From the Cancer Biology Program, Division of
Hematology Oncology, Department of Medicine, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, Massachusetts 02215 and § Jena University Hospital, Research Unit Molecular Cell
Biology, Drackendorfer Strasse 1 D-07747, Jena, Germany
Received for publication, October 4, 2002, and in revised form, November 5, 2002
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ABSTRACT |
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Receptor tyrosine kinases (RTKs) are key
regulators of cellular homeostasis. Based on in vitro and
ex vivo studies, protein tyrosine phosphatase-1B (PTP1B)
was implicated in the regulation of several RTKs, yet mice lacking
PTP1B show defects mainly in insulin and leptin receptor signaling. To
address this apparent paradox, we studied RTK signaling in primary and
immortalized fibroblasts from PTP1B Regulation of cellular proliferation, adhesion, and migration is
pivotal for maintaining homeostasis. Multiple peptide growth factors
direct these processes to differing extents in primary fibroblasts and
fibroblast cell lines. These growth factors signal via receptors with
intrinsic protein-tyrosine kinase activity, termed receptor tyrosine
kinases (RTKs).1 Ligand
binding activates the intrinsic kinase activity of these receptors,
resulting in the phosphorylation of multiple receptor tyrosyl residues.
These serve as docking sites to recruit signal relay molecules
containing src homology-2 and/or phosphotyrosine binding domains, most of which also are RTK substrates. The resultant complexes lead to activation of downstream signaling, including the
Ras-Erk and PI-3K/Akt pathways. Ultimately, downstream signaling pathways stimulate new transcription and changes in cellular behavior and/or state (reviewed in Ref. 1).
Because of their pleiotropic actions, RTKs must be regulated carefully.
Abnormally increased RTK activity can have dire consequences, including
developmental abnormalities (reviewed in Ref. 2), cancer (reviewed in
Ref. 3), or fibrosis (reviewed in Ref. 4). Classic PTPs comprise
a large family of receptor-like and non-receptor enzymes that share a
highly conserved catalytic (PTP) domain that is absolutely specific for
phosphotyrosine hydrolysis (reviewed in Ref. 5). Because tyrosyl
phosphorylation is reversible, PTPs probably play important roles in
the regulation of RTKs and/or their substrates (reviewed in Refs. 5,
6).
Based largely on experiments in which wild-type PTPs or their
catalytically impaired (dominant-negative) mutants were overexpressed, multiple PTPs, including LAR (7), DEP-1 (8), TC-PTP (9, 10), Shp-1 (11,
12), Shp-2 (13), and PTP1B (14-17) have been implicated in the
dephosphorylation of various RTKs. Which, if any, of these PTPs
regulate RTK signaling under physiologically relevant conditions of
expression has remained largely unclear. Also unknown is the extent to
which RTK dephosphorylation, as opposed to other down-regulatory
mechanisms such as RTK degradation and/or inhibitory seryl
phosphorylation, plays the (a) key role in receptor inactivation.
PTP1B is a widely expressed non-receptor PTP that is localized on
intracellular membranes via a hydrophobic C-terminal targeting sequence
(18, 19). A role for PTP1B in the regulation of many cellular functions has been suggested, including integrin (20-23), cadherin (24, 25), and cytokine receptor signaling (26-28), cell cycle
regulation (29-31), and the response to cellular stress (32). Multiple
studies indicated that PTP1B dephosphorylates the EGFR (16, 17) and
the insulin receptor (IR) (14, 33-35). Analysis of
PTP1B We used fibroblasts derived from wild-type (WT) and
PTP1B Materials--
Affinity-purified polyclonal antibodies to murine
PTP1B were described previously (37). Antibodies against murine
PDGFR Cell Culture--
Primary mouse embryonic fibroblasts (MEFs)
were generated from embryonic day 14 embryos from WT or
PTP1B Cell Stimulation, Immunoprecipitation, and
Immunoblotting--
MEFs were seeded at 2.5 × 105
cells per 60-mm tissue culture plate for 24 h, starved in
serum-free Dulbecco's modified Eagle's medium plus antibiotics for
48 h, and stimulated with EGF (50 ng/ml) or PDGF-BB (50 ng/ml). At
the indicated times after stimulation, cells were lysed in Nonidet P-40
buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM sodium orthovanadate, and protease inhibitor mixture (final concentrations, 20 µg/ml
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml
aprotinin, 1 µg/ml of pepstatin, and 1 µg/ml of antipain)). Lysates
were clarified by centrifugation at 13,000 rpm for 10 min and protein
concentrations were determined using a bicinchoninic acid protein assay
kit (Pierce Chemical). For immunoprecipitations, lysates were incubated
with the appropriate antibodies for 3 h overnight at 4 °C.
