From the Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York 11724 and the ¶ Molecular Oncology Group, McGill
University Hospital Center, Montreal, Quebec H3A 1A1, Canada
Received for publication, October 17, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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The receptor protein-tyrosine phosphatase (PTP)
DEP-1 (CD148/PTP- A variety of ligands trigger the reversible phosphorylation of
tyrosyl residues in cellular proteins, a process that underlies the
control of such fundamental cellular functions as growth and proliferation, migration, and morphogenesis. Tyrosine phosphorylation is regulated by the coordinated action of protein-tyrosine kinases (PTKs)1 and protein-tyrosine
phosphatases (PTPs). Classically it was thought that the PTKs provided
the "on-switch" to initiate a physiological response, whereas the
PTPs functioned to counteract the PTKs and to return the system to its
basal state. However, it was soon shown that PTPs may themselves
function positively to promote signaling, for example, by promoting the
dephosphorylation and activation of PTKs, thus coordinating with,
rather than antagonizing PTK function (reviewed in Ref. 1). A further
level of complexity has been introduced with the realization that
whether a defined PTP functions positively or negatively may depend
upon the signaling context. Thus, SHP-2 is an activator of signaling
through the HGF/SF receptor Met (2) and the epidermal growth
factor receptor (3), but is an inhibitor of signaling through
the platelet-derived growth factor receptor (4). Following ligand
binding, a receptor PTK may become phosphorylated on multiple tyrosine
residues, which serve as docking sites for distinct signaling proteins.
The spectrum of such signaling molecules that associate with the PTK
will determine the nature of the response that is initiated following
ligand stimulation. The possibility exists, therefore, that a PTP may dephosphorylate a particular site in a receptor PTK and thereby determine the signaling outcome of a particular stimulus. Thus, dephosphorylation of receptor PTKs by members of the PTP family may
function as a mechanism for regulating the specificity of a signaling
event rather than simply as an "off-switch."
DEP-1 is a receptor PTP whose expression is enhanced as cells approach
confluence (5). Initially cloned from human cDNA libraries (5, 6),
DEP-1 homologues were subsequently identified in rat and mouse (7, 8).
DEP-1 comprises an extracellular segment of eight fibronectin type III
repeats, a transmembrane domain, and a single cytoplasmic PTP domain.
Also known as PTP- In addition to its role in growth inhibition, DEP-1 has also been
implicated in differentiation. The levels of DEP-1 mRNA are
increased in various cell lines in response to factors that lead to
differentiation (8, 11, 13, 14). Interestingly, in rat thyroid cells
the expression of DEP-1 (rPTP- DEP-1 has recently been shown to localize at cell borders in
endothelial cells and its staining pattern overlapped with that of the
junctional protein VE-cadherin (18). Interestingly, members of the
cadherin family of cell-cell adhesion molecules function in the
suppression of cell growth and tumor invasion. Junctional components
such as Using an in vitro affinity chromatography system we
identified a set of proteins from human breast tumor cell lines
(MDA-MB-231, T-47D, and T-47D/Met) that interacted specifically with
the substrate-trapping mutant form of DEP-1. These proteins included
the junctional component p120 catenin (p120Ctn), the
adaptor protein Gab1, and the HGF/SF receptor Met. Met induces
mitogenic, motogenic, and morphogenic responses after ligand activation
by recruiting a number of signaling and docking molecules and has been
implicated in the phosphorylation of cell junction proteins. Disruption
of normal signaling through Met has been implicated in certain cancers.
Ligand-induced activation of Met by HGF/SF leads to the
autophosphorylation of specific tyrosine residues within the PTK.
Phosphorylation of Tyr1234 and Tyr1235 in the
activation loop of Met is required for kinase activity, whereas
phosphorylation of COOH-terminal tyrosine residues (Tyr1349
and Tyr1356) is required for the recruitment of signaling
and adapter molecules including Gab1 (reviewed in Ref. 23). Additional
COOH-terminal tyrosines such as Tyr1365 appear to be
important for mediating a morphogenic signal although the identity of
proteins that interact with this site is currently unknown (24). We
present evidence that DEP-1 preferentially dephosphorylates specific
tyrosine residues in the COOH-terminal domain of Met. By selectively
dephosphorylating such sites in the kinase, DEP-1 may attenuate
particular signaling events emanating from Met thus, potentially,
regulating the outcome of cellular responses induced by HGF/SF stimulation.
Generation of DEP-1 cDNA Constructs--
Full-length human
DEP-1 cDNA was isolated and subcloned into the mammalian expression
vector pMT2 (5). The nucleotide and amino acid numbers listed below
correspond to the human DEP-1 sequence reported previously (5)
(GenBankTM accession number U10886). DEP-1 point mutants
(C1239S and D1205A) were generated by overlap extension using
pMT2.DEP-1 as template. The resulting mutant PCR products were
exchanged with the wild type sequence in pMT2.DEP-1 and sequenced to
confirm the mutations.
DEP-1 cytoplasmic domain constructs were generated using the
pMT2.DEP-1 wild type or point mutant (C1239S and D1205A) constructs as template. A 5' primer introduced a BamHI site before the
DEP-1 cytoplasmic sequence at nucleotide 3338, whereas a 3' primer
added a SalI site after the DEP-1 stop codon. The resulting
PCR products (DEP-1 nucleotides 3338-4362) were cloned into the
BamHI/SalI sites of the pMAL-c2E vector from New
England Biolabs (Beverly, MA) generating wild type and point mutant
(C1239S and D1205A) pMAL.DEP-1 constructs. The fusion proteins were
expressed in Escherichia coli and purified on amylose resin
according to the manufacturer's instructions. The resulting proteins
(~84 kDa) have maltose-binding protein (MBP) fused to the
NH2 terminus of the DEP-1 cytoplasmic domain (amino acids
997-1337).
