From the
PTP1C and PTP1D are non-transmembrane protein-tyrosine
phosphatases (PTPs), which contain two src homology-2 domains. These
enzymes are believed to play a role in regulating downstream signaling
from receptors with intrinsic tyrosine kinase activity. The present
study describes the tyrosine phosphorylation and the catalytic activity
of both PTPs in CCL39 cells, a Chinese hamster lung fibroblast cell
line, upon addition of a variety of growth factors. We demonstrate that
PTP1C activity was significantly stimulated by insulin and the phorbol
ester 12- O-tetradecanoylphorbol-13-acetate but was not
influenced by serum, platelet-derived growth factor (PDGF), or
Phosphorylation and dephosphorylation of proteins on tyrosyl
residues are important reactions involved in the regulation of cell
growth and differentiation
(1, 2) . Elucidating the
mechanisms by which receptor tyrosine kinases select their targets and
thereby stimulate specific intracellular signaling pathways has been
aided by the identification of the SH2 domain, a conserved sequence
motif of approximately 100 amino acids, which is found in a diverse
group of cytoplasmic proteins
(3) . An increasing body of
evidence suggests that SH2 domains mediate specific interactions with
tyrosine-phosphorylated proteins. Upon binding of an external ligand to
receptors with intrinsic tyrosine kinase activity, receptor
autophosphorylation acts as a molecular switch to create binding sites
for the SH2 domains of a range of cytoplasmic signaling proteins
including Grb2, the regulatory subunit of phosphatidylinositol
3`-kinase (p85), phospholipase C
Several phosphotyrosine phosphatases
(PTPs)
Both PTP1C and
PTP1D are known to associate with activated receptor tyrosine kinases
or substrates of these kinases
(16, 17, 18, 19, 20, 33, 34) .
One possible role for these SH2-containing PTPs is to act as negative
regulators of receptor function by dephosphorylating either the
autophosphorylation receptor sites or their cognate substrates.
However, numerous studies have reported that PTP1C and PTP1D themselves
become and remain tyrosine phosphorylated in growth factor-stimulated
cells and are stably associated with tyrosine-phosphorylated growth
factor receptors, arguing against this simple model
(6, 11, 18) . Furthermore, PTP1C seems to play a
key role in hematopoiesis as reflected by the profound disturbances
exhibited by bone marrow cells from mice carrying the moth-eaten
mutation, which results in a total lack of PTP1C expression
(21) . It has been also proposed that PTP1D acts as a positive
mediator of growth factor-stimulated mitogenic signal transduction,
serving as an adaptor between the PDGF receptor and the Grb2-Sos
complex
(22, 23) .
In an attempt to clarify the roles
of both PTP1C and PTP1D to the mitogenic signaling cascade, we have
examined in this report the contribution of both PTP1C and PTP1D in the
mitogenic response induced by activation of tyrosine kinase receptors
(PDGF) or G protein-coupled receptors (
We demonstrate that PTP1D activity
could be modified by a range of agonists, with an increase in the level
of tyrosine phosphorylation of PTP1D correlating with inhibition of
activity. In contrast, an increase in the level of tyrosine
phosphorylation of PTP1C showed no correlation with the enzyme's
activity. We also demonstrate that overexpression of PTP1C or PTP1D had
no effect on growth factor-stimulated DNA synthesis, whereas expression
of a mutant inactive form of PTP1D inhibited the stimulatory effects of
PDGF and
Materials Highly purified human
The luciferase gene driven by
the human c- fos promoter represents a sensitive reporter of
growth factor-induced transcriptional activity
(38) . This early
gene promoter contains the well characterized serum-responsive element
(SRE), whose activity is induced upon activation of the
serum-responsive factor (30). In transiently transfected CCL39,
SRE-regulated gene expression was significantly stimulated by serum,
In the present study, we have compared the possible
involvement of PTP1C and PTP1D in the mitogenic signaling pathways of
Chinese hamster lung fibroblast cells when stimulated via either
tyrosine kinase receptors (PDGF) or G protein-coupled receptors
(
PTP1D is endogenously expressed in CCL39
cells but not PTP1C, a result in accordance with the ubiquitous
expression of PTP1D
(9, 10, 11, 12) and
the restricted expression of PTP1C to hematopoietic and epithelial
cells
(5, 6, 7, 8, 20) . In
addition, the two proteins, although sharing an overall homology, have
major regions with no similarity
(9) . Thus, we expected that
the regulation of each phosphatase, when expressed in the same cell
line, would not be identical. PTP1C, when expressed in CCL39 cells
(PTP1C-10 clone), displays a high basal activity. Addition of growth
factors such as
An
increase in the level of serine phosphorylation of PTP1C has been
previously described in vitro(20) and in response to
protein kinase C in vitro(19) . However, the overall
