1 Craniofacial Developmental Biology and Regeneration Branch, National Institute
of Dental and Craniofacial Research, National Institutes of Health, Bethesda,
MD 20892-4370, USA
2 Department of Molecular Virology and Oncology, Cancer Research Institute,
Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan
* Author for correspondence (e-mail: kenneth.yamada{at}nih.gov)
Accepted 23 April 2003
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Summary |
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Key words: CrkII, PTP1B, Cell migration, Tyrosine phosphorylation, Phosphatase
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Introduction |
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CrkII is known to be tyrosine phosphorylated after many types of
stimulation (Feller et al.,
1998). Most of the total population of CrkII molecules is tyrosine
phosphorylated in HeLa cells, but less than 40% of total CrkII is
phosphorylated on tyrosine 221 in various human cell lines
(Hashimoto et al., 1998
). The
tyrosine-phosphorylation site of CrkII is known to serve as a high-affinity
binding site for the SH2 domain of CrkII, resulting in intramolecular binding
of the SH2 domain to phosphotyrosine 221
(Rosen et al., 1995
). This
intramolecular binding results not only in a blockade of CrkII SH2-mediated
binding to phosphotyrosine residues in other molecules, but also in reduced
affinity for C3G due to masking or modification of the CrkII central SH3
domain (Okada et al.,
1998
).
Tyrosine phosphorylation of CrkII may induce negative regulation.
Substitution of tyrosine 221 to phenylalanine greatly reduces the tyrosine
phosphorylation of CrkII, yet it increases the binding activity of CrkII to
other phosphorylated proteins (Escalante
et al., 2000). Abl family tyrosine kinases phosphorylate tyrosine
221 on CrkII, resulting in decreased CrkII-to-p130cas
association; Abl kinase also inhibits cell migration
(Kain and Klemke, 2001
). CrkI,
which is an alternative spicing variant and lacks tyrosine 221 and the
C-terminal SH3 domain of CrkII, is upregulated and promotes cell migration and
invasion in glioblastoma (Takino et al.,
2003
). However, it is not yet clear whether
tyrosine-phosphorylated CrkII is dephosphorylated by cellular phosphatases,
nor how crucial phosphorylation of CrkII at this specific site is to the
process of cell migration.
In the present study, we examined whether PTP1B could dephosphorylate CrkII. PTP1B was able to directly dephosphorylate CrkII, as well as its SH2 binding proteins p130cas and paxillin. A CrkII mutant with tyrosine residue 221 substituted by phenylalanine (CrkII-Y221F) could not be tyrosine phosphorylated, and it showed significantly increased binding to p130cas and paxillin. The CrkII-Y221F mutant, but not wild-type CrkII, promoted cell migration on fibronectin concomitant with increased binding to p130cas and its SH3-binding proteins. This CrkII-Y221F-induced cell migration was suppressed by co-transfecting with dominant-negative (DN) Rac1 but not wild-type Rac1. Moreover, co-expression of CrkII and PTP1B promoted HT1080 cell migration on fibronectin, whereas expression of either regulatory protein alone did not. These findings provide evidence that the regulation of CrkII activity by tyrosine phosphatase/kinase targeting of tyrosine 221 is an important factor in cell migration mediated by the focal adhesion kinase (FAK)/p130cas/CrkII pathway.
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Materials and Methods |
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Antibodies and reagents
Mouse monoclonal anti-phosphotyrosine (PY20),
anti-p130cas, anti-Crk, anti-FAK (focal adhesion kinase),
and anti-paxillin antibodies were purchased from Transduction Laboratories.
Rabbit polyclonal anti-p130cas (C-20), anti-CrkII and
anti-GFP antibodies were from Santa Cruz Biotechnology. Mouse monoclonal
anti--tubulin, anti-VSV glycoprotein and anti-GST antibodies, and
Dulbecco's modified Eagle's medium (DMEM) were from Sigma-Aldrich. Mouse
monoclonal anti-PTP1B antibody was from Calbiochem. Cy3-conjugated mouse
immunoglobulin G from Jackson ImmunoResearch Laboratories and
rhodamine-labeled phalloidin from Molecular Probes were used at 1:1000
dilution. Recombinant human PTP1B was from Upstate Biotechnology.
Cell culture and transfection
The human embryo kidney 293-EBNA cell line was purchased from Invitrogen.
HT1080 cells were obtained from ATCC. Cells were maintained in DMEM
supplemented with 5% fetal bovine serum, 100 units/ml penicillin and 100
µg/ml streptomycin and cultured in 5% CO2 at 37°C. HT1080
cells were seeded at 5x104 cells/ml and 293-EBNA cells at
1x105 cells/ml at 24 hours before transfection. Transient
transfections were performed by standard calcium phosphate methods.
