(Received for publication, February 3, 1995; and in revised form, May 18, 1995)
From the
The SH2 domain protein tyrosine phosphatases (PTPases) PTP1C and PTP1D were found associated with epidermal growth factor (EGF) receptor which was purified from A431 cell membranes by several steps of chromatography. Both PTPases also associated with the EGF receptor upon exposure of immunoprecipitated receptor to lysates of MCF7 mammary carcinoma cells. The associated PTPases had little activity toward the bound receptor when it was autophosphorylated in vitro. Receptor dephosphorylation could, however, be initiated by treatment of the receptor-PTPase complex with phosphatidic acid (PA). When autophosphorylated EGF receptor was exposed to lysates of PTP1C or PTP1D overexpressing 293 cells, the association of PTP1C but not of PTP1D was enhanced in the presence of PA. In intact A431 cells, an association of PTP1C and PTP1D with the EGF receptor was detectable by coimmunoprecipitation experiments. PA treatment reduced the phosphorylation state of ligand activated EGF receptors in A431 cells and in 293 cells overexpressing EGF receptors together with PTP1C but not in 293 cells overexpressing EGF receptors alone or together with PTP1D. We conclude that PTP1C but not PTP1D participates in dephosphorylation of activated EGF receptors. A possible role of PA for physiological modulation of EGF receptor signaling is discussed.
Growth factor receptors of the tyrosine kinase-type undergo
rapid autophosphorylation upon ligand
stimulation(1, 2) . The autophosphorylation generates
binding sites for SH2 domain containing intracellular proteins.
Membrane recruitment of these molecules by binding to the
autophosphorylated growth factor receptors is considered an important
step for activation of several intracellular signaling pathways which
ultimately lead to the cellular responses as cell division, cell
differentiation, or cell locomotion. Dephosphorylation of the
autophosphorylated growth factor receptors by PTPases ()(3, 4, 5, 6) presumably
presents a major mechanism of negative regulation of tyrosine kinase
receptor signaling. Recently, it has become clear that PTPases comprise
a large family of structurally very diverse molecules which can broadly
be classified into cytosolic and transmembrane enzymes (see (7, 8, 9, 10) for reviews).
Different PTPases of both classes are capable to dephosphorylate
autophosphorylated growth factor receptors in
vitro(11, 12, 13) and when
overexpressed together with the receptors in animal
cells(14, 15) . Also, attenuation of growth factor
receptor signaling by overexpression of PTPases has been
demonstrated(16) . In particular, it could be shown that
cellular transformation induced by overexpression of the growth factor
receptors HER2/Neu or v-Fms can be reverted by coexpression of the
PTPases PTP1B or T-cell PTP(15, 17, 18) . The
currently available data suggest that, despite an only moderate
substrate specificity of PTPases in vitro, the interaction of
PTPases with phosphorylated substrates in intact cells is rather
specific and in addition finely tuned by various mechanisms regulating
the activity of the PTPases, including phosphorylation of the PTPases
at Ser/Thr or Tyr
residues(14, 19, 20, 21, 22) .
To date, little is known about the identity and regulation of PTPases
which physiologically interact with specific growth factor receptors
and thereby regulate the respective signaling pathways. Such knowledge
is, however, highly desirable for understanding the regulation of
tyrosine kinase receptor signaling in normal and tumor cells. Also,
characterization of PTPases which specifically regulate certain
signaling pathways will identify novel targets for pharmacological
modulation of cell growth. It might be possible to identify such
PTPases by virtue of their physical association with receptor tyrosine
kinases. Obvious candidates for a physical association with
autophosphorylated growth factor receptors are PTPases possessing SH2
domains. Two SH2 domain-containing PTPases have been identified so far
and designated (among other names) PTP1C (SH-PTP1) and PTP1D (SH-PTP2,
syp, SH-PTP3, PTP2C) (see (23) for review). Association of
PTP1D to the cytoplasmic domains of PDGF receptor, HER2/Neu, the EGF
receptor, and Kit/SCF receptor has been demonstrated when the PTPase
and the respective receptors were transiently overexpressed in 293
cells(14) . Association of PTP1D was also shown with PDGF and
EGF receptors in fibroblasts(24) , with PDGF receptors in
epithelial cells(25) , and with insulin receptors as well as
the insulin receptor substrate-1 in vitro(26) .
