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INTRODUCTION |
Dynamic changes in the extent of tyrosine phosphorylation of
cellular proteins are fundamental to signal transduction pathways that
regulate cell growth and differentiation, the cell cycle, tissue
morphogenesis, and cytoskeletal organization. Tyrosine phosphorylation
is tightly controlled by coordination of the activities of
protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs)1 (1). Although much
progress has been made in our understanding of the structure, function,
and regulation of protein-tyrosine kinases, PTPs are less well characterized.
Similar to protein-tyrosine kinases, PTPs can be divided into two
structurally distinct subgroups: cytoplasmic PTPs and
transmembrane-type (receptor-like) PTPs (RPTPs) (2). Despite their
marked diversity in overall structure, all PTPs possess a core sequence
(I/V)HCXAGXXR(S/T)G that contains conserved
cysteine and arginine residues critical for enzymatic activity (3). The
cellular functions of PTPs are thought to be determined by their
substrate specificity, subcellular localization, and interaction with
other signaling molecules.
Although initial observations of the effects of overexpression of PTPs
suggested that these enzymes might simply counteract the signaling
events elicited by protein-tyrosine kinases, more recent genetic and
biochemical evidence indicates that PTPs play more complex roles in a
wide range of cellular activities (4, 5). For example, SHP-1 and SHP-2,
two Src homology 2 domain-containing cytoplasmic PTPs play negative and
positive regulatory roles, respectively, in protein-tyrosine kinase
signaling (6). Members of the band 4.1 family of cytoplasmic PTPs,
including PTPH1 and PTPMEG, are thought to regulate cytoskeletal
reorganization (7, 8). In addition, the RPTP PTP
affects
cell-substratum adhesion and cellular transformation by regulating the
activity of the protein-tyrosine kinase Src (9). Finally, members of
the LAR family of RPTPs, including PTP
(10) and PTP
(11) as well as Drosophila DLAR and the related DPTP69D (12, 13), are
essential for axon guidance or neuronal differentiation in the
developing nervous system.
We have previously cloned a human RPTP termed SAP-1 (stomach
cancer-associated PTP-1) (14). This enzyme contains a single catalytic
domain in the cytoplasmic region, a single transmembrane domain, and
eight fibronectin type III-like domains in the extracellular region.
SAP-1 belongs to the class 2 subfamily of RPTPs (15), which includes
HPTP
(15), DEP-1 (16), PTP-U2 (17), and Drosophila
DPTP10D (18, 19). The observations that the fibronectin type III-like
domain is present in many neural cell adhesion molecules and that SAP-1
is abundant in the brain suggest that this PTP plays a role in neural
cell-cell adhesion signaling (14). SAP-1 is also abundant in a subset
of pancreatic and colorectal cancer cell lines and tissues but not in
their normal counterparts (14, 20). Furthermore, the SAP-1 gene is
located on the long arm of human chromosome 19, at position q13.4,
which is close to the locus of the gene for carcinoembryonic antigen at
19q13.2 (14). Expression of SAP-1 may thus also be associated with
cancer development. However, the biological roles of this PTP remain unknown.
The identification of physiological substrates of PTPs provides
important insight into the functions of these enzymes. However, substrate identification for this class of enzymes has proved problematic because of the low affinity and transient nature of the
interaction of PTPs with their substrates. A recently developed substrate-trapping strategy has overcome this limitation and greatly facilitated isolation of specific substrates of PTPs (21-24). With the
use of this strategy, we have now identified p130cas, a major
phosphotyrosyl protein that is localized to focal adhesions (FAs), as a
potential substrate of SAP-1. We also provide evidence that SAP-1
negatively regulates various integrin-promoted cellular responses and
that the enzymatic activity of this PTP is up-regulated by cell-cell adhesion.
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EXPERIMENTAL PROCEDURES |
Expression Vectors--
The cDNAs encoding a catalytically
inactive mutant of SAP-1 (SAP-1C/S), in which Cys1022 is
replaced by Ser, and a substrate-trapping mutant of SAP-1 (SAP-1D/A),
in which Asp988 is replaced by Ala, were generated by
site-directed mutagenesis with human SAP-1 cDNA as a template and a
transformer site-directed mutagenesis kit
(CLONTECH). The full-length wild-type and mutant SAP-1 cDNAs were then inserted separately into the
HindIII site of the pRc/CMV expression vector (Invitrogen).
The pSR
vector encoding hemagglutinin (HA)-tagged p130cas
was kindly provided by H. Hirai (University of Tokyo), the pBabe vector
encoding Myc-tagged paxillin-
was provided by H. Sabe (Osaka
Bioscience Institute), the pRc/CMV vector encoding HA-tagged focal
adhesion kinase (FAK) was provided by S. K. Hanks (Vanderbilt University, Nashville, TN), and the pcDNA3 vector encoding
HA-tagged extracellular signal-regulated kinase 2 (ERK2) was provided
by J. S. Gutkind (National Institutes of Health, Bethesda, MD).
The pRc/CMV vector encoding HA-tagged mouse p62dok was
generated as described (25).
Cells, Antibodies, and Transfection--
Chinese hamster ovary
(CHO) cell lines stably expressing wild-type SAP-1 (SAP-1WT) or
SAP-1C/S were generated as described previously (26), and the
expression level of each SAP-1 protein was determined by immunoblot
analysis with polyclonal antibodies to SAP-1 as described below. CHO-K1
cells and the established cell lines were maintained in Ham's F-12
medium supplemented with 10% fetal bovine serum. 293 human embryonic
kidney cells, Panc-1 cells, WiDr cells, Swiss 3T3 cells, and NIH 3T3
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. Transfection of CHO-K1 cells
or 293 cells (~2 × 105 cells/60-mm dish) was
performed with the use of a CellPhect Transfection Kit (Amersham
Pharmacia Biotech).