Silencing of endogenous PTP1B with small interfering RNA (siRNA)
Purified, duplexed siRNA for PTP1B and ß-actin were purchased from
Dharmacon. The siRNA sequence targeting human PTP1B (GenBank accession number
M31724) was from position 799-819. Twenty microlitres of siRNA (20 µM) plus
1 µg of pcDNA3 and 1 µg of pRK-GFP plasmids were transfected into
293-EBNA cells cultured in 60 mm diameter dishes by calcium phosphate
co-precipitation. At 36 hours after transfection, the cells were trypsinized,
suspended in DMEM containing 1 mg/ml bovine serum albumin (BSA) for 20
minutes, and replated on fibronectin-coated dishes for the indicated
periods.
Immunoprecipitation and immunoblotting
Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and
homogenized in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM
EGTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1% NP-40, 0.25%
sodium deoxycholate and protease inhibitor cocktail (Boehringer Mannheim,
Germany). Cell lysates were centrifuged at 15,000 g for 15
minutes at 4°C to remove insoluble material. Protein concentrations of
lysates were determined using a bicinchoninic acid protein assay kit (Pierce)
and samples were adjusted to equal protein concentration and volume. The
samples were used for immunoprecipitation with the indicated antibodies for 2
hours at 4°C followed by sedimentation with GammaBind Plus
SepharoseTM (Amersham Pharmacia Biotech). The immunoprecipitates were
separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were
blocked with 3% BSA in TBS-T (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for
1 hour at room temperature (RT), then probed with the indicated antibodies for
2 hours at RT. After washing the membranes in three 10 minute washes with
TBS-T, the membranes were incubated with horseradish peroxidase-linked
secondary antibody (Amersham Pharmacia Biotech) for 1 hour at RT followed by
enhanced chemiluminescence detection using SuperSignalTM (Pierce). The
membranes were then stripped with 2% SDS, 100 mM ß-mercaptoethanol in
62.5 mM Tris-HCl, pH 6.8, for 20 minutes at 70°C. Stripped membranes were
washed extensively in TBS and placed in 3% BSA blocking buffer overnight, and
then were re-probed with another antibody as indicated.
Protein phosphatase assay
PTP1B dephosphorylation of CrkII was examined using an in-blot phosphatase
assay. In brief, phosphorylated CrkII and FAK were obtained from
immunoprecipitates with anti-VSV antibody from lysates of 293-EBNA cells
co-transfected with VSV-FAK and VSV-CrkII. Immunoprecipitated FAK and CrkII
were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The
membrane was incubated with 20 U/ml recombinant PTP1B in assay buffer
containing 25 mM HEPES (pH 7.2), 50 mM NaCl, 2.5 mM EDTA, and 5 mM
dithiothreitol (DTT) at 37°C for 30 minutes. The phosphorylation state of
FAK and CrkII was determined with anti-phosphotyrosine antibody (PY20). The
membrane was then re-probed with anti-VSV antibody.
Cell motility
HT1080 cells were co-transfected with 2 µg pcDNA-CrkII-WT,
pcDNA-CrkII-W169L or pSSR-DN-p130cas, or either 0.5
µg pcDNA-CrkII-WT or pcDNA-CrkII-Y221F together with 0.5 µg GFP
(pGZ21
xZ) and pHA262pur puromycin-resistance plasmids. The cells were
subcultured at a 1:3 dilution 12 hours after transfection and maintained for
36 hours in 1.5 µg/ml puromycin-containing medium. This selection for
transient transfectants routinely resulted in 90% positive cells expressing
GFP as determined by fluorescence microscopy. After puromycin selection, cells
expressing various constructs were washed twice with DMEM containing 1 mg/ml
BSA and replated on 35 mm glass-bottom microwell dishes (MatTek) coated with
10 µg/ml fibronectin; the cells were cultured overnight in the same
serum-free medium. Cell movements were monitored using Zeiss inverted
microscopes. Video images were collected with Newvicon cameras (model 2400;
Hamamatsu Photonics) at 20 minute intervals, digitized and stored as image
stacks using MetaMorph 3.5 software (Universal Imaging). Image stacks were
converted to QuickTime movies, and the positions of nuclei were tracked to
quantify cell motility using Move-tr/2D software (Library, Tokyo, Japan).
Alternatively, migration assays were performed in 48-well chemotaxis chambers (Neuro Probe). Cells selected by puromycin were trypsinized, kept in suspension for 20 minutes to recover from the trypsinization, then added to the upper wells of chambers separated from the lower wells by a polycarbonate membrane coated with 10 µg/ml fibronectin. Cells were allowed to migrate for 10 hours at 37°C. The membrane was fixed, stained and scanned before and after wiping the upper side to remove non-migrating cells.