Immune complexes were collected onto protein A-Sepharose beads, washed
extensively, resolved by SDS-PAGE and transferred onto Immobilon-P
membranes (Millipore, Bedford, MA). Immunoblots were blocked with 5%
bovine serum albumin or Carnation non-fat dry milk in 10 mM
Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20. After
incubation with appropriate primary and secondary antibodies, blots
were visualized using enhanced chemiluminescence reagents (ECL;
Amersham Biosciences). Quantification was carried out using NIH Image
Pro Software Version 1.62; data are expressed as relative units of
phosphorylated protein per total protein for each band. Anti-PTP1B and
anti-PDGFR In-gel Phosphatase Assays--
Cells were starved overnight in
0.5% fetal calf serum, treated with 2 mM
H2O2 at 37 °C for 30 min, and stimulated
with 50 ng/ml PDGF at room temperature for 10 min or left untreated.
Anti-PDGFR immunoprecipitates were prepared from lysates of one
75-cm2 tissue culture flask. Control immunoprecipitations
were carried out using normal rabbit IgG (Santa Cruz). In-gel
phosphatase assays were performed as described by Burridge and Nelson
(41) with minor modifications. For substrate,
poly(Glu4Tyr1)n was radiolabeled with [ EGFR and PDGFR Are Hyperphosphorylated in
PTP1B Downstream Signaling Pathways Are Affected Minimally in
PTP1B
To begin to understand the molecular basis for these effects, we
focused on more immediate events after PDGFR activation. PDGF-stimulated tyrosyl phosphorylation of Shp2 (Fig.
3A) and Shc (Fig.
3B), two signal relay molecules implicated in Erk activation (reviewed in Refs. 42, 43), was enhanced significantly in PTP1B PTP1B Regulation of PDGFR Tyrosyl Phosphorylation Is
Cell-autonomous and Direct--
Conceivably, increased RTK activation
in PTP1B
These data indicate that PTP1B regulates PDGFR tyrosyl phosphorylation
in a cell-autonomous manner. To seek further evidence that PTP1B
directly regulates PDGFR tyrosyl phosphorylation, we asked whether the
PDGFR could form a stable complex with PTP1B-D/A, which is impaired
catalytically, but retains substrate-binding ability (16, 43, 45).
PTP1B immunoprecipitates prepared from starved or PDGFR-stimulated,
immortalized PTP1B
Although coimmunoprecipitation of the PDGFR and WT-PTP1B was not
observed by immunoblotting, PDGFR immunoprecipitates contained an
~50-kDa PTP activity detectable by a highly sensitive in-gel phosphatase assay (Fig. 4D). This activity is almost
certainly PTP1B, because it is absent in PDGFR immunoprecipitates
prepared from PTP1B
Although our data show clearly that PTP1B plays a role in regulating
EGFR and PGDFR phosphorylation, we have been unable to detect any
effect of PTP1B deficiency on the biological activities of these growth
factors, at least in MEFs. Consistent with the normal growth
factor-induced AKT stimulation in PTP1B
There are several possible reasons why lack of PTP1B (although it has
some biochemical effects on EGFR and PDGFR signaling) fails to reach
the "threshold" for important biological effects. In immortalized
fibroblasts, PTP1B is required for adhesion-evoked Erk activation (Ref.