Met Chimeric Construct--
The chimeric receptor colony
stimulating factor 1 (CSF)-Met comprising the extracellular domain of
human CSF-1R and the transmembrane and cytoplasmic domains of human Met
was described previously (25).
Cell Culture and Transfections--
MDA-MB-231 (ATTC HTB-26) and
T-47D (ATTC HTB-133) human breast tumor cells were cultured in
Dulbecco's modified Eagle's medium containing 5% fetal bovine serum,
100 units/ml penicillin, 100 µg/ml streptomycin, and 1% nonessential
amino acids. The T-47D/Met cell line (26) was cultured in Dulbecco's
modified Eagle's medium as above supplemented with 200 µg/ml G418.
Human embryonal kidney 293 cells (ATTC CRL-1573) were cultured in
Dulbecco's modified Eagle's medium containing 10% bovine calf serum,
100 units/ml penicillin, and 100 µg/ml streptomycin.
Transfection of 293 cells was performed using the calcium
phosphate-mediated transfection protocol. For trapping experiments, 293 cells were transfected with 20 µg of CSF-Met DNA (pXM.CSF-Met) and 20 µg of empty vector DNA (pMT2) or 20 µg of DEP-1 DNA (pMT2.DEP-1, pMT2.DEP-1(CS), and pMT2.DEP-1(DA)) per 10-cm dish. To examine dephosphorylation in 293 cells, 20 µg of CSF-Met DNA (pXM.CSF-Met) was co-transfected with increasing amounts of DEP-1 DNA (pMT2.DEP-1) (0, 1, 2, 5, and 10 µg) or 10 µg of DEP-1(CS) DNA (pMT2.DEP-1(CS)) per 10-cm dish of cells. The total amount of DNA in each transfection was normalized using empty vector DNA (pMT2).
Antibodies--
DEP-1 monoclonal antibodies A3 and 143-41 used
for immunoprecipitations were generous gifts from Dr. Gregorio Aversa
and Dr. Antoni Gayá, respectively, and were described previously
(9, 10). The DEP-1 polyclonal antibody CS895A was generated against the
DEP-1 extracellular domain peptide (CDASNTERSRAGSPTAP) corresponding to
amino acids 292-307 coupled to KLH (Pierce, Rockford, IL). The Met
polyclonal antibody 144 used for immunoprecipitations was generated
against a carboxyl-terminal peptide and was described previously (27).
The antiphosphotyrosine monoclonal antibodies G98 and G104 were
generated in our laboratory and described previously (28).
Antiphosphotyrosine-agarose (PT-66) was purchased from Sigma and
antiphosphotyrosine (4G10)-agarose conjugate was purchased from Upstate
Biotechnology (Lake Placid, NY). The Met antibody C-12 was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for
p120Ctn, E-cadherin, Grb2, and phosphotyrosine (PY20) were
purchased from BD Transduction Labs (Lexington KY). Substrate Trapping--
Prior to lysis, T-47D and T-47D/Met
cells were treated with 50 µM pervanadate for 20 min
while MDA-MB-231 cells were treated with 100 µM
pervanadate for 20 min. Cells were rinsed with phosphate-buffered saline and lysed in 1% Nonidet P-40 buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM HEPES, pH 7.5, 1 mM
EDTA, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM
benzamidine). For trapping experiments in vitro the lysis
buffer also contained 5 mM iodoacetic acid to inhibit
cellular PTPs irreversibly. After incubation on ice for 5 min
dithiothreitol was added to a final concentration of 10 mM
to inactivate any unreacted iodoacetic acid. Insoluble material was
removed by centrifugation. T-47D (1 mg) or MDA-MB-231 lysates (5 mg)
were mixed with MBP or the MBP.DEP-1 constructs bound to amylose resin
at a ratio of 1 µg of fusion to 500 µg of lysate. Lysates and
fusion proteins were incubated at 4 °C for 2 h and washed
extensively with 1% Nonidet P-40 buffer. Tyrosine-phosphorylated proteins were immunoprecipitated using 0.1 mg of T-47D cell lysate and
a combination of 5 µl each of antiphosphotyrosine antibodies PT-66
and 4G10. Lysate and antibodies were incubated at 4 °C for 2 h
and washed extensively with 1% Nonidet P-40 buffer. Protein complexes
were released by incubation in reducing Laemmli sample buffer at
95 °C subjected to SDS-PAGE on 8% gels and transferred onto
Immobilon-P membranes (Millipore, Bedford, MA) for immunoblotting.
To determine whether the tyrosine-phosphorylated proteins bound to the
substrate-trapping mutants at the PTP active site, we tested the
effects of vanadate on complex formation. MBP fusion proteins bound to
amylose were preincubated in 1% Nonidet P-40 buffer (without EDTA)
with or without 2 mM vanadate. Cells were rinsed with
phosphate-buffered saline and lysed in 1% Nonidet P-40 buffer (without
EDTA) with or without 2 mM vanadate. For vanadate-competition experiments, the lysis buffer also contained 5 mM iodoacetic acid and after 5 min on ice dithiothreitol
was added to a final concentration of 10 mM. Insoluble
material was removed by centrifugation and samples were processed as above.
Proteins bound to the DEP-1 substrate-trapping mutant were analyzed by
immunoblotting. T-47D and T-47D/Met cells were treated and lysed as
above. Lysates (30 mg) were mixed with MBP.DEP-1 or
MBP.DEP-1(DA) bound to amylose resin at a ratio of 1 µg of fusion protein to 500 µg of lysate. Lysates and fusion proteins were
incubated at 4 °C for 2 h and washed extensively with 1% Nonidet P-40 buffer. Protein complexes were released by incubation in
reducing Laemmli sample buffer at 95 °C subjected to SDS-PAGE on 8%
gels and transferred onto Immobilon-P membranes for immunoblotting. The
samples were divided into 5-mg lysate equivalents per fusion per lane.
