(Received for publication, January 16, 1996; and in revised form, February 12, 1996)
From the
PTP1C, an SH2 domain-containing protein-tyrosine phosphatase, is predominantly expressed in hematopoietic cells, in which it negatively regulates cellular signaling. However, this enzyme is also expressed in many non-hematopoietic cells. We demonstrate here that in non-hematopoietic 293 cells, overexpression of a catalytically inactive mutant of PTP1C strongly suppressed the stimulatory effects of the epidermal growth factor or serum on cell proliferation, early gene transcription, and DNA synthesis. Similarly, the phosphorylation of the mitogen-activated protein kinase and mitogen-activated protein kinase kinase activity was markedly inhibited by overexpression of mutant PTP1C. The inhibitory effect of mutant PTP1C was overcome by cotransfection with wild-type PTP1C, but not with the structurally related PTP2C. Furthermore, expression of the mutant phosphatase resulted in hyperphosphorylation on tyrosine of a 95-kDa protein that was co-immunoprecipitated with the mutant, but not with the wild-type protein. These results suggest that, unlike in hematopoietic cells, PTP1C in 293 cells plays a positive role in epidermal growth factor- or serum-activated mitogenesis. Thus, PTP1C participates in multiple signaling pathways, where the enzyme, depending on its target molecules, may function as either a positive or negative mediator.
Upon binding of external ligands, several cell-surface
receptors, such as the insulin receptor, epidermal growth factor (EGF) ()receptor, and platelet-derived growth factor (PDGF)
receptor, are activated via dimerization, resulting in
autophosphorylation on multiple tyrosine residues within the
intracellular domains of the receptors(1, 2) . The
autophosphorylated domain of the receptor then provides high affinity
binding sites for specific cellular SH2 domain-containing
proteins(3) . The interaction of these SH2-containing proteins
with specific tyrosyl-phosphorylated sites of the receptor causes other
signaling molecules to be recruited to the receptor, where these
molecules, as well as most of the SH2-containing proteins, can be
phosphorylated by the receptors and/or other recruited
proteins(3, 4) . Formation of these signal
transduction complexes coordinates the multiple intracellular programs
that initiate various changes in cell proliferation.
Previously, we and others have identified two SH2 domain-containing protein-tyrosine phosphatases, designated PTP1C (5) (also termed SH-PTP1/HCP/SHP) (6, 7, 8, 9) and PTP2C (9) (also known as SH-PTP2, Syp, and PTP1D)(10, 11, 12) . PTP2C, a ubiquitously expressed enzyme, is highly homologous to another member of this SH2-containing subfamily, the product of the Drosophila corkscrew (csw) gene (13) . The SH2 domains of PTP2C interact with activated receptor protein-tyrosine kinases, such as the EGF and PDGF receptors(11, 12) , and with the substrates of these receptor protein-tyrosine kinases, such as insulin receptor substrate-1(14) . As a result of these interactions, PTP2C itself becomes tyrosyl-phosphorylated by the receptor protein-tyrosine kinases, and its activity can be modulated by such tyrosyl phosphorylation(12, 14, 15, 16) . In these mitogen-activated protein (MAP) kinase pathways, PTP2C appears to be a positive mediator (17, 18, 19, 20, 21) . PTP1C is predominantly expressed in hematopoietic cells(6, 7) . Like PTP2C, PTP1C can be tyrosyl-phosphorylated in response to various ligands, such as colony-stimulating factor-1, stem cell factor, PDGF, insulin, thrombin, and PMA(22, 23, 24, 25, 26) . In hematopoietic cells, PTP1C was found to associate with c-Kit, the receptor for the Fc region of IgG, and the erythropoietin receptor(22, 27, 28) . The interaction of PTP1C with these receptors through the SH2 domains appears to play a role as a negative regulator in hematopoietic cells(22, 27, 28, 29) . However, recent data revealed that PTP1C is also substantially expressed in a variety of non-hematopoietic cells, especially in some malignant epithelial cells(5, 6, 12, 30) , suggesting that this enzyme plays some roles in cellular signal transduction in these cells. In this report, we demonstrate that in human embryonic kidney 293 cells, the phosphatase activity of PTP1C, like that of PTP2C, is a positive mediator in serum- and EGF-activated mitogenic signaling.
Figure 4: Effect of overexpressed PTP1C and PTP1C C455S on gel mobility shifts of MAP kinases. Serum-starved 293 cells stably expressing wild-type PTP1C and mutant PTP1C C455S (PTP1C(C-S)) were stimulated for 5 min with EGF at 10 ng/ml. Whole cell extracts (20 µg of protein sample/well) were subjected to 10% SDS-polyacrylamide gel electrophoresis, transferred to Hybond C membrane, and blotted with polyclonal anti-MAP kinase antibody (upper panel) or anti-PTP1C antibody 237 (lower panel). The positions of ERK1, ERK2, and PTP1C are indicated. Note that the level of PTP1C in cells transfected with the vector represents the endogenous phosphatase in 293 cells.