Receptor association with PTP1D leads to its tyrosine phosphorylation
and activation of the PTPase
activity(14, 24, 27) . Although PTP1D is
capable of dephosphorylating in vitro tyrosine-phosphorylated
peptides corresponding to growth factor receptor autophosphorylation
sites(28) , it has little activity on coexpressed or
coimmunoprecipitated receptors(14, 24) , with the
possible exception of Kit/SCF receptor(14) . For PTP1C,
physical association with autophosphorylated growth factor receptors
has also been demonstrated for ligand-stimulated Kit/SCF receptor in
hematopoietic cells (29) and with the cytoplasmic domain of
HER2 in a transient overexpression system(14) . Transient
coexpression of PTP1C with different tyrosine kinase receptors results
in complete or partial dephosphorylation of PDGF
and
receptors, insulin-like growth factor-I receptor, Kit/SCF receptor,
insulin receptor, EGF receptor, and HER2(14) . A role of PTP1C
for negative modulation of tyrosine kinase receptor signaling in
hematopoietic cells has been proposed in relation to various
proliferation abnormalities observed in mice carrying a defective PTP1C
gene(23, 30) . Recently, association of PTP1C with the
erythropoietin receptor (31) and with the Fc
RIIB1 receptor
in B lymphocytes (32) and a role for PTP1C in negative
regulation of erythropoiesis and B cell signaling, respectively, have
been demonstrated. Both PTP1C and PTP1D are phosphorylated and possibly
regulated by Ser/Thr specific kinases(33, 34) , and
PTP1C is potently activated in vitro by anionic phospholipids (35) including PA. Various tyrosine kinases are capable of
phosphorylating PTP1C in vitro, and it becomes tyrosine
phosphorylated in A431 cells in response to EGF treatment(36) .
It is not known if these mechanisms are important for the regulation of
the activity of the SH2 domain PTPases toward growth factor receptors
or other substrates in intact cells.
Here we demonstrate that PTP1D and PTP1C associate with EGF receptors in human epithelial tumor cells. The phosphatase(s) can be activated by PA to dephosphorylate the associated autophosphorylated receptor in vitro. PA stimulates association of PTP1C but not PTP1D to the EGF receptor. Phosphatase activity toward the EGF receptor can be activated by PA in intact A431 cells, and the activity of PTP1C toward EGF receptor, when transiently coexpressed in 293 cells, can be enhanced by PA. Our data suggest that PTP1C plays a role in EGF receptor dephosphorylation and that PA might be a physiological modulator of PTP1C activity.
For
immunoprecipitation, peak fractions of column-purified EGF receptor
were diluted with column buffer containing additionally 2 mM sodium orthovanadate and incubated with either 3 µg of
monoclonal anti-PTP1C or anti-PTP1D antibodies or 1 µg of
monoclonal anti-EGF receptor antibody 425 (kind gift of Dr. A.
Luckenbach, E. Merck, Darmstadt) for 2 h at 4 °C. The immune
complexes were collected with 20 µl of 50% suspension of protein
A-Sepharose (Pharmacia), extensively washed with column buffer
containing 2 mM sodium orthovanadate and once with 50 mM HEPES, pH 7.5, containing 3 mM MnCl and 2
mM sodium orthovanadate. Then receptor autophosphorylation was
performed as described above. For detection of PTPase association to
EGF receptors in intact A431 cells, subconfluent cultures in six-well
plates (Falcon) were kept in serum-free DMEM overnight. Then cells were
stimulated with 100 ng/ml EGF for 10 min at room temperature or left
unstimulated. The cultures were washed twice with ice-cold
phosphate-buffered saline and extracted with lysis buffer, containing 2
mM sodium orthovanadate. Lysates were clarified by
centrifugation and subjected to immunoprecipitation and
autophosphorylation as described above. Lysate corresponding to
1-2
10
cells was used per precipitation.