Immunofluorescence staining
Glass coverslips (12 mm diameter) were coated with 10 µg/ml fibronectin
in PBS overnight at 4°C, and then blocked with 5 mg/ml BSA for an
additional 1 hour at 37°C. After puromycin selection, cells expressing
GFP-PTP1B were washed twice with DMEM containing 1 mg/ml BSA and replated on
the coverslips, and were then cultured for 2 hours in DMEM containing 1 mg/ml
BSA. The cells were fixed with 4% paraformaldehyde in PBS for 20 minutes, then
were permeabilized with 0.5% Triton X-100 and 4% paraformaldehyde in PBS for 5
minutes. Focal adhesions were visualized by incubating first with mouse
anti-paxillin monoclonal antibody for 1 hour at RT, then with Cy3-conjugated
goat antibody to mouse immunoglobulin G. Actin filaments were stained with
rhodamine-labeled phalloidin. Localization was evaluated by confocal laser
microscopy (Carl Zeiss, LSM510).
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Results |
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To determine which domain of CrkII is responsible for protecting p130cas from dephosphorylation by PTP1B, 293-EBNA cells were co-transfected with GFP-PTP1B and mutants of CrkII. As shown in Fig. 1B, only the CrkII SH2 mutant (CrkII-R38L) failed to protect and bind to tyrosine-phosphorylated p130cas, whereas two other mutants including Y221F (mutation of tyrosine 221 to phenylalanine) bound similarly or better than wild-type. Thus, although GFP-PTP1B can effectively induce p130cas dephosphorylation, co-expression of CrkII restores tyrosine phosphorylation of p130cas by binding to p130cas through its SH2 domain. These results indicate that CrkII binding to p130cas can protect its phosphotyrosine from PTP1B; paxillin was less effectively protected.
PTP1B dephosphorylates CrkII
We tested the hypothesis that PTP1B can directly dephosphorylate CrkII
using an in-blot phosphatase assay. Incubation with recombinant human PTP1B
significantly reduced the tyrosine phosphorylation of CrkII compared with
controls (Fig. 2A). By
contrast, PTP1B did not dephosphorylate FAK, indicating that PTP1B can
directly and specifically dephosphorylate CrkII in vitro. To determine whether
expression of PTP1B could also reduce the tyrosine-phosphorylation level of
CrkII in vivo, either 0.5 µg of CrkII-WT or CrkII-Y221F was co-expressed in
293-EBNA cells with or without 2 µg of GFP-PTP1B, and both CrkII tyrosine
phosphorylation and the tyrosine-phosphorylated proteins co-precipitating with
CrkII were analyzed (Fig. 2B).
Although overexpressed CrkII-WT was tyrosine phosphorylated, co-expression
with GFP-PTP1B abrogated tyrosine phosphorylation of CrkII. In parallel with
the loss of phosphorylated CrkII, the electrophoretic mobility of CrkII-WT in
cells co-expressing GFP-PTP1B shifted to a single band with faster migration,
due to loss of the more-slowly migrating band of a doublet; this result is
consistent with dephosphorylation. CrkII-Y221F migrated with slightly higher
mobility, and it did not stain with anti-phosphotyrosine antibody; it remained
as a single band regardless of GFP-PTP1B co-expression. Thus, PTP1B could
dephosphorylate CrkII and shift its mobility, unless CrkII was mutated at
Y221.
|
Overexpression of the catalytically inactive mutant of PTP1B (PTP1B-C215S) had no effect on CrkII phosphorylation (Fig. 2C), further supporting a role for PTP1B as a phosphatase for CrkII in vivo. Tyrosine-phosphorylated proteins of approximately 130 kDa and 67 kDa (same sizes as p130cas and paxillin) were found to co-precipitate substantially more with CrkII-Y221F than with CrkII-WT (Fig. 2B). The identity of these proteins co-precipitating with CrkII-Y221F was confirmed by immunoprecipitation using anti-p130cas and anti-paxillin antibodies. As shown in Fig. 2D, CrkII-Y221F was considerably more effectively co-precipitated with both p130cas and paxillin compared with CrkII-WT. These data suggest a negative role for Y221 phosphorylation in these CrkII interactions.
Fibronectin promotes partial CrkII dephosphorylation
Although the adhesion of cells to fibronectin is associated with well-known
increases in tyrosine phosphorylation, including phosphorylation of FAK and
p130cas, our findings with the CrkII-Y221F mutant
suggested that there might actually be a concomitant loss of phosphorylation
of CrkII. As shown in Fig. 3AB,
attachment of 293-EBNA and HT1080 cells to fibronectin induced tyrosine
phosphorylation and binding of p130cas to CrkII compared
with detached cells in suspension. Concomitantly, however, CrkII was
dephosphorylated as indicated by western immunoblotting. This loss of
phosphorylation was accompanied by an electrophoretic mobility shift of CrkII,
which formed a doublet band with a faster-migrating component lacking tyrosine
phosphorylation (Fig. 3A,B,C).