effect on enzyme activity was not clear. Additional serine/threonine
kinases, such as MAP kinase, may phosphorylate PTP1C in vitro,
as has recently been demonstrated for PTP1D in epidermal growth
factor-stimulated PC12 cells (32). This threonine phosphorylation of
PTP1D was found to correlate with a pronounced inhibition of PTP1D
activity (32). Our in vitro experiments show that PTP1C and
PTP1D can be phosphorylated by MAP kinase with a concomitant loss of
activity, suggesting that the failure to detect any change in PTP1C
activity in response to growth factors could be the result of a balance
between a stimulation by tyrosine phosphorylation and an inhibition by
threonine phosphorylation promoted by activation of MAP kinase
(40) . Indeed, serum,
Transient
expression of PTP1C in 293 cells leads to partial or complete
dephosphorylation of epidermal growth factor, PDGF, and insulin-like
growth factor-1 receptors
(12) . Hence, we were surprised to
note that the expression of PTP1C in CCL39 cells did not modify the
cell's ability to reinitiate DNA synthesis in response to serum,
CCL39 cells express endogenous PTP1D; hence, to follow a transfected
PTP1D molecule, we added an epitope tag. The transfected tagged
molecule (PTP1D-VSVG) behaved in an identical manner to endogenous
PTP1D in resting and stimulated cells. In PTP1D-3 cells, stable clones
expressing high levels of PTP1D-VSVG, all agonists tested except
insulin and TPA inhibited PTP1D activity. This inhibitory effect could
be explained by MAP kinase-mediated threonine phosphorylation since we
observed that PTP1D, in common with PTP1C, is phosphorylated and
inhibited by MAP kinase in vitro. Previous studies showed
either no change in PTP1D activity
(10, 11, 15) or a weak activation upon growth factor stimulation
(12) . These discrepancies could be explained as follows: (i)
Feng et al.(11) were studying Syp, an alternatively
spliced mouse homologue of PTP1D, which lacks the putative
phosphorylation sites for MAP kinases; ii) Vogel et al.(12) overexpressed both growth factor receptor tyrosine kinase
and PTP1D, enhancing the extent of tyrosine phosphorylation and hence
catalytic activity of the phosphatase.
Our results demonstrate for
the first time that the level of PTP1D phosphorylation is notably
increased following PDGF and
To investigate the role of PTP1D in growth factor
signaling, we performed a series of experiments using a catalytically
inactive mutant, PTP1D/C459S. We hypothesized that this inactive PTP1D
could act as a competitive inhibitor of specific PTP1D-mediated
tyrosine dephosphorylation of target proteins in vitro. In
transiently transfected CCL39 cells, expression of PTP1D/CS but not the
wild type phosphatase reduced PDGF and
As a more direct examination of the role of PTP1D in
mitogenic signaling, we examined growth factor-stimulated DNA synthesis
in cells transiently transfected with PTP1D/CS. Expression of the
inactive phosphatase completely inhibited the stimulatory effect of
PDGF and partially inhibited the effect of
The
present study does not demonstrate the mechanism by which PTP1D
functions as a positive growth regulator. Two reports recently noted
that activation of the PDGF receptor leads to tyrosine phosphorylation
of Syp (PTP1D) on residue(s), which then acts as a binding site for the
Grb2-Sos complex
(22, 23) . This could then provoke an
increase in GTP-bound p21. In addition to this important role, it is
likely that other substrates exist for PTP1D. An alternative
possibility is that PTP1D acts as a positive regulator by
dephosphorylating phosphotyrosine sites that negatively regulate the
signaling potential of other polypeptides, such as the Src-type
tyrosine kinases.
Finally, this report clarifies the involvement of
PTP1C and PTP1D in mitogenic signaling pathways induced by different
growth factors. We have demonstrated that these two PTPs may
participate in signal transduction but in different manners. We have
also demonstrated for the first time that PTP1D may act as a positive
mediator of mitogenic signals induced by both tyrosine kinase receptors
and G protein-coupled receptors. Our data suggest that PTP1D is able to
specifically interact with PDGF receptor-mediated signaling cascades.
Hence, current experiments in our laboratory involve the use of
chimeric PTP1C and PTP1D molecules in an attempt to precisely define
the regions of PTP1D required for its specificity of interaction.
We thank Drs. G. L'Allemain for kindly providing
the antiphosphotyrosine antiserum and A. Brunet for providing the MAP
kinase kinase constitutive active mutant. We also thank Prof. R.
Stanley for helpful discussions of this work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-thrombin. However, tyrosine phosphorylation of PTP1C was
increased in response to insulin, PDGF, and
-thrombin. PTP1D
activity was slightly stimulated by insulin and
12- O-tetradecanoylphorbol-13-acetate but was significantly
inhibited by serum, PDGF, and
-thrombin, although tyrosine
phosphorylation is increased in response to these agonists.