Rabbit polyclonal antibodies to SAP-1 (14) and the mouse monoclonal
antibody (mAb) 3G5 to SAP-1 were generated in response to a glutathione
S-transferase (GST) fusion protein containing the
cytoplasmic region of SAP-1 in our laboratory. Rabbit polyclonal antibodies to p62dok were described previously (25). The mAb
12CA5 to the HA epitope tag and the mAb 9E10 to the Myc tag were
purified from the culture supernatants of mouse hybridoma cells. Mouse
mAbs to p130cas, to paxillin, and to
-catenin were obtained
from Transduction Laboratories; horseradish peroxidase-conjugated mouse
mAb PY20 to phosphotyrosine and rabbit polyclonal antibodies to
p130cas or to FAK were from Santa Cruz Biotechnology; rabbit
polyclonal antibodies that react specifically with
tyrosine-phosphorylated ERK or with total ERK protein were from New
England BioLabs; a mouse mAb to vinculin was from Sigma; fluorescein
isothiocyanate-conjugated sheep polyclonal antibodies to mouse
immunoglobulin and Texas red-conjugated donkey polyclonal antibodies to
rabbit immunoglobulin were from Amersham Pharmacia Biotech.
Immunoprecipitation and Immunoblot Analysis--
Cells were
thawed on ice in 1 ml of ice-cold lysis buffer (20 mM
Tris-HCl (pH 7.6), 140 mM NaCl, 1 mM EDTA, 1%
(v/v) Nonidet P-40) containing 5 mM NaF, 1 mM
phenylmethylsulfonyl fluoride, aprotinin (10 µg/ml), and 1 mM sodium vanadate. The cell lysates were centrifuged at
10,000 × g for 15 min at 4 °C, and the resulting supernatants were subjected to immunoprecipitation and immunoblot analysis. For immunoprecipitation, the supernatants were incubated for
3 h at 4 °C with antibody-coupled protein G-Sepharose beads (20 µl of beads) (Amersham Pharmacia Biotech). The beads were washed
three times with 1 ml of WG buffer (50 mM Hepes-NaOH (pH 7.6), 150 mM NaCl, 0.1% (v/v) Triton X-100) and then
suspended in Laemmli sample buffer. Immunoblot analysis with various
antibodies was performed with the use of the ECL detection system
(Amersham Pharmacia Biotech).
Expression and Purification of Recombinant PTPs--
The
cytoplasmic regions of SAP-1WT, SAP-1C/S, and SAP-1D/A were produced as
GST fusion proteins. The polymerase chain reaction was performed with
wild-type or mutant SAP-1 cDNA as template and with
5'-TAGGATCCCCAGGGGACATCCCAGCTGAAG (nucleotides 2436-2457 of SAP-1
cDNA) and 5'-TCGAATTCGGGCTGCCGACCCAGCCCCCTCG (nucleotides 3400-3422) as sense and antisense primers, respectively. The
amplification products were digested with BamHI and
EcoRI and inserted in-frame into the BamHI and
EcoRI sites of pGEX-2T (Amersham Pharmacia Biotech). The
encoded GST fusion proteins were then expressed in Escherichia
coli and purified with the use of glutathione-Sepharose beads
(Amersham Pharmacia Biotech). The recombinant full-length SHP-2 was
prepared as described previously (26).
Substrate Trapping--
Panc-1 cells in one 100-mm dish were
treated with 100 µM pervanadate for 30 min and then lysed
in 1 ml of buffer A (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1%
(v/v) Triton X-100) containing 1 mM phenylmethylsulfonyl
fluoride, 1 mM benzamidine, 10 µM leupeptin,
aprotinin (10 µg/ml), and 5 mM iodoacetic acid. After
incubation of the lysate for 30 min at 4 °C with gentle agitation,
dithiothreitol was added to a final concentration of 10 mM
to inactivate any unreacted iodoacetic acid. The lysate was then
centrifuged at 100,000 × g for 30 min at 4 °C, and
the resulting supernatant was incubated for 2 h at 4 °C with
glutathione-Sepharose beads conjugated with 10 µg of recombinant
SAP-1 protein. The beads were washed three times with 1 ml of buffer A,
and bound material was subjected to immunoblot analysis. In some
experiments, 1 mM orthovanadate was included during the
incubation of beads with lysate.
In-blot Dephosphorylation Assay--
Lysates from
pervanadate-treated Panc-1 cells were subjected to immunoprecipitation;
the resultant precipitates containing tyrosine-phosphorylated proteins
were fractionated by SDS-polyacrylamide gel electrophoresis, and the
separated proteins were transferred to a nitrocellulose filter. The
dephosphorylation reaction was performed by incubating the filter in
the absence or presence of recombinant SAP-1 or SHP-2 for 20 min at
30 °C in a solution containing 50 mM Hepes-NaOH (pH
7.1), 150 mM NaCl, 10 mM dithiothreitol, and 2 mM EDTA. The filter was then washed for 5 min at room
temperature with phosphate-buffered saline (PBS) containing 500 mM NaCl, 0.5% (w/v) SDS, and 0.1% (v/v) Triton X-100. The
extent of tyrosine phosphorylation of each protein was determined by
immunoblot analysis with mAb PY20 to phosphotyrosine.
Cell-spreading Assay--
Cells were detached from the culture
dish by treatment with 0.025% trypsin, collected by centrifugation,
and washed once with serum-free Ham's F-12 medium. The cells were
replated in serum-free Ham's F-12 medium at a density of 1 × 105 cells/ml on 60-mm culture dishes coated with
fibronectin (10 µg/ml) (Sigma) or poly-L-lysine (10 µg/ml) (Sigma). The cells were then incubated for 2 h at
37 °C in a humidified incubator containing 5% CO2,
after which they were examined under a light microscope equipped with
phase-contrast optics (model IX 70; Olympus), and random fields were
photographed at a total magnification of 200×.