26).2 In our experiments,
"growth factor"-evoked Erk activation was carried out on attached
cells; thus, the requirement of PTP1B for adhesion signaling may limit
the increase in Erk activation that results in loss of negative
regulation of RTK signaling by PTP1B. However, in
PTP1B
Although dephosphorylation has a role in EGFR and PDGFR inactivation,
additional mechanisms may be of equal or greater importance. Chief
among these is receptor degradation via lysosomal targeting, promoted
by the Cbl family of ubiquitin ligases (reviewed in Ref. 54). Classic
studies show that the half-lives of the EGFR and PDGFR decrease
dramatically after ligand stimulation. Although recent work indicates
that dephosphorylation precedes degradation under normal conditions
(46, 55), it is not clear whether the former is obligate for the
latter. If not, (or if alternative PTPs can substitute for this
function of PTP1B), the EGFR and/or PDGFR may be targeted for
degradation even if the receptor remains phosphorylated for an
abnormally long time after growth factor stimulation in
PTP1B
Why PTP1B deficiency has a larger effect on Erk than on Akt activation
also remains to be determined. PTP1B might prefer certain phosphorylation sites on the PDGFR and EGFR; thus, PTP1B deficiency could have greater consequences for downstream signaling pathways emanating from those sites. However, a previous study indicated that
the isolated catalytic domain of PTP1B had no preference for individual
sites on the PDGFR (49). Compensatory PTPs also could prefer certain
PDGFR/EGFR sites. Negative regulatory mechanisms for the Akt pathway
could be more robust than those for the Erk pathway, such that a mild
increase in upstream RTK activation has a bigger effect on the latter
than the former. A final, intriguing possibility is that Akt and Erk
activation are directed by RTKs present within different cellular
compartments. For example, Akt activation may involve activated RTKs at
the plasma membrane, whereas full activation of the Erk pathway occurs
inside the cell. This model is consistent with the observation that
blocking endocytosis using a dominant negative mutant of dynamin
impairs only some signals emanating from the EGFR, notably including
Erk activation (57, 58). Likewise, preventing internalization of NGF
has no effect on TrkA-stimulated Akt activation, whereas Erk activation is impaired (59). Notably, we have shown recently that PDGFR and EGFR
dephosphorylation by PTP1B requires RTK endocytosis and occurs on the
surface of the endoplasmic reticulum (46). Conceivably, PTP1B
deficiency preferentially affects the Erk pathway because PTP1B only
dephosphorylates endocytosed RTKs. Most of the above models are not
mutually exclusive, and a combination of explanations may account for
our findings.
Our results have important implications for understanding the
physiological function of PTP1B. PTP1B/
mice. After growth
factor treatment, cells lacking PTP1B exhibit increased and sustained
phosphorylation of the epidermal growth factor receptor (EGFR) and the
platelet-derived growth factor receptor (PDGFR). However, Erk
activation is enhanced only slightly, and there is no increase in Akt
activation in PTP1B-deficient cells. Our results show that PTP1B does
play a role in regulating EGFR and PDGFR phosphorylation but that other
signaling mechanisms can largely compensate for PTP1B deficiency.
In-gel phosphatase experiments suggest that other PTPs may help to
regulate the EGFR and PDGFR in PTP1B
/
fibroblasts. This
and other compensatory mechanisms prevent widespread, uncontrolled
activation of RTKs in the absence of PTP1B and probably explain the
relatively mild effects of PTP1B deletion in mice.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
Results and Discussion
REFERENCES
/
mice confirmed that the IR is a key
physiological target of PTP1B (36, 37). However, these mice lack any
obvious signs of increased activity of the EGFR or PDGFR, such as
increased tumor incidence or fibrosis.
/
mice to address the role of PTP1B in the
regulation of RTK signaling. We find that PTP1B
/
cells
exhibit enhanced and sustained tyrosyl phosphorylation of both RTKs.
