that there was
significant expression of Syk in cells transfected with LipofectAMINE
Plus alone (Fig. 2A, lane 1) or SSyk transfected cells (lane 2), but
the level of Syk was reduced drastically when cells were transfected
with ASSyk (lane 3). Similarly, the MDA-MB-231 cells were
transfected with wild type or SykK
cDNA in the
presence of LipofectAMINE Plus, and expression of Syk was determined by
Western blot analysis. The expression of Syk was absent in cells
transfected with LipofectAMINE Plus alone (Fig. 2B,
lane 1), whereas a significant level of Syk expression was
observed in both wild type (lane 2) and SykK
-transfected cells (lane 3) . The level of Syk was also
quantified densitometrically and analyzed statistically (Fig. 2,
A and B, lower panels). These
transfected cells were used for cell migration assays and for the
detection of downstream signaling molecules.

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Fig. 2.
Detection of Syk in Syk-specific
S-oligonucleotide-transfected MCF-7 and Syk cDNA
transfected-MDA-MB-231 cells by Western blot analysis. A,
MCF-7 cells were transiently transfected with SSyk or ASSyk with
LipofectAMINE Plus. The cell lysates containing equal amounts of total
proteins were resolved by SDS-PAGE and analyzed by Western blot using
anti-Syk antibody. Lane 1, with LipofectAMINE Plus;
lane 2, with SSyk; lane 3, with ASSyk.
B, MDA-MB-231 cells were transiently transfected with wild
type or SykK cDNA in pcDNA 3.1 with LipofectAMINE
Plus. Equal amounts of total proteins in cell lysates were separated by
SDS-PAGE and analyzed by Western blot using anti-Syk antibody.
Lane 1, with LipofectAMINE Plus; lane 2, with
wild type Syk cDNA; lane 3, with SykK
cDNA. The arrow indicates the Syk-specific band. The
expression of Syk was quantified by densitometric analysis and is
represented in the form of a bar graph. The mean value of
triplicate experiments is indicated in the lower panels of
A and B.
|
|
Syk and PI 3'-kinase Play Critical Roles in Cell Migration--
To
ascertain the roles of Syk and PI 3'-kinase in the regulation of cell
migration, both MDA-MB-231 and MCF-7 cells were used for the migration
assay. The MCF-7 cells were transfected with Syk-specific
S-oligonucleotides and performed the migration assay. The data
demonstrated that ASSyk-transfected cells showed a dramatic increase of
cell migration (252%) compared with the cells transfected with
LipofectAMINE Plus alone (100%) or SSyk-transfected cells (98%) (Fig.
3A). Similarly, in the highly
invasive MDA-MB-231 cells, when transfected with wild type Syk
cDNA, there was drastic reduction of cell migration (24%) compared
with cells transfected with LipofectAMINE Plus alone (100%) or
SykK
-transfected cells (97%) (Fig. 3B). These
data suggest that wild type Syk suppressed cell migration, whereas
SykK
had no effect on suppression of cell migration.
Pretreatment of MCF-7 cells with increasing concentrations of
piceatannol (0-20 µM), a Syk inhibitor followed by
migration assays, enhanced cell migration (100-258%) in a
dose-dependent manner (Fig. 3C). To prove
further the role of piceatannol on migration of wild type Syk-transfected MDA-MB-231 cells, the transfected cells were treated with different doses of piceatannol (0-10 µM), and then
the migration assay was conducted. The results indicated that there was
enhancement of cell migration (37-98%) when the cells were
transfected with wild type Syk followed by treatment with increasing
concentrations of piceatannol compared with piceatannol-untreated, wild
type Syk-transfected (25%) cells (Fig. 3D). The
SykK
-transfected cells showed 98% migration, whereas the
number of cells that migrated using LipofectAMINE Plus-transfected
cells were considered as 100% (Fig. 3D). The
SykK
-transfected cells had no effect on suppression of
cell migration in MDA-MB-231 cells. We also studied the effects of
wortmannin and LY294002 (PI 3'-kinase inhibitors) on the migration of
MCF-7 and MDA-MB-231 cells. Both MCF-7 and MDA-MB-231 cell lines were treated individually with different doses of wortmannin (0-100 nM) or LY294002 (0-10 µM) as described
earlier and used for cell migration assays. The results indicated that
both of these inhibitors independently suppressed the cell migration of
MCF-7 (Fig. 3E) and MDA-MB-231 (Fig. 3F) cells in
a dose-dependent manner. These data further suggested that
PI 3'-kinase plays significant roles in regulating cell migration in
these cells. To investigate whether pV, a tyrosine phosphatase
inhibitor, regulates PI 3'-kinase dependent cell migration, both cell
lines were treated with 250 µM pV in the absence or
presence of 0-100 nM wortmannin or 0-10 µM
LY294002 and used for the migration assay. The data demonstrated that
pV induced migration in both cell lines. However, pV-induced migration is suppressed by wortmannin or LY294002 (PI 3'-kinase inhibitors) in
these cells (Fig. 3, E and F).

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Fig. 3.
A, effect of transfection of
MCF-7 cells with Syk-specific S-oligonucleotides on cell migration. The
cells were transiently transfected with SSyk or ASSyk using
LipofectAMINE Plus. The transfected cells (5 × 105
cells/well) were used for cell migration assay as described under
"Experimental Procedures." The cells transfected with ASSyk showed
a dramatic increase of cell migration compared with cells transfected
with SSyk or LipofectAMINE Plus alone. B, effect of
transfection of MDA-MB-231 cells with wild type or SykK
cDNA on cell migration. The cells were transfected with wild type
or SykK cDNA, and the transfected cells were used for
the cell migration assay. The cells transfected with wild type Syk
cDNA showed a drastic reduction of cell migration compared with
cells transfected with SykK or with LipofectAMINE Plus
alone. C, effect of piceatannol, a Syk inhibitor, on
migration of MCF-7 cells. The cells were pretreated with 0-20
µM piceatannol at 37 °C for 30 min and then used for
migration assay. Piceatannol enhanced the migration in a
dose-dependent manner. D, effect of piceatannol
on migration of Syk cDNA transfected MDA-MB-231 cells. The cells
transfected with wild type Syk cDNA were treated with increasing
concentrations (0-10 µM) of piceatannol and used for the
migration assay as described above. Wild type Syk cDNA-transfected
cells treated with increasing concentrations of piceatannol showed
enhancement of cell migration compared with nontreated, wild type Syk
cDNA-transfected cells. Cells transfected with SykK
or with LipofectAMINE Plus alone showed maximum migration.