In HT1080 cells transfected with PTP1B, this fibronectin-induced mobility
shift involved nearly all CrkII molecules (as indicated by the loss of nearly
the entire upper band of the doublet), and the tyrosine phosphorylation of
p130cas induced by fibronectin stimulation was reduced
(Fig. 3C).
|
PTP1B is essential for fibronectin-induced CrkII
dephosphorylation
Our results showing that dephosphorylation of CrkII is induced by PTP1B
expression and fibronectin stimulation suggests that PTP1B may be involved in
fibronectin-promoted dephosphorylation of CrkII. To test the ability of PTP1B
to dephosphorylate CrkII with fibronectin stimulation, we used a short
interfering dsRNA (siRNA) RNA interference approach to achieve knockdown of
endogeneous PTP1B levels. PTP1B was downregulated in the cells transfected
with siRNA for PTP1B but not with actin siRNA
(Fig. 4A). By contrast, actin
was decreased in the cells transfected with siRNA for actin but with PTP1B
siRNA, confirming specific downregulation of PTP1B by siRNA transfection. As
shown in Fig. 4B, attachment of
293-EBNA cells to fibronectin induced dephosphorylation of CrkII compared with
detached cells in suspension, which was consistent with the previous results
in Fig. 3. Concomitant with the
decreased levels of PTP1B, the faster-migrating component (dephosphorylated
CrkII) was decreased in PTP1B siRNA knockdown cells compared with control
cells, both when cells were kept in suspension and when plated onto
fibronectin (Fig. 4B).
|
Subcellular localization of PTP1B
PTP1B contains two proline-rich domains, which are consensus sequences for
SH3 domain-binding motifs, and it selectively binds to the SH3 domains of
Grb2, Crk and p130cas
(Liu et al., 1996). We next
investigated the subcellular distribution of PTP1B and CrkII in cells
stimulated with fibronectin. HT1080 cells transfected with GFP-PTP1B were
plated on fibronectin-coated coverslips and analyzed by confocal
immunofluorescence microscopy. As shown in
Fig. 5, CrkII was mainly
distributed in the cytoplasm with some membrane-associated staining.
Consistent with a previous study, PTP1B was not only strongly localized to the
endoplasmic reticulum, but also extended to the cell periphery. This pattern
of PTP1B localization was at times parallel to microtubules, and it ended at
focal adhesions. Co-localization between CrkII and PTP1B was observed faintly
at plasma membrane-associated sites (Fig.
5).
|
Effects of CrkII phosphorylation on cell migration
The association of CrkII with p130cas has been shown by
Klemke et al. (Klemke et al.,
1998) to regulate FG pancreatic carcinoma cell migration on
fibronectin. HT1080 cells were transfected with high or low amounts of
plasmids encoding CrkII-WT or CrkII-Y221F, and the dominant-negative
p130cas (p130casDSD) and the central
SH3 mutant of CrkII (CrkII-SH3M) were tested for effects on rates of cell
migration on fibronectin. Both p130casDSD and CrkII-SH3M
reduced cell migration to 60% of control cells (P<0.001). High but
not low levels of CrkII-WT overexpression enhanced HT1080 cell migration by
35% compared with control cells (P<0.001). By contrast, low but
not high levels of nonphosphorylated CrkII-Y221F expression significantly
increased the average rate of cell migration 47% above controls
(Fig. 6A; P<0.001).
Fig. 6B shows the morphology of
the transfected cells at 12 hours after replating onto fibronectin.
Concomitant with the increase of migration, the cells expressing high levels
of CrkII-WT (high) and low levels of CrkII-Y221F (low) shows pseudopodial
extension and membrane ruffling compared with control and low-level expressors
of CrkII-WT (low). By contrast, high levels of CrkII-Y221F expression were
accompanied by random pseudopodial extension and a flattened, more-spread
morphology consistent with increased cell-substrate adhesion.
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Co-expression of CrkII and PTP1B promotes cell migration
Although our mutational analysis of CrkII at residue Y221 strongly
suggested that lack of phosphorylation of this site mimicking
fibronectin-induced reduction of CrkII phosphorylation can regulate migration,
the direct experimental test would be to induce CrkII dephosphorylation by
PTP1B and to examine its effects on cell migration. HT1080 cells were
co-transfected with CrkII and various concentrations of the GFP-PTP1B
expression plasmid. After 36 hours of puromycin selection, the cells were
serum-starved for 12 hours and replated onto culture dishes coated with
fibronectin for 2 hours; tyrosine phosphorylation of
p130cas and paxillin were analyzed by immunoprecipitation
and immunoblotting. As shown in Fig.
