From the
PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is
recruited to the growth factor receptors upon stimulation of cells. To
investigate its role in growth factor signaling, we have overexpressed
by approximately 6-fold the native PTP2C and a catalytically inactive
mutant of the enzyme in 293 human embryonic kidney cells. The native
PTP2C was located entirely in the cytosol, while the inactive mutant
was nearly equally distributed in cytosolic and membrane fractions.
Expression of the latter caused hyperphosphorylation on tyrosine of a
43-kDa protein, which was co-immunoprecipitated and co-partitioned in
the plasma membrane fraction with the inactive PTP2C mutant. This
protein may represent a physiological substrate of PTP2C.
Overexpression of the native PTP2C enhanced epidermal growth factor
(EGF)-stimulated mitogen-activated protein (MAP) kinase kinase activity
by 30%, whereas expression of the inactive mutant reduced the
stimulated activity by 50%. Similar effects were observed for the
activation of MAP kinase as determined by activity assay, gel mobility
shift, and tyrosine phosphorylation. The data suggest that the
phosphatase activity of PTP2C is partly required for MAP kinase
activation by EGF and that PTP2C may function by dephosphorylating the
43-kDa membrane protein.
External signals, such as growth factors, initiate their signals
by binding to ligand-specific growth factor receptors on the cellular
membrane
(1) . Receptor autophosphorylation on multiple tyrosyl
residues, as a result of ligand binding, provides high-affinity binding
sites for specific cellular proteins containing SH2
MAP kinase activation via the Ras signaling
pathway in mammalian cells has been well
defined
(18, 19) . GRB2, an SH2 and SH3 domain-containing
protein, works as an adaptor by binding directly or indirectly via
another protein such as SHC to upstream autophosphorylated growth
factor receptors or their substrates and to downstream nucleotide
exchange factor mSOS, thus activating Ras. GTP-bound Ras facilitates
the recruitment of Raf-1 to the plasma membrane, resulting in its
activation followed by activation of MAP kinase kinase. MAP kinase
kinase, in turn, activates MAP kinase which requires phosphorylation on
both tyrosine and threonine residues.
To investigate the role of
PTP2C in growth factor signaling, we have expressed the native PTP2C
and a catalytic inactive form of the enzyme in 293 human embryonic
kidney cells. Our study demonstrated that expression of the mutant of
PTP2C had a profound impact on protein tyrosine phosphorylation in the
cells. Furthermore, while overexpression of the native enzyme increased
EGF-stimulated MAP kinase activation, expression of the catalytically
inactive mutant decreased such activation.
Buffer A: 50 mM
For PTP activity assay, sodium
vanadate was omitted from all the buffers used. Assays were performed
with anti-PTP2C immunoprecipitates using 1 µM
To
immunoprecipitate ERK1 and ERK2 with anti-MAP kinase antibody, cell
extracts were brought to 0.5% SDS and then boiled for 10 min. After
cooling down, samples were diluted 5-fold with the extraction buffer
and incubated with anti-MAP kinase serum 7884 overnight. The cell
extracts and immunoprecipitates were separated on 10% SDS gel. Western
blotting was carried out as described above.
Increased tyrosine phosphorylation of
proteins caused by expression of the catalytically inactive mutant of
PTP2C has also been observed by others. In one study,
hyperphosphorylation of a 120-kDa protein which associated with PTP2C
was observed in insulin-stimulated NIH 3T3 cells (14). In another
study, increased tyrosine phosphorylation of the 125-kDa focal adhesion
kinase was demonstrated in CHO cells, but the phosphorylation of this
protein decreased upon insulin treatment of the cells
(16) . It
is not known whether the 43-kDa and 100-kDa proteins we observed are
related to the 120-kDa protein and focal adhesion kinase.
Overexpression of PTP2C had no effect on the basal or unstimulated
MAP kinase activity, indicating that PTP2C may be necessary but not
sufficient for turning on the signaling pathway. This may also suggest
that the localization or compartmentalization of the phosphatase is
crucial for its function. In nonstimulated cells, PTP2C stays in the
cytosol, where it probably remains inactive. Upon growth factor
stimulation of the cells, PTP2C associates with the growth factor
receptors allowing it to interact with phospholipids in the plasma
membrane, which have been shown to activate the enzyme in
vitro(20, 25) . It is tempting to postulate that
the associations of PTP2C with activated growth factor receptors, which
direct the enzyme to the plasma membrane, lead to its activation in a
way analogous to the activation of SOS and Raf mediated by activated
Ras (26-28).