Despite increased RTK phosphorylation, EGF- and PDGF-evoked Akt
activation is not enhanced, whereas Erk activation is only minimally
increased. Our results show that PTP1B is a bona fide
regulator of EGF and PDGF signaling in vivo, but other cellular regulatory mechanisms, including other PTPs, can largely compensate for loss of PTP1B regulation of these RTKs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
Results and Discussion
REFERENCES
were provided by Dr. D. DeMaio (Yale Medical School, New
Haven, CT). Monoclonal antibodies against phosphotyrosine (4G10) were from Upstate Biotechnology, Inc (Lake Placid, NY) and anti-PTP1B monoclonal antibodies (FG6) were from Calbiochem. Rabbit polyclonal antibodies against total Akt, phosphorylated (Ser-473) Akt, and phosphorylated (Thr-202/Tyr-204) Erk were from Cell Signaling (Beverly,
MA). Anti-Erk2, -Shp2, and -EGFR antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA).
/
(exon 1) mice (C57/Bl6J X SV129 background)
(37). Embryos were incubated in trypsin/EDTA (Invitrogen) for 30 min at 37 °C, and dissociated cells were collected by centrifugation
and cultured in Dulbecco's modified Eagle's medium supplemented with
15% fetal calf serum,100 units/ml penicillin, and 10 mg/ml
streptomycin at 37 °C in a 5% humidified CO2
atmosphere. Four independent WT and PTP1B
/
MEF strains
were used for experiments, with similar results. For immortalization
with simian virus 40 large T antigen, PTP1B
/
MEFs were
infected with pZipTex (38), and these cells were maintained as a pool
and used for subsequent experiments. WT human PTP1B (hPTP1B-WT) or the
substrate-trapping mutant PTP1B-D181A (hPTP1B-D/A) (16) cloned into the
retroviral vector pWZL (39, 40) were the generous gifts of Dr. N. Tonks
(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). To generate
viral stocks, these vectors were transiently transfected into the
Phoenix-Eco retroviral packaging line
(http://www.stanford.edu/group/nolan/index.html), and supernatants were
collected 48 h later. Immortalized PTP1B
/
cells
were infected in the presence of polybrene (1 µg/ml) and selected in
Dulbecco's modified Eagle's medium, 10% fetal calf serum plus
hygromycin (700 µg/ml). Pools of hygromycin-resistant cells were
maintained in Dulbecco's modified Eagle's medium plus 10% fetal calf
serum, 100 units/ml penicillin, 10 mg/ml streptomycin, and 200 µg/ml hygromycin.
antisera were used at 1/1000 for immunoblotting.
Anti-human PTP1B monoclonal antibodies (FG6) were used at 1 µg/ml for
immunoprecipitations and immunoblotting. All other antibodies were used
at concentrations as recommended by the supplier.
32P]ATP (PerkinElmer Life
Sciences) using recombinant human p60c-Src, expressed as a
glutathione S-transferase-fusion protein in
Escherichia coli. The substrate was cast in a 10%
SDS-polyacrylamide gel (acrylamide/bisacrylamide, 30:0.8). Instead of
Tween 40, Tween 20 was used throughout, and the final renaturation step
was carried out overnight in a buffer containing 50 mM
Tris-HCl, pH 7.4, 0.3% 2-mercaptoethanol, 1 mM EDTA, and
0.04% Tween 20.
Results and Discussion
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
Results and Discussion
REFERENCES
/
MEFs--
Previous reports indicated
that PTP1B dephosphorylates the EGFR, at least when one or both of
these proteins are overexpressed. To investigate whether the EGFR is a
PTP1B target under physiological conditions, primary MEFs were
generated from PTP1B (exon 1)
/
mice and WT control
mice. Starved MEFs were stimulated with EGF (50 ng/ml) for various
times or left unstimulated. Total cell lysates or EGFR
immunoprecipitates were resolved by SDS-PAGE and subjected to
anti-phosphotyrosine immunoblotting (Fig.