Immunoprecipitations--
Transfected cells were rinsed with
phosphate-buffered saline and lysed in 1% Nonidet P-40 buffer (1%
Nonidet P-40, 150 mM NaCl, 20 mM HEPES, pH 7.5, 1 mM EDTA, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM benzamidine, 50 mM NaF, 5 mM
iodoacetic acid) and processed as above. For substrate-trapping
experiments, DEP-1 was immunoprecipitated from 1 mg of lysate with the
DEP-1 antibodies A3 and 143-41 and Met was immunoprecipitated from 1 mg
of cell lysate using the Met antibody 144.
For dephosphorylation and recruitment experiments transfected cells
were rinsed with phosphate-buffered saline and lysed in 1% Nonidet
P-40 buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM HEPES, pH 7.5, 1 mM EDTA, 5 µg/ml
aprotinin, 5 µg/ml leupeptin, 1 mM benzamidine, 50 mM NaF, 5 mM iodoacetic acid, 1 mM
vanadate) and processed as above. Met was immunoprecipitated from 1 mg
of lysate using the Met antibody 144. Lysate and antibody were
incubated at 4 °C for 1 h. Protein A-Sepharose 4 fast flow
(Amersham Biosciences) was added for 45 min at 4 °C. Immune
complexes were washed extensively with 1% Nonidet P-40 buffer,
released by incubation in reducing Laemmli sample buffer at 95 °C
subjected to SDS-PAGE on 8% gels, and transferred onto Immobilon-P
membranes for immunoblotting.
The DEP-1(DA) Substrate-trapping Mutant Interacted with a Subset of
Tyrosine-phosphorylated Proteins from Two Human Breast Tumor
Lines--
We employed two human breast tumor lines (T-47D and
MDA-MB-231), which express DEP-1, for in vitro studies to
identify potential physiological substrates of the PTP. The cells were
treated with pervanadate to generate the broadest spectrum of potential
phosphotyrosine-containing substrates for analysis. DEP-1 fusion
proteins comprising the MBP fused to the NH2
terminus of the DEP-1 cytoplasmic domain (amino acids 997-1337) were
generated. Wild type (MBP.DEP-1), catalytically inactive
(MBP.DEP-1(CS)), and substrate-trapping (MBP.DEP-1(DA)) mutant forms of
DEP-1 were used for purification of potential substrates by affinity
chromatography in vitro. DEP-1 fusion proteins were
incubated with lysate of pervanadate-treated T-47D cells.
Tyrosine-phosphorylated proteins that interacted with the fusion
proteins were visualized by immunoblotting with antiphosphotyrosine
antibodies. Only the substrate-trapping mutant form of DEP-1
(MBP.DEP-1(DA)) bound tyrosine-phosphorylated proteins (Fig.
1A). In addition, when a
comparison was made between the tyrosine-phosphorylated proteins that
bound to the DEP-1 substrate-trapping mutant and the proteins
immunoprecipitated with antiphosphotyrosine antibodies, we observed
that MBP.DEP-1(DA) recognized only a small subset of the
tyrosine-phosphorylated proteins from the lysate of pervanadate-treated
T-47D cells (Fig. 1A). To determine whether the proteins
that interacted with MBP.DEP-1(DA) were potential substrates, the
fusion proteins were preincubated with vanadate. Vanadate is a
competitive inhibitor that blocks the PTP active site and prevents
substrate binding and phosphatase activity (29). The interaction
between the tyrosine-phosphorylated proteins and MBP.DEP-1(DA) was
inhibited by vanadate, suggesting that they bound to the active site
and may represent substrates of DEP-1 (Fig. 1B).
Similarly, DEP-1 fusion proteins were incubated with the lysate of
pervanadate-treated MDA-MB-231 cells. As was seen with the T-47D cell
lysates, only the substrate-trapping mutant form of DEP-1
(MBP.DEP-1(DA)) interacted with tyrosine-phosphorylated proteins from
MDA-MB-231 cell lysates (Fig. 1C) and only a small subset of
the pool of available tyrosine-phosphorylated proteins was recognized
by the PTP (data not shown). This interaction was also inhibited by
vanadate (Fig. 1D). Pervanadate treatment resulted in the
accumulation of tyrosine-phosphorylated proteins in both cell lines,
however, a lower concentration of pervanadate was needed to induce high
levels of tyrosine phosphorylation in T-47D cells compared with
MDA-MB-231 cells. Similar results were obtained for both cell lines and
we present data obtained from T-47D cells in the subsequent figure.
Identification of Proteins That Interacted with the DEP-1
Substrate-trapping Mutant--
Although the tyrosine-phosphorylated
proteins that interacted with MBP.DEP-1(DA) were easily detected by
immunoblotting with antiphosphotyrosine antibodies, they were difficult
to detect on Coomassie-stained gels, suggesting that they were not
abundant proteins. After a large scale preparation of DEP-1 substrates from T-47D cells we detected a 100-kDa protein on Coomassie-stained gels (data not shown) that corresponded to a 100-kDa
tyrosine-phosphorylated protein detected by immunoblotting (Fig.
1A, arrow). Peptides derived from this protein
were sequenced by mass spectrometry. Two individual peptides (NLSYQVHR,
SQSSHSYDDSTLPLIDR) matched sequences in the Src substrate and adherens
junction component p120Ctn (Table
I). Both sequences can be found in all
the p120Ctn isoforms identified to date (30).
Based on the identification of p120Ctn as a potential
substrate of DEP-1, we sought to determine whether the PTP interacted
with other junctional components. Immunoblot analysis revealed that the
DEP-1 substrate-trapping mutant did not interact with the transmembrane
protein E-cadherin from pervanadate-treated T-47D cell lysates (Fig.