E, effect of pV and a PI 3'-kinase inhibitor (wortmannin or
LY294002) on migration of MCF-7 cells. MCF-7 cells were treated
individually with pV, wortmannin, LY294002, or with a combination of pV
and wortmannin or pV and LY294002 and used for migration assay as
described under "Experimental Procedures." Wortmannin and LY294002
independently reduced the migration in a dose-dependent
manner, whereas pV induced the migration. pV-induced migration was also
blocked by wortmannin or LY294002. F, effect of pV and PI
3'-kinase inhibitor (wortmannin or LY294002) on migration of MDA-MB-231
cells. MDA-MB-231 cells were treated individually with pV, wortmannin,
and LY294002 under different conditions as described above and used for
migration assay. Wortmannin and LY294002 reduced migration, whereas pV
induced it. The pV-induced migration was also inhibited by wortmannin
or LY294002. In all of these experiments, the results are expressed as
the means ± S.E. of three determinations.
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Syk Down-regulates PI 3'-Kinase Activity in MCF-7 (Low Invasive)
and MDA-MB-231 (Highly Invasive) Cells--
To check the role of Syk
in suppression of PI 3'-kinase activity in MCF-7 and MDA-MB-231 cells,
a PI 3'-kinase assay was performed under different conditions. These
cells were lysed individually in lysis buffer, and the lysates
containing equal amounts of total proteins were immunoprecipitated with
anti-p85
antibody. The immunoprecipitated samples were used for the
kinase assay. The radioactive PIP was separated by TLC and visualized
by autoradiography. The activity of PI 3'-kinase was higher in
MDA-MB-231 cells (Fig. 4A,
lane 2) compared with MCF-7 cells (lane 1). The
MCF-7 cells were transfected with Syk-specific S-oligonucleotides,
immunoprecipitated with anti-p85
antibody, and used for the PI
3'-kinase assay. The MCF-7 cells transfected with ASSyk showed a higher
level of PI 3'-kinase activity (Fig. 4B, lane 3)
compared with LipofectAMINE Plus alone (lane 1) or
SSyk-transfected (lane 2) cells. These data suggested that
Syk suppressed PI 3'-kinase activity in MCF-7 cells. Moreover, our
previous data indicated that MCF-7 cells transfected with ASSyk showed
enhancement of cell migration compared with SSyk-transfected cells.
These data further demonstrated that Syk suppressed the cell migration
by inhibiting the PI 3'-kinase activity in MCF-7 cells (Figs.
3A and 4B).

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Fig. 4.
PI 3'-kinase activity. A,
equal amounts of total proteins from the lysates of MCF-7 and
MDA-MB-231 cells were immunoprecipitated with mouse monoclonal
anti-p85 antibody, and the immunocomplexes were assayed for their
ability to phosphorylate PI to PIP using [ -32p]ATP at
30 °C for 10 min. The PIP was resolved by TLC and autoradiographed.
Lane 1, MCF-7 cells; lane 2, MDA-MB-231 cells.
B, the serum-starved MCF-7 cells were transfected with
Syk-specific S-oligonucleotides, and then PI 3'-kinase activity was
measured as described under "Experimental Procedures." Lane
1, with LipofectAMINE Plus; lane 2, with SSyk;
lane 3, with ASSyk. C, MDA-MB-231 cells were
transfected with Syk cDNA and used for PI 3'-kinase assay.
Lane 1, with LipofectAMINE Plus; lane 2, with
SykK ; and lane 3, with wild type Syk.
D, MDA-MB-231 cells were transfected with wild type Syk
cDNA followed by treatment with increasing concentrations (0-10
µM) of piceatannol at 37 °C for 30 min and used for PI
3'-kinase assay. Lane 1, with LipofectAMINE Plus alone;
lanes 2-4, cells were transfected with wild type Syk
cDNA and then treated with increasing concentrations of
piceatannol. Lane 2, without piceatannol; lane 3,
with 5 µM piceatannol; lane 4, with 10 µM piceatannol. E, MDA-MB-231 cells were
treated with 250 µM pV at room temperature for 0-30 min,
and the cell lysates were used for the PI 3'-kinase assay. Lane
1, untreated cells; lane 2, with pV for 5 min;
lane 3, with pV for 15 min; lane 4, with pV for
30 min. F, MCF-7 cells were treated with 0-10
µM piceatannol for 30 min or with 250 µM pV
for 0-30 min and used for the PI 3'-kinase assay. Lane 1,
untreated cells; lane 2, with 5 µM
piceatannol; lane 3, with 10 µM piceatannol;
lane 4, with pV for 15 min; lane 5, with pV for
30 min. In all the cases, the upper arrows indicate the
PIP-specific bands. All of these bands in A-F were
quantified by densitometric analysis and are represented in the form of
a bar graph. The mean value of triplicate experiments is
indicated.
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The MDA-MB-231 cells were transfected with SykK
or wild
type Syk cDNA, cell lysates were immunoprecipitated with
anti-p85
antibody, and the kinase assay was performed. The cells
transfected with wild type Syk cDNA reduced the PI 3'-kinase
activity (Fig. 4C, lane 3) compared with
LipofectAMINE Plus-transfected cells (lane 1) or cells
transfected with SykK
(lane 2). Because Syk
suppressed the PI 3'-kinase activity, we sought to determine whether
piceatannol, a Syk inhibitor, reversed the PI 3'-kinase activity.