Figure 1:
Association of PTPase
activity with the EGF receptor (EGFR). A,
copurification of EGF receptor and PTPase activity from A431 cell
membranes. A particulate fraction from A431 cells was extracted with
detergent and subjected to affinity chromatography on wheat germ
agglutinin as described under ``Experimental Procedures.''
The glycoprotein fraction eluted from the wheat germ agglutinin column
was then chromatographed over a tyrosine-Sepharose column as described
by Akiyama et al.(37) . Fractions were monitored for
the presence of EGF receptor by an autophosphorylation assay. The peak
fractions were concentrated and either directly subjected to
chromatography on a Superdex 200HRC sizing column (squares) or
incubated with 20 mM phenylphosphate and subsequently
separated on the same column (triangles). The points represent
the PTPase activity toward P-labeled Raytide. The elution
position of EGF receptor (hatched area) was detected by
autophosphorylation assays and found to be identical (fractions
9-11) under both conditions. B, binding of PTPase
to immunoprecipitated EGF receptor. EGF receptor was immunoprecipitated
with anti-EGF receptor antibodies 108 from cell lysates of EGF receptor
overexpressing 293 cells, as described under ``Experimental
Procedures.'' The precipitates were incubated in the absence of
any agents (1) or in the presence of ATP (2) or
ATP
S (3-5), washed, and exposed to lysates of MCF7
human mammary carcinoma cells in the presence (4) or absence (1-3, 5) of 20 mM phenylphosphate (PhP). After extensive washing, the receptor-associated PTPase
activity was measured using exogenously added
P-labeled
EGF receptor, as described under ``Experimental Procedures,''
in the absence (lanes 1-4) or presence (lane 5)
of 1 mM sodium orthovanadate (V). The results are
expressed in arbitrary units.
In an
alternative approach, EGF receptors were immunoprecipitated from
lysates of 293 cells which overexpressed the receptor. Then, the
immunoprecipitates were incubated with lysates of MCF7 human mammary
carcinoma cells, thereafter washed extensively and checked for
associated PTPase activity using autophosphorylated EGF receptor as a
substrate. As shown in Fig. 1B, PTPase activity was
associated with the immunoprecipitated receptors. The extent of PTPase
association was dependent on the pretreatment of the immunoprecipitated
receptors. Only little PTPase activity associated in the absence of any
pretreatment. A clear PTPase association was, however, detectable by
allowing autophosphorylation of the receptors in the presence of ATP
and even more in the presence of ATPS before exposing it to the
cells lysates. The associated PTPase activity was almost completely
inhibited by sodium orthovanadate (Fig. 1B). When
immunoprecipitated autophosphorylated EGF receptor was exposed to MCF7
lysate in the presence of phenylphosphate, the amount of
receptor-associated PTPase activity was greatly reduced (Fig. 1B), again indicating the involvement of SH2
domains in the receptor-PTPase association. We therefore analyzed the
EGF receptor preparations containing PTPase activity for the presence
of SH2 domain PTPases by immunoblotting. Clearly, PTP1C and PTP1D were
detectable by immunoblotting with specific antibodies in peak fractions
of the column-purified EGF receptor (Fig. 2) as well as
associated from MCF7 cell lysates to immunoprecipitated EGF receptor
(not shown). To determine whether a PTPase-EGF receptor-association
also occurs in intact A431 cells, A431 cell lysates were subjected to
immunoprecipitation with anti-PTP1C and anti-PTP1D antibodies, and the
immunoprecipitates were analyzed by autophosphorylation in the presence
of [
-
P]ATP and EGF, followed by SDS-PAGE
and autoradiography. When lysates of serum-deprived but not
EGF-stimulated cells were subjected to immunoprecipitation, weak
autophosphorylation signals at the expected size of EGF receptors were
obtained upon precipitation with both PTP1C and PTP1D antibodies (Fig. 3, lanes 1 and 2), whereas no signal was
seen when nonspecific IgG was employed for the immunoprecipitation (Fig. 3, lane 3). When the A431 cells were stimulated
with EGF prior to lysis and subsequent immunoprecipitation, the
intensity of the phosphorylated 170-kDa band which was
immunoprecipitated with anti-PTP1C and anti-PTP1D antibodies was
greatly enhanced (Fig. 3, lanes 4 and 5). Only
a small amount of EGF receptor was precipitated with nonspecific IgG (lane 6). These results clearly demonstrate an association of
EGF receptor with both PTP1C and PTP1D in intact A431 cells and also
indicate a more efficient interaction of PTP1C and PTP1D with the
stimulated EGF receptor versus the nonstimulated receptor.