2). The cytoplasmic proteins
The DEP-1 substrate-trapping mutant bound several
tyrosine-phosphorylated proteins from both T-47D and MDA-MB-231 cell
lines (Fig. 1). Based on the molecular weights of these proteins and our observation that DEP-1 interacted with components of adherens junctions, we probed for signaling molecules known to localize to
cell-cell junctions. We observed that MBP.DEP-1(DA) trapped Met, the
HGF/SF receptor, from pervanadate-treated MDA-MB-231 cells (data not
shown). Because Met is expressed at low levels in T-47D cells we
employed a T-47D stable cell line ectopically expressing the PTK
(T-47D/Met), which has been used previously in analysis of Met function
(26). MBP.DEP-1(DA) also trapped Met from pervanadate-treated T-47D/Met
cell lysate and this interaction was not seen with the wild type DEP-1
(MBP.DEP-1) (Fig. 2). This suggests a transient interaction between
DEP-1 and Met consistent with that of enzyme and substrate.
Met exerts its pleiotropic effects by recruiting a number of
docking and signaling molecules (reviewed in Ref. 23).
MBP.DEP-1(DA) trapped the docking protein Gab1 from T-47D/Met
cell lysates (Fig. 2). Following activation of Met, Gab1 is recruited
to the kinase and phosphorylated on tyrosine residues, which allows for
the recruitment of other signaling and adapter molecules thereby
amplifying downstream signals. Interestingly MBP.DEP-1(DA) also trapped
Gab1 from T-47D cells suggesting that the Gab1-DEP-1 interaction is at
least partially direct and does not require Met (Fig. 2).
The Full-length DEP-1(DA) Substrate-trapping Mutant Trapped Met
from 293 Cells--
DEP-1 is a transmembrane PTP, however, in the
substrate-trapping experiments described above we utilized only the
cytoplasmic domain of the enzyme and observed an interaction with Met.
To determine whether the trapping mutant form of full-length DEP-1 also
trapped Met, we co-expressed full-length DEP-1 and the mutants DEP-1(CS) and DEP-1(DA) with a chimeric Met construct CSF-Met. This
chimeric receptor, which comprises the extracellular domain of human
CSF-1R and the transmembrane and cytoplasmic domains of human Met (25),
is constitutively active when expressed in 293 cells, bypassing the
requirement for ligand stimulation. DEP-1 was immunoprecipitated under
conditions that would preserve protein complexes. Immunoblots showed
that similar levels of DEP-1, DEP-1(CS), and DEP-1(DA) were
immunoprecipitated from 293 cell lysates (Fig. 3A). No endogenous DEP-1 could
be detected in immunoprecipitates from 293 cells expressing the Met
chimera alone. As with the DEP-1(DA) cytoplasmic domain fusion protein,
full-length DEP-1(DA) formed a stable complex with Met (Fig.
3A). Interestingly, the full-length DEP-1(CS) mutant also
bound Met, but less efficiently than the DEP-1(DA) mutant. Similar
results were observed in the interaction between PTP-PEST and its
substrate p130 (28). There appeared to be no stable interaction between
wild type DEP-1 and Met when they were co-expressed in 293 cells (Fig.
3A).
Full-length Wild Type DEP-1 Dephosphorylated Met in 293 Cells--
Because full-length substrate-trapping mutant forms of
DEP-1 bound Met when co-expressed in 293 cells (Fig. 3A) we
investigated whether full-length wild type DEP-1 could dephosphorylate
Met. Full-length DEP-1 and the mutants DEP-1(CS) and DEP-1(DA) were co-expressed with the CSF-Met chimera in 293 cells, as above. The Met
chimera was immunoprecipitated from cell lysates with an antibody
directed toward the Met portion of the chimera. Immunoblots revealed
that similar levels of CSF-Met were immunoprecipitated in each
condition (Fig. 3B). We observed that the Met chimera was
tyrosine phosphorylated when it was expressed alone in 293 cells,
however, we did not detect the presence of tyrosine phosphorylation when it was co-expressed with wild type DEP-1 (Fig. 3B).
Although the DEP-1(CS) and DEP-1(DA) mutants interacted with the Met
chimera (Fig. 3A), Met was not dephosphorylated in the cells
expressing these mutants, suggesting that dephosphorylation required
DEP-1 catalytic activity.
DEP-1 Preferentially Dephosphorylated COOH-terminal Tyrosine
Residues in Met--
When equal amounts of DEP-1 and CSF-Met plasmid
DNA were transfected into 293 cells, the level of DEP-1 protein
expressed was sufficient to dephosphorylate Met (Fig. 3B).
We performed a dose-response analysis to determine whether varying the
expression level of DEP-1 would affect its ability to dephosphorylate
Met. We transfected 293 cells with a constant concentration of CSF-Met DNA (20 µg) and increasing amounts of wild type DEP-1 DNA (0, 1, 2.5, 5, and 10 µg) or 10 µg of the catalytically inactive DEP-1(CS) mutant DNA (Fig. 4A).
Immunoblots showed that as the levels of DEP-1 plasmid DNA used for
transfection was increased, the level of DEP-1 protein that was
expressed also increased, whereas the levels of Met protein were
similar (Fig. 4A). Although similar amounts of Met were
immunoprecipitated from 293 cell lysates, a gradual decrease in the
level of phosphorylation of Met was detected with increasing expression
of wild type DEP-1 (Fig. 4B). The phosphorylation of Met was
similar when Met was expressed either alone or with the catalytically
inactive form of DEP-1 (DEP-1(CS).
Met contains three tyrosines in the activation loop of the catalytic
domain (Tyr1230, Tyr1234, and
Tyr1235) and phosphorylation of Tyr1234 and
Tyr1235 is required for full activation of the kinase (31).
