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INTRODUCTION |
The cell adhesion molecule, CD44, is one of the major hyaluronic
acid (HA)1 receptors (1-3).
It belongs to a family of transmembrane glycoproteins which contain a
variable extracellular domain, a single spanning 23-amino acid
transmembrane domain, and a 70-amino acid cytoplasmic domain (4).
Nucleotide sequence analyses reveal that many CD44 isoforms (derived
from alternative splicing mechanisms) are variants of the standard
form, CD44s (4). CD44s (molecular mass ~85 kDa) is the most common
isoform of CD44 found in many cell types including human ovarian
carcinoma cells (5-9). The presence of high levels of CD44s (often
together with CD44v) is emerging as an important metastatic tumor
marker in a number of carcinomas, and is also implicated in the
unfavorable prognosis of a variety of cancers including human ovarian
cancers (5-9).
The invasive phenotype of CD44s-positive epithelial tumor cells has
been linked to HA-mediated CD44 signaling and cytoskeletal activation.
CD44s contains several HA-binding sites in their extracellular domain
(1-3). The binding of HA to CD44s causes cells to adhere to the
extracellular matrix (ECM) components (1-3), and has also been
implicated in the stimulation of several different biological activities (10-16). The intracellular domain of CD44 binds to
signaling proteins such as RhoGTPases (e.g. RhoA) (17);
Tiam1, a guanine nucleotide exchange factor for Rac1 (18); and
cytoskeletal proteins, including ankyrin (2, 3, 9, 17-21) and
the ERM proteins (ezrin, radixin, and moesin) (23). Recent studies
indicate that the binding of ECM components (e.g. HA)
promote CD44-mediated Tiam1-Rac1 signaling and cytoskeleton function
leading to specific structural changes in the plasma membrane and tumor
cell migration in metastatic tumor cells (18). These findings strongly
suggest that the CD44 molecule provides a direct linkage between the
ECM and the cytoskeleton. In particular, the coordinated oncogenic signaling processes contributed by HA-dependent and
CD44-mediated cytoskeleton activation is considered to be a possible
mechanism underlying tumor cell motility and migration: an obvious
prerequisite for metastasis.
The Src family kinases are classified as oncogenic proteins due to
their ability to activate cell proliferation (24, 25), spreading (26,
27), and migration (27-30) in many cell types including epithelial
tumor cells (30). The amino terminus of Src contains a myristoylation
(or palmitoylation) site, which is important for membrane association
(31, 32). Src also contains several functional domains including Src
homology (SH) 3 and SH2 domains, the catalytic protein-tyrosine kinase
core, and a conserved regulatory tyrosine phosphorylation site (31,
32). Certain amino acid residues in the c-Src molecule play an
important role in modulating its kinase activity. Mutations of specific
key amino acids result in either up-regulation or down-regulation of
c-Src kinase activity. For example, replacement of tyrosine 527 with phenylalanine (e.g. Y527F, the dominant-active form of c-Src
kinase) strongly activates c-Src kinase tranforming capability and
enzyme activities (33). Mutation of lysine 295 to arginine
(e.g. K295R, the dominant-negative form of c-Src kinase)
renders c-Src kinase defective and reduces c-Src kinase-mediated
biological activities (33, 34).
In addition, it has been observed that the interaction between Src
kinase and membrane-linked molecules regulates receptor signaling and
various cellular functions (31, 32). In fact, CD44s-mediated cellular
signaling has been suggested to involve Src kinase family members (35).
For example, Lck, one of the Src kinase family members, is found to be
closely complexed with CD44s during T-cell activation (35). CD44 also
selectively associates with active Src family tyrosine kinases
(e.g. Lck and Fyn) in glycosphingolipid-rich plasma membrane
domains of human peripheral blood lymphocytes (36). Moreover, the
cytoplasmic domain of CD44s has been shown to be involved in the
recruitment of the Src family (e.g. Src, Yes, and Fyn) in
prostate tumor cells during anchorage-independent colony growth (20).
Collectively, all these observations support the notion that c-Src
kinases participate in CD44-mediated cellular signaling.
The questions of (i) whether CD44s-mediated c-Src kinase signaling
plays a direct role in regulating ovarian tumor cell activation and
(ii) which cytoskeletal protein(s) is (are) most likely involved in
CD44-c-Src kinase-regulated downstream effector function leading to
human ovarian cell migration are specifically addressed in this study.
We have determined that CD44s (the standard form) and c-Src kinase are
physically linked and functionally coupled in human ovarian tumor cells
(SK-OV-3.ipl cell line). Furthermore, our data show that the
cytoplasmic domain of CD44s is important for the association with c-Src
kinase. HA treatment of ovarian tumor cells recruits c-Src kinase to
CD44s and activates c-Src kinase activity. We have also demonstrated
that c-Src kinase phosphorylates the cytoskeletal protein cortactin
both in vivo and in vitro. Most importantly,
cortactin phosphorylation by c-Src kinase attenuates its cross-linking
ability of filamentous actin. Overexpression of a dominant-active form
of c-Src kinase, by transfecting SK-OV-3.ipl cells with c-Src
(Y527F)cDNA in these ovarian tumor cells, promotes the onset of
CD44s/c-Src kinase-regulated cortactin function and tumor cell
migration. Transfection of SK-OV-3.ipl cells with a dominant-negative
mutant of c-Src kinase (K295R-kinase dead) effectively blocks the tumor
cell-specific phenotype. Therefore, we believe that CD44-activated
c-Src kinase signaling is directly involved in stimulating
cortactin-cytoskeleton interaction and HA-mediated tumor cell migration
during the progression of human ovarian cancer.
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MATERIALS AND METHODS |
Cell Lines and Culture--
The SK-OV-3.ipl cell line was
established from ascites that developed in a nu/nu mouse given an
intraperitoneal injection of SK-OV-3 human ovarian carcinoma cell line
(obtained from the American Type Culture Collection) as described
previously (37). Cells were grown in Dulbecco's modified Eagle's
medium/F-12 medium supplement (Life Technologies, Inc.) supplemented
with 10% fetal bovine serum.
Immunoreagents--
Monoclonal rat anti-CD44 antibody (clone
020; isotype IgG2b; obtained from CMB-TECH, Inc., Miami,
FL) used in this study recognizes the standard form of CD44 (CD44s).
Monoclonal mouse anti-phosphotyrosine antibody (PY-plusTM; clone PY20;
IgG2b) was purchased from Zymed Laboratories
Inc. (South San Francisco, CA). Rabbit polyclonal antibody
against human c-Src kinase (recognizing a peptide sequence corresponding to amino acids 509-533 at the carboxyl terminus of human
c-Src p60) and rabbit anti-Src (Tyr(P)418) phosphospecific
antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA)
and BIOSOURCE International (Camarillo, CA), respectively. Mouse monoclonal anti-green fluorescent protein (GFP)
antibody and mouse monoclonal anti-cortactin antibody (clone 4F11) were
purchased from PharMingen (San Diego, CA) and Upstate Biotechnology
Inc. (Lake Placid, NY), respectively.
Cloning, Expression, and Purification of CD44 Cytoplasmic Domain
(CD44cyt) from Escherichia coli--
The procedure for preparing the
fusion protein of CD44's cytoplasmic domain was the same as described
previously (9, 17).