Mitogen-activated protein kinase phosphorylated PTP1C and PTP1D in
in vitro kinase assays, suggesting that both PTPs are target
proteins for mitogen-activated protein kinase. We also show that
overexpression of PTP1C or PTP1D had no effect on DNA synthesis
stimulated by different growth factors. However, a mutated inactive
form of PTP1D strongly inhibited the stimulatory effects of both PDGF
and
-thrombin on early gene transcription and DNA synthesis. These
results demonstrate for the first time that PTP1C and PTP1D may
participate in signal transduction but in different manners and that
only PTP1D is a positive mediator of mitogenic signals induced by both
tyrosine kinase receptors and G protein-coupled receptors in
fibroblasts.
- and pp60
. This
specific association then permits activation of their respective
signaling pathways
(3) .
(
)
appear to contain SH2 domains
(4) . Two of the SH2-containing PTPs that have been studied in
some detail are PTP1C (also termed SH-PTP1, HCP, and SHP), which is
expressed predominantly in hematopoietic cells
(5, 6, 7, 8) , and PTP1D (also termed
SH-PTP2, Syp, PTP2C, and SH-PTP3), a ubiquitously expressed protein,
which is the homologue of the Drosophilacsw gene
product, Csw
(9, 10, 11, 12) . Although
considerable progress has been made in determining PTP structure
(13, 14, 15) , little is known about the
participation of these PTPs in signal transduction.
-thrombin). This was
performed in a Chinese hamster lung fibroblast cell line (CCL39), whose
growth factor requirements are well defined (24). The potential
physiological importance of such involvement is emphasized by the
recent finding that PTP1C is expressed in many non-hematopoietic cells
and notably in malignant epithelial cell lines
(7, 20) ,
suggesting that this enzyme, as for PTP1D, could play a prominant role
in growth factor-mediated signal transduction within non-hematopoietic
cells
(18, 19) .
-thrombin on early gene transcription and DNA synthesis.
In light of these findings, the importance of PTP1C and PTP1D to
mitogenic signaling is discussed.
-thrombin was a generous gift of Dr. J.
Fenton II (New York State Department of Health, Albany, NY). Human
recombinant PDGF was from Boehringer Mannheim.
[ methyl-
H]thymidine and the enhanced
chemiluminescence (ECL) immunodetection system were obtained from
Amersham Corp. [
-
P]ATP was from ICN. The
P4D5 monoclonal antibody directed against an epitope of the vesicular
stomatitus virus glycoprotein (VSVG) (25) was kindly provided by Dr B.
Goud (Institut Pasteur, Paris, France). The monoclonal 12CA5 antibody,
raised against a peptide from influenza hemagglutinin HA1 protein, was
purchased from Babco (Emeryville, CA). The monoclonal antibodies
directed against PTP1C and PTP1D were from UBI (Lake Placid, NY) and
Affiniti (Nottingham, United Kingdom), respectively.
Antiphosphotyrosine antibodies were a gift of Dr. G. L'Allemain
(Centre de Biochimie, UMR 134, Nice). CCL39 is an established line of
Chinese hamster lung fibroblasts (ATCC). All other materials were
obtained from Sigma unless otherwise stated. Methods
Cell Culture
CCL39 and its derivative,
PS200, which lacks Na/H
antiporter
activity (26), were cultured in Dulbecco's modified Eagle's
medium (DMEM) (Life Technologies, Inc.) containing 7.5% fetal bovine
serum, penicillin (50 units/ml), and streptomycin (50 µg/ml).
Growth-arrested cells were obtained by total serum deprivation for 24
h.
Construction of Expression Vectors and in Vitro
Mutagenesis of PTP cDNAs
The full-length human PTP1D cDNA
(B. Neel, Molecular Medicine Unit, Beth Israel Hospital, Boston) was
subcloned into the expression vector pcDNAneo (Invitrogen) in frame
with the cDNA encoding for VSVG epitope. Mutation of the critical
cysteine of the catalytic site of the molecule to serine (C459S) was
performed in the pBluescript SK vector by site-directed mutagenesis of
double-stranded DNA according to the Clontech strategy (27) and
thereafter subcloned into the expression vector pcDNAneo. This inactive
PTP1D construct will thereafter be referred to as PTP1D/CS. The
full-length human PTP1C cDNA (M. Thomas, Howard Hughes Medical
Institute, St. Louis, MO) was also subcloned into pcDNAneo.
Stable Expression of PTP1C and PTP1D
For
stable expression, a H-killing selection
technique previously described
(26) was employed. PS200 cells
(10
per 10-cm plate) were cotransfected by the calcium
phosphate precipitation technique with 2 µg of pEAP expression
vector (Na
/H
antiporter cDNA) (26)
and 18 µg of pcDNAneo or PTP cDNA constructs. 48 h after
transfection, cells were subjected to an acid-load selection that
killed non-transfected cells (usually >90% of the cell population).