Generation of and Infection with Recombinant
Adenoviruses--
The cDNAs encoding SAP-1WT or SAP-1C/S were
cloned separately into the SwaI site of pAxCAwt (27), which
contains the CAG promoter, and the resulting constructs were
introduced together with DNA-terminal protein complex by
transfection into 293 cells. The resulting recombinant adenoviruses
were screened by immunoblot analysis and cloned by limiting dilution.
Adenoviral vectors were propagated by a standard procedure, purified by
two rounds of CsCl density gradient centrifugation, and then titrated
with a limiting dilution assay in 293 cells. Swiss 3T3 cells seeded on glass coverslips in a 6-well plate were infected with viruses at
37 °C for 1 h, with brief agitation every 20 min, and were then
exposed to normal growth medium.
Immunofluorescence Staining--
Cells were washed with PBS,
fixed with 3.7% formaldehyde for 20 min, permeabilized with 0.5%
(v/v) Triton X-100 in PBS for 5 min, and incubated for 2 h with
TBS-T (20 mM Tris-HCl (pH 7.6), 150 mM NaCl,
0.05% (v/v) Tween 20) containing 5% (w/v) nonfat dry milk, 10% fetal
bovine serum, and 1% (w/v) bovine serum albumin. The cells were then
incubated for 1 h at room temperature with mAb 3G5 to SAP-1 (1 µg/ml) or with polyclonal antibodies to SAP-1 (1 µg/ml), together
with either rhodamine-labeled phalloidin (0.1 µg/ml) (Sigma) or a mAb
to vinculin (20 µg/ml). Cells were then washed twice with PBS and
incubated with fluorescein isothiocyanate- or Texas red-conjugated
secondary antibodies for 30 min. After washing three times with PBS,
the cells were examined under a laser-scanning confocal microscope
(model MRC-1024; Bio-Rad), and built-up images were constructed.
Assay of ERK2 activation--
Transfected 293 cells (60-mm
dishes) were deprived of serum for 12 h and then incubated for 5 min with 10 µM lysophosphatidic acid (LPA) (Sigma) or
epidermal growth factor (EGF) (100 ng/ml) (Calbiochem) or for 10 min
with 1 µM 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma). Cells were then lysed in 400 µl of a solution
containing 50 mM Hepes-NaOH (pH 7.8), 150 mM
NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.1% (v/v) Triton X-100, 20 mM
-glycerophosphate, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, aprotinin (10 µg/ml),
and 1 mM sodium vanadate. Recombinant ERK2 was
immunoprecipitated with mAb 12CA5 to the HA tag and then subjected to
immunoblot analysis with antibodies specific for activated ERK or for
total ERK protein. The extent of ERK2 phosphorylation was quantified by
scanning densitometry with the NIH Image program.
Colony Formation Assay--
CHO-K1 cells or NIH 3T3 cells
(~2 × 105 cells/60-mm dish) were transiently
transfected with 3 µg of pRc/CMV containing (or not) SAP-1WT or
SAP-1C/S cDNA. After 48 h, transfected cells were diluted 1:20
with normal growth medium supplemented with G418 (400 µg/ml) and
cultured in 100-mm dishes. The medium was changed every 3 days, and,
after 14 days, cells that had formed colonies were fixed with 3.7%
formaldehyde and stained with crystal violet. The efficiency of colony
formation was quantified by scanning densitometry with the NIH Image program.
Determination of PTP Activity--
The enzymatic activity of
recombinant SAP-1 was assayed with p-nitrophenylphosphate
(pNPP) as a substrate as described previously (14). Cellular SAP-1
activity immunopurified with mAb 3G5 was assayed with pNPP as described
previously (28).
Reproducibility--
All data presented are representative of at
least three independent experiments.
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RESULTS |
Identification of p130cas as a Potential
Substrate of SAP-1--
To identify physiological substrates of SAP-1,
we first generated two types of SAP-1 mutant: a catalytically inactive
mutant, in which the invariant Cys1022 residue is replaced
by Ser, and a substrate-trapping mutant, in which the invariant
Asp988 residue is replaced by Ala. The latter mutant was
expected to be a more powerful tool than was the former because it
should not only be catalytically inactive but also retain the ability to bind SAP-1 substrates (22). We then prepared GST fusion proteins that contain the catalytic domains of wild-type SAP-1 (GST-SAP-1WT), catalytically inactive SAP-1 (GST-SAP-1C/S), or the substrate-trapping mutant of SAP-1 (GST-SAP-1D/A). Each recombinant protein was expressed in and purified from bacteria as a 60-kDa polypeptide, as revealed by
SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (Fig. 1A).

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Fig. 1.
Association of p130cas and paxillin
with a substrate-trapping mutant of SAP-1. A,
expression and purification of recombinant SAP-1 proteins. GST fusion
proteins containing the cytoplasmic region of either wild-type SAP-1
(GST-SAP-1WT), a catalytically inactive mutant of SAP-1 (GST-SAP-1C/S),
or a substrate-trapping mutant of SAP-1 (GST-SAP-1D/A) were expressed
in bacteria, purified with the use of glutathione-Sepharose beads, and
subjected to SDS-polyacrylamide gel electrophoresis and Coomassie Blue
staining. The position of each recombinant protein is indicated by the
arrow labeled SAP-1. B, lysates prepared from
pervanadate-treated Panc-1 cells were incubated for 2 h at 4 °C
with glutathione-Sepharose beads conjugated with each recombinant SAP-1
protein or with GST alone. The bead-bound proteins were then subjected
to immunoblot analysis with horseradish peroxidase-conjugated mAb PY20
to phosphotyrosine ( PY). The positions of bound
phosphotyrosyl proteins are indicated. C, effect of vanadate
on the association between mutant SAP-1 and phosphotyrosyl proteins.