7A, the co-expression of PTP1B with CrkII significantly decreased
the binding of CrkII to paxillin, but not its binding to
p130cas. Although the tyrosine phosphorylation of paxillin
was attenuated by PTP1B regardless of co-expression with CrkII, tyrosine
phosphorylation of p130cas was protected by CrkII
expression, suggesting that CrkII dephosphorylated by PTP1B preferentially
bound to and protected tyrosine-phosphorylated p130cas,
but not paxillin. As shown in Fig.
7B, HT1080 cell migration rates on fibronectin were increased in
the cells co-expressed with CrkII and PTP1B, and CrkII-Y221F with or without
PTP1B (to 119%, 125% and 121% of controls, respectively; P<0.05),
but not in the cells expressed CrkII or PTP1B alone. These results indicate
that CrkII dephosphorylated by PTP1B preferentially binds to and protects
tyrosine-phosphorylated p130cas (but not paxillin)
associated with promotion of cell migration.
|
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Discussion |
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The CrkII adaptor protein has been implicated in signal transduction
cascades by its association with SH2- and SH3-binding partner proteins.
Tyrosine dephosphorylation of p130cas was effectively
prevented by binding of CrkII (Fig.
1 of this study). CrkII itself is phosphorylated at residue
tyrosine 221 after many types of stimuli
(Matsuda and Kurata, 1996).
Abl tyrosine kinase, which binds to the N-terminal SH3 domain of CrkII, is a
negative regulator of cell migration through its ability to regulate the
p130cas/CrkII complex accompanied by phosphorylation of
the tyrosine 221 residue of CrkII, suggesting the importance of CrkII tyrosine
phosphorylation in cell migration (Kain
and Klemke, 2001
). By mutational analysis, we show in this study
that tyrosine 221 can be directly linked to the rate of cell migration (Figs
6,
7). The mechanism by which
CrkII might be dephosphorylated and its binding activated has not been clear.
Here, we provide, to the best of our knowledge, the first evidence that PTP1B
can directly dephosphorylate CrkII both in vitro and in intact cells (Figs
2,
3,
4).
PTP1B is a ubiquitous endoplasmic reticulum-associated enzyme, which is
also localized at focal adhesions
(Frangioni et al., 1992;
Arregui et al., 1998
). We also
found that PTP1B was mainly localized at the endoplasmic reticulum, but also
extended to the cell periphery. This peripheral population was parallel to
some microtubules, ended at focal adhesions, and was accompanied by faint
co-localization between CrkII and PTP1B when the cells were plated onto
fibronectin (Fig. 5). PTP1B has
been implicated in the negative regulation of cell growth, differentiation and
transformation (Byon et al.,
1997
). Liu et al. (Liu et al.,
1998
) reported that overexpression of PTP1B in Rat-1 fibroblasts
resulted in markedly reduced migration on fibronectin. By contrast, several
lines of evidence suggest an opposite role for PTP1B in integrin-mediated
signaling. Expression of a catalytically inactive mutant of PTP1B in L cells
decreases fibronectin-mediated cell spreading and FAK phosphorylation, whereas
wild-type PTP1B had no effect (Arregui et
al., 1998
). We also could not show any negative effect of
overexpressing PTP1B alone on fibronectin-induced cell migration and spreading
in two other cell types, U87-MG and HT1080 cells
(Tamura et al., 1998
)
(Fig. 7B). Furthermore,
overexpression of PTP1B in breast cancer cells (MDA-MB-435S) and HEK293 cells
decreases phosphorylation of the c-Src inhibitory site (Tyr-527), resulting in
an increase in Src kinase activity, which is essential for adhesion-dependent
p130cas phosphorylation
(Bjorge et al., 2000
). It is
likely, therefore, that the different phenotypes resulting from PTP1B
expression may be due to differences in cell types and/or expression levels of
PTP1B. Interestingly, embryonic fibroblasts from PTP1B knockout mice display
significant delays in p130cas phosphorylation and cell
spreading induced by attachment to fibronectin
(Cheng et al., 2001
). Our
findings that CrkII phosphorylation is reduced by fibronectin stimulation and
further reduced by PTP1B expression, and that 293-EBNA cells with PTP1B
knockdown by siRNA show decreased CrkII dephosphorylation induced by
attachment to fibronectin suggest a positive role of PTP1B in
integrin-mediated signaling (Figs
3,
4). Indeed, Src Tyr-527 is
hyperphosphorylated in PTP1B-deficient fibroblasts compared with wild-type,
but only when the cells are held in suspension and not when plated onto
fibronectin (Cheng et al.,
2001
). We found that detachment of cells (cells maintained in
suspension) can induce CrkII phosphorylation, which is also elevated in PTP1B
knockdown cells and is reduced by PTP1B expression (Figs
3,
4). These data suggest that the
high levels of phosphorylation of CrkII in cells kept in suspension may result
in the attenuation of fibronectin-mediated events. However, PTP1B knockdown
did not increase cell migration (data not shown), implying the involvement of
other phosphatases or small remnants of PTP1B in dephosphorylation of CrkII.