Expression Constructs--
The c-Src (Y527F) mutant or the c-Src
(K295R) mutant cDNAs (kindly provided by Dr. David Shalloway
(Cornell University, Ithaca, NY) was cloned into pEGFPN1 vector
(CLONTECH) using PCR-based cloning strategy.
Specifically, c-Src (Y527F) mutant cDNA or c-Src (K295R) mutant
cDNA was amplified by PCR with two specific primers (left,
5'-GCCTCGAGATGGGGAGCAGCAAGAGCAAG-3'; right,
5'-GCAAGCTTTAGGTTCTCTCCAGGCTGGTA-3') linked with a specific enzyme
digestion site (XhoI or HindIII). PCR product
digested with XhoI and HindIII was purified with
QIAquick PCR purification kit (Quagen). The cDNA was cloned into
pEGFPN1 vector digested with XhoI and HindIII.
Subsequently, both c-Src (Y527F) mutant cDNA and c-Src (K295R)
mutant cDNA sequences were confirmed by nucleotide sequencing analyses.
Cell Transfection--
To establish a transient expression
system, SK-OV-3.ipl cells were transfected with various plasmid DNAs
(e.g. GFP-tagged c-Src (Y527F) or GFP-c-Src (K295R) or
vector alone) using electroporation methods according to those
procedures described previously (38). Various transfectants were then
analyzed for their protein expression (e.g. c-Src-related
proteins) by immunoblot, c-Src kinase activity, and tumor cell
migration assays as described below.
In Vitro Binding of c-Src Recombinant Protein to
CD44--
Purified constitutively activated c-Src kinase recombinant
protein (39) was first bound to anti-c-Src kinase-conjugated immunoaffinity beads. Subsequently, aliquots (10-20 ng of proteins) of
these beads were incubated with 0.5 ml of a binding buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% bovine
serum albumin, and 0.05% Triton X-100) in the presence of various
concentrations (10-400 ng/ml) of 125I-labeled FLAG-CD44cyt
fusion protein (5,000 cpm/ng protein) at 4 °C for 5 h.
Specifically, equilibrium binding conditions were determined by
performing a time course (1-10 h) of 125I-labeled
FLAG-CD44cyt binding to c-Src kinase at 4 °C. The binding equilibrium was found to be established when the in vitro
CD44-c-Src kinase binding assay was conducted at 4 °C after 4 h. Following binding, beads were washed extensively in the binding
buffer and the bead-bound radioactivity was counted.
As a control, 125I-labeled FLAG-CD44cyt fusion protein was
also incubated with uncoated beads to determine the binding observed due to the nonspecific binding of the ligand. Nonspecific binding, which represented ~15-20% of the total binding, was always
subtracted from the total binding. The values expressed under
"Results" represent an average of triplicate determinations of
three to five experiments with S.D. less than ±5%.
Immunoprecipitation and Immunoblotting
Techniques--
SK-OV-3.ipl cells were solubilized in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton
X-100 buffer and immunoprecipitated using rat anti-CD44s antibody or
rabbit anti-c-Src kinase antibody followed by goat anti- rat IgG or
goat anti-rabbit IgG, respectively. The immunoprecipitated material was
solubilized in SDS sample buffer, electrophoresed, and immunoblotted
with rabbit anti-c-Src kinase antibody (5 µg/ml) or rat anti-CD44s
antibody (5 µg/ml), followed by incubation with horseradish
peroxidase-conjugated goat anti-rabbit IgG or goat anti-rat IgG
(1:10,000 dilution) at room temperature for 1 h. The blots were
developed using ECL chemiluminescence reagent (Amersham Pharmacia
Biotech) according to the manufacturer's instructions.
Cell lysates were also prepared from SK-OV-3.ipl cells treated with HA
at various concentrations (e.g. 0, 5, 10, 25, 50, and 100 µg/ml) for various time intervals (e.g. 0, 2, 5, 10, 15, 20, 30, and 60 min) and immunoprecipitated with anti-CD44s IgG to isolate CD44s-c-Src kinase complex (as described above).
Immunoprecipitates were immunoblotted with either anti-Src
(Tyr(P)418) (5 µg/ml) or anti-c-Src kinase (5 µg/ml).
In some cases, SK-OV-3.ipl cells (treated with HA (50 µg/ml) for
10-60 min in the absence or the presence of PP2, an inhibitor for
c-Src family kinases (purchased form Calbiochem-Novabiochem, San Diego,
CA); or untreated) were solubilized in 50 mM Tris-HCl (pH
7.4), 150 mM NaCl, 1% Triton X-100 buffer. These materials
were then immunoprecipitated by mouse anti-cortactin, followed by
immunoblotting with mouse anti-phosphotyrosine antibody/or reblotting
with mouse anti-cortactin plus peroxidase-conjugated goat anti-mouse
IgG and ECL chemiluminescence reagent.
In some experiments, SK-OV-3.ipl cells (e.g. untransfected
or transfected by GFP-tagged c-Src (Y527F)cDNA or GFP-tagged c-Src (K295R)cDNA or vector alone) were treated with HA (50 µg/ml) for 10-60 min. Cell lysates of these transfectants were immunoprecipitated by rat anti-CD44s antibody (5 µg/ml). The anti-CD44s-mediated immunoprecipitated material was then immunoblotted with mouse anti-GFP
antibody (5 µg/ml) for 1 h at room temperature. Some cell lysate
was either immunoblotted with mouse anti-GFP antibody (5 µg/ml) or
immunoprecipitated with mouse anti-GFP antibody (5 µg/ml), followed
by immunoblotting with rabbit anti-c-Src antibody (5 µg/ml). In some
cases, the cell lysate of these cells was immunoprecipitated by mouse
anti-cortactin (5 µg/ml), followed by immunoblotting with mouse
anti-phosphotyrosine antibody/reblotting with mouse anti-cortactin
antibody plus horseradish peroxidase-conjugated goat anti-mouse IgG
(1:10,000 dilution) and ECL chemiluminescence reagent.
Immunofluorescence Staining--
SK-OV-3.ipl cells
(untransfected or transfected with various plasmid DNAs such as
GFP-tagged c-Src (Y527F)cDNA or GFP-tagged c-Src (K295R)cDNA or
vector alone) were first washed with PBS (0.1 M phosphate
buffer (pH 7.5) and 150 mM NaCl) buffer and fixed by 2%
paraformaldehyde. Subsequently, untransfected/vector-transfected cells
or GFP-tagged SK-OV-3.ipl transfectants were stained with Texas
Red-labeled or cyanine (Cy5)-labeled rat anti-CD44s antibody. In some
cases, GFP-tagged and cyanine-labeled cells were then rendered
permeable by ethanol treatment, followed by incubating with Texas
Red-conjugated mouse anti-cortactin IgG. To detect nonspecific antibody
binding, cyanine-CD44s labeled cells were incubated with Texas
Red-conjugated normal mouse IgG. No labeling was observed in such
control samples. These labeled samples were examined with a confocal
laser scanning microscope (MultiProbe 2001 Inverted CLSM system,
Molecular Dynamics, Sunnyvale, CA). Cells displaying membrane
projections were counted under the microscope. Specifically, every cell
in the field was examined for the occurrence of the cell phenotypes
(e.g. with or without membrane projections). At least
200-300 cells (in 12 different fields) were examined in each sample.