Cultures were subsequently changed to complete growth medium and were
allowed to proliferate for 2-3 days before repeating two cycles
of acid-load selection at 2-3-day intervals. Acid-load-resistant
populations or independent clones were then isolated and screened by
Western blot for expression of transfected PTP1C or PTP1D.
Western Blot Analysis
Transfected cells
were washed twice with cold phosphate-buffered saline and lysed in
Triton X-100 lysis buffer (1% (w/v) Triton X-100, 50 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 5
mM EDTA, 500 µM sodium orthovanadate, 30
mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml leupeptin, 1 µM pepstatin, 5
µg/ml aprotinin) for 15 min at 4 °C. Insoluble material was
removed by centrifugation at 12 000 g for 5 min at 4
°C. Proteins (50 µg) from cell lysates were separated in SDS,
7.5% polyacrylamide gels and electrophoretically transferred to
Hybond-C membranes (Amersham) in 25 mM Tris, 192 mM
glycine. Membranes were blocked in Tris-buffered saline (20 mM
Tris-HCl, pH 7.5, 137 mM NaCl) containing 5% nonfat dry milk
for anti-PTP1C, anti-VSVG, and anti-PTP1D antibodies or 10% bovine
serum albumin for antiphosphotyrosine antibodies. The blots were then
incubated with polyclonal anti-PTP1C (UBI), monoclonal anti-PTP1D
(Transduction Laboratories, Affinity), anti-VSVG, or
antiphosphotyrosine in blocking solution for 2-4 h at 25 °C
and then incubated with horseradish peroxidase-conjugated goat
anti-rabbit (1:1000) or anti-mouse (1:500) IgG in blocking solution for
1 h. The blots were visualized by the Amersham ECL system.
Immune Complex Phosphatase Assay
Cell
lysates were prepared as described above and incubated with 3 µg of
anti-PTP1C (UBI) or anti-VSVG (PTP1D) antibodies at 4 °C. After 3
h, protein A-Sepharose was added and allowed to form a complex for 1 h
at 4 °C. Immune complexes were washed 3 times with Triton X-100
lysis buffer. An aliquot of these immunoprecipitates was retained and
added to 2 Laemmli buffer for Western blotting. Immune
complexes were then washed 3 times with phosphatase buffer (50
mM Hepes, pH 7.0, 60 mM NaCl, 60 mM KCl, 0.1
mM phenylmethylsulfonyl fluoride, 1 µM pepstatin,
5 µg/ml aprotinin, 1 µg/ml leupeptin). Phosphatase activity was
assayed by resuspending the final pellet in a total volume of 80 µl
of phosphatase buffer (pH 5.5) containing 1 mg/ml bovine serum albumin,
5 mM EDTA, 10 mM dithiothreitol. The reaction was
initiated by the addition of para-nitrophenyl phosphate (pNPP) (10
mM, final concentration) for 30 min at 30 °C. The reaction
was stopped by the addition of 0.9 ml of 1 N NaOH, and the
absorbance of the samples was measured at 410 nm. Measurement of DNA Synthesis Reinitiation
([
H]Thymidine
Incorporation)-Quiescent cells in 24-well plates were
stimulated in a serum-free DMEM/Ham's F-12 medium (1:1)
containing [
H]thymidine (3 µM, 0.5
µCi/ml) with the hormones and growth factors indicated. After 24 h
of incubation, the cells were fixed and washed three times with
ice-cold trichloroacetic acid (5%). Cells were then recovered with 0.1
N NaOH, and the radioactivity incorporated was counted.
In Vitro Phosphorylation of PTP1C and PTP1D by
Activated MAP Kinase
Quiescent CCL39 cells overexpressing
HA-epitope-tagged MAP kinase (28) were stimulated in Hepes-buffered
DMEM with 20% serum for 10 min at 37 °C. Cells were washed with
ice-cold phosphate-buffered saline and lysed in Triton X-100 lysis
buffer supplemented with 40 mM -glycerophosphate and 10
mM pNPP for 15 min at 4 °C. Insoluble material was removed
by centrifugation at 12,000
g for 5 min at 4 °C.
Proteins from cell lysates were incubated with 12CA5 antibody
preabsorbed to protein A-Sepharose-coated beads for 2 h at 4 °C.
Immune complexes were washed three times with Triton X-100 buffer and
mixed with the beads coming from PTP1C and PTP1D-VSVG immune complexes.