In fact, a recent study has reported that even though PTP1B-/-
cells can exhibit enhanced tyrosine phosphorylation of epidermal growth factor
(EGF) receptor or platelet-derived growth factor (PDGF) receptor in response
to EGF or PDGF stimulation, Akt and ERK activation are only minimally or not
enhanced (Haj et al., 2003
).
The authors suggest that even though PTP1B plays a role in regulating EGFR and
PDGFR, other regulatory mechanisms come into play when it is missing; they
speculate that this finding might explain why no classical protein tyrosine
phosphatases have been found to be tumor suppressor genes. PTP1B might be
important in cell migration by activating CrkII and Src in a process dependent
on their relative intracellular concentrations.
CrkII has been identified as a mediator of cell migration through its
association with p130cas and paxillin
(Klemke et al., 1998). The
p130cas/CrkII/DOCK180 pathway is reported to promote cell
migration and to activate Rac1 (Kiyokawa
et al., 1998a
; Kiyokawa et
al., 1998b
). As documented in
Fig. 2, CrkII-Y221F can bind
more effectively to paxillin and p130cas than wild-type
CrkII, suggesting that the expression of CrkII-Y221F might facilitate this
pathway. At a relatively low expression level where wild-type CrkII could not
stimulate cell migration, nonphosphorylated CrkII-Y221F could readily promote
HT1080 cell migration on fibronectin. By contrast, high expression of
CrkII-Y221F did not elevate rates of cell migration in these cells, which
displayed enhanced random pseudopodial extension and a flattened, more-spread,
apparently more-adhesive morphology. This result may be explained by Crk
activation of C3G by tyrosine phosphorylation and C3G-dependent Rap1
activation that promotes cell adhesion and spreading, but repression of cell
migration (Ohba et al., 2001
).
The authors also suggest that CrkII-C3G may function downstream of paxillin,
but not p130cas, to suppress cell migration. CrkII-Y221F
fails to induce JNK activation and cell migration on fibronectin in COS-7
cells (Abassi et al., 2002). Girardin and Yaniv (Girardin and Yaniv, 2001)
reported that the p130cas/CrkII complex serves as a
scaffolding structure for JNK signaling pathway. With scaffold proteins for
mitogen-activated protein kinase cascades, even though the presence of an
optimal scaffold concentration can increase the signaling output, if the
scaffold concentration is greater than optimal, a significant decrease in
signaling can occur (Levchenko et al.,
2000
). A well-studied scaffold for the JNK signaling cascade is
the JNK interacting protein (JIP) family. Indeed, JIP-1 was originally
identified as an inhibitor of the JNK signaling pathway, whereas the JIP
family can function as scaffolding for the JNK signaling cascade
(Dickens et al., 1997
;
Ito et al., 1999
).
Overexpression of the JNK binding domain of JIP-1 can inhibit JNK activation
(Harding et al., 2001
). Our
results that a relatively low expression level, but not high expression, of
CrkII-Y221F promoted HT1080 cell migration on fibronectin is consistent with
the ability of CrkII to function as a scaffold structure binding
p130cas in cell migration. Consequently, it appears that
the differing roles of Y221F-CrkII in cell migration may reflect differing
expression and optimal concentration in different cell types.
Recent research has revealed that tyrosine phosphorylation of paxillin
reduces cell migration, whereas tyrosine phosphorylation of
p130cas facilitates cell migration
(Yano et al., 2000). In this
study, we found that co-expression of CrkII and PTP1B preferentially protects
against dephosphorylation of p130cas, but not of paxillin,
concomitant with promoting cell migration on fibronectin. The promotion of
cell migration by PTP1B associated with dephosphorylation of paxillin and
CrkII might involve a change in the balance of tyrosine phosphorylation
between p130cas and paxillin. v-Crk has been implicated in
the activation of Rho and phosphatidylinositol 3-kinase, which are required
for focal adhesion formation and cell migration
(Altun-Gultekin et al., 1998
;
Akagi et al., 2000
), although
it is not known whether nononcogenic CrkII also participates in Rho and
phosphatidylinositol 3-kinase activation. In general, CrkII may act as a
molecular switch for cell migration after binding to
p130cas or paxillin and activation of Rac1
(Klemke et al., 1998
).
The data presented here provide the first evidence that PTP1B directly dephosphorylates CrkII in vitro and in vivo, with tyrosine residue 221 of CrkII serving as a regulatory element in binding to tyrosine-phosphorylated proteins and transducing a signal to induce cell migration on fibronectin. We conclude that the regulation of CrkII activity by tyrosine kinases and phosphatases can be an important factor in regulating cell migration mediated by the FAK/p130cas/CrkII pathway.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abassi, Y. A. and Vuori, K. (2002). Tyrosine 221 in CrkII regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling. EMBO J. 17,4571 -4582.[CrossRef]
Akagi, T., Shishido, T., Murata, K. and Hanafusa, H.