1A). As expected, in starved
cells, there was no detectable tyrosyl phosphorylation of the EGFR in
total cell lysates or EGFR immunoprecipitates from either WT or
PTP1B
/
MEFs. However, after stimulation, EGFR tyrosyl
phosphorylation was enhanced and sustained in PTP1B
/
MEFs compared with control MEFs (KO/WT ratio ± S.E., 1.73 ± 0.22, 1.46 ± 0.18, 1.83 ± 0.13, and 1.55 ± 0.21 at 1, 5, 15, and 30 min, respectively, as quantified by scanning
densitometry). Reprobing these blots with anti-EGFR antibodies revealed
that the difference in EGFR tyrosyl phosphorylation was not caused by
increased EGFR expression in PTP1B
/
MEFs, but instead
reflected increased specific tyrosyl phosphorylation. Together with the
earlier PTP1B overexpression/dominant-negative mutant studies, these
data show that PTP1B plays a role in dephosphorylating the EGFR, at
least in MEFs. Although to our knowledge PTP1B has not been shown to
regulate the PDGFR, PDGF-stimulated PDGFR tyrosyl phosphorylation also
was increased in the absence of PTP1B (Fig. 1B), (KO/WT
ratio ± S.E., 1.53 ± 0.22, 1.72 ± 0.12, 1.94 ± 0.35, and 2.1 ± 0.44 for 1, 5, 15, and 30 min, respectively),
suggesting that PTP1B also regulates the PDGFR. Anti-PTP1B immunoblots
confirmed the absence of PTP1B protein in the PTP1B
/
MEFs (Fig. 1C).
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Fig. 1.
Hyperphosphorylation of the EGFR and
PDGFR in PTP1B
/
MEFs. Primary MEFs were derived from wild-type
(WT) and PTP1B
/
(
/
) mice, as described.
MEFs were stimulated with either 50 ng/ml of EGF (A) or 50 ng/ml of PDGF-BB (B) for the indicated times. Total cell
lysates (TCL) and immunoprecipitates (IP) of
either EGFR or PDGFR
were subjected to SDS-PAGE in 8% gels and
immunoblotted for either anti-phosphotyrosine (pTyr) or
receptor levels, as indicated. C, immunoblots of PTP1B,
confirming its absence in the lysates of MEFs derived from PTP1B
/
mice.
/
MEFs--
Although EGFR and PDGFR
tyrosyl phosphorylation are increased in the absence of PTP1B, mice
lacking PTP1B exhibit no obvious hypermorphism of EGFR or PDGFR
pathways. To begin to understand this apparent paradox, we examined
signaling pathways downstream of these RTKs in WT and
PTP1B
/
MEFs. Activation of the Erk pathway, as assessed
by immunoblotting with phospho-specific anti-Erk antibodies, was
enhanced slightly in response to EGF stimulation of
PTP1B
/
, compared with WT MEFs (KO/WT ratio ± S.E., 1.02 ± 0.08, 1.31 ± 0.1, 1.36 ± 0.25, and
1.39 ± 0.04 at 1, 5, 15, and 30 min, respectively). Erk
activation was enhanced to an even greater extent in PDGF-stimulated PTP1B
/
MEFs compared with WT (Fig.
2A) (KO/WT ratio ± S.E.,
1.13 ± 0.15, 1.2 ± 0.07, 1.58 ± 0.14, and 1.76 ± 0.36 at 1, 5, 15, and 30 min, respectively). In contrast, growth
factor-stimulated Akt activation (as assayed by pAkt immunoblotting)
was not increased in PTP1B
/
MEFs (compared with WT
MEFs) in response to either growth factor, in fact there was a tendency
for decreased activation (Fig. 2B) (e.g. KO/WT
ratio ± S.E., 0.93 ± 0.21, 0.85 ± 0.04, 0.95 ± 0.11, and 0.99 ± 0.12 for 1, 5, 15, and 30 min of PDGF
stimulation, respectively; 0.73 ± 0.28 and 0.98 ± 0.15 for
1 and 5 min post-EGF stimulation, respectively). These results
suggested that MEFs compensate for the increased EGFR or PDGFR
activation caused by PTP1B deficiency somewhere between the
hyperphosphorylated RTK and Akt activation. The small enhancement of
Erk activation in PTP1B
/
cells suggests compensatory
mechanisms for the effects of increased RTK activation on this
signaling pathway as well.
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Fig. 2.