Quantitative values describing the percentage of cells displaying
membrane projections in each sample were expressed as "percentage of
total cells."
Tumor Cell Migration Assays--
Twenty-four transwell units
were used for monitoring in vitro cell migration as
described previously (9, 17, 18, 21). Specifically, the 5-µm porosity
polycarbonate filters (CoStar Corp., Cambridge, MA) were used for the
cell migration assay. SK-OV-3.ipl cells (untransfected or transfected
with GFP-tagged c-Src (Y527F)cDNA or GFP-tagged c-Src
(K295R)cDNA or vector alone) (~1 × 104
cells/well) were placed in the upper chamber of the transwell unit. The
growth medium containing high glucose DMEM supplemented with 200 µg/ml hyaluronic acid was placed in the lower chamber of the
transwell unit. After 18 h of incubation at 37 °C in a humidified 95% air, 5% CO2 atmosphere, vital stain MTT
(Sigma) was added at a final concentration of 0.2 mg/ml to both the
upper and the lower chambers and incubated for additional 4 h at
37 °C. Migrative cells at the lower part of the filter were removed by swabbing with small pieces of Whatman filter paper. Both the polycarbonate filter and the Whatman paper were placed in dimethyl sulfoxide to solubilize the crystal. Color intensity was measured in
450 nm.
Cell migration processes were determined by measuring the cells that
migrate from then upper chambers to the lower side of the polycarbonate
filters by standard cell number counting methods as described
previously (9, 17, 18, 21). The CD44-specific cell migration was
determined by subtracting nonspecific cell migration (i.e.
cells migrate to the lower chamber in the presence of anti-CD44s
antibody treatment) from the total migrative cells in the lower
chamber. The CD44-specific cell migration in vector-transfected cells
(control) is designated as 100%. Each assay was set up in triplicate
and repeated at least three times. All data were analyzed statistically
using Student's t test, and statistical significance was
set at p < 0.01.
In Vitro c-Src Kinase Assay--
An in vitro c-Src
kinase assay using enolase as a substrate was performed as described
previously (40). Briefly, lysates from SK-OV-3.ipl cells (treated with
HA (50 µg/ml) for 10-60 min; or pretreated with anti-CD44 followed
by HA (50 µg/ml) treatment for 10-60 min; or untreated) were
prepared and processed for anti-CD44-mediated or anti-c-Src
kinase-mediated immunoprecipitation to obtain the CD44s-c-Src kinase
complexes. For assaying c-Src kinase activity, CD44s-c-Src kinase
complexes were incubated with 10 µl of reaction buffer (20 mM PIPES (pH 7.0), 10 mM MnCl2, 10 µM Na3VO4)), 1 µl of freshly
prepared acid-denatured enolase (Sigma) (5 µg of enolase plus 1 µl
of 50 mM HCl incubated at 30 °C for 10 min and then neutralized with 1 µl of 1 M PIPES (pH 7.0)), and 20 mM ATP. After 10 min of incubation at 30 °C, reactions
were terminated by adding 2× SDS sample buffer. Samples were then
electrophoresed and immunoblotted with mouse anti-phosphotyrosine
antibody plus horseradish peroxidase-conjugated goat anti-mouse IgG and
ECL chemiluminescence reagent.
Phosphorylation of Cortactin by Endogenous c-Src, c-Src
(Y527F)/c-Src (K295R) in Vitro--
Untransfected SK-OV-3.ipl cells
and various transfectants were treated with HA (50 µg/ml) for various
time intervals (0, 2, 5, 10, 15, 20, 30, and 60 min). At each time
point, 100 mM c-Src-related proteins (isolated from
HA-treated untransfected cells using anti-c-Src-conjugated immunoaffinity beads, or SK-OV-3.ipl cells transfected with either c-Src (Y527F) or c-Src (K295R) or vector alone using
anti-GFP-conjugated Sepharose beads) were incubated with 20 µl of the
reaction buffer (20 mM PIPES (pH 7.0), 10 mM
MnCl2, 10 µM
Na3VO4)), 1 µl of recombinant cortactin (a
gift from Dr. Alan Mak, Queen's University, Kingston, Ontario, Canada)
(5 µg of cortactin dissolved in 2 µl of 1 M PIPES (pH
7.0)), and 10 µCi of [
-32P]ATP (5000 Ci/mmol) at
30 °C for 1 h. To quantitate phosphorylated cortactin, aliquots
of the reactions were analyzed by SDS-PAGE. The radioactivity
associated with GFP-tagged cortactin band was analyzed by
autoradiograms quantitated by a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA) or scintillating counting methods.
F-actin Cross-linking Assay--
The procedures for conducting
F-actin cross-linking experiments were the same as those described
previously (41, 42) with some modifications. Unphosphorylated or c-Src
kinase phosphorylated recombinant cortactin (as described above)
(50-100 nM) in 50 µl of TKM buffer (50 mM
Tris-HCl (pH7.4), 134 mM KCl, and 1 mM
MgCl2) was mixed with an equal volume of 8 µM
125I-labeled F-actin followed by a 30-min incubation at
room temperature. Subsequently, the mixture was centrifuged at
25,000 × g for 10 min at room temperature. The
supernatant was then collected, and the radioactivity in this fraction
was counted. The decrease (or loss) of radioactivity in the supernatant
fraction reflects F-actin precipitation due to the cross-linking
reaction (41, 42). The F-actin cross-linking reaction in the presence
of unphosphorylated cortactin (treated with a control protein isolated
from untransfected cells using anti-GFP conjugated Sepharose beads)
(control) is designated as 100%. Each assay was set up in triplicate
and repeated at least three times. All data were analyzed statistically
using Student's t test, and statistical significance was
set at p < 0.01.
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RESULTS |
Physical Association between CD44s (the Standard Form) and c-Src
Kinase in Human Ovarian Tumor Cells--
CD44 family of transmembrane
glycoproteins have been implicated in multiple oncogenic signaling
pathways during tumor progression of various human cancers (2, 3, 9,
17, 18, 20). One of the CD44 signaling-related regulatory molecules is
the Src family kinases (20, 35, 36). The question of whether the Src
family kinases also participate in CD44-linked ovarian tumor
cell-specific function is addressed in this study.
Immunoblotting with anti-CD44 antibody (recognizing an epitope located
at the NH2-terminal region of CD44s (the standard form)) indicates that a single CD44s protein (mass ~ 85 kDa) is
expressed in the human ovarian tumor cell line, SK-OV-3.ipl (Fig.
1, lane 1). In
addition, we have used anti-c-Src kinase antibody-mediated immunoblot
to detect the expression of c-Src kinase as a single polypeptide
(mass ~ 60 kDa) (Fig. 1, lane 2) in
SK-OV-3.ipl cells (Fig. 1, lane 2). Both CD44s
and c-Src kinase detected in SK-OV-3.ipl cells by anti-CD44s and
anti-c-Src kinase-mediated immunoblot is specific since no protein is
detected in these cells using preimmune rat IgG or rabbit IgG,
respectively (Fig. 1, lanes 5 and
6).

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Fig. 1.