After mixing, the beads were washed again with kinase buffer (20
mM Hepes, pH 7.4, 20 mM MgCl
, 1
mM MnCl
, 1 mM dithiothreitol, 10
mM pNPP). MAP kinase activity was finally assayed by
resuspending the final pellet in 50 µl of kinase buffer containing
5 µCi [
-
P]ATP (5000 cpm/pmol) and 50
µM ATP per sample. The reaction mixture was incubated for
30 min at 30 °C. A positive control was also performed by inclusion
of myelin basic protein at a concentration of 0.25 µg/ml with
HA-MAP kinase immune complexes. The reactions were stopped by Laemmli
sample buffer and then heated at 95 °C for 5 min. Proteins were
separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide),
and the gels were subjected to autoradiography. In some experiments,
the effect of MAP kinase-mediated phosphorylation of both PTP1C and
PTP1D on enzyme activity was assessed after in vitro phosphorylation of each phosphatase as described above but in the
presence of only unlabeled ATP, followed by an immune complex
phosphatase assay.
Transient Transfection of PTP1D (WT), PTP1D/CS,
PTP1C, and Luciferase Assays
CCL39 cells were seeded at a
density of 300,000 cells per well in a 12-well plate and cotransfected
by the calcium phosphate technique with 0.33 µg of
c- fos-luciferase reporter vector (Dr. P. Czernilofsky, Bender
& Co., Vienna, Austria) and different amounts of the relevant
expression vector pcDNAneo containing epitope-tagged PTP1D-VSVG (WT or
CS) or PTP1C. 1 day following transfection, cells were rendered
quiescent by media aspiration, followed by two rinses in
phosphate-buffered saline and 24 h of serum starvation. Luciferase
activity was measured according to the Promega protocol.
Transient Transfection of PTP1D (WT) and PTP1D/CS and
DNA Synthesis Reinitiation
PS200 cells were seeded at a
density of 300,000 cells per well in a 12-well plate and cotransfected
by the calcium phosphate technique with 4 µg of pEAP and 5 µg
of PTP1D (WT or CS). 48 h after transfection, cells were subjected to
an acid-load selection that killed non-transfected cells, usually
>90% of the cell population. Cultures were subsequently changed to
complete growth medium for 12 h and thereafter deprived of growth
factors for 24 h in a 1:1 mixture of DMEM and Ham's/F-12 medium.
Cells were then stimulated with different concentrations of serum,
-thrombin, or PDGF in fresh DMEM/F-12 medium containing 0.25
µCi/ml and 3 µM
[ methyl-
H]thymidine (Amersham). After 24
h of stimulation, radioactivity incorporated in acid-precipitable
material was measured by liquid scintillation spectrometry.
Data Presentation
Assays were performed
in either duplicate or triplicate. The data presented are from
representative experiments performed at least twice.
Stable Expression of PTP1C and PTP1D in PS200
Cells
The biological consequences of PTP1C and PTP1D
overexpression were examined in cultured fibroblasts after transfection
of their cDNA cloned in the mammalian expression vector pcDNAneo. The
method of selection based on co-expression of the
Na/H
antiporter gene was employed to
isolate transfected cells that express each phosphatase. After three
consecutive acid-load recovery tests, cells stably expressing the
Na
/H
antiporter were analyzed for
PTP1C or PTP1D-VSVG expression. Thereafter, phenotypic stability of
transfected clones was maintained by applying the acid-load selection
weekly. For PTP1C, one stable clone (PTP1C-10) was chosen to perform
all the experiments. This clone expressed a high level of PTP1C, as
determined by Western blot (Fig. 1); however, we were unable to
detect endogenous expression of PTP1C in PS200 cells. For PTP1D, one
stable clone (PTP1D-3) was chosen to perform all the experiments. This
clone expressed a high level of PTP1D-VSVG, as determined by Western
blot (Fig. 1); furthermore, as shown in Fig. 1, we were
able to detect a low level of the endogenous form of PTP1D.
Immunofluorescence studies confirmed that these two phosphatases were
cytoplasmic enzymes (data not shown).
Figure 1:
Expression of PTP1C and PTP1D-VSVG in
PS200 cells. Cells were stably transfected with pcDNAneo expression
vector alone or containing PTP1C or PTP1D-VSVG. Different clones were
obtained in each case. Cell lysates (50 µg) of three independent
clones were analyzed by Western blotting and probed with polyclonal
anti-PTP1C antibody (UBI) ( lanes1 and 2)
and monoclonal anti-PTP1D antibody ( lanes3 and
4). The positions of PTP1C, PTP1D (endogenous), and PTP1D-VSVG
are indicated.