Accordingly, MDA-MB-231 cells were transfected with wild type Syk
cDNA, treated with different doses of piceatannol (0-10
µM), and then the kinase assay was conducted. The results
demonstrated that piceatannol dose-dependently increased the PI 3'-kinase activity in these cells (Fig. 4D,
lanes 3 and 4) compared with untreated, wild type
Syk-transfected cells (lane 2). The cells transfected with
LipofectAMINE Plus alone showed a higher level of PI 3'-kinase activity
(lane 1). To check whether pV, a tyrosine phosphatase
inhibitor, regulates PI 3'-kinase activity, the MDA-MB-231 cells were
treated with 250 µM pV for 0-30 min, and then PI
3'-kinase activity was measured. The results indicated that pV induces
the PI 3'-kinase activity in a time-dependent manner in
these cells (Fig. 4E, lanes 1-4). Similarly,
MCF-7 cells were also treated with either piceatannol or pV and then
used for the kinase assay. The data showed that both piceatannol (Fig. 4F, lanes 2 and 3) and pV (lanes
4 and 5) individually enhanced PI 3'-kinase activity
compared with untreated (lane 1) MCF-7 cells. These data
strongly suggested that Syk down-regulates the PI 3'-kinase activity in
both MCF-7 and MDA-MB-231 cells. All of these PIP-specific bands were
quantified by densitometric analysis and are represented in the form of
a bar graph. The mean value of triplicate experiments is indicated.
Syk Suppresses pV-induced Tyrosine Phosphorylation of I
B
and
Subsequent Interaction between Tyrosine-phosphorylated I
B
and PI
3'-Kinase--
To assess the role of pV on tyrosine phosphorylation of
I
B
in breast cancer cells, both MCF-7 and MDA-MB-231 cells were treated individually with 250 µM pV for 0-30 min and
immunoprecipitated with anti-I
B
antibody. Half of the
immunoprecipitated samples were separated by SDS-PAGE and immunoblotted
with anti-phosphotyrosine antibody. The remaining half of the samples
was analyzed by Western blot analysis using anti-I
B
(phosphoserine-specific) antibody. The data demonstrated that pV
induces the tyrosine phosphorylation of I
B
in both MCF-7 (Fig.
5A, upper panel,
lanes 1-4) and MDA-MB-231 (lanes 5-8) cells,
but the pV-induced tyrosine phosphorylation was higher in MDA-MB-231
cells. In contrast, the serine phosphorylation of I
B
was
unchanged in both cell lines (Fig. 5A, lower
panel, lanes 1-8), suggesting that pV regulates the
tyrosine phosphorylation but not serine phosphorylation of I
B
in
these cells. The bands were analyzed by densitometry, and the values of
-fold changes are indicated (Fig. 5A).

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Fig. 5.
A, role of pV in stimulation of
interaction between I B and PI 3'-kinase through tyrosine
phosphorylation of I B . Both MCF-7 and MDA-MB-231 cells were
individually treated with pV for 0-30 min, and the cell lysates
containing equal amounts of total proteins were immunoprecipitated with
nonphosphorylated anti-I B antibody. Half of the
immunoprecipitated samples were resolved by SDS-PAGE and analyzed by
Western blot using anti-phosphotyrosine antibody (upper
panel in A), and the remaining half of the samples were
immunoblotted with phosphoserine-specific anti-I B antibody
(lower panel in A). Lanes 1-4, MCF-7
cells. Lane 1, control; lane 2, with pV for 5 min; lane 3, with pV for 15 min; lane 4, with pV
for 30 min. Lanes 5-8, MDA-MB-231 cells. Lane 5,
control; lane 6, with pV for 5 min; lane 7, with
pV for 15 min; lane 8, with pV for 30 min. The
arrow in the upper panel of A
indicates a tyrosine-phosphorylated I B -specific band, and in the
lower panel of A the arrow shows a
serine-phosphorylated I B -specific band. Note that the level of
tyrosine phosphorylation of I B is increased in presence of pV
(upper panel of A), but pV has no effect on
serine phosphorylation of I B (lower panel of
A) in both cell lines, indicating that pV induces tyrosine
phosphorylation of I B . More tyrosine phosphorylation is observed
in MDA-MB-231 cells. All of these bands were quantified by
densitometric analysis, and the values of -fold changes are indicated.
MCF-7 cells (B) and MDA-MB-231 cells (C) cells
were treated individually with 250 µM pV at room
temperature for a period of 0-30 min. In a separate experiment, the
cells were also pretreated with ST 638, a protein-tyrosine kinase
inhibitor (400 nM) and then treated with 250 µM pV. The cell lysates were immunoprecipitated with
monoclonal anti-p85 antibody. The immunocomplexes were resolved by
SDS-PAGE and analyzed by Western blot using anti-phosphotyrosine
antibody. Lane 1, control; lane 2, with pV for 15 min; lane 3, with pV for 30 min; lane 4, with ST
638 and then with pV for 30 min. The arrows indicate the
tyrosine-phosphorylated I B -specific band. As loading controls,
both of these blots were reprobed with anti-actin antibody (lower
panels in B and C). D, effect of
Syk-specific S-oligonucleotides on suppression of pV-induced tyrosine
phosphorylation of I B and subsequent interaction between
tyrosine-phosphorylated I B and PI 3'-kinase. MCF-7 cells were
transfected with Syk-specific S-oligonucleotides, and MDA-MB-231 cells
were transfected with Syk cDNA in presence of LipofectAMINE Plus.
Both of these transfected cell lines were treated individually with 250 µM pV and lysed in lysis buffer. The lysates were
immunoprecipitated with anti-p85 antibody. The immunoprecipitated
samples were separated by SDS-PAGE and analyzed by Western blot using
anti-phosphotyrosine antibody. Lane 1, with LipofectAMINE
Plus alone; lane 2, with SSyk; lane 3, with ASSyk
(upper panel of D, lanes 1-3, MCF-7
cells); lane 4, with LipofectAMINE Plus alone; lane
5, with SykK ; lane 6, with wild type Syk
(upper panel of D, lanes 4-6,
MDA-MB-231 cells). Note that MCF-7 cells transfected with ASSyk
induced, but MDA-MB-231 cells transfected with wild type Syk reduced,
the tyrosine phosphorylation of I B . As loading controls, the same
blots were reprobed with anti-actin antibody (lower panel of
D). E and F, role of pV in regulation
of colocalization of I B and PI 3'-kinase. MCF-7 (E)
and MDA-MB-231 (F) cells were treated with 250 µM pV at room temperature for 0-30 min, fixed, and
incubated with a mixture of monoclonal anti-PI 3'-kinase, p85 , and
rabbit polyclonal anti-I B antibodies. These cells were incubated
further with a mixture of FITC-conjugated anti-rabbit IgG and
TRITC-conjugated anti-mouse IgG antibodies and analyzed under confocal
microscopy. a-c, untreated cells; d-f, pV for
15 min and g-i, pV for 30 min. a, d,
and g are stained with anti-PI 3'-kinase, p85 , and
TRITC-conjugated antibodies, respectively; b, e,
and h are stained with anti-I B and FITC-conjugated
antibodies, respectively; and c, f, and
i are overlapping forms of TRITC and FITC stained cells.