Detection of CD44s and c-Src kinase complex
in SK-OV-3.ipl cells. SK-OV-3.ipl cells (5 × 105
cells) were solubilized by 1% Nonidet P-40 buffer followed by
immunoprecipitation and/or immunoblot by anti-CD44s antibody or
anti-c-Src kinase antibody, respectively as described under
"Experimental Procedures." Lane 1, detection
of CD44s with rat anti-CD44s-mediated immunoblot of SK-OV-3.ipl cells.
Lane 2, detection of c-Src kinase with rabbit
anti-c-Src kinase-mediated immunoblot of SK-OV-3.ipl cells.
Lane 3, detection of c-Src kinase in the complex
by anti-CD44s-immunoprecipitation followed by immunoblotting with
anti-c-Src kinase antibody. Lane 4, detection of
CD44s in the complex by anti-c-Src kinase-mediated immunoprecipitation
followed by immunoblotting with anti-CD44s antibody. Lane
5, immunoblot of SK-OV-3.ipl cells with preimmune rat IgG.
Lane 6, immunoblot of SK-OV-3.ipl cells with
preimmune rabbit IgG.
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We then addressed the question of whether there is a physical linkage
between CD44s and c-Src kinase in human ovarian tumor cells. First, we
carried out anti-CD44s-mediated and anti-c-Src kinase-mediated
precipitation followed by anti-c-Src kinase immunoblot (Fig. 1,
lane 3) or anti-anti-CD44s immunoblot (Fig. 1,
lane 4), respectively. Our results indicate that
the c-Src kinase band is revealed in anti-CD44s-immunoprecipitated
materials (Fig. 1, lane 3), and the CD44s band is
detected in the anti-c-Src kinase-immunoprecipitated materials (Fig. 1,
lane 4). These findings establish the fact that
CD44s and c-Src kinase are closely associated with each other in
ovarian tumor cells.
We have also used purified, FLAG-tagged cytoplasmic domain of CD44
fusion protein (FLAG-CD44cyt) and a constitutively activated c-Src
kinase recombinant protein to conduct in vitro binding
assays. Specifically, the binding of 125I-labeled
FLAG-CD44cyt to c-Src kinase under equilibrium binding conditions was
tested. Scatchard plot analyses presented in Fig. 2 indicate that c-Src kinase binds to the
cytoplasmic domain of CD44 (CD44cyt) at a single site with high
affinity (an apparent dissociation constant (Kd) of
~2.0 nM). These findings clearly indicate that the
cytoplasmic domain of CD44 interacts directly with c-Src kinase.

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Fig. 2.
Binding of 124I-labeled
FLAG-CD44cyt to c-Src recombinant protein. Various concentrations
of 125I-labeled FLAG-CD44cyt were incubated with c-Src
recombinant protein-coupled beads at 4 °C for 4 h. Following
binding, beads were washed extensively in binding buffer and the
bead-bound radioactivity was counted. As a control,
125I-labeled FLAG-CD44cyt was also incubated with uncoated
beads to determine the binding observed due to the nonspecific binding
of the ligand. Nonspecific binding, which represented ~20% of the
total binding, was always subtracted from the total binding. Our
binding data are highly reproducible. The values expressed under
"Results" represent an average of triplicate determinations of
three to five experiments with S.D. less than ±5%. The data represent
Scatchard plot analysis of the equilibrium binding data between
125I-labeled FLAG-CD44cyt and c-Src kinase.
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Stimulation of CD44s-associated c-Src Kinase Activity by HA in
SK-OV-3.ipl Cells--
Previous studies show that full catalytic
activity of c-Src kinase requires phosphorylation of tyrosine 418 (43).
Using specific anti-phospho-Src antibody (i.e.
anti-Src(Tyr(P)418), designed to detect the activated form
of c-Src kinase), we have found that c-Src kinase activation (detected
in the CD44s-c-Src kinase complex) is HA-dependent (Fig.
3A). A relatively low level of
c-Src kinase activation is detected when SKOV-3.ipl cells were treated
with either a low HA concentration (~5-10 µg/ml) or a high HA
concentration (100 µg/ml) (Fig. 3A). If the concentration of HA increases to 25-50 µg/ml, c-Src kinase activation becomes significantly elevated (Fig. 3A).

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Fig. 3.
Detection of c-Src kinase activity in
CD44s-c-Src complex. Cell lysates were prepared from SK-OV-3.ipl
cells treated with HA at various concentrations (e.g. 0, 5, 10, 25, 50, and 100 µg/ml) for 10 min and immunoprecipitated with
anti-CD44s antibody (to isolate CD44s-c-Src kinase complex) followed by
immunoblotting with various antibodies (e.g.
anti-phospho-Src (Tyr(P)418) antibody, anti-Src antibody or
anti-phosphotyrosine antibody. A, detection of the activated
form of c-Src kinase by anti-phospho-Src
(Tyr(P)418)-mediated immuno blot using anti-CD44s-mediated
immunoprecipitated materials prepared from SK-OV-3 cells treated with
HA at various concentrations (e.g. 0, 5, 10, 25, 50, and 100 µg/ml). B, tyrosine phosphorylation of enolase by c-Src
kinase in the CD44s complex detected by anti-phosphotyrosine antibody
(panel a). (Note that an equal amount of c-Src
kinase (revealed by anti-Src-mediated immunoblot) was used in this
kinase assay (panel b).) C,
recruitment of c-Src kinase into CD44s complex detected by
anti-CD44s-mediated immunoprecipation followed by anti-c-Src
kinase-mediated immunoblot using SK-OV-3.ipl cells treated with 50 µg/ml HA (right lane) or without any HA
(left lane).
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The kinase activity was also measured by the ability of c-Src kinase
(using an equal amount of c-Src kinase protein) (Fig. 3B,
panel b, right and left
lanes) to tyrosine-phosphorylate the substrate, enolase,
in vitro (Fig. 3B). Our results indicate that CD44s-associated c-Src kinase isolated from HA-treated cells shows a
significantly higher level of kinase activity (Fig. 3B,
panel a, right lane) than
that obtained from untreated cells (Fig. 3B, panel a, left lane) or
HA-treated cells in the presence of anti-CD44s antibody (data not
shown). Furthermore, we have observed that HA treatment recruits a
significant amount of c-Src kinase (Fig. 3C,
right lane) into the CD44s-c-Src kinase complex
(Fig. 3C, left lanes). Since CD44 does
not contain intrinsic catalytic properties, HA must activate and
recruit cellular tyrosine protein kinases such as c-Src kinase to
regulate tyrosine protein phosphorylation.
The cytoskeletal protein, cortactin, is a known c-Src kinase substrate,
which is composed of six and a half 37-amino acid tandem repeats and an
SH3 domain at the carboxyl terminus (41, 42). Between the SH3 and the
repeat domains, there is an
-helical structure plus a sequence rich
in proline residues (41, 42). Tyrosine phosphorylation of cortactin by
c-Src kinase occurs in the region between the proline-rich sequence and
the SH3 domain (41, 42, 44). In this study, we have found that HA
treatment of ovarian tumor cells (SK-OV-3.ipl cells) stimulates
tyrosine phosphorylation of the 85-kDa protein, cortactin, in
vivo (Fig. 4, lane
3, a and b). In contrast, the level of
cortactin tyrosine phosphorylation appears to be very low in those
cells without any HA treatment (Fig. 4, lane 1,
a and b) or treated with HA in the presence of
PP2 (an inhibitor for the Src family kinases) (Fig. 4, lane
2, a and b). Kinetic analyses show
that c-Src kinase activation (measured by c-Src-mediated cortactin
phosphorylation) occurs as early as 2-5 min and reaches the maximal
level ~30-60 min after the addition of HA to SKOV-3.ipl cells (Fig.