All PTPs contain a highly conserved cysteinyl
residue within their catalytic centers. Mutation of this cysteine to
serine impairs the phosphatase activity, which was proved to be true
for PTP2C in this study. The catalytically inactive enzyme presumably
has two negative effects. First, the catalytic domain of the mutant
enzyme can still bind substrates and thus block their dephosphorylation
by the native enzyme. Second, the mutant enzyme can compete with the
native enzyme for SH2 domain binding sites and prevent the latter from
accessing its targets. Both effects can cause hyperphosphorylation of
the target proteins. In this regard, the hyperphosphorylation of the
43-kDa protein resulting from the expression of PTP2C(C-S) may indicate
that it is a physiological substrate of the enzyme. In addition,
co-immunoprecipitation and co-localization of this protein with
PTP2C(C-S) on the plasma membrane suggest that p43 serves as an anchor
protein for PTP2C(C-S) at this site. In any case, this protein may play
a crucial role for the function of PTP2C. It might also be involved in
MAP kinase activation by functioning as a negative regulator of the
process.
(
)
domains. These SH2 domain-phosphopeptide interactions
recruit other signaling molecules to the receptors where they can be
phosphorylated by the receptors and/or recruit still other
proteins
(2) . Among the SH2 domain-containing proteins recruited
to the growth factor receptors is a protein-tyrosine phosphatase (PTP)
designated as PTP2C (also termed as SH-PTP2, SH-PTP3, PTP1D, and
Syp)
(3, 4, 5, 6, 7) . PTP2C is a
widely expressed enzyme. It shares high homology with corkscrew, which
is required for normal development of Drosophila by playing a
positive role in the transduction of the torso signal acting
in concert with D-Raf
(8) . The parallel nature of the
torso signal transduction pathway to MAP kinase activation in
mammalian cells suggests that PTP2C may play a similar role in
mammalian systems. Recent studies indeed suggest that PTP2C is involved
in growth factor signaling and in MAP kinase activation. First, PTP2C
binds to EGF and PDGF receptors
(6, 9, 10) and
insulin receptor substrate-1
(11) . Second, phosphorylation of
PTP2C at its C terminus couples GRB2 to PDGF receptor, providing a
mechanism for PDGF-induced activation of Ras
(12, 13) .
Third, overexpression of catalytically inactive forms of PTP2C blocks
insulin-stimulated MAP kinase activation (14-16). Fourth, as a
feedback reaction, PTP2C is phosphorylated and inhibited by MAP
kinase
(17) .
Materials
Human 293 cells were obtained from the
American Type Culture Collection. Polyclonal anti-PTP2C (serum 1263)
and anti-MAP kinase (serum 7884) were raised in rabbits against an SH2
domain-truncated form of PTP2C expressed in Escherichia coli (20) and against a 22-amino acid peptide derived from the
subdomain XI of MAP kinase
(21) , respectively. Monoclonal
ribophorin II antibody S.D.1 was kindly provided by Dr. David Meyer
(UCLA). Epidermal growth factor (EGF) and monoclonal
anti-phosphotyrosine and polyclonal sheep-anti-EGF receptor antibodies
were purchased from Upstate Biotechnology Inc. Myelin basic protein
(MBP) and [-
P]ATP were purchased from Sigma
and Amersham, respectively.
-glycerophosphate (pH 7.3) and 2 mM EDTA. Buffer B: 25
mM Tris-HCl (pH 7.5), 100 mM NaCl, 50 mM
KCl, and 1.5 mM MgCl
. Both buffers were also
supplemented with 5 mM
-mercaptoethanol, 1 mM
EGTA, 0.2 mM Na
VO
, 0.1 µM
microcystin, 1.0 mM benzamidine, 0.1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 1
µM pepstatin A, and 1 µg/ml aprotinin.