Effects of Different Agonists on Tyrosine
Phosphorylation and Catalytic Activity of PTP1C and
PTP1D
In an initial series of experiments, the effects of
different agonists on PTP1C and PTP1D activities were evaluated. As
shown in Fig. 2 A, insulin (1 µg/ml) and the phorbol
ester, TPA (0.1 µM), caused a transient stimulation of
PTP1C activity, which was evident as early as 1 min after addition of
agonist and was maintained for at least 30 min. However, serum (10%),
-thrombin (1 unit/ml), and PDGF (30 ng/ml) had no significant
effect. In resting cells, in the absence of agonist, the presence of
phosphotyrosine on PTP1C was barely detectable
(Fig. 3 A). In addition, in a range of experiments, no
phosphotyrosine was detected (Fig. 3 C). Hence, in
resting cells, we cannot routinely detect the presence of
phosphotyrosine on PTP1C. However, tyrosine phosphorylation of PTP1C
was readily observed in cells stimulated with PDGF,
-thrombin
(Fig. 3 A), and insulin (Fig. 3 C). In
response to insulin, two tyrosine-phosphorylated proteins
co-immunoprecipitated with PTP1C; although no experiments have been
performed to confirm this assumption, one of these proteins may
correspond to the insulin receptor
subunit (95 kDa) and the other
to IRS1 (180 kDa). In contrast to PTP1C, PTP1D activity was
significantly inhibited (30-65%) by serum,
-thrombin, and
PDGF after 1, 5, and 30 min of stimulation (Fig. 2 B).
Addition of insulin and TPA to the cells had only a slight stimulatory
effect on PTP1D. Tyrosine phosphorylation of PTP1D was also readily
observed in cells stimulated with PDGF and
-thrombin
(Fig. 4) but was not detectable in response to insulin (data not
shown). The presence of phosphotyrosine residues on PTP1D correlated
with the inhibition of activity of the protein. An amino acid sequence
analysis revealed that PTP1C and PTP1D contain putative phosphorylation
sites for MAP kinase
(7, 9) . An in vitro kinase assay revealed that MAP kinase was able to phosphorylate
both immunopurified PTP1C and PTP1D to a significant level and that
phosphorylation of both PTP1C and PTP1D by MAP kinase in vitro led to an inhibition of each phosphatase's activity by 22
and 25%, respectively (data not shown). These results suggest that both
PTP1C and PTP1D are potential substrates of MAP kinase in
vitro.
Figure 2:
PTP1C ( A) and PTP1D ( B)
activities in response to different agonists. Serum-starved PTP1C-10
( A) and PTP1D-3 cells ( B) were stimulated with 10%
serum, 30 ng/ml PDGF, 1 unit/ml -thrombin, 1 µg/ml insulin,
and 0.1 µM TPA for 0, 1, 5, and 30 min at 37 °C.
PTP1C-10 cell lysates were immunoprecipitated with 4 µg/ml of
polyclonal anti-PTP1C antibody (UBI), whereas PTP1D-3 cell lysates were
immunoprecipitated with 2 µl/ml monoclonal anti-VSVG antibody.
Immunoprecipitates were assayed for PTP activity as described under
``Experimental Procedures.'' Results are the means ±
S.E. of at least three experiments. *, significantly different from
control at p < 0.05 (Student's t test).
Figure 3:
Tyrosine phosphorylation of PTP1C by PDGF,
-thrombin, and insulin in PTP1C-10 cells. Serum-starved PTP1C-10
cells were stimulated with PDGF (30 ng/ml),
-thrombin (1 unit/ml),
and insulin (1 µg/ml) for 0, 1, 5, and 30 min at 37 °C. Cell
lysates were immunoprecipitated with 4 µg/ml polyclonal anti-PTP1C
(UBI) and immunoblotted with antiphosphotyrosine ( PY) antibody
( panelsA and C) or anti-PTP1C antibody
(UBI) ( panelsB and D). PanelB, experiment with PDGF gave an identical result in terms
of PTP1C protein levels immunoprecipitated.
Figure 4:
Tyrosine phosphorylation of PTP1D-VSVG by
PDGF and -thrombin in PTP1D-3 cells. Serum-starved PTP1D-3 cells
were stimulated with 30 ng/ml PDGF or 1 unit/ml
-thrombin for 0,
1, 5, and 30 min at 37 °C. Cell lysates were immunoprecipitated
with 2 µl/ml monoclonal anti-VSVG and then immunoblotted with
antiphosphotyrosine antibody ( PY) ( panelA)
or anti-VSVG ( panelB). PanelB,
experiment with PDGF gave an identical result in terms of PTP1D protein
levels immunoprecipitated.
Effect of PTP1C and PTP1D Overexpression on DNA
Synthesis Reinitiation
CCL39 and its derivative, PS200, are
highly dependent upon growth factor addition for reinitiation of DNA
synthesis (24). We measured the dose response of fetal calf serum
(FCS), -thrombin, and PDGF on induction of DNA synthesis in PS200
overexpressing PTP1C (PTP1C-10 cells) or PTPD-VSVG (PTP1D-3 cells).
Fig. 5
shows that the serum, PDGF, and
-thrombin dose
responses from G
-arrested cells, transfected with either
the empty vector or expressing a high level of PTP1C or PTP1D, are
identical. These results demonstrate that overexpression of PTP1C or
PTP1D had no detectable effect on the ability of a range of mitogenic
agents to stimulate the reinitiation of DNA synthesis in CCL39 or PS200
cells.