5). These findings clearly indicate that
the binding of HA to these CD44s containing SK-OV-3.ipl cells promotes
c-Src kinase activation leading to cortactin phosphorylation.

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Fig. 4.
Detection of cortactin tyrosine
phosphorylation in HA-treated SK-OV-3.ipl cells. SK-OV-3.ipl cells
(treated with HA (50 µg/ml) for 10 min in the absence or the presence
of PP2 (an inhibitor for c-Src family kinases) or untreated) were
solubilized in 50 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 1% Triton X-100 buffer. Cell lysates were then
immunoprecipitated by anti-cortactin antibody followed by
immunoblotting with mouse anti-phosphotyrosine antibody or reblotting
with anti-cortactin plus peroxidase-conjugated goat anti-mouse IgG
(1:10,000 dilution) as described under "Experimental Procedures."
Lane 1, anti-cortactin-mediated
immunoprecipitated materials (isolated from untreated SK-OV-3.ipl
cells) were immunoblotted with anti-phosphotyrosine antibody
(panel a) or reblotted with anti-cortactin
(panel b). Lane 2,
anti-cortactin-mediated immunoprecipitated materials (isolated from
SK-OV-3.ipl cells treated with HA in the presence of PP2 (an inhibitor
for the Src family kinases)) were immunoblotted with
anti-phosphotyrosine antibody (panel a) or
reblotted with anti-cortactin (panel b).
Lane 3, anti-cortactin-mediated
immunoprecipitated materials (isolated from SK-OV-3.ipl cells treated
with HA) were immunoblotted with anti-phosphotyrosine antibody
(panel a) or reblotted with anti-cortactin
(panel b).
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Fig. 5.
Kinectic analysis of cortactin
phosphorylation by c-Src kinase in SK-OV-3.ipl cells treated with
HA. SK-OV-3.ipl cells were treated with HA (50 µg/ml) for
various time intervals (0, 2, 5, 10, 15, 20, 30, and 60 min). At each
time point, c-Src kinase (isolated from anti-c-Src-conjugated
immunoaffinity beads) were assayed for its activity, by incubating with
10 µl of the reaction buffer (20 mM PIPES (pH 7.0), 10 mM MnCl2, 10 µM
Na3VO4)), 1 µl of recombinant cortactin (5 µg of cortactin dissolved in 2 µl of 1 M PIPES (pH
7.0)), and 10 µCi of [ -32P]ATP at 30 °C for
1 h. To quantitate phosphorylated cortactin, aliquots of the
reactions were analyzed by SDS-PAGE. The radioactivity associated with
recombinant cortactin band was analyzed by autoradiograms quantitated
by a PhosphorImager or scintillating counting methods as described
under "Experimental Procedures."
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Our preliminary data indicate that both Yes and Fyn are at least 5-fold
less than c-Src kinase in SK-OV-3.ipl cells. Using specific anti-Fyn or
anti-Yes-mediated immunoblot on anti-CD44s-precipitated material, we
are unable to detect the association of these kinase molecules with
CD44s or phosphorylation of cortactin (data not shown). Therefore,
c-Src appears to be the major Src kinase family member (not Fyn or Yes)
that is associated with CD44s and thus solely responsible for cortactin phosphorylation.
Effects of CD44s-c-Src Kinase Signaling on Cortactin Function and
Ovarian Tumor Cell Migration--
The invasive phenotype of tumor
cells (characterized by membranous projections and tumor cell
migration) is closely associated with CD44s-mediated membrane motility
and cytoskeleton function (9, 18, 21). To correlate CD44s-c-Src kinase
signaling with ovarian tumor cell-specific behaviors, we have
transiently transfected the ovarian tumor cells (SK-OV-3.ipl cells)
with a dominant active form of GFP-tagged c-Src (Y527F)cDNA or a
dominant-negative mutant of GFP-tagged c-Src kinase (K295R)cDNA
(lacking kinase activity), respectively. Our results indicate that both
GFP-tagged c-Src (Y527F) (Fig.
6A, lane
2) and GFP-tagged c-Src (K295R) (Fig. 6A,
lane 3) are expressed as a 80-kDa protein
detected by anti-GFP-mediated immunoprecipitation followed by
anti-c-Src immunoblot (Fig. 6A). No detectable endogenous
c-Src is detected in the anti-GFP-mediated immunoprecipitation followed
by anti-c-Src-mediated immunoblot (Fig. 6A, lanes
1-3). The expression of these two GFP-tagged c-Src mutant
proteins can also be detected by anti-GFP-mediated immunoblot (Fig.
7A, lanes
3 and 4). No protein band was detected in
untransfected (Fig. 7A, lane 1) or
vector-transfected SK-OV-3.ipl cells by anti-GFP-mediated immunoblotting (Fig. 7A, lane 2) or by
anti-GFP-mediated immunoprecipitation followed by anti-c-Src immunoblot
(Fig. 6A, lane 1).

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Fig. 6.
Detection of c-Src kinase mutant proteins and
CD44s complex in SK-OV-3.ipl cells transfected with c-Src
(Y527F)cDNA or c-Src (K295R)cDNA. A,
SK-OV-3.ipl transfected with GFP-tagged c-Src (Y527F)cDNA or
GFP-tagged c-Src (K295R)cDNA or vector alone were
immunoprecipitated with mouse anti-GFP antibody followed by
immunoblotting with anti-c-Src antibody. Lane 1,
detection of cellular protein(s) associated with mouse
anti-GFP-mediated immunoprecipitation and anti-c-Src kinase-mediated
immunoblot in vector-transfected cells. Lane 2,
detection of GFP-c-Src expression by mouse anti-GFP-mediated
immunoprecipitation followed by anti-c-Src kinase-mediated immunoblot
of c-Src (Y527F)cDNA-transfected cells. Lane
3, detection of GFP-c-Src expression by mouse
anti-GFP-mediated immunoprecipitation followed by anti-c-Src
kinase-mediated immunoblot of c-Src (K295R)cDNA-transfected cells.
B, SK-OV-3.ipl transfected with GFP-tagged c-Src
(Y527F)cDNA or GFP-tagged c-Src (K295R)cDNA or vector alone
were immunoprecipitated with rat anti-CD44s-mediated
immunoprecipitation followed by immunoblotting with anti-GFP antibody.
Lane 1, detection of GFP-c-Src association with
CD44s isolated from vector-transfected cells treated with
(lane 2) or without HA (lane 1).
Lane 2, detection of GFP-c-Src association with
CD44s isolated from c-Src (Y527F)cDNA-transfected cells treated
with (lane 4) or without HA (lane
3). Lane 3, detection of GFP-c-Src
association with CD44s isolated from c-Src (K295R)cDNA-transfected
cells treated with (lane 6) or without HA
(lane 5).
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Fig. 7.