Minimal alteration of downstream signaling
after growth factor stimulation of PTP1B /
MEFs. MEFs were stimulated with either EGF or PDGF
for
the indicated times. Lysates were subjected to SDS-PAGE and
immunoblotted with either phospho-specific Erk (pErk),
followed by anti-Erk-2 (Erk-2) antibodies (A) or
phospho-specific AKT (pAkt) followed by anti-total Akt
(Akt) antibodies (B).
/
MEFs compared with WT. Association of Shp2 with
the PDGFR is probably increased as well (Fig.
4). Activation of the Akt pathway is
mediated, at least in part, by recruitment of phosphatidyl inositol-3
kinase (PI-3K) to the PDGFR (reviewed in Ref. 44). Consistent with the
lack of enhanced Akt activation in PDGF-stimulated PTP1B
/
MEFs, association of the p85 (targeting) subunit
of PI-3K with the PDGFR was not increased in the absence of PTP1B (Fig.
3C). In addition, PDGFR-associated PI-3K activity was not
altered significantly in PTP1B
/
, compared with WT cells
(data not shown).
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Fig. 3.
Phosphorylation of PDGFR targets in wild type
and PTP1B /
MEFs. Lysates of MEFs,
stimulated with PDFGF
for the indicated times, were
immunoprecipitated with anti-Shp2 (A), anti-Shc
(B), or anti-PDGFR
(C) antibodies, and immune
complexes were resolved by SDS-PAGE. Shp2 and Shc immunoprecipitates
were immunoblotted with anti-phosphotyrosine (pTyr), -Shp2,
and -Shc antibodies, as indicated. PDGFR immunoprecipitates were
immunoblotted for PDGFR and for the p85 subunit of PI-3K.
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Fig. 4.
Reconstitution of PTP1B in immortalized
murine PTP1B /
fibroblasts. Immortalized
PTP1B
/
cells were generated and then reconstituted with
hPTP1B-WT, hPTP1B-D/A, or the parental pWZL vector, as described under
"Experimental Procedures." A, lysates from
randomly growing cells were subjected to SDS-PAGE and then
immunoblotted for hPTP1B and Shp2. B, lysates of
PDGF-stimulated PTP1B
/
and hPTP1B-WT fibroblasts were
subjected to SDS-PAGE and then immunoblotted with either
anti-phosphotyrosine (Ptyr), anti-phospho-Erk
(pMAPK), anti-total Erk-2 (ERK 2),
anti-phospho-Akt (pAKT) and anti-total Akt
(AKT) antibodies, respectively. C, lysates
of unstimulated and PDGF-stimulated (15 min) PTP1B
/
,
hPTP1B-WT-, and hPTP1B-D/A-reconstituted cells were immunoprecipitated
with anti-hPTP1B antibodies and then subjected to anti-phosphotyrosine
(Ptyr) immunoblotting. Right panel,
multiple PTPs, including PTP1B, associate with the PDGFR in murine
fibroblasts. Lysates of immortalized PTP1B
/
fibroblasts, reconstituted with hPTP1B or the parental vector, were
subjected to immunoprecipitation with anti-PDGFR antibodies or normal
rabbit IgG as control, as indicated. The immunoprecipitates were
separated by SDS-PAGE in 10% gels containing
32P-tyrosine-phosphorylated
poly(Glu4Tyr1), and PTPs were visualized as
activity bands (white) after renaturation. Identification of
PTPs is based on immunodepletion experiments and comparison of wild
type and PTP1B
/
cells. Note that several fragments of
PTP1B are detectable. A component of about 40 kDa is present in the
PTP1B
/
cells and is therefore not a PTP1B fragment.
Different parts of the same gel have been exposed for different lengths
of time to optimize PTP detection. Lower panels show
controls for PDGFR immunoprecipitation (PDGFR-IP) and PDGFR
tyrosine phosphorylation (pTyr).
/
MEFs could reflect an indirect effect of
PTP1B on fibroblast differentiation, rather than an effect of PTP1B on
RTK signaling per se. To exclude this possibility, we
restored PTP1B expression to PTP1B
/
cells (Fig.