Cell lysates prepared as in B were incubated with
GST-SAP-1C/S or GST-SAP-1D/A in the absence ( ) or presence (+) of 1 mM orthovanadate, after which the bead-bound proteins were
subjected to immunoblot analysis with mAb PY20. The positions of bound
phosphotyrosyl proteins are indicated. The immunoreactive material
apparent immediately above p55 (B and C) and
p75-85 (C) in the GST-SAP-1D/A lanes corresponds
to cross-reactive polypeptides of unknown origin. D,
recognition of p125-135 and p75-85 by specific antibodies to
p130cas and to paxillin, respectively. The precipitates
prepared as described in B were subjected to immunoblot
analysis with polyclonal antibodies to p130cas
( Cas) or with a mAb to paxillin ( paxillin),
as indicated.
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Whereas GST-SAP-1WT exhibited substantial catalytic activity with the
artificial substrate pNPP, GST-SAP-1C/S and GST-SAP-1D/A possessed
virtually no activity (Fig.
2A). Each of the three
recombinant proteins bound to glutathione-Sepharose beads was then
incubated with lysates prepared from pervanadate-treated Panc-1 cells,
which express SAP-1 (14). Immunoblot analysis with the mAb PY20 to phosphotyrosine revealed that GST-SAP-1D/A, but not GST-SAP-1WT or GST
alone, precipitated several phosphotyrosyl proteins with molecular
sizes of 190, 125-135, 75-85, and 55 kDa (hereafter referred to as
p190, p125-135, p75-85, and p55, respectively) (Fig. 1B).
GST-SAP-1C/S precipitated a similar group of proteins, although to a
much lesser extent than did GST-SAP-1D/A (Fig. 1, B and
C). The amounts of p190, p125-135, p75-85, and p55 bound to GST-SAP1D/A were markedly reduced by incubation of cell lysates with
the recombinant protein in the presence of vanadate, a potent inhibitor
of PTPs; in contrast, the amounts of these proteins bound to
GST-SAP-1C/S were substantially increased by incubation with vanadate
(Fig. 1C). Vanadate ions interact directly with the thiolate
anion of the cysteine residue in the catalytic core sequence of PTPs
and thereby block enzyme-substrate association (29, 30). These results
thus suggest that all of the detected phosphotyrosyl proteins might
bind, either directly or indirectly, to the active site of SAP-1.

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Fig. 2.
Dephosphorylation of p130cas by SAP-1
in vitro. A, catalytic activities of
recombinant wild-type and the mutant SAP-1 proteins, a GST fusion
protein containing full-length SHP-2 (GST-SHP-2), and GST with pNPP as
the substrate. Data are expressed as absorbance units at 410 nm and are
the means of triplicates from a representative experiment.
B, PTP activities toward p130cas, paxillin, and
-catenin. Lysates of pervanadate-treated Panc-1 cells were subjected
to immunoprecipitation with antibodies to p130cas (top
panel), to paxillin (middle panel), or to -catenin
(bottom panel). The resultant precipitates were fractionated
by SDS-polyacrylamide gel electrophoresis, after which the separated
proteins were transferred to a nitrocellulose membrane and incubated
with GST-SAP-1WT (50 ng/ml), GST-SHP-2 (5 µg/ml), or GST (5 µg/ml)
for 20 min at 30 °C. The extent of tyrosine phosphorylation of each
protein was determined by immunoblot analysis with mAb PY20 to
phosphotyrosine ( PY).
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To identify p125-135, we subjected the proteins precipitated by
GST-SAP-1D/A to immunoblot analysis with antibodies specific for either
FAK, Ras GTPase-activating protein, or p130cas, all of which
are in the size range of 125-135 kDa and are phosphorylated on
tyrosine residues. Of these antibodies, the polyclonal antibodies to
p130cas specifically recognized a 125- to 135-kDa protein that
bound to GST-SAP-1D/A (Fig. 1D). A mAb to p130cas
that recognizes a distinct epitope of the protein also reacted with
p125-135 (data not shown). These results suggest that p125-135 might
be p130cas. We also tested the ability of antibodies to
paxillin, cortactin, or SHP-2 to recognize p75-85. A mAb to paxillin
specifically reacted with a 75-85-kDa protein that bound to
GST-SAP-1D/A (Fig. 1D), suggesting that p75-85 might be
paxillin. However, we cannot exclude the possibility that the p125-135
and p75-85 bands contain additional unidentified proteins. The
identities of p190 and p55 remain unknown.
We next examined whether SAP-1 dephosphorylates p130cas and
paxillin in vitro. The catalytic activity of GST-SAP-1WT
with pNPP as substrate was markedly greater than that of the
recombinant SHP-2 used as a control (Fig. 2A).
Tyrosine-phosphorylated forms of p130cas and paxillin
immunopurified from pervanadate-treated Panc-1 cells were also
incubated with equal amounts of activity of GST-SAP-1WT and GST-SHP-2,
after which their phosphorylation status was determined. GST-SAP-1WT
effectively dephosphorylated p130cas and, to a lesser extent,
paxillin; in contrast, GST-SHP-2 or GST alone had virtually no effect
on the extent of phosphorylation of these proteins (Fig.
2B). The human RPTP PTP
dephosphorylates
-catenin
(31); however, SAP-1 did not exhibit detectable catalytic activity with
-catenin as substrate (Fig. 2B). Together, these results
indicate that SAP-1 selectively dephosphorylates p130cas and,
to a lesser extent, paxillin in vitro.