Note that there was more colocalization of I B and PI 3'-kinase,
p85 in pV-treated cells (d-i) compared with untreated
cells (a-c), but higher levels of colocalization were seen
in MDA-MB-231 cells.
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To confirm further the effects of pV on tyrosine phosphorylation of
I
B
and regulation of interaction between phosphorylated I
B
and the p85
subunit of PI 3'-kinase, both cell lines were treated
individually with pV under the same conditions described above. The
cell lysates were immunoprecipitated with anti-p85
antibody and
immunoblotted with anti-phosphotyrosine antibody. In separate
experiments, cells were pretreated with ST 638, a protein-tyrosine
kinase inhibitor, then treated with pV, immunoprecipitated with
anti-p85
antibody, and detected by immunoblotting using anti-phosphotyrosine antibody. The data indicate that pV induces the
interaction between I
B
and p85
subunit of PI 3'-kinase through
tyrosine phosphorylation of I
B
in MCF-7 (Fig. 5B,
upper panel, lanes 1-3) and MDA-MB-231 (Fig.
5C, upper panel, lanes 1-3) cells,
whereas ST 638 suppresses pV-induced tyrosine phosphorylation in both
cell lines (Fig. 5, B and C, upper
panel, lane 4). As loading controls, both blots were
reprobed with anti-actin antibody (Fig. 5, B and
C, lower panels).
To delineate the role of Syk on pV-induced tyrosine phosphorylation of
I
B
and subsequent interaction between tyrosine-phosphorylated I
B
and the p85
subunit of PI 3'-kinase, MCF-7 cells were
transfected with Syk-specific S-oligonucleotides, and MDA-MB-231 cells
were transfected with wild type or SykK
cDNA. Both
cell lines were treated individually with 250 µM pV and
lysed in lysis buffer. The cell lysates containing equal amounts of
total proteins were immunoprecipitated with anti-p85
antibody and
detected by Western blot analysis using anti-phosphotyrosine antibody.
MCF-7 cells transfected with ASSyk showed a higher level of tyrosine
phosphorylation of I
B
(Fig. 5D, upper
panel, lane 3), but this phosphorylation was not
detected in SSyk-transfected cells (lane 2) or the cells
transfected with LipofectAMINE Plus alone (lane 1).
Similarly, MDA-MB-231 cells transfected with wild type Syk cDNA
showed a reduction of tyrosine phosphorylation of I
B
(lane
6) compared with SykK
-transfected cells (lane
5) or cells transfected with LipofectAMINE Plus alone (lane
4). As loading controls, the same blots were reprobed with
anti-actin antibody (lower panel). These data suggest that
Syk suppresses the tyrosine phosphorylation of I
B
and subsequent interaction between I
B
and PI 3'-kinase in both cell lines.
To determine whether pV induces the colocalization of I
B
with the
p85 subunit of PI 3'-kinase, both cell lines were treated individually
in absence or presence of 250 µM pV, fixed, and incubated with a mixture of anti-I
B
and anti-p85
antibodies. These cells were incubated again with a mixture of anti-rabbit FITC- and anti-mouse TRITC-conjugated IgG. The immunofluorescence-labeled cells were detected by confocal microscopy. The data indicated that pV enhances the colocalization of I
B
and PI 3'-kinase in both cell lines (Fig. 5, E and F, d-i) compared with
untreated cells (a--c), but a higher level of
colocalization was observed in MDA-MB-231 cells (Fig.
5F).
Syk Inhibits Transcriptional Activity of NF
B--
Because Syk
suppressed the tyrosine phosphorylation of I
B
, we sought to
determine whether Syk has any role in the transactivation of NF
B in
breast cancer cells. Accordingly, MCF-7 cells were transfected with
Syk-specific S-oligonucleotides and luciferase reporter construct
pNF
B-Luc. Similarly, MDA-MB-231 cells were transfected with wild
type or SykK
cDNA followed by transfection with
pNF
B-Luc. The transfection efficiency was normalized by
cotransfecting the cells with pRL vector. Changes in luciferase
activity with respect to control were calculated. The -fold changes
were calculated, and the means of triplicate determinations were
plotted. In separate experiments, these transfected cells were also
treated individually with increasing concentrations of piceatannol. The
data indicated that there was at least an 8-fold increase of NF
B
luciferase activity in MCF-7 cells transfected with ASSyk compared with
LipofectAMINE Plus or SSyk-transfected cells (Fig.
6A). In contrast, MDA-MB-231
cells transfected with wild type Syk showed a 10-fold decrease in
NF
B luciferase activity compared with cell transfected with
SykK
or LipofectAMINE Plus alone (Fig. 6B).
Piceatannol enhanced the NF
B activity in both of these cell lines in
a dose-dependent manner (Fig. 6, A and
B). These data clearly demonstrated that Syk suppressed the
transcriptional activity of NF
B in both cell lines.

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Fig. 6.
A and B, effect of Syk on
NF B transactivation in MCF-7 (A) and MDA-MB-231
(B) cells. A, MCF-7 cells were transiently
transfected with Syk-specific S-oligonucleotides and luciferase
reporter construct (pNF B-Luc) in the presence of LipofectAMINE Plus
as described under "Experimental Procedures." The transfection
efficiency was normalized by cotransfecting the cells with pRL vector.