Figure 2:
Detection of PTP1D and PTP1C in column-
purified EGF receptor (EGFR) preparations. EGF receptor was
purified from A431 cell membranes, as described under
``Experimental Procedures,'' to the stage of
tyrosine-Sepharose chromatography. Peak fractions were highly
concentrated (Microcon) and subjected to SDS-PAGE and immunoblotting
with monoclonal anti-PTP1D or PTP1C antibodies as indicated. The amount
of sample applied per lane corresponds to EGF receptor isolated from
about 5 10
A431 cells (lanes 3 and 4). For reference, aliquots of lysates of 293 cells
overexpressing either PTP1C (lane 1) or PTP1D (lane
6) were also analyzed. Only SDS-PAGE sample buffer was loaded in lanes 2 and 5. The positions of the phosphatases and
of molecular mass markers are indicated.
Figure 3:
Detection of EGF receptor (EGFR)
in anti-PTP1C and anti-PTP1D immunoprecipitates. Serum-deprived
subconfluent cultures of A431 cells were left unstimulated (lanes
1-3) or were stimulated with 100 ng/ml EGF for 10 min at
room temperature (lanes 4-6). Cell lysates were prepared
and subjected to immunoprecipitation with anti-PTP1C antibodies (lanes 1 and 4), anti-PTP1D antibodies (lanes 2 and 5) or nonspecific IgG (lanes 3 and 6). Subsequently, the samples were incubated on ice in the
presence of [-
P]ATP, as described under
``Experimental Procedures,'' and then analyzed by SDS-PAGE
and autoradiography.
Figure 4: PA activates EGF receptor (EGFR) dephosphorylation by the receptor-associated PTPase(s). Column-purified EGF receptor was incubated in the absence (A) or presence of 50 µg/ml PA (B) for 20 min on ice. Then, the receptor was autophosphorylated, as described under ``Experimental Procedures,'' the phosphorylation was quenched by addition of EDTA, the samples were transferred to 37 °C, and the radioactivity in the receptor was monitored at the indicated time points by SDS-PAGE and autoradiography. Numbers underneath the lanes represent the percentage of radioactivity (mean of the duplicates, 100% at time point 0 min) as obtained by densitometric quantification.
We next wished to determine whether an activation of phosphatase(s) for the autophosphorylated EGF receptor by PA might occur in intact cells and further to differentiate whether PTP1C or PTP1D (both present in the investigated receptor preparations) might mediate the effect of PA. To this end, PTP1C and PTP1D were transiently coexpressed with the EGF receptor in 293 cells. The cells were left untreated or were treated with PA, then stimulated with EGF. Subsequently the phosphorylation state of the EGF receptors was analyzed by immunoblotting with antiphosphotyrosine antibodies. When EGF receptor was expressed in the absence of PTPase, no effect of PA treatment on the phosphorylation state of the receptor was seen (Fig. 5, upper panel as indicated). As described earlier(14) , expression of PTP1D had no effect on the phosphorylation state of coexpressed EGF receptors (Fig. 5, upper panel), and PA treatment of cells expressing EGF receptors and PTP1D had no effect (Fig. 5, upper panel). In contrast, PTP1C expression reduced the phosphorylation state of coexpressed EGF receptors to 32% of the respective control. When cells overexpressing EGF receptors and PTP1C were in addition treated with PA, the extent of EGF receptor phosphorylation was further reduced after 1 h of treatment to 21% and even further after 24 h of PA treatment to 13%. Control experiments (Fig. 5, lower panels) confirmed that comparable amounts of EGF receptor and the respective PTPases entered the analysis in all cases. We conclude that PA treatment enhances the EGF receptor-directed activity of PTP1C in this system.