(2000). v-Crk activates the phosphoinositide 3-kinase/Akt pathway
in transformation. Proc. Natl. Acad. Sci. USA
97,7290
-7295.
Altun-Gultekin, Z. F., Chandriani, S., Bougeret, C., Ishizaki,
T., Narumiya, S., de Graaf, P., van Bergen en Henegouwen, P., Hanafusa, H.,
Wagner, J. A. and Birge, R. B. (1998). Activation of
Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk
adaptor protein. Mol. Cell. Biol.
18,3044
-3058.
Arregui, C. O., Balsamo, J. and Lilien, J.
(1998). Impaired integrin-mediated adhesion and signaling in
fibroblasts expressing a dominant-negative mutant PTP1B. J. Cell
Biol. 143,861
-873.
Birge, R. B., Fajardo, J. E., Mayer, B. J. and Hanafusa, H.
(1992). Tyrosine-phosphorylated epidermal growth factor receptor
and cellular p130 provide high affinity binding substrates to analyze
Crk-phosphotyrosine-dependent interactions in vitro. J. Biol.
Chem. 267,10588
-10595.
Bjorge, J. D., Pang, A. and Fujita, D. J.
(2000). Identification of protein-tyrosine phosphatase 1B as the
major tyrosine phosphatase activity capable of dephosphorylating and
activating c-Src in several human breast cancer cell lines. J.
Biol. Chem. 275,41439
-41446.
Byon, J. C., Kenner, K. A., Kusari, A. B. and Kusari, J. (1997). Regulation of growth factor-induced signaling by protein-tyrosine-phosphatases. Proc. Soc. Exp. Biol. Med. 216,1 -20.[Abstract]
Cheng, A., Bal, G. S., Kennedy, B. P. and Tremblay, M. L.
(2001). Attenuation of adhesion-dependent signaling and cell
spreading in transformed fibroblasts lacking protein tyrosine phosphatase-1B.
J. Biol. Chem. 276,25848
-25855.
Dickens, M., Rogers, J. S., Cavanagh, J., Raitano, A., Xia, Z.,
Halpem, J. R., Greenberg, M. E., Sawyers, C. L. and Davis, R.
(1997). A cytoplasmic inhibitor of the JNK signal transduction
pathway. Science 277,693
-696.
Escalante, M., Courtney, J., Chin, W. G., Teng, K. K., Kim, J.
I., Fajardo, J. E., Mayer, B. J., Hempstead, B. L. and Birge, R. B.
(2000). Phosphorylation of c-Crk II on the negative regulatory
Tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis.
J. Biol. Chem. 275,24787
-24797.
Feller, S. M., Posern, G., Voss, J., Kardinal, C., Sakkab, D., Zheng, J. and Knudsen, B. S. (1998). Physiological signals and oncogenesis mediated through Crk family adapter proteins. J. Cell. Physiol. 177,535 -552.[CrossRef][Medline]
Frangioni, J. V., Beahm, P. H., Shifrin, V., Jost, C. A. and Neel, B. G. (1992). The nontransmembrane tyrosine phosphatase PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence. Cell 68,545 -560.[Medline]
Giradin, S. E. and Yaniv, M. (2001). A deirect
interaction between JNK1 and CrkII is critical for Rac1-induced JNK
activation. EMBO J. 20,3437
-3446.
Haj, F. G., Markova, B., Klaman, L. D., Bohmer, F. D. and Neel,
B. G. (2003). Regulation of receptor tyrosine kinase
signaling by protein tyrosine phosphatase-1B. J. Biol.
Chem. 278,739
-744.
Harding, T. C., Xue, L., Bienemann, A., Haywood, D., Dickens,
M., Tolkovsky, A. M. and Uney, J. B. (2001). Inhibition of
JNK by overexpression of the JNK binding domain of JIP-1 prevents apoptosis in
sympathetic neurons. J. Biol. Chem.
276,4531
-4534.
Hashimoto, Y., Katayama, H., Kiyokawa, E., Ota, S., Kurata, T.,
Gotoh, N., Otsuka, N., Shibata, M. and Matsuda, M. (1998).
Phosphorylation of CrkII adaptor protein at tyrosine 221 by epidermal growth
factor receptor. J. Biol. Chem.
273,17186
-17191.
Ito, M., Yoshioka, K., Akechi, M., Yamashita, S., Takamatsu, N.,
Sugiyama, K., Hibi, M., Nakabeppu, Y., Shiba, T. and Yamamoto, K.
(1999). JSAP1, a novel Jun N-terminal protein kinase
(JNK)-binding protein that functions as a scaffold factor in the JNK signaling
pathway. Mol. Cell. Biol.