Characterization of c-Src mutant proteins in
SK-OV-3.ipl cells transfected with c-Src (Y527F)cDNA or c-Src
(K295R)cDNA, and measurement of the F-actin cross-linking activity
of cortactin (phosphorylated by c-Src (Y527F) or c-Src (K295R)).
SK-OV-3.ipl cells (e.g. untransfected or transfected by
GFP-tagged c-Src (Y527F)cDNA or GFP-tagged c-Src (K295R)cDNA or
vector alone) were immunoblotted with mouse anti-GFP antibody, or
immunoprecipitated with mouse anti-cortactin followed by immunoblotting
with mouse anti-phosphotyrosine antibody/reblotting with mouse
anti-cortactin antibody as described under "Experimental
Procedures." A, detection of GFP protein expression in
untransfected cells (lane 1), vector-transfected
cells (lane 2), GFP-tagged c-Src
(Y527F)cDNA-transfected cells (lane 3), or
GFP-tagged c-Src (K295R)cDNA-transfected cells (lane
4) by immunoblotting with mouse anti-GFP antibody.
B, panel a, detection of cortactin
tyrosine phosphorylation in untransfected cells (lane
1), vector-transfected cells (lane 2),
GFP-tagged c-Src (Y527F)cDNA-transfected cells (lane
3), or GFP-tagged c-Src (K295R)cDNA-transfected cells
(lane 4) by immunoprecipitating various cell
lysates with mouse anti-cortactin followed by immunoblotting with mouse
anti-phosphotyrosine antibody. B, panel
b, detection of cortactin expression in untransfected cells
(lane 1), vector-transfected cells
(lane 2), GFP-tagged c-Src
(Y527F)cDNA-transfected cells (lane 3), or
GFP-tagged c-Src (K295R)cDNA-transfected cells (lane
4) by immunoprecipitating cell lysates with mouse
anti-cortactin followed by reblotting with mouse anti-cortactin
antibody. C, analysis of cortactin phosphorylation by
c-Src-related proteins. Purified cortactin recombinant protein was
incubated with c-Src-related proteins isolated from untransfected cells
(a) or SK-OV-3.ipl cells (transfected with either GFP-tagged
c-Src (Y527F) (c) or GFP-tagged c-Src (K295R) (d)
or vector alone (b) using anti-GFP-Sepahrose beads) in the
kinase reaction buffer at 30 °C for 1 h as described under
"Experimental Procedures." To quantitate phosphorylated cortactin,
aliquots of the reactions were analyzed by SDS-PAGE. The radioactivity
associated with cortactin band were analyzed by Scintillating counting.
D, measurement of the F-actin cross-linking activity of
cortactin. Purified cortactin recombinant protein treated with
c-Src-related proteins was subjected to F-actin cross-linking analysis.
Specifically, unphosphorylated cortactin (treated with c-Src-related
proteins isolated from untransfected cells (a) or
vector-transfected cells (b) or SK-OV-3.ipl cells
transfected with c-Src (K295R)cDNA (d) using
anti-GFP-conjugated beads); or phosphorylated cortactin (isolated from
SK-OV-3.ipl cells transfected with c-Src (Y527F)cDNA using anti-GFP-conjugated beads (c))
was incubated with a TKM buffer (50 mM Tris-HCl (pH7.4),
134 mM KCl, and 1 mM MgCl2).
Unphosphorylated or phosphorylated cortactin was then mixed with
125I-labeled F-actin followed by a 30-min incubation at
room temperature. Subsequently, the mixture was centrifuged at
25,000 × g for 10 min at room temperature. The
supernatant was then collected, and the radioactivity in this fraction
was counted. The decrease (or loss) of radioactivity in the supernatant
fraction reflects F-actin precipitation due to the cross-linking
reaction (41, 42). The F-actin cross-linking reaction in the presence
of unphosphorylated cortactin (treated by anti-GFP-conjugated beads
prepared from untransfected cells) (control) is designated as 100%.
Each assay was set up in triplicate and repeated at least three times.
All data were analyzed statistically using the Student's t
test, and statistical significance was set at p < 0.01.
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Transfected GFP-c-Src forms (e.g. GFP-c-Src (Y527F) or
GFP-c-Src (K295R)) are also associated with CD44s (Fig. 6B).
HA treatment promotes an accumulation of the dominant active form of
c-Src (GFP-c-Src (Y527F)) (Fig., 6B, lanes
3 and 4) (but not the dominant-negative form of
c-Src (GFP-c-Src (K295R)) (Fig. 6C, lanes
5 and 6) into anti-CD44s-mediated
immunoprecipitated materials. No GFP-related protein was observed in
anti-CD44s immunoprecipitates blotted with anti-GFP antibody using
vector-transfected cells treated with (lane 2) or
without HA (lane 1). These results suggest that the activated c-Src kinase (but not the inactivated c-Src kinase) is
involved in the recruitment of c-Src kinase to CD44s during HA-mediated
cellular signaling.
In addition, we have demonstrated that transfection of SK-OV-3.ipl
cells with c-Src (Y527F)cDNA stimulates cortactin tyrosine phosphorylation in vivo (Fig. 7B, lane
3, a and b), as compared with
cortactin in untransfected (Fig. 7B, lane
1, a and b) or vector-transfected
cells (Fig. 7B, lane 2, a
and b). Our results also demonstrate that overexpression of
c-Src (K295R) does not induce cortactin tyrosine phosphorylation (Fig.
7B, lane 4, a and
b). These data support the conclusion that cortactin serves as one of the cellular substrates for c-Src kinase in
vivo.
To determine whether there is any kinase activity associated with
GFP-tagged c-Src (Y527F) or GFP-tagged c-Src (K295R), we have incubated
these c-Src (Y527F)/(K295R) proteins with purified recombinant
cortactin in the presence of [
-32P]ATP. As shown in
Fig. 7C, a significant amount of cortactin phosphorylation
occurs in the presence of c-Src (Y527F) (Fig. 7C,
c), but not c-Src (K295R) (Fig. 7C,
d). In contrast, very little tyrosine phosphorylation of
cortactin is detected in control proteins using anti-GFP-conjugated
beads prepared from untransfected/vector-transfected cells (Fig.
7C, a and b). Apparently, tyrosine
phosphorylation of cortactin activated by c-Src (Y527F) (but not by
c-Src (K295R)) can also occur in vitro.
Tyrosine phosphorylation of cortactin by c-Src kinase has been shown to
down-regulate its cross-linking with filamentous actin (F-actin)
in vitro (41, 42). Here, we have shown that unphosphorylated cortactin (treated by anti-GFP-conjugated beads isolated from untranfected cells (Fig. 7C, a) or
vector-transfected cells (Fig. 7C, b) or c-Src
(K295R)cDNA-transfected cells (Fig. 7C, d))
promotes F-actin cross-linking activity in vitro (Fig.
7D, a, b, and d). In
contrast, c-Src (Y527F)-phosphorylated cortactin (Fig. 7C, c) significantly reduces its ability to cross-link F-actin
(Fig. 7D, c). These results support the notion
that tyrosine phosphorylation of cortactin by c-Src kinase inhibits its
interaction with F-actin, which may be required for
cytoskeleton-mediated function. Using double immunolabel staining, we
have observed that both c-Src kinase (Fig.
8A) and CD44s (Fig.