The non-S-oligonucleotide-transfected cells were also treated with
varying concentrations (0-10 µM) of piceatannol. These
cells were harvested, and luciferase activity was measured. The cells
transfected with ASSyk showed a dramatic increase of NF B luciferase
activity compared with SSyk-transfected cells or cells transfected with
LipofectAMINE Plus alone. Piceatannol enhanced the luciferase activity
in a dose-dependent manner. B, MDA-MB-231 cells
were transfected with Syk cDNA and pNF B-Luc using LipofectAMINE
Plus as described under "Experimental Procedures." The wild type
Syk cDNA-transfected cells were also treated with varying
concentrations of piceatannol. MDA-MB-231 cells transfected with wild
type Syk drastically suppressed the luciferase activity compared with
SykK -transfected cells or cells transfected with
LipofectAMINE Plus alone. Piceatannol enhanced the luciferase activity
in wild type Syk-transfected cells. C and D,
effects of pV and PI 3'-kinase inhibitors on NF B transactivation in
MCF-7 (C) and MDA-MB-231 (D) cells. Both MCF-7
and MDA-MB-231 cells were transfected individually with pNF B-Luc.
These cells were then treated with pV alone, pV with 1-100
nM wortmannin or 1-10 µM LY294002, and
luciferase activity was measured. In both of these cell lines, pV
enhanced the luciferase activity, but pV-induced luciferase activity
was blocked by wortmannin or LY294002 in a dose-dependent
manner. The values were normalized to Renilla luciferase
activity. The -fold changes were calculated, and the results are
expressed as the means ± S.E. of three determination.
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To check the role of PI 3'-kinase inhibitors (wortmannin or LY294002)
on pV-induced NF
B activity, both cell lines were transfected individually with pNF
B-Luc and pRL in the presence of LipofectAMINE Plus. These transfected cells were then treated with pV alone, pV with
wortmannin, or pV with LY294002. The data indicated that pV
up-regulates the NF
B activity, but PI 3'-kinase inhibitors (wortmannin and LY294002) blocked the pV-induced NF
B activity in
both cell lines (Fig. 6, C and D).
PI 3'-Kinase and NF
B Play Crucial Roles in uPA Secretion and
Cell Migration--
To delineate whether PI 3'-kinase plays any role
in uPA secretion, both MCF-7 and MDA-MB-231 cells were treated
individually with 250 µM pV alone or pV with 100 nM wortmannin, or pV with 10 µM LY294002. The
cells were lysed, and the lysates containing equal amounts of total
proteins were resolved by SDS-PAGE and analyzed by Western blot
analysis using anti-uPA antibody. The data indicated that MCF-7 cells
treated with pV showed a higher level of uPA secretion (Fig.
7A, lane 2)
compared with untreated cells (lane 1), whereas wortmannin
(lane 3) and LY294002 (lane 4) separately
inhibited the pV-induced uPA secretion in these cells. Similarly, pV
also induced uPA secretion (lane 6) compared with control
(lane 5) in MDA-MB-231 cells, whereas both wortmannin (lane 7) and LY294002 (lane 8) blocked the
pV-induced uPA secretion in these cells.

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Fig. 7.
A, effects of pV and PI 3'-kinase
inhibitors on uPA secretion. MCF-7 and MDA-MB-231 cells were either
treated with 250 µM pV alone, pV with 100 nM
wortmannin or 10 µM LY294002, and lysed. The level of uPA
in the lysates was detected by Western blot analysis using anti-uPA
antibody. Lanes 1-4, MCF-7 cells. Lane 1,
untreated cells; lane 2, with pV; lane 3, with
wortmannin and pV; lane 4, with LY294002 and pV. Lanes
5-8, MDA-MB-231 cells. Lane 5, untreated cells;
lane 6, with pV; lane 7, with wortmannin and pV;
and lane 8, with LY294002 and pV. The arrow
indicates the uPA-specific band. B and C, effects
of NF B modulators and other agents on uPA secretion.
B, MCF-7 cells were treated individually with 100 µg/ml SN-50, 100 µg/ml SN-50M, 10 µg/ml actinomycin-D, 50 µM curcumin, and 5 ng/ml PMA. The cells were lysed, and
uPA expression in the lysates was detected by Western blot analysis.
Lane 1, untreated cells; lane 2, with SN-50M;
lane 3, with SN-50; lane 4, with actinomycin-D;
lane 5, with curcumin; lane 6, with PMA.
C, MDA-MB-231 cells were treated under the same conditions
as those described above. Lane 1, untreated cells;
lane 2, with SN-50M; lane 3, with SN-50;
lane 4, with actinomycin-D; lane 5, with
curcumin; lane 6, with PMA. The level of uPA was decreased
significantly when both of these cell lines were pretreated with SN-50,
actinomycin-D, or curcumin. As expected, PMA induced uPA expression.
Note that the constitutive expression of uPA was much higher in
MDA-MB-231 cells compared with MCF-7 cells. The level of uPA was
quantified by densitometric analysis and is represented as -fold
changes (A-C). D and E, effects of
NF B modulators and other agents on cell migration. Both MCF-7
(D) and MDA-MB-231 (E) cells were pretreated
individually with SN-50M, SN-50, actinomycin-D, PMA, and curcumin under
the same conditions as described above. These treated cells were used
for the migration assay as described under "Experimental
Procedures." The number of untreated cells migrated was considered as
100%. The cells pretreated with SN-50, actinomycin-D, or curcumin
showed a dramatic reduction of cell migration compared with untreated
cells or cells treated with SN-50M. As expected, PMA induced cell
migration. The results are expressed as the means ± S.E. of three
determinations.
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We have also examined the effects of NF
B modulators and other agents
on uPA secretion upon treating these cells (MCF-7 and MDA-MB-231) with
SN-50, SN-50M, actinomycin-D, curcumin, and PMA. These treated cell
lysates containing equal amounts of total proteins were separated by
SDS-PAGE and immunoblotted with anti-uPA antibody. The level of uPA
expression was reduced significantly when MDA-MB-231 cells were treated
individually with SN-50 (NF
B inhibitory peptide) (Fig.