We also examined whether SAP-1 dephosphorylates p130cas and
paxillin in intact cells. We subjected 293 cells to transient
transfection with expression vectors encoding SAP-1 and either
HA-tagged p130cas (HA-Cas) or Myc epitope-tagged paxillin-
and then evaluated the extent of tyrosine phosphorylation of each
tagged recombinant protein. Wild-type SAP-1 (SAP-1WT) expressed in
these cells possessed substantial catalytic activity, whereas the
catalytically inactive mutant (SAP-1C/S) or the substrate-trapping
mutant (SAP-1D/A) did not, as revealed by an immunocomplex phosphatase
assay using mAb 3G5 to SAP-1 (data not shown). Coexpression of SAP-1WT,
but not that of SAP-1C/S, markedly reduced the extent of tyrosine phosphorylation of HA-Cas (Fig.
3A), suggesting that SAP-1
dephosphorylates p130cas in intact cells. In contrast,
coexpression of SAP-1D/A markedly increased the extent of tyrosine
phosphorylation of HA-Cas. The presence of equal amounts of recombinant
SAP-1 proteins in the different transfected cells was confirmed by
immunoblot analysis with antibodies to SAP-1 (Fig. 3F).
Given that substrate-trapping mutants of PTPs often induce
hyperphosphorylation of the corresponding substrates (21-24), these
results indicate that p130cas serves as a direct substrate of
SAP-1 in vivo. Coexpression of SAP-1WT also reduced the
extent of tyrosine phosphorylation of paxillin-
; however, the
substrate-trapping mutant did not increase the extent of tyrosine
phosphorylation of this protein (Fig. 3B). Thus, paxillin
might not be a physiological substrate of SAP-1.

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Fig. 3.
Effects of overexpression of SAP-1 on
tyrosine phosphorylation of p130cas (A),
paxillin (B), FAK (C), p62dok
(D), and ERK2 (E). 293 cells
were transiently transfected with 1 µg of pSR encoding HA-tagged
p130cas (A), pBabe encoding Myc epitope-tagged
paxillin- (B), pRc/CMV encoding HA-tagged FAK
(C) or HA-tagged p62dok (D), or
pcDNA3 encoding HA-tagged ERK2 (E) together with 4 µg
either of pRc/CMV encoding SAP-1WT (WT), SAP-1C/S
(C/S), or SAP-1D/A (D/A) or
of the empty vector (Vector). Forty-eight hours after
transfection, whole cell lysates were prepared and subjected to
immunoprecipitation (IP) with mAb 12CA5 to the HA tag
( HA) or with mAb 9E10 to the Myc tag ( Myc),
as indicated. The resulting precipitates were then subjected to
immunoblot analysis either with horseradish peroxidase-conjugated mAb
PY20 to phosphotyrosine ( PY) (A-D,
upper panels) or with polyclonal antibodies specific for
tyrosine-phosphorylated ERK ( pMAPK) (E,
upper panel). Duplicate samples were subjected to immunoblot
analysis with antibodies to p130cas ( Cas)
(A, lower panel), to paxillin
( paxillin) (B, lower panel), to FAK
( FAK) (C, lower panel), to
p62dok ( Dok) (D, lower
panel), or to total ERK protein ( MAPK)
(E, lower panel) to determine the amounts of each
protein in the immunoprecipitates. F, total cell lysates
were also subjected to immunoblot analysis with polyclonal antibodies
to SAP-1 ( SAP-1) to determine the amount of each SAP-1 protein
expressed. The positions of p130cas (Cas), paxillin,
FAK, p62dok (Dok), ERK2, and the phosphorylated
forms of each of these proteins (Cas-P,
Paxillin-P, FAK-P, Dok-P, and
ERK2-P) as well as of SAP-1 are indicated.
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Effects of Overexpression of Wild-type or Mutant SAP-1 on Tyrosine
Phosphorylation of FA-associated Proteins--
p130cas
undergoes tyrosine phosphorylation during integrin-mediated cell
adhesion and mediates the assembly of FAs (32, 33). If SAP-1-mediated
dephosphorylation of p130cas induces the disassembly of FAs,
then it might also affect the tyrosine phosphorylation status of other
FA-associated proteins. To test this hypothesis, we examined the
effects of SAP-1 overexpression on the tyrosine phosphorylation of two
other components of the integrin-signaling pathway, FAK and
p62dok (25, 34). Coexpression of SAP-1WT, but not that of
SAP-1C/S, substantially reduced the extent of tyrosine phosphorylation
of HA-tagged forms of FAK (Fig. 3C) and p62dok (Fig.
3D). These recombinant proteins also underwent
hyperphosphorylation in cells expressing SAP-1D/A, indicating that this
latter mutant exerts a dominant negative effect on tyrosine
dephosphorylation of FAK and p62dok. Thus, SAP-1 also mediates
the dephosphorylation of FAK and p62dok in vivo;
however, these effects may be indirect given that neither FAK nor
p62dok were detected as potential substrates of SAP-1 in the
substrate-trapping assay. We also examined the tyrosine phosphorylation
of ERK, which localizes predominantly to the cytosol and is not
associated with FAs. Immunoblot analysis with antibodies specific for
tyrosine-phosphorylated (activated) ERK revealed that, although SAP-1WT
reduced the extent of tyrosine phosphorylation of HA-tagged ERK2, the
latter did not undergo hyperphosphorylation when coexpressed with
SAP-1D/A, again demonstrating the specificity of the dominant negative
effect of this mutant (Fig. 3E).
SAP-1-induced Disruption of Actin Stress Fibers and FAs--
We
next examined the effect of SAP-1 on the actin-based cytoskeleton.
Since WiDr cells or Panc-1 cells, which normally express SAP-1, were
not suitable for examination of cytoskeletal organization, we could not
determine subcellular localization of endogenous SAP-1 by
immunofluorescence staining. We therefore subjected Swiss 3T3 cells to
infection with adenoviruses encoding SAP-1WT or SAP-1C/S and then
examined the cells by confocal microscopy. Infection with a control
virus encoding
-galactosidase had no marked effect on cytoskeletal
architecture, with the infected cells showing a staining pattern for
filamentous actin similar to that of noninfected cells (data not
shown). Immunostaining with mAb 3G5 to SAP-1 revealed that SAP-1WT was
distributed diffusely throughout the cell periphery (Fig.