Figure 5:
Reinitiation of DNA synthesis in PTP1C-10
and PTP1D-3 cells. Confluent PS200 stably expressing the vector alone
(), PTP1C (
), or PTP1D-VSVG (
) were arrested for 24 h
in serum-free DMEM/F-12 medium. Reinitiation of DNA synthesis in
response to increasing concentrations of FCS, PDGF, or
-thrombin
was measured as described under ``Experimental Procedures.''
Each point represents a duplicate value. Errorbars are not shown to improve clarity, but errors were
less than 5% of the mean. Results are expressed as the percent of
maximal [
H]thymidine incorporation obtained with
10% FCS.
Effect of PTP1D, PTP1C, and PTP1D/CS on c-fos
Promoter-dependent Luciferase Activity
To further clarify
the role of PTP1D in growth factor-stimulated mitogenic signal
transduction, we generated a catalytically inactive PTP1D by mutating
the catalytic cysteine 459 to serine. Mutation of the analogous
cysteine in PTP1B results in a catalytically inactive enzyme, which
still binds but does not hydrolyze tyrosine phosphate (29). This
mutation completely abolished the phosphatase activity of PTP1D as
determined by incubation of the immunoprecipitated mutant with pNPP
(results not shown). This construct (pcDNAneo-PTP1D/CS) was then
transiently transfected in CCL39 cells, and its expression was verified
by Western blotting (data not shown).
-thrombin, and PDGF (Fig. 6 A). This response is not
affected by the overexpression of PTP1D/WT. In contrast, expression of
PTP1D/CS markedly blocked the stimulatory effect of PDGF and to a
lesser extent that of
-thrombin and serum. Interestingly,
PTP1D/CS-mediated inhibition of SRE-regulated gene expression
stimulated by PDGF could be overcome by cotransfection of the wild type
PTP1D construct but not by the PTP1C construct (data not shown).
Figure 6:
Effect of PTP1C, PTP1D, and PTP1D/CS on
c- fos promoter-dependent luciferase activity. CCL39 cells were
transfected with 0.33 µg of reporter vector pADneo
fos-luciferase and 1 µg of pcDNAneo expression vector
alone or containing PTP1D (WT or PTP1D/CS) ( panelA)
or 1 µg of pcDNAneo expression vector alone or containing PTP1D/CS
with or without 1 µg of S218D/S222D, (SS/DD) a constitutively
active mutant of MAP kinase kinase ( panelB). 24 h
after transfection, cells were arrested in DMEM for 24 h and then
stimulated with 10% FCS, 30 ng/ml PDGF, or 1 unit/ml -thrombin
( panelA) or with 30 ng/ml PDGF ( panelB). After 24 h, cells were lysed, and luciferase activity
was determined according to the Promega protocol. Each point represents the mean of duplicate values of at least three
independent experiments. Results are the means ± S.E. of at
least three experiments. *, significantly different from control at
p < 0.05 (Student's t test). PanelB, errorbars are not shown to improve
clarity, but errors were less than 5% of the
mean.
To
determine where in the signaling pathway leading from both the PDGF or
-thrombin receptor to SRE stimulation the PTP1D/CS construct was
exerting its inhibitory effect, we evaluated the effect of PTP1D/CS on
the stimulatory effect of a constitutive active mutant of MAP kinase
kinase (S218D/S222D). This mutant has been demonstrated to induce
growth factor relaxation and oncogenicity when expressed in CCL39 cells
(31). Cotransfection of the reporter gene with this constitutively
active mutant caused a significant increase in basal luciferase
activity (Fig. 6 B); this high level of luciferase
activity could still be increased by stimulation with PDGF. However,
cotransfection of PTP1D/CS had no effect on the high basal luciferase
activity induced by the active MAP kinase kinase mutant alone but
totally abolished the stimulatory effect of PDGF
(Fig. 6 B). Hence, PTP1D/CS would seem to inhibit the
signaling pathways leading to SRE stimulation at a level in the cascade
upstream of MAP kinase kinase.
Effect of PTP1D and PTP1D/CS on DNA Synthesis
Reinitiation
As cotransfection of PTP1D/CS was able to
inhibit the PDGF-mediated stimulation of SRE-regulated gene expression
(Fig. 6), we hypothesized that expression of PTP1D/CS would
antagonize the mitogenic potential of PDGF. This was determined in
CCL39 cells transiently transfected with PTP1D/CS, followed by a
determination of PDGF-mediated reinitiation of DNA synthesis. These
experiments demonstrate that PTP1D/CS exerted a strong inhibitory
effect on PDGF-stimulated DNA synthesis in transiently transfected
cells (Fig. 7 A). However, the mitogenic signal of serum
and -thrombin was only partially inhibited (Fig. 7, B and C).