Construction and Expression of PTP2C in 293
Cells
A catalytically inactive form of PTP2C, designated as
PTP2C(C-S), was generated by substituting the highly conserved cysteine
459 with a serine in a polymerase chain reaction. The site of mutation
was confirmed by DNA sequencing. The cDNA inserts corresponding to the
native and the Cys-to-Ser mutant were constructed into the pRC/CMV
vector (Invitrogen). Transfection was carried out according to the
standard calcium phosphate co-precipitation technique. To obtain stable
cell lines, cells were selected in medium containing 0.5 mg/ml
geniticin (G418 sulfate, Life Technologies, Inc.), and single colonies
expressing high level PTP2C or PTP2C(C-S) were obtained after 2-3
weeks. Wild type 293 cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum, 50
µg/ml streptomycin, and 50 units/ml penicillin. The transfected
cells were grown in the same medium supplemented with 0.5 mg/ml G418.
Immunoprecipitation, Western Blotting, and PTP
Assay
Confluent 293 cells were lysed in Buffer A supplemented
with 1% Triton X-100 and 0.1 M NaCl. The extract was cleared
by centrifugation at full speed in a microcentrifuge. For
immunoprecipitation with anti-PTP2C antibody, the extracts were
preincubated with 50 µl of a 50% slurry of protein A-Sepharose
(Pharmacia Biotech Inc.) for 2 h at 4 °C. After a brief
centrifugation, the supernatant was incubated with antibody prebound to
protein A-Sepharose overnight. The beads were washed three times with
the cold lysis buffer. For Western blot analyses, samples were
separated by 10% SDS-PAGE and transferred to a polyvinylidene
difluoride membrane (Millipore). The membranes were probed with various
primary antibodies and detected using the ECL system with horseradish
peroxidase-conjugated secondary antibodies (Amersham) according to the
manufacturer's protocol.
P-labeled ENDpYINASL peptide (800 cpm/pmol) as the
substrate at pH 5.0 as described
(20, 22) .
Fractionation of Cell Extracts
Cells were
collected, washed with cold phosphate-buffered saline, and suspended in
Buffer B. Lysis was performed by nitrogen cavitation for 20 min at 350
p.s.i. Nuclear pellets were removed by centrifugation at 800
g for 20 min. The postnuclear extract was further centrifuged
at 100,000
g for 45 min to give a clear cytosolic
supernatant and a pelleted membrane fraction. The latter pellet, washed
once with Buffer B and then dissolved in Buffer B supplemented with 1%
Triton X-100, was referred to as the membrane extract. Further
fractionation of the membrane fraction was performed by employing a
17.5% Percoll density gradient. The postnuclear extract (1 ml) was
loaded on the top of a 9-ml Percoll suspension made in buffer B.
Sedimentation was performed for 1 h at 18,000 rpm with a Beckman 50 Ti
rotor. A total of 20 fractions were collected from bottom to top.
Stimulation of Cells and Assays for MAP Kinase and MAP
Kinase Kinase
Cells (80-90% confluency) were starved at 0%
serum for 24 h and then stimulated with EGF for various periods of
time. The reactions were stopped by washing with ice-cold
phosphate-buffered saline. Cells were then collected and lysed in
Buffer A by brief sonication. The extracts were cleared by
centrifugation at 100,000 g for 25 min. For protein
kinase activity assay, samples were passed through a DEAE 52 minicolumn
equilibrated with Buffer A. The column was washed with the same buffer
and eluted with 0.3 M NaCl. The flow-through was collected for
MAP kinase kinase assay and the elute for MAP kinase assay. The
activity of MAP kinase kinase was determined by the ability of the
enzyme to stimulate the MBP kinase activity of recombinant ERK2 in a
coupled assay system as described
(21) . MAP kinase was assayed
with MBP as substrate in the presence of PKI peptide and
calmidizolium
(21) . Blanks were run without ERK2 and MBP for MAP
kinase and the kinase kinase assays, respectively.