7C, lane 3), actinomycin-D (protein synthesis
inhibitor) (lane 4), and curcumin (lane 5)
compared with untreated cells (lane 1). No changes of uPA
secretion were observed in cells treated with SN-50M (NF
B control
peptide) (lane 2). As expected, PMA induces uPA secretion in
these cells (lane 6). Similar results were obtained in MCF-7
cells (Fig. 7B, lanes 1-6). The constitutive expression of uPA was much higher in MDA-MB-231 cells compared with
MCF-7 cells (Fig. 7, B and C, lane 1).
In all of these experiments, the uPA-specific bands were quantified by
densitometric analysis, and the values of -fold changes are indicated.
Because the NF
B-responsive element is present in the promoter region
of uPA, we sought to determine whether NF
B-regulated uPA expression
has any role in the migration of breast cancer cells. Accordingly, both
cell lines were pretreated individually with SN-50, SN-50M,
actinomycin-D, curcumin, and PMA and used for the migration assay. The
results indicated that SN-50, actinomycin-D, and curcumin separately
inhibited the cell migration in both MCF-7 (Fig. 7D) and
MDA-MB-231 (Fig. 7E) cells compared with untreated cells.
The cells treated with SN-50M had no effect on suppression of cell
migration. As expected, PMA induced cell migration in both cell lines
(Fig. 7, D and E). These and previous data
suggested that uPA secretion and cell migration are regulated by PI
3'-kinase and NF
B (30).
Syk Suppresses PI 3'-Kinase-mediated uPA Secretion and Cell
Migration--
To ascertain whether Syk regulates PI
3'-kinase-dependent uPA secretion, MCF-7 cells were either
transfected with Syk-specific S-oligonucleotides or treated with
piceatannol. These cells were used for the detection of uPA by Western
blot analysis using anti-uPA antibody. The cells transfected with ASSyk
showed a higher level of uPA expression (Fig.
8A, lane 3)
compared with Syk-transfected cells (lane 2) or cells
transfected with LipofectAMINE Plus alone (lane 1). The
level of uPA secretion was increased dose-dependently when
the cells were treated with increasing concentrations of piceatannol
(Fig. 8B, lanes 2-4) and decreased when the
cells were incubated with increasing doses of wortmannin (Fig.
8C, lanes 2-4) or LY294002 (data not shown). The
very low level of constitutive uPA expression was observed in untreated
cells (Fig. 8, B and C, lane 1). The
MDA-MB-231 cells transfected with wild type Syk showed significant
reduction of uPA secretion (Fig. 8E, lane 2) compared with SykK
-transfected cells (lane 3)
or cells transfected with LipofectAMINE Plus alone (lane 1).
Wortmannin inhibited the expression of uPA in these cells in a
dose-dependent manner (Fig. 8F, lanes
1-4). The constitutive expression of uPA was much higher in
highly invasive MDA-MB-231 cells rather than low invasive MCF-7 cells
(Fig. 8).

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Fig. 8.
effects of Syk and PI 3'-kinase inhibitor on
uPA secretion and cell migration. A, MCF-7 cells were
transfected with Syk-specific S-oligonucleotides, lysed, and the level
of uPA was detected by Western blot analysis. Lane 1, with
LipofectAMINE Plus alone; lane 2, with SSyk; lane
3, with ASSyk. B, MCF-7 cells were treated with 0-20
µM piceatannol, and uPA was detected by Western blot
analysis. Lane 1, untreated cells; lane 2, with 1 µM piceatannol; lane 3: with 10 µM piceatannol; lane 4, with 20 µM piceatannol. C, MCF-7 cells were treated
with 0-100 nM wortmannin, a PI 3'-kinase inhibitor, and
the level of uPA was analyzed. Lane 1, untreated cells;
lane 2, with 10 nM wortmannin; lane
3, with 50 nM wortmannin; lane 4, with 100 nM wortmannin. Note that ASSyk and piceatannol enhanced the
uPA secretion, whereas wortmannin reduced the uPA secretion in these
cells (A-C). D, MCF-7 cells were transfected
with Syk-specific S-oligonucleotides as described above, treated with
or without 10 µg/ml anti-uPA antibody, and used for migration assay.
The cells transfected with ASSyk showed a dramatic increase in cell
migration compared with cells transfected with SSyk or LipofectAMINE
Plus alone. Pretreatment of nontransfected cells with anti-uPA antibody
showed a drastic reduction of cell migration, whereas ASSyk-transfected
cells, when treated with anti-uPA antibody, showed moderate inhibition
of cell migration. The number of cells migrated in LipofectAMINE
Plus-transfected cells was considered as 100%. The results are
expressed as the means ± S.E. of three determinations.
E, MDA-MB-231 cells were transfected with Syk cDNA, and
expression of uPA was detected by Western blot analysis. Lane
1, with LipofectAMINE Plus; lane 2, with wild type Syk;
lane 3, with SykK . F, MDA-MB-231
cells were treated with increasing concentrations of wortmannin (0-100
nM), and the level of uPA was detected. Lane 1,
untreated cells; lane 2, with 10 nM wortmannin;
lane 3, with 50 nM wortmannin; lane
4, with 100 nM wortmannin. Wild type Syk and
wortmannin suppressed the uPA secretion in these cells (E
and F). Note that the constitutive expression of uPA was
higher in MDA-MB-231 cells compared with MCF-7 cells. G,
MDA-MB-231 cells were transfected with wild type or SykK
cDNA, treated in the absence or presence of 10 µg/ml anti-uPA
antibody, and used for the cell migration assay. The wild type
Syk-transfected cells showed a dramatic reduction of cell migration
compared with cells transfected with LipofectAMINE Plus alone or
SykK . The cells transfected with wild type Syk followed
by treatment with anti-uPA antibody showed maximum inhibition of cell
migration, whereas the nontransfected cells treated with anti-uPA
antibody showed moderate inhibition of cell migration. The number of
cells migrated in LipofectAMINE Plus-transfected cells was used as
100%. The results are expressed as the means ± S.E. of three
determinations. In all Western blot experiments, the arrows
indicate the uPA-specific band. The bands were quantified by
densitometry and are represented in the form of a bar graph.