Figure 7:
Effect
of PTP1D and PTP1D/CS on DNA synthesis reinitiation. Cells were
transfected with 4 µg of pEAP expression vector
(Na/H
antiporter cDNA) and 5 µg
of pcDNAneo expression vector alone or containing PTP1D (WT or
PTP1D/CS). 48 h after transfection, cells were subjected to an
acid-load selection that killed non-transfected cells. Cultures were
subsequently changed to complete growth medium for 12 h and were
thereafter deprived of growth factors for 24 h in serum-free DMEM/F-12
medium. Cells were then stimulated with increasing concentrations of
FCS, PDGF, and
-thrombin. DNA reinitiation was then measured as
described under ``Experimental Procedures.'' Results are the
means ± S.E. of at least three experiments. *, significantly
different from control at p < 0.05 (Student's t test).
-thrombin). For this goal, we have produced CCL39 clones that
express high levels of PTP1C (clone PTP1C-10) or overexpress an
epitope-tagged PTP1D (clone PTP1D-3) and determined the activity of
each ectopically expressed enzyme in response to mitogenic and
non-mitogenic stimuli.
-thrombin or PDGF failed to further increase the
basal activity, although the level of tyrosine phosphorylation was
increased by both agonists. However, insulin and TPA were both able to
enhance PTP1C activity. Two studies have previously demonstrated that
PTP1C can be phosphorylated on at least one tyrosine residue (Tyr-538)
in response to growth factor stimulation
(19, 20) .
Moreover, purified PTP1C was phosphorylated in vitro at the
same residue by protein tyrosine kinases. The biological significance
of tyrosine phosphorylation of PTP1C remains to be established but may
directly modify the activity of the enzyme. It has been reported that
the activity of PTP1D was activated by tyrosine phosphorylation
(12) . However, a similar modulation of enzyme activity has not
been observed for PTP1C, probably due to rapid autodephosphorylation
(6, 17, 19) . In the present study, we show that
insulin stimulates both the tyrosine phosphorylation and the activity
of PTP1C, whereas
-thrombin and PDGF increased tyrosine
phosphorylation without any detectable change in the activity even
under conditions that do not allow autodephosphorylation of the
protein, i.e. at 10-fold lower substrate concentrations or
upon incubation of the enzyme at 37 °C prior to substrate addition.
Hence, the relationship between the level of PTP1C tyrosine
phosphorylation and catalytic activity is not readily apparent.
-thrombin, and PDGF are strong
stimulators of MAP kinase activity in CCL39 cells, whereas TPA and
insulin are much less potent
(35, 36) .
-thrombin, or PDGF. As insulin was one of the few agonists able to
stimulate PTP1C activity in PTP1C-10 cells, we hypothesized that
insulin may be able to antagonize serum or PDGF-mediated
[
H]thymidine incorporation. However, such an
effect was not reliably reproducible (results not shown). We thus
conclude that in CCL39 cells, the ectopically expressed functional
PTP1C does not play a major role in the control of mitogenic signaling.
-thrombin stimulation. PTP1D has
previously been shown to be a substrate for the PDGF receptor
(11, 22, 23) . However, the mechanism by which
-thrombin promotes the tyrosine phosphorylation of this PTP is
unknown; a possible candidate would be a Src-type tyrosine kinase.
Indeed, we recently demonstrated that
-thrombin stimulated the
kinase activity of Src and Fyn in CCL39 cells (37) and that PTP1D is
constitutively tyrosine phosphorylated in v- src-transformed
cells
(11) . Thus,
-thrombin may promote tyrosine
phosphorylation of PTP1D in CCL39 cells by activating a Src-type
tyrosine kinase.
-thrombin-stimulated
transcription of a reporter gene linked to the c- fos promoter.
This inhibition could be reverted by transfection of PTP1D construct
but not by the PTP1C construct, suggesting that these two PTPs have
different target specificities. Previous studies have clearly
demonstrated that upon PDGF stimulation, PTP1D binds to the PDGF
receptor and becomes phosphorylated. The major site of PTP1D tyrosine
phosphorylation is present in a sequence conforming to the consensus
binding site for the SH2 domain of Grb2
(22) , which in
association with Sos 1, couples the PDGF receptor to Ras. Thus, PTP1D
could act as an adaptor between the PDGF receptor and the Grb2-Sos
complex and thus be situated upstream of MAP kinase in the signaling
cascade
(22, 23) . Our data are in agreement with this
model since PTP1D/CS was unable to block the high basal transcriptional
activity of the c- fos promoter induced by a constitutively
active MAP kinase kinase mutant but totally abolished the stimulatory
effect of PDGF.
-thrombin and serum.
These data confirm the results of two previous studies demonstrating
that expression of a catalytically inactive PTP1D phosphatase in 3T3-L1
cells (41) or microinjection of neutralizing PTP1D antibody in Rat-1
cells (42) dramatically decreased the mitogenic effect of insulin,
epidermal growth factor, and insulin-like growth factor-1.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.