4, A and G).
Expression of SAP-1WT resulted in marked disruption of actin stress
fibers, with disorganized, thin actin filaments apparent in the
subcortical region (Fig. 4C). Furthermore, the number of
vinculin-positive patches was markedly decreased in these cells,
reflecting disruption of FAs (Fig. 4I). In contrast, SAP-1C/S was predominantly apparent at the leading edge of cells (Fig.
4, B and H), where it was colocalized with
filamentous actin (Fig. 4F). The numbers of actin stress
fibers and vinculin-positive FAs in the cells expressing SAP-1C/S were
similar to those in cells infected with the control virus (Fig. 4,
D and J; data not shown). These results together
with those of the coexpression experiments indicate that SAP-1
associates with FAs and that it disrupts both actin stress fibers and
FAs by mediating the dephosphorylation of FA-associated proteins.

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Fig. 4.
Effects of overexpression of SAP-1 on
cytoskeletal architecture. Swiss 3T3 cells seeded on coverslips
were infected with adenoviruses encoding SAP-1WT
(Ax-SAP-1WT) (A, C, E,
G, I, and K) or SAP-1C/S
(Ax-SAP-1C/S) (B, D, F,
H, J, and L) at a multiplicity of
infection of 200 plaque-forming units per cell. Twelve hours after
infection, cells were fixed and stained with mAb 3G5 to SAP-1
(A-F) or with polyclonal antibodies to SAP-1
(G-L) together with either rhodamine-labeled phalloidin
(A-F) or a mAb to vinculin (G-L). For detection
of SAP-1, cells were further incubated with appropriate secondary
antibodies conjugated with either fluorescein isothiocyanate
(A-F) or Texas red (G-L). For detection of
vinculin, cells were also incubated with secondary antibodies
conjugated with fluorescein isothiocyanate (G-L). Cells
were examined with a confocal microscope. Arrowheads in
B and H indicate SAP-1C/S present at the leading
edge of a cell. Original magnification, 630×.
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Effect of SAP-1 on Cell Spreading on Fibronectin--
To examine
the effect of SAP-1 on cell spreading, we established CHO cell lines
that stably express SAP-1WT (CHO-SAP-1WT) or SAP-1C/S (CHO-SAP-1C/S)
(Fig. 5A). The number and size
of colonies formed by cells expressing SAP-1WT were substantially
smaller than were those of colonies formed by cells expressing SAP-1C/S or those of colonies formed by cells transfected with the empty vector
(see Fig. 7). Furthermore, the amount of SAP-1 in each CHO-SAP-1WT cell
line was consistently lower than that in the CHO-SAP-1C/S cell lines
(Fig. 5A; data not shown). Exponential growth rates were
similar for all cell lines established (data not shown); however, a
marked morphological difference was observed. Thus, under normal
culture conditions, CHO-SAP-1WT cells were less polarized than were
CHO-SAP-1C/S cells or CHO-K1 cells (data not shown). CHO-K1 and
CHO-SAP-1C/S cells exhibited an elongated, spindle-like morphology when
cultured on fibronectin, a specific integrin ligand; in contrast,
CHO-SAP-1WT cells exhibited a flattened, less polarized morphology
(Fig. 5B). None of the cells spread substantially when
cultured on the nonspecific substrate poly-L-lysine (Fig.
5B). These results suggest that SAP-1 affects
integrin-mediated cell spreading in a manner dependent on its catalytic
activity.

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Fig. 5.
Effect of ectopic expression of SAP-1 on cell
spreading on fibronectin. A, whole cell lysates
prepared from CHO-K1 cells or from CHO-K1 cells stably expressing
SAP-1WT (CHO-SAP-1WT) cells (two independent lines assayed)
or SAP-1C/S (CHO-SAP-1C/S) cells were subjected to
immunoblot analysis with polyclonal antibodies to SAP-1
( SAP-1). As a positive control, lysates from WiDr cells
were similarly analyzed. B, CHO-K1, CHO-SAP-1WT, or
CHO-SAP-1C/S cells were detached from their culture dishes and replated
in serum-free Ham's F-12 medium on dishes coated with fibronectin or
with poly-L-lysine. After incubation for 2 h at
37 °C, the cells were photographed with a light microscope equipped
with phase-contrast optics. Original magnification, 200×.
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Inhibition by SAP-1 of Growth Factor-induced ERK
Activation--
Cell adhesion to extracellular matrix (ECM) proteins
promotes, in an FAK-dependent manner, the activation of ERK
induced by various growth factors (35-38). We therefore examined
whether SAP-1 affects growth factor-induced ERK activation. 293 cells
were transiently cotransfected with vectors encoding HA-ERK2 and SAP-1
and were then exposed to LPA, EGF, or TPA. All of these growth
stimulants induced substantial activation of HA-ERK2 in cells
cotransfected with an empty vector (Fig.
6). The activity of HA-ERK2 in
unstimulated cells was reduced by coexpression of SAP-1WT, suggesting
that SAP-1 may inactivate an upstream element required for basal ERK2 activity. Coexpression of SAP-1WT also prevented activation of HA-ERK2
by LPA (Fig. 6A), substantially inhibited HA-ERK2 activation by EGF (Fig. 6B), and inhibited to a lesser extent HA-ERK2
activation by TPA (Fig. 6C). In contrast, coexpression of
SAP-1C/S did not markedly affect the activity of HA-ERK2 under basal or
stimulated conditions. Whereas LPA and EGF bound to specific membrane
receptors and activated ERKs in a protein-tyrosine
kinase-dependent manner, TPA activated these enzymes
predominantly by a protein-tyrosine kinase-independent pathway that
includes protein kinase C and Raf. Together, these results suggest that
SAP-1 negatively regulates growth factor-induced ERK activation and
that the extent of this effect depends on the signaling pathways
triggered by each growth factor.