RESULTS
Overexpression of PTP2C and PTP2C(C-S) in 293
Cells
Constructs containing PTP2C or the mutant PTP2C(C-S) and
the pRC/CMV vector alone were used to transfect 293 cells. Clonal cell
lines were isolated by G418 selection. Most of the clones selected an
overexpressed substantial level of exogenous PTP2C or PTP2C(C-S) as
compared with the vector control and the parental cells. Typically,
about 6-8-fold overexpression was obtained as determined by
Western blot with anti-PTP2C serum (Fig. 1) followed by
densitometric analyses. The activity of PTP2C in the transfected cells
was measured following immunoprecipitation of the cell extract with
anti-PTP2C serum. To precipitate approximately equal portions of
enzyme, extracts from cells overexpressing PTP2C or PTP2C(C-S) were
diluted 6-fold with the extraction buffer while that from the vector
control cells was undiluted. Upon incubation of an equal volume of cell
extracts with anti-PTP2C serum and protein A-Sepharose, each
immunoprecipitate gave about an equal amount of anti-PTP2C
cross-reactivity in Western blotting analyses (data not shown). PTP
activity in the immunoprecipitates was then assayed by employing the
P-labeled peptide substrate ENDpYINASL in a liquid-solid
phase reaction mixture (). As expected, nearly equal
amounts of activity were detected in the immunoprecipitates from the
control cells and cells over-expressing PTP2C, corresponding to an
approximately 6.5-fold total increase of PTP2C activity in the
transfected cells. This suggests that the exogenously expressed PTP2C
possessed the same activity as the endogenous enzyme. In contrast, the
activity per mg of cell extract from the cells overexpressing
PTP2C(C-S) was similar to that obtained from the control cells,
indicating that the Cys-to-Ser mutant of PTP2C is totally inactive.
Control experiments with preimmune serum showed essentially no PTP
activity (data not shown).
Figure 1:
Overexpression of PTP2C and PTP2C(C-S)
in 293 cells and the effects on protein tyrosine phosphorylation. Cell
extracts (25 µg) from wild type 293 cells (lane W) and
cells transfected with the vector alone (lane V), native PTP2C
(lane N), or catalytically inactive PTP2C(C-S) (lane
M) were separated on 10% SDS gel, transferred to polyvinylidene
difluoride membrane, and blotted with anti-PTP2C or
anti-phosphotyrosine (Anti-PY) as indicated. The
hyperphosphorylated protein p43 is indicated by an
arrow.
Effects of the Altered PTP2C Expression on the Tyrosine
Phosphorylation of Proteins in 293 Cells
Protein tyrosine
phosphorylation was detected by Western blotting analyses employing
anti-phosphotyrosine antibody as shown in Fig. 1. Overexpression
of the native PTP2C showed essentially no effect on protein tyrosine
phosphorylation at the basal level. No significant effect was observed
in serum-stimulated cells either (data not shown). However,
overexpression of PTP2C(C-S) resulted in changes of tyrosine
phosphorylation of proteins in the cells. In particular, a protein of
approximately 43 kDa (designated as p43) was hyperphosphorylated on
tyrosine. This protein ran as a rather broad band on SDS gel, probably
due to differing degrees of phosphorylation on multiple sites. Note
that tyrosine phosphorylation of another protein of approximately 100
kDa slightly increased also. Stimulation of the cells with serum did
not cause significant change in the tyrosine phosphorylation of these
proteins (data not shown).