To determine whether DEP-1 acted on these tyrosine residues,
phospho-specific antibodies were employed. Fig. 4B shows
that similar to the effects on the overall levels of Met
phosphorylation, there was a gradual decrease in the level of
phosphorylation of the activation loop tyrosine residues with
increasing expression of wild type DEP-1 and no effect on
phosphorylation with the expression of DEP-1(CS). Phosphorylation of
Tyr1349 and Tyr1356 in the multisubstrate
docking site of Met is required for the transduction of downstream
signals. Tyr1349 is a binding site for the adapter protein
Gab1 whereas Tyr1356 is primarily responsible for binding
Grb2, phosphatidylinositol 3-kinase, phospholipase C (PLC)- Increased Expression of DEP-1 Attenuated the Interaction between
Met and Grb2--
Ligand-induced activation of Met results in the
recruitment of a number of proteins that are important for transmitting
downstream signals. The dephosphorylation of a docking site tyrosine
residue in Met prompted us to look at the recruitment of Grb2. Met was immunoprecipitated from 293 cells as above co-expressing CSF-Met and
varying amounts of DEP-1 and the immunoprecipitates were probed for the
presence of the Grb2 adapter protein. Grb2 binds to Met directly via
Tyr1356 (32, 33). Analysis of the cell lysates revealed
that the level of Grb2 was not affected by the expression of DEP-1 and Met in these cells (Fig. 4C). However, we observed that with
increasing levels of DEP-1 there was a gradual decrease in the amount
of Grb2 that co-immunoprecipitated with Met (Fig. 4C)
coincident with the changes in overall tyrosine phosphorylation status
of the PTK.
Aberrant regulation of tyrosine phosphorylation, for example
caused by disruption of the normal balance of activities of PTKs and
PTPs, has been implicated in the manifestation of many aspects of the
transformed phenotype. An understanding of which PTKs and PTPs
contribute to this process will provide important insights into the
etiology, and potential avenues for treatment, of cancer. DEP-1 was
identified originally as a PTP whose expression is enhanced as cells
approach confluence, suggesting a role in contact dependent growth
inhibition. DEP-1 has also been implicated in differentiation and loss
of DEP-1 expression may contribute to cellular transformation. Identification of physiological substrates of DEP-1 will be crucial to
understanding the role that this enzyme plays in these processes. Using
substrate-trapping mutant forms of DEP-1 in vitro we
identified several potential substrates including the PTK Met, the
adapter protein Gab1, and the junctional component p120Ctn
(Fig. 2). In addition to trapping Met in vitro, full-length
DEP-1 substrate-trapping mutants trapped a Met chimeric receptor when co-expressed in 293 cells (Fig. 3A). Furthermore, wild type
DEP-1 dephosphorylated Met in this cellular context.
Met is the prototypic member of a small subfamily of receptor PTKs that
includes Ron and the chicken homologue of Ron, Sea. HGF/SF is the
ligand for Met, whereas macrophage-stimulating protein is the ligand
for Ron and Sea. Members of this subfamily of PTKs are expressed in a
variety of cell types including epithelial, endothelial, and
hematopoietic cells. Interestingly, the expression pattern of DEP-1
overlaps with the expression pattern of these receptor PTKs consistent
with a possible interaction between these enzymes under physiological conditions.
Following activation by HGF/SF, Met is able to exert a variety of
effects by recruiting docking and signaling molecules. Phosphorylation of the tyrosine residues in the activation loop of the PTK domain potentiates the intrinsic kinase activity of Met, whereas
phosphorylation of the two docking site tyrosine residues
(Tyr1349 and Tyr1356) allows for the
recruitment of adaptor molecules including Grb2, SHC, and Gab1 and
signaling enzymes including phosphatidylinositol 3-kinase, PLC- The role of specific adaptor and signaling molecules in transducing Met
signals has been studied extensively. The adapter protein Grb2 recruits
SOS to activated receptor PTKs to induce Ras-mitogen-activated protein
kinase signaling. In Met signaling Ras stimulation is necessary and
sufficient to induce proliferation (39). Grb2 binds to Met directly at
a binding site that contains phosphorylated Tyr1356 (32,
33, 40). In addition Grb2 can be recruited to Met via the adapter
protein SHC (41). We observed a gradual decrease in the recruitment of
Grb2 to the Met chimera with increasing expression of DEP-1 (Fig.
4C) coincident with overall dephosphorylation of the PTK
(Fig. 4B). These data suggest that unlike
Tyr1349 and Tyr1365, Tyr1356 may
not be preferentially dephosphorylated by DEP-1 thereby allowing sustained recruitment of Grb2 to Met. In addition, we did not detect a
change in mitogen-activated protein kinase activation with increasing
expression of DEP-1 (data not shown) suggesting that the level of Grb2
recruited to the Met chimera may be sufficient to activate
mitogen-activated protein kinase. As cells reach confluence DEP-1 expression levels increase suggesting a role for this PTP in
contact-dependent growth inhibition (5). Interestingly, it
was at higher levels of DEP-1 expression that we detected a decrease in
the association between Grb2 and Met suggesting that the ability of
DEP-1 to affect mitogenic signals may depend upon the level of
expression of the PTP. In our system the use of a kinase that was
constitutively active bypassed the need for ligand stimulation,
however, under conditions in which Met is activated by its
physiological ligand the effects of DEP-1 on the phosphorylation of Met
at Tyr1356 and the recruitment of Grb2 may be more pronounced.