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Fig. 6.
Effects of overexpression of SAP-1 on growth
factor-induced ERK activation. 293 cells were transiently
cotransfected with 1 µg of pcDNA3 encoding HA-ERK2 and 4 µg
either of pRc/CMV encoding SAP-1WT (WT) or SAP-1C/S
(C/S) or of the empty vector (Vector).
Forty-eight hours after transfection, cells were deprived of serum for
12 h and then incubated in the absence ( ) or presence (+) of 10 µM LPA (A) or EGF (100 ng/ml) (B)
for 5 min or of 1 µM TPA for 10 min (C). Whole
cell lysates were prepared and subjected to immunoprecipitation with
mAb 12CA5 to the HA tag, and the resulting precipitates were subjected
to immunoblot analysis with antibodies specific for
tyrosine-phosphorylated ERK (top left panels). Duplicate
samples were subjected to immunoblot analysis with antibodies to total
ERK (left middle panels) to ensure that equal amounts of
ERK2 were present in each precipitate. The cell lysates were also
subjected to immunoblot analysis with antibodies to SAP-1 to determine
the amount of each recombinant SAP-1 protein (left bottom
panels). The phosphorylation of HA-ERK2 was quantified by scanning
densitometry with the NIH Image program and expressed as a percentage
of the value for cells transfected with the empty vector and exposed to
each stimulant (right panels).
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Inhibition by SAP-1 of Colony Formation--
To gain further
insight into the biological consequences of SAP-1-mediated protein
dephosphorylation, we next examined the effect of this PTP on colony
formation. CHO-K1 or NIH 3T3 cells were transiently transfected with
vectors encoding SAP-1WT or SAP-1C/S, and the efficiency of colony
formation was evaluated. Expression of SAP-1WT reduced the number of
G418-resistant colonies formed by CHO-K1 or NIH 3T3 cells by 65 and
54%, respectively, compared with the value for cells transfected with
the empty vector (Fig. 7). Expression of
SAP-1C/S also reduced the extent of colony formation by CHO-K1 and NIH
3T3 cells by 30 and 38%, respectively. These results are consistent
with the notion that SAP-1 inhibits cell proliferation by reducing ERK
activity, but they also suggest that this effect may not depend
entirely on the catalytic activity of SAP-1.

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Fig. 7.
Effects of overexpression of SAP-1 on colony
formation. CHO-K1 or NIH 3T3 cells were transiently transfected
with 3 µg either of pRc/CMV encoding SAP-1WT or SAP-1C/S or of the
empty vector (Vector). Forty-eight hours after transfection,
cells were exposed to normal growth medium containing G418 (400 µg/ml) for 14 days. Cells were then fixed and stained with crystal
violet (A). The efficiency of colony formation by each cell
line was quantified by scanning densitometry with the NIH Image program
and is expressed as a percentage of the value for cells transfected
with the empty vector (B).
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Activation of SAP-1 by Cell-Cell Adhesion--
Tyrosine
phosphorylation of p130cas is regulated by cell adhesion to the
ECM (34). Our observation that SAP-1 catalyzes the dephosphorylation of
p130cas suggests that the activity of this PTP might also be
regulated by cell-substratum adhesion. The presence of multiple
fibronectin type III-like domains in the extracellular region of SAP-1
(14) also suggests that its activity might be regulated by cell-cell adhesion. To investigate these possibilities, we measured cellular SAP-1 activity under adherent or suspension conditions with the in vitro assay. The mAb 3G5 immunoprecipitated PTP activity
from an adherent monolayer of either CHO-SAP-1WT or WiDr cells (Fig. 8A). SAP-1 activity in each
cell line decreased markedly as a function of time in suspension,
reaching a minimal value of ~50% of that for adherent cells after
~15 min (Fig. 8A; data not shown). This effect was not
attributable to cell damage because the experimental treatment did not
substantially affect the viability of either cell line, as revealed by
trypan blue exclusion, for up to 60 min (data not shown).

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Fig. 8.
Up-regulation of SAP-1 activity by cell-cell
contact. A, confluent monolayers of CHO-SAP-1WT or WiDr
cells were maintained adherent (Ad), or the cells were
detached from the culture dish and maintained in suspension for 0-60
min in normal growth medium at 37 °C. Cell lysates were then
prepared, normalized for protein concentration, and subjected to
immunoprecipitation with mAb 3G5 to SAP-1. The immunopurified SAP-1 was
assayed for PTP activity with pNPP as substrate. B,
CHO-SAP-1WT or WiDr cells were cultured until the indicated extent of
confluence was achieved, after which immunopurified SAP-1 was assayed
for PTP activity as in A. The apparent PTP activity obtained
with precipitates prepared with an unrelated mouse mAb was subtracted
from the activity observed with precipitates prepared with mAb 3G5.
Data are means of triplicates from a representative experiment.
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Because detachment from the culture dish disrupts not only
cell-substratum adhesion but also cell-cell adhesion, we could not
conclude from this experiment which type of adhesion regulates SAP-1
activity. We therefore measured the activity of SAP-1 immunopurified from cells cultured at various densities; at the high density, all
cells achieved cell-cell adhesion, whereas at the low density, virtually none did. For both cell lines studied, SAP-1 activity in the
high density culture was substantially greater than that in the low
density culture (Fig. 8B). Thus, cell-cell adhesion may be
responsible for up-regulation of SAP-1 activity, although we cannot
exclude the possibility that cell-substratum adhesion also has a
regulatory role.