Co-fractionation and Co-immunoprecipitation of PTP2C(C-S)
with p43
Cytosolic and membrane fractions of postnuclear
extracts were separated by high speed centrifugation. While almost all
the PTP2C in the control cells and cells overexpressing the native
PTP2C distributed in the cytosolic fraction, nearly 50% of the enzyme
was associated with the membrane fraction in cells expressing
PTP2C(C-S) (Fig. 2). PTP assays following immunoprecipitation of
the membrane fraction with anti-PTP2C serum yielded essentially no
activity, indicating that the anti-PTP2C cross-reactivity detected in
the membrane fraction represents the mutant enzyme only. For all cases,
almost no PTP2C was found in the nuclear fraction (data not shown). The
hyperphosphorylated p43 was also found in the membrane fraction
(Fig. 2). In addition, it was co-immunoprecipitated with
PTP2C(C-S), which might indicate a direct association of these two
proteins. Separation of the postnuclear extract from cells
overexpressing PTP2C(C-S) on a Percoll density gradient was shown in
Fig. 3
. Nearly half of the anti-PTP2C cross-reactivity was
detected on the top of the gradient (fractions 16-20), which
presumably represent the cytosolic fraction. Most of the rest was found
in the middle of the gradient (fractions 6-12) where p43 was also
detected. The distribution pattern of p43 and associated PTP2C(C-S)
followed that of the EGF receptor, a plasma membrane marker, but
differed from that exhibited by the endoplasmic reticulum maker
ribophorin II
(23) . The data indicate that PTP2C(C-S) and the
hyperphosphorylated p43 were partitioned in the plasma membrane
fraction. Based on the results, one can postulate that PTP2C(C-S)
caused the hyperphosphorylation of p43 by preventing its
dephosphorylation by the endogenous native enzyme and that
tyrosine-phosphorylated p43, in turn, serves as an anchor for
PTP2C(C-S) on the plasma membrane.
Figure 2:
Distribution of PTP2C, PTP2C(C-S), and the
hyperphosphorylated p43 in cell fractions. Lanes V,
N, and M represent 293 cells transfected with the
vector alone, native PTP2C, and mutant PTP2C(C-S), respectively.
Cytosolic (lane 1) and membrane (lane 2) fractions
were separated by centrifugation at 100,000 g. Cell
extracts and their immunoprecipitates with anti-PTP2C were subject to
SDS-PAGE and Western blotting analyses as described in Fig. 1. The
arrow indicates the co-precipitated, hyperphosphorylated
p43.
Figure 3:
Fractionation of cell extracts from 293
cells expressing PTP2C(C-S). Postnuclear extracts were fractionated
using a Percoll density gradient as described under ``Experimental
Procedures.'' Fractions were subject to SDS-PAGE and Western
blotting analyses with anti-EGF receptor, anti-PTP2C, anti-ribophorin
II, and anti-phosphotyrosine (for p43). The number for each
fraction is indicated.
Effects of the Altered PTP2C Expression on MAP Kinase
Activation
To examine the role of PTP2C in the MAP kinase
activation pathway, 293 cells with altered PTP2C expression were
stimulated with EGF. Fig. 4illustrates the time courses of MAP
kinase and MAP kinase kinase activation. The activity profile of both
kinases followed a typical biphasic pattern with peak activity
occurring at 5 min. Altered PTP2C expression did not affect the time
course of activation significantly. However, there was a small but
significant impact on its magnitude. Overexpression of the native PTP2C
increased the peak of EGF-stimulated MAP kinase kinase activity by
about 30% while the expression of the catalytically inactive mutant
decreased it by approximately 50% (Fig. 4A). Similar
effects were observed for the activity of MAP kinase
(Fig. 4B). These results were reproduced in other clonal
cell lines selected.
Figure 4:
Activation of MAP kinase kinase
(A) and MAP kinase (B) in 293 cells transfected with
vector alone (--
), PTP2C (
-
- -
), and PTP2C(C-S) (
- -
-
). Serum-starved cells were stimulated with 20 ng/ml EGF
for the indicated time. MAP kinase activity was determined by
incubating 5-µg samples with MBP and
[
-
P]ATP (1000 cpm/pmol) for 10 min. MAP
kinase kinase was analyzed by incubating 2-µg samples with ERK2 and
cold ATP for 10 min followed by the addition of MBP and
P-labeled ATP and further incubating for 10 min. See
``Experimental Procedures'' for
details.
The effects of the altered PTP2C expression
were also apparent in MAP kinase gel shift assays and Western blot
analyzed with anti-phosphotyrosine (Fig. 5). Mobility shifts of
MAP kinase (ERK1 and ERK2) on SDS-gel reflect the phosphorylations on
the enzyme, which are required for its activation. As shown in
Fig. 5A, 5- and 10-min stimulation of cells with EGF
resulted in a significant portion of both ERK1 and ERK2 shifted in
cells with the vector control and the native PTP2C, while much less was
observed in the cells expressing the mutant PTP2C. Although the gel
shifts were not perfectly correlated with the activity as has been
reported
(21) , the data indicate that the decreased MAP kinase
activity in cells with PTP2C(C-S) was due to reduced phosphorylation.