8B) are colocalized at the cellular membranes of
untransfected SK-OV-3.ipl cells (Fig. 8C). In c-Src (Y527F)cDNA-transfected cells, both GFP-tagged c-Src kinase (Y527F) (Fig. 8D) and CD44s (Fig. 8E) are closely
colocalized in the plasma membranes and long membrane projections (Fig.
8F). In contrast, vector-transfected cells expressing CD44s
on their surface (Fig. 8, inset b) (with no
detectable GFP label (Fig. 8, inset a)) are not
able to induce long membrane projections (Fig. 8, inset
c). It is also noted that overexpression of GFP-tagged c-Src
(K295R) significantly reduces c-Src kinase (Fig. 8G)
colocalization (Fig. 8I) with CD44s (Fig. 8H) and
the formation of membrane projections (Fig. 8, G-I).

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Fig. 8.
Immunofluorescence staining of c-Src kinase
and CD44s in untransfected SK-OV-3.ipl cells or SK-OV-3.ipl
transfectants. SK-OV-3.ipl cells (untransfected or transfected
with GFP-tagged c-Src (Y527F)cDNA or GFP-tagged c-Src
(K295R)cDNA or vector alone) were fixed by 2%
paraformaldehyde. Subsequently, cells were rendered permeable by
ethanol treatment and stained with various immunoreagents as described
under "Experimental Procedures." A-C, FITC-labeled
anti-c-Src kinase staining (A), Texas Red-labeled anti-CD44s
staining (B), and colocalization of c-Src kinase and CD44s
(C) in untransfected SK-OV-3.ipl cells. D-F,
GFP-tagged c-Src (Y527F) (D), Texas Red-labeled anti-CD44s
staining (E), and colocalization of GFP-tagged c-Src (Y527F)
and CD44s (F) in GFP-tagged c-Src
(Y527F)cDNA-transfected SK-OV-3.ipl cells. G-I,
GFP-tagged c-Src (K295R) (G), Texas Red-labeled anti-CD44s
staining (H), and colocalization of GFP-tagged c-Src (K295R)
and CD44s (I) in GFP-tagged c-Src
(K295R)cDNA-transfected SK-OV-3.ipl cells. Insets
a-c, No detectable staining of GFP (a), Texas
Red-labeled anti-CD44s (b), and a merged image
(c) of a and b in vector-transfected
SK-OV-3.ipl cells.
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Further analyses indicate that transfection of SK-OV-3.ipl cells with
GFP-tagged c-Src kinase (Y527F)cDNA induces a recruitment of
cortactin (Fig. 9C) to c-Src
kinase (Fig. 9A) and CD44s (Fig. 9B)-linked
membrane projections. Our results also indicate that these c-Src
(Y527F) transfectants display a significant increase in
HA-dependent and a CD44s-specific ovarian tumor cell
migration (Table I) as compared with
untransfected/vector-transfected tumor cells (Table I). In contrast,
overexpression of the dominant-negative form of c-Src (K295R) in
SK-OV-3.ipl cells not only blocks the membrane localization of c-Src
kinase (Fig. 9D) to CD44s (Fig. 9E) but also
inhibits cortactin membrane association (Fig. 9F) with CD44s
(Fig. 9E) and the formation of membrane projections (Fig. 9,
D-F). Consequently, HA and CD44s-specific tumor cell migration is also inhibited in these c-Src mutant (K295R) transfectants (Table I). These findings suggest that CD44s-mediated c-Src kinase signaling plays a pivotal role in regulating cortactin-cytoskeleton function and HA-mediated tumor cell migration during human ovarian cancer progression.

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Fig. 9.
Immunofluorescence staining of c-Src kinase,
CD44s and cortactin in SK-OV-3.ipl transfectants. SK-OV-3.ipl
cells transfected with GFP-tagged c-Src (Y527F)cDNA or GFP-tagged
c-Src (K295R)cDNA were fixed by 2% paraformaldehyde. Subsequently,
cells were rendered permeable by ethanol treatment and stained with
various immunoreagents as described under "Experimental
Procedures." A-C, GFP-tagged c-Src kinase staining
(A, green color), cyanine
(Cy5)-labeled anti-CD44s staining (B, blue
color), and Texas Red-labeled anti-cortactin staining
(C, red color) in SK-OV-3.ipl cells
transfected with c-Src (Y527F)cDNA. D-F, GFP-tagged
c-Src kinase staining (D, green
color), Cyanine (Cy5)-labeled anti-CD44s staining
(E, blue color), and Texas Red-labeled
anti-cortactin staining (F, red color)
in SK-OV-3.ipl cells transfected with c-Src (K295R)cDNA.
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DISCUSSION |
HA is the major glycosaminoglycan of the ECM. It is known to cause
cell aggregation of a number of different cell types and has been
implicated in the stimulation of cell proliferation, cell migration,
cell adhesion, and angiogenesis (9-21, 29). Overexpression of HA often
occurs at sites of tumor attachment and invasion (45). The adhesion
molecule, CD44 is one of the major HA receptors on the surface of a
number of different cell types including human ovarian tumor cells
(5-9). In the ovary, HA is present in large amounts in the mesothelial
lining (7). In fact, it has been postulated that CD44 interaction with
HA may be one of the important requirement for the spread of ovarian cancer. Nevertheless, the cellular and molecular mechanisms affecting the ability of CD44-positive ovarian tumor cells to spread and implant
at HA-enriched locations within the peritoneal cavity remain unclear.
A number of studies indicate that transmembrane interaction between the
cytoplasmic domain of CD44 and cytoskeletal proteins (in particular,
ankyrin) plays an important role in CD44-mediated oncogenic signaling
(2, 3). In particular, a 15-amino acid sequence located in the
cytoplasmic domain of CD44 appears to be required for high affinity
ankyrin binding (19-21). Several factors, including protein kinase C
(46, 47) and Rho kinase-mediated phosphorylation (17), palmitoylation
(48), and GTP binding (49), are required for the up-regulation of
CD44-ankyrin interaction. Furthermore, we have found that the S2
subdomain (but not other subdomains) of the ankyrin repeat domain binds
to CD44 directly (9); and overexpression of the S2 subdomain of ankyrin
repeat domain promotes CD44-mediated human ovarian tumor cell migration (9). Most recently, Tiam1 (T lymphoma invasion
and metastasis), one of the guanine nucleotide
(GDP/GTP) exchange factors for RhoGTPases (e.g. Rac1) has
also been found to bind CD44 (18) and to be regulated by the
cytoskeletal proteins (e.g. ankyrin) (50) during metastatic
tumor cell migration. Therefore, the selective interaction of CD44 with
various oncogenic signaling molecules and/or cytoskeletal proteins
could be one of the critical steps in promoting abnormal tumor cell motility.
A number of nonreceptor cytoplasmic tyrosine kinases (e.g.
Src, Abl, Fps, Syk/ZAP70, Jak subfamilies, and several unclassified kinases) are involved in signal transduction pathways by coupling with
surface receptors during cellular responses (51). In this study we have
found that c-Src kinase and the cell surface adhesion molecule, CD44s
are closely associated as a complex in human ovarian tumor cells (Figs.