After Met activation the adapter molecule Gab1 is strongly
tyrosine-phosphorylated and recruited to Met directly through
Tyr1349 (42) and indirectly via Grb2 bound to
Tyr1356 (43-45). Gab1 can amplify and diversify Met
signaling by recruiting additional signaling proteins such as
phosphatidylinositol 3-kinase, PLC- The acquisition of a motile phenotype occurs during normal development
as well as in tumor progression and requires dissolution of cell-cell
junctions. Growth factors such as HGF/SF are able to disrupt cell
adhesion and induce phosphorylation of junctional components such as
In this study, we have shown for the first time that the PTP DEP-1
recognizes the receptor PTK Met as a substrate. Furthermore, our data
suggest that DEP-1 displays selectivity for particular phosphorylation
sites within this PTK and therefore, may function in determining which
signaling outcomes result from stimulation of Met. Under normal
conditions, Met has been implicated in the control of mitogenesis,
morphogenesis, and migration. However, aberrant signaling initiated
either as a result of mutations in Met, or overexpression of the
kinase, have been described in a variety of human cancers (50).
Interestingly, frequent deletions, loss of heterozygosity, and missense
mutations in the DEP-1 gene have been identified in several
human cancers including colon, lung, and breast (17), which have also
been associated with aberrant Met signaling. Our demonstration of a
functional interaction between DEP-1 and Met as enzyme and substrate
raises the intriguing possibility that the up-regulation of Met may be
coupled with the down-regulation of DEP-1 in the progression of certain
human cancers.
) has been implicated in the regulation of cell
growth, differentiation, and transformation, and most recently has been identified as a potential tumor suppressor gene mutated in colon, lung,
and breast cancers. We have generated constructs comprising the
cytoplasmic segment of DEP-1 fused to the maltose-binding protein to
identify potential substrates and thereby suggest a physiological
function for DEP-1. We have shown that the substrate-trapping mutant
form of DEP-1 interacted with a small subset of tyrosine-phosphorylated proteins from lysates of the human breast tumor cell lines MDA-MB-231, T-47D, and T-47D/Met and have identified the hepatocyte growth factor/scatter factor receptor Met, the adapter protein Gab1, and the junctional component p120 catenin as potential substrates. Following ligand stimulation, phosphorylation of specific tyrosyl residues in Met induces mitogenic, motogenic, and morphogenic responses. When co-expressed in 293 cells, the full-length
substrate-trapping mutant form of DEP-1 formed a stable complex with
the chimeric receptor colony stimulating factor 1 (CSF)-Met and
wild type DEP-1 dephosphorylated CSF-Met. Furthermore, we observed that
DEP-1 preferentially dephosphorylated a Gab1 binding site
(Tyr1349) and a COOH-terminal tyrosine implicated in
morphogenesis (Tyr1365), whereas tyrosine residues in the
activation loop of Met (Tyr1230, Tyr1234, and
Tyr1235) were not preferred targets of the PTP. The ability
of DEP-1 preferentially to dephosphorylate particular tyrosine residues that are required for Met-induced signaling suggests that DEP-1 may
function in controlling the specificity of signals induced by this PTK,
rather than as a simple "off-switch" to counteract PTK activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(6) and CD148 (9, 10), DEP-1 is expressed in a
variety of tissues and cell types. There is a growing body of evidence suggesting a role for DEP-1 in the inhibition of cell growth. After
vascular injury DEP-1 expression is down-regulated in migrating and
proliferating rat endothelial cells (8). Attempts have been made to
express DEP-1 constitutively in breast cells and macrophages (11, 12),
however, this inhibited development of stable cell lines, further
reinforcing a role for DEP-1 in growth inhibition.
) mRNA decreases with increasing
levels of transformation (13, 15). Re-introduction of DEP-1 into the
transformed cells leads to reduced growth rates, stabilization of
the cyclin-dependent kinase inhibitor p27, and partial
re-acquisition of a differentiated phenotype (16). Loss of DEP-1
expression has also been observed in human thyroid tumors (16).
Furthermore, the DEP-1 gene Ptprj was identified as a positional candidate for the mouse colon-cancer susceptibility locus
Scc1 (17). Frequent deletions, loss of heterozygosity, and missense
mutations in the human Ptprj gene have also been identified
in colon, lung, and breast cancers (17). Taken together these data
indicate that DEP-1 may be a critical factor in controlling cellular
growth and transformation.
-catenin, however, can also promote cell growth by inducing
the transcription of genes involved in proliferation and cancer
progression (reviewed in Ref. 19). The growth inhibitory effects of
cadherins may involve binding and sequestration of the signaling pool
of the catenins (20, 21). Reversible tyrosine phosphorylation is an
important aspect of the regulation of junctional integrity and the
control of signals emanating from these sites (reviewed in Ref. 22).
The identification of the PTKs and PTPs that act upon the components of
cell junctions will be important for understanding the regulation of
cell morphology and the control of gene expression, events that
ultimately influence growth and migration.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Catenin (6F9)
and plakoglobin (15F11) antibodies were purchased from Sigma and the Gab1 COOH-terminal antibody was purchased from Upstate Biotechnology. Anti-c-Met
(Tyr(P)1230-Tyr(P)1234-Tyr(P)1235
and Tyr(P)1365) antibodies were purchased from BioSource
International (Camarillo, CA) and phospho-Met (Tyr1349) was
purchased from Cell Signaling Technology (Beverly, MA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Fig. 1.