Further supporting this were the results from Western blot analyses of
the immunoprecipitated MAP kinase (ERK1 and ERK2) with
anti-phosphotyrosine antibody. EGF stimulated transient tyrosine
phosphorylation of ERK1 and ERK2. Compared with the cells containing
the vector alone, a higher level of phosphotyrosine was observed for
the cells overexpressing the native PTP2C, while a lower level was
obtained with cells expressing the mutant PTP2C. At basal level,
neither significant gel shift nor tyrosine phosphorylation was
detected, suggesting that the basal MBP kinase activity at zero time
(see Fig. 4B) was probably due to protein kinases other
than MAP kinase. Additional experiments indicated that the altered
PTP2C expression had essentially no effect on the dose response of 293
cells to EGF and autophosphorylation of the EGF receptor upon
stimulation (data not shown).
Figure 5:
Gel
mobility shift (A) and tyrosine phosphorylation of MAP kinase
(B). Serum-starved 293 cells transfected with the vector
control, native PTP2C, and mutant PTP2C(C-S) were stimulated with 20
ng/ml EGF for the indicated time. Cell extracts (A) or
anti-MAP kinase immunoprecipitates (B) were separated on 10%
SDS gel, transferred to polyvinylidene difluoride membrane, and blotted
with polyclonal anti-MAP kinase antibody (A) or
anti-phosphotyrosine monoclonal antibody (B). Positions of
ERK1 and ERK2 are indicated.
DISCUSSION
In this study, we found that overexpression of PTP2C slightly
increased EGF-induced activation of MAP kinase while overexpression of
the catalytically inactive mutant of PTP2C modestly decreased such
activation. The remaining activation observed in the latter case could
be attributed to endogenous PTP2C in the cells and/or a parallel
activation pathway independent of PTP2C. In any case, the data suggest
that the phosphatase activity of PTP2C is at least partly required for
MAP kinase activation. These results are consistent with the recent
reports on the positive role of corkscrew in Drosophila torso signaling
(8) and the blockage of insulin-induced MAP
kinase activation by catalytically inactive forms of Syp and SH-PTP2 in
3T3 and CHO cells
(14, 15, 16) . The precise
mechanism by which PTP2C regulates the MAP kinase activation pathway is
unknown. Binding of PTP2C to growth factor receptors, including the EGF
receptor, has been well documented
(6, 7) , but it is not
known whether PTP2C can specifically dephosphorylate the
autophosphorylated receptors. If it does, overexpression of PTP2C might
reduce phosphorylation of the receptor and thus down-regulate MAP
kinase activation. Conversely, expression of the inactive mutant of
PTP2C should stabilize such phosphorylation and thus potentiate the
signaling pathway. Opposite effects were observed in this study, with
the native enzyme increasing the signal and the mutant decreasing the
signaling. Therefore, it is unlikely that the PTP2C acts at the
receptor level but instead probably functions downstream of the
receptor. Recent studies demonstrated that PTP2C can couple GRB2 to the
PDGF receptor by binding to the PDGF receptor via its SH2 domains and
to GRB2 via its tyrosine-phosphorylated C-terminal segment. The
GRB2-SOS complex would activate Ras and finally turn on the MAP kinase
pathway
(12, 13) . However, according to this model, one
should expect that the Cys-to-Ser mutation of PTP2C, which diminishes
auto-dephosphorylation of the enzyme and thus increases its
phosphorylation
(24) , should enhance MAP kinase activation.
Therefore, the present results suggest a more complicated role for
PTP2C than serving as an adaptor for GRB2 binding. PTP2C might play a
more important role in the aforementioned signaling pathway by
dephosphorylating its physiological substrates. Altered expression of
PTP2C affected MAP kinase kinase as well as MAP kinase activation.
Given that the activity of MAP kinase kinase is controlled by
phosphorylation of seryl residues
(18, 19) , PTP2C
apparently acts at another step upstream of MAP kinase kinase.
Table:
Activity of PTP2C in 293 cell
extracts
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.