1, 8, and 9). Abnormal regulation of Src-related enzymes is known to
lead to oncogenic cellular transformation. This was first demonstrated
by the identification of three Src family members (e.g.
c-Src, c-Yes, and c-Fgr) as the transforming elements of acutely
transforming retroviruses (52). Several tyrosine phosphorylation
substrates (e.g. PLC
(53), Vav (54, 55),
phosphatidylinositol 3-kinase (56), etc.) have been detected in
activated cells. In most cases, the kinase(s) directly phosphorylating these polypeptides is (are) not known. It is assumed that c-Src kinase
or tyrosine protein kinases activated by Src-like enzymes (e.g. Syk/ZAP70) (57, 58) is responsible for the tyrosine phosphorylation of these cellular proteins. In addition, some cytoplasmic tyrosine kinases, such as Csk, are considered to serve important regulatory functions by modulating the activity of Src subfamily kinases (59, 60).
The activity of c-Src kinase is required for epithelial cell scattering
(29, 61, 62), migration (30), and organization of the cortical
cytoskeleton (63). The cytoskeletal protein, cortactin (encoded by the
EMS1 gene) is a prominent substrate for Src-related
protein-tyrosine kinases (41, 42, 64). Overexpression of cortactin
correlates with cancer progression and poor patient prognosis (65). A
number of ligands (including fibroblast growth factor (66), epidermal
growth factor (67), and thrombin (68)), integrin activation (69),
phagocytosis (70), and mechanical strain (71) have all been shown to
stimulate tyrosine phosphorylation of cortactin leading to
cytoskeleton-regulated cell motility (42). Here, we have determined
that cortactin is a cellular substrate for c-Src kinase in human
ovarian tumor cells (SK-OV-3.ipl cell line) activated by HA binding to
CD44 (Figs. 4 and 5). Two structural features of cortactin, a repeat
domain and a COOH-terminal SH3 domain resemble neufectin, a
F-actin-associated protein (45). In fact, cortactin is considered to be
an actin binding protein (41, 42, 44). Our data indicate that the
unphosphorylated form of cortactin (in the presence of the
dominant-negative mutant protein of c-Src kinase (K295R)) is capable of
cross-linking the actin filaments into bundles in vitro
(Fig. 7). In contrast, tyrosine phosphorylation of cortactin by the
dominant-active form of c-Src kinase (Y527F) down-regulates its ability
to cross-link filamentous actin (Fig. 7). These results are consistent
with previous findings suggesting cortactin plays an important role as
an F-actin modulator in c-Src kinase-regulated cytoskeleton reorganization.
To further elucidate cortactin interaction with c-Src kinase and CD44s
in vivo, we have used immunocytochemical staining and confocal microscopy to monitor morphological changes and the
intracellular distribution of various proteins in SK-OV-3.ipl
transfectants expressing GFP-tagged c-Src mutants (e.g.
c-Src (Y527F) or c-Src (K295R)). We have observed that overexpression
of the dominant-active form of GFP-tagged c-Src kinase (Y527F) (by
transfecting SK-OV-3.ipl cells with c-Src (Y527F)cDNA) promotes
colocalization of cortactin (Fig. 9C) with CD44s (Fig.
9B) and c-Src kinase (Fig. 9A) at membrane projections (Fig. 9, A-C). In contrast, transfection of
SK-OV-3.ipl cells with the dominant-negative c-Src kinase mutant
(K295R)cDNA effectively inhibits the recruitment of cortactin (Fig.
9F) or c-Src kinase (Fig. 9D) to CD44s (Fig.
9E) at the cellular membranes and impairs the formation
membrane projections (Fig. 9, D-F) (Table I). These
findings suggest that the ability of cortactin to remodel actin
filaments (Fig. 9) or become recruited into CD44s-associated membrane
projections is tightly regulated by c-Src kinase activity.
There is increasing evidence that the ability of CD44 to transmit
information from the cell's exterior to its interior depends on
HA-CD44 interaction and selective down-stream molecular switches (1-3). During ovarian tumor cell transformation and spreading, overexpressed CD44s is tightly coupled with at least two different tyrosine kinase-based oncogenic regulators such as p185HER2
(8) and c-Src kinase (20, 35, 36). In SK-OV-3.ipl cells, CD44s and the
receptor tyrosine kinase, p185HER2, are physically linked
to each other via interchain disulfide bonds, and HA is capable of
stimulating CD44s-associated p185HER2 tyrosine kinase
activity, causing an increase in the ovarian carcinoma cell growth (8).
In this study we have described a CD44s-related nonreceptor c-Src
tyrosine kinase signaling pathway in human ovarian tumor cells.
Specifically, we have determined that CD44s binds to c-Src kinase both
in vivo (Figs. 1, 6, 8, and 9) and in vitro (Fig.
2). In particular, the cytoplasmic domain of CD44s binds to c-Src
kinase at a single site with high affinity (an apparent dissociation
constant (Kd) of ~2.0 nM) (Fig. 2).
Preliminary data indicate that the SH3 domain of c-Src kinase may be
involved in the binding of a proline-containing sequence in the
cytoplasmic domain of CD44s (data not shown).
Most Src family kinases are modified with specific lipids that direct
them to subdomains of cell membrane that have high cholesterol and
glycolipid content, called membrane "rafts" (31). In fact, the Src
kinases, Lck and Fyn, have been shown to be associated with CD44s in
glycosphingolipid-rich plasma membrane domains of human peripheral
blood lymphocytes (36). Therefore, it is possible that the interaction
between CD44 and c-Src kinase at the membrane rafts facilitates
HA-mediated stimulation of the catalytic activity of c-Src kinase
(Figs. 3-5). This, in turn, modifies cortactin structure/function (Figs. 4, 5, and 7-9) and induces membrane projections and tumor cell
migration (Table I). Several lines of evidence also indicate that
activation of c-Src activity was correlated with its ability to
interact with tyrosine-phosphorylated p185HER2 both
in vitro and in vivo in mammary tumors (72, 73).
The questions of (i) whether "cross-talk" occurs between
CD44s-c-Src kinase and CD44s-p185HER2 tyrosine kinase; and
(ii) what other tyrosine-phosphorylated substrate(s) is(are) involved
in ovarian tumor development are currently under investigation in our laboratory.
In normal cells, such as human endothelial cells, the level of CD44
expression is relatively low and HA does not bind CD44 very well. In
fact, HA binds to RHAMM (another HA binding receptor) better than it
does to CD44 in normal cells (75). Consequently, the c-Src kinase
activity associated with CD44 in normal cells fails to be activated by
HA. In the ovary, large amounts of HA accumulation in the mesothelial
lining (7) are involved in tumor attachment/invasion (45). CD44 is
overexpressed on the surface of ovarian tumor cells and mediates cell
migration in response to HA (9). Therefore, CD44 interaction with HA
has been postulated to be one of the important requirements for the spread of ovarian cancer. In this study we have provided new evidence that the binding of HA to CD44s on the surface of ovarian tumor cells
not only promotes c-Src kinase recruitment to CD44s, but also activates
c-Src kinase activity to phosphorylate the cytoskeletal protein,
cortactin, leading to tumor cell migration. This new information may
establish HA-inducible CD44s-c-Src kinase signaling as an important
functional indicator to evaluate oncogenic potential, and allow the
development of new drug targets to inhibit tumor cell motility during
the progression of human ovarian cancer.