Tyrosine-phosphorylated proteins trapped by
DEP-1(DA) from pervanadate-treated breast tumor cells. A,
immunoblot of tyrosine-phosphorylated proteins trapped by DEP-1(DA)
from T-47D cells. T-47D cells were treated with 50 µM
pervanadate for 20 min prior to lysis. MBP or MBP.DEP-1 fusion proteins
were incubated with cell lysates and protein complexes were analyzed by
SDS-PAGE and immunoblotting using antiphosphotyrosine antibodies. An
antiphosphotyrosine immunoprecipitation was also performed on
pervanadate-treated cell lysates to illustrate the full compliment of
tyrosine-phosphorylated proteins (PY IP). B,
effects of vanadate on the interaction between tyrosine-phosphorylated
proteins with the DEP-1(DA) substrate-trapping mutant. T-47D cells were
treated as in A. Cells were lysed in lysis buffer (see
"Experimental Procedures") with (+) or without ( ) 2 mM vanadate. MBP and MBP.DEP-1 fusion proteins were
preincubated with (+) or without (
) 2 mM vanadate and
added to cell lysates. Protein complexes were analyzed by SDS-PAGE and
immunoblotting using antiphosphotyrosine antibodies. C,
immunoblot of tyrosine-phosphorylated proteins trapped by DEP-1(DA)
from MDA-MB-231 cells. MDA-MB-231 cells were treated with 100 µM pervanadate for 20 min prior to lysis. MBP or
MBP.DEP-1 fusion proteins were incubated with cell lysates and samples
were processed as in A. D, effects of vanadate on the
interaction between tyrosine-phosphorylated proteins with the DEP-1(DA)
substrate-trapping mutant. MDA-MB-231 cells were treated as in
C. Cells were lysed and samples processed as in
B, and protein complexes were analyzed by SDS-PAGE and
immunoblotting using antiphosphotyrosine antibodies.
Identification of p120Ctn as a substrate of DEP-1
-catenin
and plakoglobin, however, were found in a complex with MBP.DEP-1(DA). Although p120Ctn only interacted with the DEP-1
substrate-trapping mutant,
-catenin and plakoglobin also interacted
with the wild type form of the enzyme (MBP.DEP-1) (Fig. 2) suggesting
that
-catenin and plakoglobin may interact with DEP-1
constitutively.
View larger version (29K):
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Fig. 2.
Identification of tyrosine-phosphorylated
proteins that interacted with the DEP-1(DA) substrate-trapping
mutant. Immunoblot analysis of proteins associated with wild type
DEP-1 and substrate-trapping mutant DEP-1(DA). T-47D and T-47D/MET
cells were treated with 50 µM pervanadate for 20 min
prior to lysis. MBP.DEP-1 or MBP.DEP-1(DA) fusion proteins
were incubated with cell lysates and protein complexes were analyzed by
SDS-PAGE and immunoblotting using antibodies directed toward E-cadherin
(E-cad), -catenin (
-cat), plakoglobin
(Pg), p120Ctn (p120), Met
(Met), and Gab1 (Gab1). Cell lysate (50 µg) was
loaded to confirm the expression and molecular weight of each of the
proteins analyzed by immunoblotting (Lysate).
View larger version (18K):
[in a new window]
Fig. 3.
Co-expression of DEP-1 and Met in 293 cells.
A, immunoblot analysis of Met associated with full-length
substrate-trapping mutants of DEP-1. 293 cells were transfected with
CSF-Met alone or in combination with wild type or mutant forms of
DEP-1. Cells were serum starved and DEP-1 was immunoprecipitated from
half of the cell lysate using monoclonal antibodies A3 and 143-41. Immunoblots using the polyclonal antibody CS895A revealed the levels of
DEP-1 in the immunoprecipitates (DEP-1). Blots were striped
and reprobed for Met (Met). B, immunoblot
analysis of the phosphorylation state of Met in the presence of wild
type or mutant forms of DEP-1. Met was immunoprecipitated from the
other half of the cell lysate from panel A using polyclonal
antibody 144. Immunoblots using polyclonal antibody C-12 directed to
the intracellular segment of Met revealed the levels of CSF-Met in the
immunoprecipitates (Met). Immunoblots were striped and
reprobed with antiphosphotyrosine antibodies (PY).
View larger version (23K):
[in a new window]
Fig. 4.
The effects of expression of DEP-1 on the
phosphorylation of Met and its association with Grb2.
A, levels of expression of DEP-1 and Met. 293 cells were
transfected with 20 µg of CSF-Met DNA and 0, 1, 2.5, 5, and 10 µg
of DEP-1 DNA or 10 µg of DEP-1(CS) DNA. Cell lysates (50 µg) were
analyzed for the expression levels of DEP-1 and Met by immunoblots with
appropriate antibodies. B, site-specific dephosphorylation
of Met by DEP-1. Met was immunoprecipitated from the serum-starved 293 cell lysates shown in panel A using polyclonal antibody 144 and the immunoprecipitates were run in duplicate. Immunoblots using
polyclonal antibody C-12 revealed a constant level of Met
immunoprecipitated from the cell lysates (Met). This blot
was stripped and reprobed with the phospho-specific antibody to
Tyr1349 in Met (P-Met Y1349). A
duplicate blot was probed with antiphosphotyrosine antibodies to
illustrate the total phosphotyrosine content (PY), then
sequentially stripped and reprobed with phospho-specific antibodies to
examine the phosphorylation status of Tyr1230,
Tyr1234, and Tyr1235 (P-Met
Y1230/34/45), and Tyr1365 (P-Met
Y1365). C, immunoblot analysis of the
association of Grb2 with Met. Blots from panel B were probed
with an antibody to Grb2 to reveal the level of Grb2 associated with
Met (Met IP/Grb2 IB). Cell lysates (50 µg) were run to
determine the level of expression of Grb2 in the transfected cells
(Lysate).
, and
SHP2 (reviewed in Ref. 23). Phospho-specific antibodies toward
Tyr1349 were used to determine whether DEP-1
dephosphorylated this site. Interestingly, unlike the gradual reduction
in phosphorylation seen for the activation loop tyrosine residues,
Tyr1349 was nearly completely dephosphorylated in the
presence of low levels of DEP-1 (Fig. 4B). This
dephosphorylation also required DEP-1 catalytic activity because no
change in the phosphorylation level of Tyr1349 was observed
in the presence of DEP-1(CS). The phosphorylation status of the other
docking site tyrosine residue (Tyr1356) could not be
determined because of the lack of phospho-specific antibodies toward
this residue. In addition to the docking site tyrosine residues, other
tyrosine residues have been shown to impact Met signaling. For example,
Tyr1365 is important for mediating a morphogenic signal
(24). Interestingly, phospho-specific antibodies directed toward this
site revealed that Tyr1365 was nearly completely
dephosphorylated in the presence of low levels of DEP-1 (Fig.