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DISCUSSION |
We have now shown that p130cas, a major FA-associated
phosphotyrosyl protein, appears to be a substrate of SAP-1. A
substrate-trapping mutant of SAP-1, but not wild-type SAP-1,
selectively precipitated p130cas from pervanadate-treated
Panc-1 cells, which express endogenous SAP-1 (14). The association of
this mutant with p130cas was disrupted by vanadate, indicating
that p130cas interacts with the active site of SAP-1. Wild-type
SAP-1 effectively and selectively dephosphorylated p130cas both
in vitro and in vivo, whereas the
substrate-trapping mutant of SAP-1 induced hyperphosphorylation of
p130cas in intact cells. These results indicate that
p130cas is a physiological substrate of SAP-1. We also showed
that 190- and 55-kDa phosphotyrosyl proteins bound to the
substrate-trapping mutant of SAP-1, suggesting that these unidentified
proteins also might be substrates of this PTP. In addition,
pervanadate-treated cells are not thought to contain a complete
spectrum of all possible phosphotyrosyl proteins. Thus, additional
targets of SAP-1 may remain to be detected.
We showed that overexpression of SAP-1 disrupts both actin stress
fibers and FAs. Evidence suggests that p130cas plays an
important role in the maintenance of these structures. Thus, the
association of p130cas with Src facilitates phosphorylation of
tyrosine residues in the substrate domain of p130cas, resulting
in the binding of Src homology 2 domain-containing molecules to this
protein (39). The Src homology 2 domain-containing adapter protein Crk
binds to tyrosine-phosphorylated p130cas and thereby induces
the rearrangement of the actin-based cytoskeleton (40). Furthermore,
p130cas associates with the actin-binding protein tensin (41),
and fibroblasts derived from p130cas knockout mice exhibit
impaired formation of actin stress fibers (42). Finally, YopH, a
Yersinia PTP capable of dephosphorylating p130cas,
disrupts actin stress fibers and FAs (43, 44). Together with these
observations, our results indicate that SAP-1 catalyzes the tyrosine
dephosphorylation of p130cas and thereby induces the
dissociation of this protein from its effector molecules, resulting in
the disruption of actin stress fibers and FAs.
Integrin-mediated cell adhesion to the ECM regulates many important
cellular responses including activation of protein kinase cascades,
cytoskeletal reorganization, proliferation, and survival (45, 46). Our
results have now shown that SAP-1 affects cell spreading on
fibronectin, a process mediated by integrin engagement. Given that
p130cas contributes to cell spreading on the ECM (42),
SAP-1-mediated dephosphorylation of p130cas might result in
impaired cell spreading on fibronectin. Consistent with this notion,
PTP-PEST, which also dephosphorylates p130cas, has been shown
to regulate FA disassembly and cell spreading (47, 48).
We have also shown that SAP-1 inhibits growth factor-induced ERK
activation, a process supported by cell adhesion to the ECM (35-38).
This inhibitory effect of SAP-1 was most pronounced with LPA, less
marked with EGF, and marginal with TPA. Given that activation of ERKs
by LPA appears to depend on FAs to a greater extent than does that by
EGF or by TPA (49), these results indicate that SAP-1 specifically
blocks ERK activation mediated through FAs. We further showed that
ectopic expression of SAP-1 inhibits colony formation, indicative of a
negative effect of this PTP on cell proliferation. In addition to
promoting cell growth through ERK activation, integrins initiate
various signals required for cell proliferation. Thus, in cooperation
with soluble growth factors, they promote cell survival through
activation of phosphatidylinositol 3-kinase and Akt, and they activate
c-Jun NH2-terminal kinases, which regulate cell cycle
progression (46). SAP-1 therefore likely inhibits cell proliferation by
inactivating both growth-promoting and survival-promoting signals
supported by integrin-mediated cell adhesion.
Our data indicate that cell-cell adhesion up-regulates the enzymatic
activity of SAP-1. Cell-cell contact increases the expression or the
catalytic activity of various RPTPs, including DEP-1, PTP
, and
PTPµ (16, 50, 51). Although such effects have been attributed to
density-dependent growth arrest or down-regulation of
growth factor signaling (52, 53), a lack of knowledge of the specific substrates of each PTP has hindered elucidation of the underlying mechanisms. The activity of Src and the extent of tyrosine
phosphorylation of its substrates, including p130cas, paxillin,
and FAK, increase in response to cell-cell contact, although again the
mechanism and the functional significance of these effects have
remained unclear (54, 55). Given that we have now shown that SAP-1
mediates the dephosphorylation of these Src substrates in
vivo, this PTP may function to counteract mitogenic signaling
initiated by Src. SAP-1 activation induced by cell-cell adhesion may
thus contribute to the mechanism by which contact-inhibited cells enter
growth arrest.
Expression of the genes for the RPTPs DEP-1 and PTP-U2, which are
structurally similar to SAP-1, increases during differentiation of
breast cancer cells and monoblastoid leukemia cells (56, 57). Ectopic
expression of DEP-1 or PTP-U2 in these cells resulted in growth
inhibition or enhanced apoptosis subsequent to terminal differentiation
(56, 57). The abundance of SAP-1 mRNA also increases during cancer
cell differentiation (56). Together with the inhibitory effect of SAP-1
on cell proliferation, these observations suggest a model in which
SAP-1, by mediating the tyrosine dephosphorylation of FA-associated
substrates, activates a signal that leads to growth arrest and
differentiation of cancer cells. According to this model, cancer
progression would be facilitated if SAP-1 expression is lost.
In conclusion, our data indicate that p130cas is a substrate of
SAP-1. Overexpression of SAP-1 disrupted the actin-based cytoskeleton. It also inhibited a wide range of cellular responses promoted by
integrin-mediated cell adhesion, including cell spreading on fibronectin, growth factor-induced ERK activation, and colony formation. Furthermore, activation of SAP-1 in response to cell-cell adhesion indicates a role for this PTP in contact inhibition of cell
growth and motility.