Figure 5: Effect of transient expression of PTP1C or PTP1D on the phosphorylation state of co-expressed EGF receptor (EGFR) in the absence or presence of PA. The indicated genes were transiently expressed under control of a CMV promotor in 293 fibroblasts. Cells were left untreated or were treated for 1 or 24 h with 50 µg/ml PA and were then stimulated (or not) with EGF as indicated. Then cell lysates were analyzed by SDS-PAGE and immunoblotting with antiphosphotyrosine antibodies (upper panel). Numbers underneath the lanes represent the percentage of intensity of the EGF receptor phosphorylation signal compared to the respective treatment variants of cells expressing only EGF receptors (100%) as obtained by densitometric quantification. The blot was stripped and reprobed with anti-EGF receptor antibodies and polyclonal anti-PTP1D antibodies which weakly cross-react with PTP1C (lower panels as indicated) to verify that comparable amounts of the respective proteins were expressed. All lanes are from the same blot, but were rearranged for better clarity.
We further considered whether PA would also modulate the EGF receptor phosphorylation state in intact A431 cells. Cells were pretreated for different length of time with PA or left untreated, then stimulated with EGF and analyzed for the phosphotyrosine content of the EGF receptors. We consistently found that PA treatment for 1 h reduced the phosphotyrosine content of the EGF receptors (Fig. 6A). A similar reduction after 15 min and 24 h of treatment was observed occasionally but not in all experiments (not shown). To check the involvement of PTPases in this reduction of receptor phosphorylation, parallel cultures were treated with 50 µM pervanadate prior to EGF stimulation. No differences in the phosphorylation state between samples from PA-treated or nontreated cells were seen in presence of pervanadate (Fig. 6B). These data suggest that the reduction in phosphotyrosine content of EGF receptors induced by PA in A431 cells is mediated by PTPases.
Figure 6: Effect of PA treatment on the phosphorylation state of EGF receptors (EGFR) in A431 cells. A, subconfluent A431 cells were cultivated for 1 h in serum-free medium in the absence or presence of 50 µg/ml PA as indicated. Thereafter, the cells were stimulated with EGF as indicated, and cell extracts were analyzed by SDS-PAGE and immunoblotting with antiphosphotyrosine antibodies. Numbers underneath the lanes represent the percentage of intensity of the EGF receptor signal compared to untreated cells. Arrows indicate the position of the autophosphorylated EGF receptor. B, same experiment as in A; however, the cells were treated in addition with 50 µM pervanadate prior to EGF stimulation.
Figure 7: Effect of PA on the association of PTP1C and PTP1D with the EGF receptor (EGFR). EGF receptor was immunoprecipitated from lysates of receptor overexpressing 293 cells using the monoclonal anti-EGF receptor antibody Ab425 coupled to Affi-Prep Hz beads. The receptor was allowed to autophosphorylate and the beads with bound phosphorylated receptor (lanes 2-4 and 6-8) or beads without bound receptor (lanes 1 and 5) were then incubated with lysates of either PTP1C (lanes 1-4) or PTP1D (lanes 5-8) overexpressing 293 cells. Incubation was performed, as described under ``Experimental Procedures,'' in the absence of further additives (lanes 2 and 6) or in the presence of solvent vehicle (lanes 3 and 7) or 50 µg/ml PA (lanes 4 and 8). Thereafter, the beads were washed and bound proteins were analyzed by SDS-PAGE and immunoblotting with monoclonal antibodies specific for either PTP1C or PTP1D as indicated. The asterisks indicate the bands of some IgG which bled from beads despite the coupling. Lanes 1-4 and 5-8 are from the same blots, but were rearranged for better clarity.