The mean values of triplicate experiments are indicated in the
bar graph.
|
|
To delineate the effect of Syk on uPA-mediated cell migration, the
MCF-7 cells were either treated with anti-uPA antibody or transfected
with Syk-specific S-oligonucleotides and then treated with anti-uPA
antibody. The data showed that the cell migration was reduced
drastically when the cells were treated with anti-uPA antibody (47%).
The cells transfected with ASSyk showed enhancement of cell migration
(252%) compared with SSyk-transfected cells or cells transfected with
LipofectAMINE Plus alone, whereas the ASSyk-transfected cells, when
treated with anti-uPA antibody, showed moderate cell migration (153%)
(Fig. 8D).
Similarly, MDA-MB-231 cells transfected with wild type Syk cDNA
followed by treatment with anti-uPA antibody reduced the migration dramatically (22%) compared with wild type Syk-transfected cells (24%) or cells treated with anti-uPA antibody (47%) (Fig.
8G). The cells transfected with LipofectAMINE Plus alone or
SykK
showed maximum migration (100%). These data
demonstrated that Syk suppresses the PI 3'-kinase-dependent
uPA secretion and uPA-mediated cell migration in both MCF-7 and
MDA-MB-231 cells.
 |
DISCUSSION |
In this study, we have demonstrated that Syk, a nonreceptor
protein-tyrosine kinase, inhibited PI 3'-kinase activity and
subsequently suppressed cell motility in both highly invasive
(MDA-MB-231) and low invasive (MCF-7) breast cancer cells. Syk is
expressed in MCF-7 cells, but its expression is not detectable in
highly invasive MDA-MB-231 cells. The wild type Syk cDNA was
transfected in MDA-MB-231 cells, and its expression and
autophosphorylation activity in these cells were comparable with the
endogenous Syk activity in MCF-7 cells (data not shown). Furthermore,
we have demonstrated that Syk suppresses the pV-induced interaction of p85 subunit of PI 3'-kinase and tyrosine-phosphorylated I
B
. Syk
reduced the NF
B luciferase activity and piceatannol, a Syk inhibitor, enhanced the NF
B activity in both MCF-7 and MDA-MB-231 cells. The inhibition of PI 3'-kinase activity down-regulates the
NF
B transactivation as well as cell motility in these cells. Moreover, inhibition of PI 3'-kinase and NF
B activities by their specific inhibitors reduced uPA secretion and cell motility in these
cells. Syk also inhibits the uPA secretion in both MCF-7 and MDA-MB-231
cells. These data demonstrated that Syk suppresses cell motility and
down-regulates NF
B activity by inhibiting the PI 3'-kinase activity
and uPA secretion in both MCF-7 and MDA-MB-231 cells.
ZAP-70, a nonreceptor protein-tyrosine kinase that shares the same
tandem SH2 domains with Syk at the amino terminus, was not detected in
the breast cancer cells (data not shown). Previous reports have
indicated that pV, a phosphotyrosine phosphatase inhibitor, induces
autophosphorylation of Syk in MCF-7 cells (13). However, our data
revealed that pV had no effect on activation of Syk in MCF-7 cells.
Earlier reports have shown that PI 3'-kinase signaling is required for
depolarization and cell migration of MCF-7 cells by insulin-like growth
factor I (31). The data also suggested that increased PI 3'-kinase
activity is correlated with the migratory potential and metastatic
activity of highly invasive breast cancer (MDA-MB-231) cells (30).
Using genetic (wild type Syk and SykK
) and
pharmacological (piceatannol) inhibitors of Syk, we have demonstrated
that Syk is involved in the suppression of cell motility, PI 3'-kinase
activity, and NF
B-mediated uPA secretion in both MCF-7 and
MDA-MB-231 cells. The p110 isoforms of PI 3'-kinase played significant
roles in cell migration, and differential activation of specific p110
isoforms is responsible for particular signaling events in different
cell types (32, 33). Recently, Sliva et al. (30) reported
that the regulatory p85
subunit of PI 3'-kinase is essential for
enhanced migration of metastatic tumor cells because overexpression of
a dominant negative regulatory subunit (p85DN) drastically reduced the
cell migration.
It has been documented recently that PI 3'-kinase plays a significant
role in NF
B activation in different cell types (34, 35). Tumor
necrosis factor
-induced NF
B activation is not affected by PI
3'-kinase inhibitors (wortmannin and LY294002). Similarly, a PI
3'-kinase inhibitor has no effect on
interleukin-1-dependent I
B
degradation, nuclear
translocation of NF
B, and NF
B-DNA binding (22). Our results
demonstrated that Syk down-regulates NF
B transactivation by
inhibiting the interaction between the tyrosine-phosphorylated I
B
and the p85 subunit of PI 3'-kinase. Earlier reports have shown that pV
and tumor necrosis factor
induced NF
B activation in Jurkat
cells, and only pV-induced activation of NF
B is inhibited by
wortmannin (21). Both wortmannin and LY294002 abrogated the
transactivation of NF
B but had no effect on NF
B-DNA binding in
MDA-MB-231 cells (30). Our data also revealed that Syk and PI 3'-kinase
inhibitors down-regulate the transactivation of NF
B but not
NF
B-DNA binding (data not shown) in both MCF-7 and MDA-MB-231 cells.
Thus we conclude that Syk-regulated transactivation of NF
B is
independent of NF
B-DNA binding in breast cancer cells.
We have shown that MDA-MB-231 cells transfected with wild type Syk but
not with SykK
suppressed the cellular migration and
treatment of wild type Syk-transfected cells with increasing
concentrations of piceatannol enhanced the cell migration in these
cells. Both wortmannin and LY294002 inhibited, but pV induced the
migration of MCF-7 and MDA-MB-231 cells. The pV-induced migration is
blocked by wortmannin or LY294002 in these cells. Similarly, the NF
B
inhibitory peptide SN-50, curcumin, and protein synthesis inhibitor
(actinomycin-D) reduced cell migration. Pretreatment of nontransfected
or Syk-transfected cells with anti-uPA antibody reduced the migration
of these cells. These data suggested that Syk suppressed the cell
migration by down-regulating the constitutively active NF
B
activation and uPA secretion by inhibiting the PI 3'-kinase activity in
breast cancer cells.