4A). A large T Ag-immortalized PTP1B
/
fibroblast cell line was generated and reconstituted with human PTP1B
(hPTP1B-WT), the PTP1B substrate-trapping mutant D181A (hPTP1B-D/A), or
the parental pWZL expression vector (see "Experimental
Procedures"). Immunostaining revealed that >90% of hPTP1B-WT- and
hPTP1B-D/A-infected, hygromycin-resistant cells expressed hPTP1B
protein (data not shown); thus, these pools can be studied directly,
avoiding potential clone-to-clone variation. Notably, PDGF-stimulated
(but not basal) PDGFR tyrosyl phosphorylation was greater in the vector
control-infected PTP1B
/
cell pool, compared with the
cells reconstituted with hPTP1B-WT (Fig. 4B). Restoration of
hPTP1B-WT expression in immortalized PTP1B
/
fibroblasts
diminished PDGF-evoked Erk activation but left PDGF-evoked Akt
activation unaffected (Fig. 4B). The effects of restoring hPTP1B expression on PDGFR tyrosyl phosphorylation were observed only
at submaximal doses of PDGF. The reason for this is not clear, but may
be caused, at least in part, by the increased level of PDGFR expression
that accompanied immortalization (data not shown). The resultant
increase in the concentration of activated PDGFR may allow access to a
secondary PDGFR phosphatase with a higher Km
value (see below).
/
cells reconstituted with hPTP1B-WT,
hPTP1B-D/A, or the vector controls were immunoblotted with
anti-phosphotyrosine antibodies. An ~180-kDa band corresponding in
size to the PDGFR coimmunoprecipitated with hPTP1B-D/A but not
hPTP1B-WT (Fig. 4C); this interaction was detected only upon
PDGF stimulation. Unfortunately, available anti-PDGFR antibodies were
insufficiently sensitive to unambiguously identify this band as the
PDGFR. However, this is highly likely; using a highly sensitive
fluorescence resonance energy transfer approach, we recently
demonstrated complex formation between the PDGFR and PTP1B in
hPTP1B-D/A-expressing cells (46). Previous studies reported
associations between the C215S and/or D181A substrate trapping mutants
of PTP1B and the PDGFR (17) as well as the EGFR (16) and IR (15),
respectively. Unlike the inducible interaction we observed, in these
earlier studies, constitutive complex formation was observed. This most
probably reflects the high level of overexpression of PTP1B trapping
mutant and/or the RTK used in those studies.
/
cells. Interestingly, PTP1B/PDGFR
association was detected only when cells were pretreated with hydrogen
peroxide (data not shown). Much recent work suggests that specific
PTPs, including PTP1B, undergo physiological oxidation (and thus
transient inactivation) by peroxide (reviewed in Ref. 47). Conceivably,
PDGFR/PTP1B association requires a conformational change in PTP1B that
is induced upon oxidation. Alternatively, transient PDGFR/PTP1B
interactions may be "trapped" by the peroxide-induced generation of
one or more intermolecular disulfide bonds. A
peroxide-dependent association between the EGFR and PTP1B
also was detected using similar assays (data not shown).
/
MEFs, we
observed no increase in PDGF-induced ruffling in PTP1B
/
MEFs compared with WT, whereas EGF did not evoke significant ruffling
in either WT or PTP1B
/
MEFs. Likewise, there was no
significant difference in the ability of PTP1B
/
and WT
MEFs to fill an artificial "wound" created by scraping off a strip
of cells from a MEF monolayer (data not shown). We were unable to study
direct effects of PTP1B deficiency on EGF- or PDGF-stimulated
mitogenesis, because neither of these growth factors acts as a sole
mitogen in MEFs.
/
MEFs, integrin-evoked Erk activation is
normal,2 so additional compensatory mechanisms must limit
the increase in RTK-evoked Erk activation in primary cells. Other PTPs
may be able to substitute for PTP1B, thereby limiting the effect of PTP1B deficiency on EGFR and PDGFR signaling. Consistent with this
notion, using the in-gel phosphatase assay, we detected multiple PTPs,
including PTP1B, in PDGFR immunoprecipitates (Fig. 4D). One
of these activities, corresponding to an ~70-kDa species, was
increased in PDGFR immunoprecipitates from PDGF-stimulated PTP1B
/
compared with WT cells. We have identified, by
means of immunodepletion, this increased PTP activity as Shp2 (data not
shown). Although some evidence suggests that Shp2 has a positive
(i.e. signal-enhancing) function in PDGFR signaling
(48-51), other work indicates a negative regulatory role (52, 53).