The activated and autophosphorylated receptor for EGF is subject to a rapid dephosphorylation by PTPases(3, 6) . Receptor dephosphorylation is believed to present a major mechanism of negative regulation of receptor function, and the identification of the involved PTPases is therefore important for understanding of EGF receptor signaling. Also, since mitogenic signaling of EGF receptors and related receptors has been implicated in unwanted growth of certain human tumors, characterization of the receptor dephosphorylation mechanisms might be of significance for a pharmacological modulation of receptor signaling. As one approach to characterize such PTPases we tried to identify those which are capable of physically associating with EGF receptors. PTPase activity which associated from MCF7 cell lysates to immunoprecipitated EGF receptors and PTPase activity which was detectable in EGF receptor preparations obtained with several purification steps from A431 cell membranes could at least in part be attributed to receptor-associated PTP1C and PTP1D. We could also demonstrate the capability of PTP1D and PTP1C to interact with the EGF receptor in intact A431 cells. A physiological interaction of these PTPases with EGF receptors has been predicted from in vitro experiments and from data obtained in cells overexpressing the phosphatases(14, 24) . Here we show that an interaction of PTP1C and PTP1D with EGF receptors is detectable in nontransfected cells, i.e. the human tumor cell line A431. As expected, the association seems largely to depend on the interaction of the PTPase SH2 domains with the receptors, since significantly more PTPase could be bound to activated phosphorylated receptors, and the association was largely abrogated by phenylphosphate. Although the displacement of a large fraction of the receptor-associated PTPase activity by phenylphosphate suggests that SH2 domain PTPases account for most if not all of the activity, our data do not completely rule out the possibility that, in addition to PTP1C and PTP1D, other, not yet identified, PTPases contribute to the receptor-associated PTPase activity.
Despite the activity of the EGF
receptor-bound PTPases against exogenous substrates, little activity
against the bound receptor was detectable in vitro. In the
presence of PA, however, dephosphorylation activity could clearly be
observed. PA also enhanced EGF receptor dephosphorylation in intact
A431 cells and in 293 cells transiently coexpressing EGF receptors and
PTP1C, but not in 293 cells expressing EGF receptors alone or EGF
receptors and PTP1D. Taken together, these data suggest that PTP1C but
not PTP1D is capable of dephosphorylating EGF receptors and that this
activity can be enhanced by PA. EGF stimulation of A431 cells has
previously been shown to increase cellular PA levels up to 0.17% of the
total cellular phospholipids largely via a pathway involving
phosphorylation of diacylglycerol(40, 41) . One might
therefore speculate that the activation of the
receptor-dephosphorylating PTPase PTP1C by PA could present part of a
negative feedback regulation of EGF receptor signaling. A role for
PTP1C in negative control of receptor activity has also been suggested
in case of Kit/SCF receptor(24, 30) , and recently
PTP1C has been shown to be involved in negative regulation of
erythropoietin receptor (31) and FcRIIB1 receptor
signaling(32) .
The activation of PTP1C by various acidic phospholipids in vitro against artificial substrates has previously been shown by Zhao et al.(35) . The data in this study suggest that the lipid-PTP1C interaction leading to PTP1C activation is direct and involves a conformational change of the phosphatase which facilitates proteolytic degradation(35) . When we examined the effect of PA on the association of both SH2 domain PTPases to the autophosphorylated EGF receptor in vitro, association of PTP1C, but not PTP1D, was increased in the presence of PA. It is possible that the conformational change of PTP1C which is induced by PA leads to the increased association with the EGF receptor and also forms the basis for activation toward the receptor as observed in our dephosphorylation experiments. Recently, a structural model for PTP1C was proposed (42) based on the kinetic analysis of differently truncated enzyme variants and enzyme activation by phosphorylated peptides which interact with the PTP1C SH2 domains. According to this model, the PTP1C SH2 domains are capable of interacting with the PTP1C C terminus in a phosphotyrosine-independent manner and thereby drive the PTPase domain in an inactive conformation(42) . One could speculate that PA has the capacity to disrupt this intramolecular interaction. Further experiments are required to clarify the activation mechanism.
In our experiments no evidence for a role of EGF receptor-associated PTP1D in receptor dephosphorylation was obtained. PTP1D has previously been demonstrated to mediate mitogenic signals of the receptors for PDGF, insulin, EGF, and insulin-like growth factor-I (43, 44, 45) and a positive role of PTP1D in tyrosine kinase receptor signaling is also likely from its homology to Drosophila csw(14, 25, 46) . The association of PTP1D with EGF receptors in human tumor cells therefore defines an interesting target for interference with EGF-driven tumor cell growth.