Our data also revealed that pV induces tyrosine phosphorylation of
I
B
and subsequently enhances the interaction between tyrosine-phosphorylated I
B
with the p85
domain of PI 3'-kinase in a time-dependent manner in both cell lines. ST 638, a
tyrosine kinase inhibitor, blocked the pV-induced tyrosine
phosphorylation of I
B
. However, pV had no effect on serine
phosphorylation of I
B
in these cells, suggesting that pV-induced
transactivation of NF
B occurs through tyrosine phosphorylation of
I
B
. Syk suppressed the tyrosine phosphorylation of I
B
and
reduced the interaction between tyrosine-phosphorylated I
B
and
p85 subunit of PI 3'-kinase. These data demonstrated that Syk regulates
the transactivation of NF
B by inhibiting the direct interaction of
tyrosine-phosphorylated I
B
and the p85 domain of PI 3'-kinase and
suggested an alternative pathway not involving the phosphorylation and
degradation of I
B
pathways.
Matrix metalloproteinases play a major role in the regulation of cancer
cell migration, extracellular matrix invasion, and metastasis by
degrading the extracellular matrix proteins (3, 4, 36). We and others
have shown recently that NF
B plays significant roles in the
activation of matrix metalloproteinases-1, -2, -3, and -9 (27, 37, 38).
uPA is also responsible for the migration and regulation of matrix
metalloproteinases activation through NF
B-mediated pathways (39). In
this study, we have detected the level of uPA in both highly invasive
(MDA-MB-231) and low invasive (MCF-7) breast cancer cells. The
constitutive secretion of uPA is significantly higher in MDA-MB-231
cells; however, a low level of uPA expression is observed in MCF-7
cells. The data also indicate that Syk down-regulates whereas pV
up-regulates the uPA production in these cells. The PI 3'-kinase
inhibitors wortmannin and LY294002 or the NF
B inhibitor SN-50
reduced uPA secretion, especially in MDA-MB-231 cells.
In summary, we have demonstrated for the first time that overexpression
of wild type Syk kinase but not the SykK
suppresses cell
motility and reduces the activation of PI 3'-kinase in MDA-MB-231
cells. In contrast, in ASSyk but not SSyk, when transfected to the
MCF-7 cells, the level of PI 3'-kinase activity as well as cell
motility were increased. In the wild type Syk cDNA-transfected
MDA-MB-231 cells, when treated with piceatannol, a Syk inhibitor, PI
3'-kinase activity increased. pV, a tyrosine phosphatase inhibitor,
enhances the activity of PI 3'-kinase and induces the interaction
between p85
, the regulatory subunit of PI 3'-kinase, and I
B
through tyrosine phosphorylation of I
B
. Syk suppresses the
transactivation of NF
B by inhibiting the tyrosine phosphorylation of
I
B
. PI 3'-kinase inhibitor reduces the pV-induced transactivation
of NF
B. Both PI 3'-kinase inhibitors wortmannin and LY294002 and
NF
B inhibitory peptide SN-50 suppress cell motility, indicating that
PI 3'-kinase and NF
B play significant roles in this process.
ASSyk-transfected MCF-7 cells enhanced uPA secretion, whereas wild type
Syk cDNA-transfected MDA-MB-231 cells reduced uPA production,
indicating that Syk down-regulates uPA secretion. These data suggested
that Syk suppresses the NF
B transactivation by inhibiting the direct
interaction of tyrosine-phosphorylated I
B
with the p85
domain
of PI 3'-kinase in breast cancer cells. Finally, these data
demonstrated that Syk down-regulates PI 3'-kinase activity and
suppresses the constitutive NF
B activity and uPA secretion that
ultimately lead to the suppression of cell motility of breast cancer
cells (Fig. 9). These findings may be
useful in designing novel therapeutic interventions using Syk as a
target molecule that will disrupt the PI 3'-kinase and NF
B signaling pathways, resulting in reduction of uPA secretion and consequent blocking of invasiveness, migration, and metastatic spread of breast
cancer.

View larger version (19K):
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|
Fig. 9.
Molecular mechanism of Syk-regulated
NF B activation and uPA secretion through
activation of PI 3'-kinase in breast cancer cells. Syk suppresses
cell motility and PI 3'-kinase activity in breast cancer cells. Syk
also inhibits NF B activation by blocking the interaction between
p85 subunits of PI 3'-kinase and tyrosine-phosphorylated I B
and subsequently reduces the uPA secretion in these cells. Wortmannin,
LY294002, SN-50, and curcumin specifically disrupt these signaling
pathways.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. Susette C. Mueller for providing
wild type and SykK
cDNAs and Dr. Rainer de Martin for
providing the pNF
B-Luc containing five tandem repeats of the NF
B
binding site. We also thank Riku Das and Hema Rangaswami for critically
reading this manuscript.
 |
FOOTNOTES |
*
This work was supported by funds from the Department of
Biotechnology (to the National Center for Cell Science) and by an extramural fund from the Department of Biotechnology of the government of India (to G. C. K.).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. Tel.:
91-20-569-0931 (ext. 203); Fax: 91-20-569-2259; E-mail:
gopalkundu@hotmail.com.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M208905200
 |
ABBREVIATIONS |
The abbreviations used are:
SH2 domain, Src
homology 2 domain;
ASSyk, Syk-specific antisense phosphorothioate oligonucleotide(s);
FITC, fluorescein isothiocyanate;
I
B
, inhibitor of nuclear factor-
B;
Luc, luciferase;
NF
B, nuclear
factor-
B;
PI 3'-kinase, phosphatidylinositol 3'-kinase;
PIP, phosphatidylinositol phosphate;
PMA, phorbol 12-myristate 13-acetate;
pV, pervanadate;
SSyk, Syk-specific sense phosphorothioate oligonucleotide(s);
SykK
, kinase-negative Syk(s);
TRITC, tetramethylrhodamine isothiocyanate;
uPA, urokinase type plasminogen
activator.
 |
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