4B). These observations suggest that DEP-1 may display
specificity for certain sites within Met.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, the
PTK Src, the PTP SHP2, as well as the transcription factor STAT3
(reviewed in Ref. 23). This multisubstrate docking site sequence is
primarily responsible for Met-mediated signal transduction and chimeric
receptors containing this sequence can induce mitogenic, motogenic, and
morphogenic responses similar to Met (25, 34-36). Cells expressing Met
with mutations at Tyr1349 and Tyr1356 are
unresponsive to HGF/SF stimulation in vitro (33), and
transgenic mice with these mutations display a lethal phenotype that
resembles the phenotype of mice lacking Met or HGF/SF (37). Modulating the phosphorylation status of the multisubstrate docking site represents an important mechanism for regulating HGF/SF-induced cellular responses. Using phospho-specific antibodies we found that
DEP-1 preferentially dephosphorylated the docking site residue Tyr1349 (Fig. 4B). Because of the lack of
phospho-specific antibodies we were unable to determine the
phosphorylation status of the other docking site residue
Tyr1356. In addition, DEP-1 preferentially dephosphorylated
Tyr1365, a residue important for mediating a morphogenic
signal (24). The tyrosine residues in the activation loop, however,
were not preferred sites and were only dephosphorylated when DEP-1 was expressed at higher levels. A recent study on the effect of DEP-1 on
platelet-derived growth factor
-receptor phosphorylation has shown
that wild type DEP-1 dephosphorylates the platelet-derived growth
factor
-receptor in porcine aortic endothelial cells ectopically expressing both proteins (38). Similar to our results with Met, DEP-1
exhibited site selectivity and preferentially dephosphorylated tyrosine
residues other than the tyrosine residue in the activation loop of
platelet-derived growth factor
-receptor. Taken together these
results suggest that DEP-1 may not act to inhibit kinase activity, but
rather by dephosphorylating specific docking sites for signaling
molecules it may modulate specific signaling pathways emanating from
the receptor PTK.
, SHP-2, and the adapter protein
Crk. Tyrosine phosphorylation of Gab1 at specific residues is required
for the recruitment of the signaling molecules. Transgenic mice lacking
Gab1 display a lethal phenotype that resembles the phenotype of mice
lacking Met or HGF/SF suggesting that Gab1 is important for Met
signaling in vivo (46). Based on the ability of DEP-1 to
trap Gab1 (Fig. 2) and specifically dephosphorylate a Gab1 docking site
in Met (Fig. 4B) it is tempting to speculate that DEP-1 may
act to modulate signals downstream of Gab1. A recent study on the
effects of DEP-1 on T-cell receptor signaling has shown that DEP-1
induction in a Jurkat cell line results in reduced tyrosine
phosphorylation of the adapter protein LAT and the enzyme PLC-
1
(47). Interestingly Gab1 and LAT are members of the same family of
docking proteins. After receptor engagement LAT is phosphorylated on
tyrosine residues and recruits additional molecules including PLC-
.
Although we observed an interaction between DEP-1 and Gab1, Baker
et al. (47) did not detect an interaction between a
substrate-trapping mutant form of DEP-1 and LAT or PLC-
1. It will be
important to determine whether DEP-1 can preferentially dephosphorylate
specific residues in Gab1 thereby influencing specific cellular responses.
-catenin and plakoglobin (48). Interestingly, DEP-1 interacted with
several known Met substrates including Gab1,
-catenin, and
plakoglobin (Fig. 2). In addition DEP-1 trapped the Src substrate and
adherens junction component p120 catenin (p120Ctn). The
interaction between p120Ctn and DEP-1 was restricted to the
substrate-trapping form of DEP-1 while
-catenin and plakoglobin
interacted with both the wild type and the mutant form of DEP-1 (Fig.
2). This suggests that p120Ctn may interact with DEP-1 in a
phosphorylation-dependent manner whereas
-catenin and
plakoglobin may interact with DEP-1 constitutively. A detailed analysis
of the human p120Ctn gene predicts that up to 32 isoforms
of the protein may exist based on alternative splicing (30). In
addition, most cell types express multiple isoforms of
p120Ctn. The sequences we identified by mass spectrometry
are present in all the isoforms of p120Ctn identified to
date, thus it is unclear whether DEP-1 interacts with specific
isoforms. While this article was in preparation, Holsinger
et al. (49) also showed that DEP-1 interacts with p120Ctn,
-catenin, and plakoglobin and dephosphorylates
p120Ctn in vitro (49).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Gregorio Aversa and Dr. Antoni Gayá for supplying DEP-1 antibodies A3 and 143-41. We are grateful to our colleagues Drs. L. Holsinger and B. Jallal from SUGEN Inc. for insightful discussions. We also thank Dr. R. DelVecchio for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant RO1-GM55989 (to N. K. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Cold Spring Harbor Laboratory Cancer Center Training Grant T32-CA09311 in Cancer Cell Biology and Tumor Virology from the National Institutes of Health.
To whom correspondence should be addressed: Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY 11724. Tel.: 516-367-8846; Fax:
516-367-6812; E-mail: tonks@cshl.org.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M210656200
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ABBREVIATIONS |
---|
The abbreviations used are:
PTK, protein-tyrosine kinases;
PTP, protein-tyrosine phosphatases;
HGF/SF, hepatocyte growth factor/scatter factor;
CSF-1R, colony-stimulating
factor 1 receptor;
MBP, maltose-binding protein;
PLC-, phospholipase
C
.
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