Alternatively, the increase in Shp2 tyrosine phosphorylation might help
explain the increased Erk activation in PTP1B
/
cells
(Fig. 2). Activities of ~110 kDa, which represents PTP-PEST, and
possibly 48 kDa, which represents
TC-PTP,3 associate with the
PDGFR under our conditions. Because receptor-like PTPs tend to be
detected poorly by in-gel assays (41), this approach may underestimate
the number of PTPs that play a role in PDGFR tyrosyl dephosphorylation.
Taken together, however, our results support the idea that one reason
for the mild effect of PTP1B deficiency on PDGFR (and, probably, EGFR)
signaling is that multiple PTPs can compensate for the lack of PTP1B.
These other PTPs are likely to be less efficient (at dephosphorylating
the PDGFR and EGFR) than PTP1B; thus, PDGFR (and EGFR) tyrosyl
phosphorylation are somewhat increased in PTP1B
/
MEFs.
/
cells. Comparing EGFR and PDGFR turnover rates
in WT and PTP1B
/
MEFs should help address these issues.
We also cannot exclude the possibility that other RTK-inactivating
mechanisms, such as inhibitory seryl phosphorylation events (56), help
compensate for the absence of PTP1B.
/
mice are, in
general, healthy; although they are hypersensitive to insulin (36, 37)
and leptin (27, 28), they show no evidence of increased EGFR or PDGFR
activity. Our results clearly show that although PTP1B plays a role in
regulating both of these receptors, other regulatory mechanisms come
into play when it is missing. These redundant signaling mechanisms may
be designed to prevent the dire consequences of a generalized increase
in RTK signaling that would occur were a single PTP
or a single RTK inactivation mechanism
to regulate the EGFR, PDGFR, and/or other RTKs.
If this explanation can be generalized to other RTK/PTP interactions,
it might explain why no classic PTPs have been found to be tumor
suppressor genes. Why, then, is the IR different? That is, why is PTP1B
a biologically limiting regulator of IR signaling? An important clue
may be provided by the different fates of the respective
ligand-stimulated RTKs. Whereas most activated EGFRs and PDGFRs are
targeted for degradation (60), the vast majority of IRs are recycled
(61). A recycling receptor may be especially dependent on
dephosphorylation for its inactivation. Further work is needed to
determine whether differential trafficking accounts for the
differential effects of PTP1B deficiency on RTKs.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. N. Tonks (Cold Spring Harbor Laboratory) for providing PTP1B retroviral constructs, Dr. D. DeMaio (Yale Medical School) for the anti-PDGFR antibodies, and Saskia Brachmann for help with the PI-3K assay.
![]() |
FOOTNOTES |
---|
* 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.
¶ Present address: Wyeth Pharmaceuticals, Metabolic Diseases, 200 Cambridge Park Dr., Mailstop 4001K, Cambridge, MA 02140.
Supported by Grant BO-1043-4 from the Deutsche Forschungsgemeinschaft.
** Supported by National Institutes of Health Grants CA49152 and DK60838 and a grant from the American Diabetes Association. To whom correspondence should be addressed: Harvard Medical School, HIM-1043, 330 Brookline Ave., Boston, Massachusetts 02215. Tel.: 617-667-0600; Fax: 617-975-5617; E-mail: bneel@caregroup.harvard.edu.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M210194200
2 F. G. Haj and B. G. Neel, unpublished observations.
3 B. Markova and F. D. Bohmer, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RTK, receptor tyrosine kinase; PTP, protein tyrosine phosphatase; IR, insulin receptor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; WT, wild type; MEF, mouse embryo fibroblast; hPTP1B-WT, wild-type human PTP1B; hPTP1B-D/A, substrate-trapping mutant PTP1B-D181A; KO, knock-out; PI-3K, phosphatidyl inositol-3 kinase.
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