19,7539
-7548.
Kain, K. H. and Klemke, R. L. (2001).
Inhibition of cell migration by Abl family tyrosine kinases through uncoupling
of Crk-CAS complexes. J. Biol. Chem.
276,16185
-16192.
Kiyokawa, E., Hashimoto, Y., Kobayashi, S., Sugimura, H.,
Kurata, T. and Matsuda, M. (1998a). Activation of Rac1 by a
Crk SH3-binding protein, DOCK180. Genes Dev.
12,3331
-3336.
Kiyokawa, E., Hashimoto, Y., Kurata, T., Sugimura, H. and
Matsuda, M. (1998b). Evidence that DOCK180 up-regulates
signals from the CrkII-p130(Cas) complex. J. Biol.
Chem. 273,24479
-24484.
Klemke, R. L., Leng, J., Molander, R., Brooks, P. C., Vuori, K.
and Cheresh, D. A. (1998). CAS/Crk coupling serves as a
"molecular switch" for induction of cell migration. J.
Cell Biol. 140,961
-972.
Levchenko, A., Bruck, J. and Sternberg, P. W.
(2000). Scaffold proteins may biphasically affect the levels of
mitogen-activated protein kinase signaling and reduce its threshold
properties. Proc. Natl. Acad. Sci. USA
97,5818
-5823.
Liu, F., Hill, D. E. and Chernoff, J. (1996).
Direct binding of the proline-rich region of protein tyrosine phosphatase 1B
to the Src homology 3 domain of p130(Cas). J. Biol.
Chem. 271,31290
-31295.
Liu, F., Sells, M. A. and Chernoff, J. (1998). Protein tyrosine phosphatase 1B negatively regulates integrin signaling. Curr. Biol. 8,173 -176.[Medline]
Matsuda, M. and Kurata, T. (1996). Emerging components of the Crk oncogene product: the first identified adaptor protein. Cell. Signal. 8,335 -340.[CrossRef][Medline]
Mayer, B. J., Hamaguchi, M. and Hanafusa, H. (1988). A novel viral oncogene with structural similarity to phospholipase C. Nature 332,272 -275.[CrossRef][Medline]
Ohba, Y., Ikuta, K., Ogura, A., Matsuda, J., Mochizuki, N.,
Nagashima, K., Kurokawa, K., Mayer, B. J., Maki, K., Miyazaki, J. et al.
(2001). Requirement for C3G-dependent Rap1 activation for cell
adhesion and embryogenesis. EMBO J.
20,3333
-3341.
Okada, S., Matsuda, M., Anafi, M., Pawson, T. and Pessin, J.
E. (1998). Insulin regulates the dynamic balance between Ras
and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and
CrkII-C3G complexes. EMBO J.
17,2554
-2565.
Rosen, M. K., Yamazaki, T., Gish, G. D., Kay, C. M., Pawson, T. and Kay, L. E. (1995). Direct demonstration of an intramolecular SH2-phosphotyrosine interaction in the Crk protein. Nature 374,477 -479.[CrossRef][Medline]
Smith, J. J., Richardson, A., Kopf, J., Yoshida, M.,
Hollingsworth, R. E. and Kornbluth, S. (2002). Apoptotic
regulation by the Crk adapter protein mediated by interactions with Wee1 and
Crm1/Exportin. Mol. Cell. Biol.
22,1412
-1423.
Takino, T., Nakada, M., Miyamori, H., Yamashita, J., Yamada, K.
M. and Sato, H. (2003). CrkI apaptor protein modulates cell
migration and invasion in glioblastoma. Cancer Res.
63,2335
-2337.
Tamura, M., Gu, J., Matsumoto, K., Aota, S., Parsons, R. and
Yamada, K. M. (1998). Inhibition of cell migration,
spreading, and focal adhesions by tumor suppressor PTEN.
Science 280,1614
-1617.
Tamura, M., Gu, J., Danen, E. H., Takino, T., Miyamoto, S. and
Yamada, K. M. (1999a). PTEN interactions with focal adhesion
kinase and suppression of the extracellular matrix-dependent
phosphatidylinositol 3-kinase/Akt cell survival pathway. J. Biol.
Chem. 274,20693
-20703.
Tamura, M., Gu, J., Takino, T. and Yamada, K. M.
(1999b). Tumor suppressor PTEN inhibition of cell invasion,
migration, and growth: differential involvement of focal adhesion kinase and
p130Cas. Cancer Res. 59,442
-449.
Yano, H., Uchida, H., Iwasaki, T., Mukai, M., Akedo, H.,
Nakamura, K., Hashimoto, S. and Sabe, H. (2000). Paxillin
alpha and Crk-associated substrate exert opposing effects on cell migration
and contact inhibition of growth through tyrosine phosphorylation.
Proc. Natl. Acad. Sci. USA
97,9076
-9081.