Figure 1: Tyrosine phosphorylation of PTP1C in 293 cells. Serum-starved semiconfluent 293 cells grown in 90-mm dishes were stimulated with EGF at 10 ng/ml for 0, 1, 2, 3, 5, 10, and 20 min at 37 °C and lysed in 1 ml of cell lysis buffer. Cell lysates containing equal amounts of total protein were immunoprecipitated by anti-PTP1C antibody and detected by Western blot analysis with anti-phosphotyrosine antibody (Anti-Ptyr) or anti-PTP1C antibody 237 as indicated.
Figure 2: Effects of PTP1C and PTP1C C455S on the growth rate of 293 cells stimulated by serum and EGF. The concentrations of serum and EGF for stimulation are indicated. The results shown are representative of three separate experiments. PTP1C(C-S), PTP1C C455S.
Figure 3: Inhibition of DNA synthesis in 293 cells by mutant PTP1C C455S. 293 cells stably expressing PTP1C or PTP1C C455S (PTP1C(C-S)) were seeded in 96-well plates (5000 cells/well) and serum-starved for 18 h. DNA synthesis in response to various concentrations of EGF and serum was measured as described under ``Experimental Procedures.'' Data are presented as means ± S.E. from three independent experiments, each performed in triplicate. FBS, fetal bovine serum.
The effects of different forms of PTP1C on the MAP
kinase cascade were further examined by directly measuring MAP kinase
kinase activity. The activity assay was performed 5 min after EGF
stimulation. The time courses showed that the peak activity for all
transfected cells occurred at approximately this point (data not
shown). Expression of wild-type PTP1C slightly increased the MAP kinase
kinase activity (15%) when compared with control cells. In
contrast, overexpression of the catalytically inactive mutant PTP1C
C455S decreased MAP kinase kinase activity by
30% (Fig. 5).
Figure 5:
Inhibition of MAP kinase kinase activity
by expression of mutant PTP1C C455S. Whole cell lysates from
EGF-treated cultures (5 min) were prepared as described under
``Experimental Procedures.'' The samples were then assayed
for MAP kinase kinase (MAPKK) activity by incubating 2 µg
of protein sample with ERK2 and unlabeled ATP for 10 min, followed by
the addition of myelin basic protein and P-labeled ATP and
further incubation for 10 min. Results are expressed as relative
activity of MAP kinase kinase, in which the activity obtained from the
vector-transfected cells was calculated as 1.0. PTP1C(C-S),
PTP1C C455S.
Figure 6: Effect of mutant PTP1C C455S on SRE-Luc activity. Semiconfluent 293 cells expressing PTP1C or PTP1C C455S (PTP1C(C-S)) were transiently cotransfected with the SRE-Luc and GFP reporter plasmids. The cells were then serum-starved and either left untreated or treated with 10% serum (upper panel), 10 ng/ml EGF (middle panel), or 5 nM PMA (TPA; lower panel) as described under ``Experimental Procedures.'' The SRE-Luc (SRE-Luc) activity was normalized for the efficiency of transfection based on the expression of GFP as well as on the total protein concentration of different cell lysates. The SRE-Luc activity is presented as the intensity of fluorescent lights. Data are presented as means ± S.E. from three independent experiments, each performed in triplicate.
Figure 7: Effect of mutant PTP1C C455S cotransfected with wild-type PTP1C or PTP2C on SRE-Luc activity. Cells stably expressing PTP1C C455S (PTP1C(C-S)) were cotransfected either with wild-type PTP1C or with PTP2C and selected by an additional marker, hygromycin B. Cells were transiently transfected with the SRE-Luc and GFP reporter plasmids and treated for EGF stimulation as described for Fig. 6. The SRE-Luc activity is presented as the intensity of fluorescent lights. Data are presented as means ± S.E. from three independent experiments, each performed in triplicate.
Figure 8:
Detection of tyrosine-hyperphosphorylated
proteins in PTP1C C455S-transfected cells. Serum-starved 293 cells
transfected with vector alone (lane 1), wild-type PTP1C (lane 2), and mutant PTP1C C455S (lane 3) were
stimulated for 5 min with EGF at 10 ng/ml. Cell extracts (20 µg of
total proteins) and their immunoprecipitates (IP) from 1
10
cells with anti-PTP1C antibody were subjected to
SDS-polyacrylamide gel electrophoresis and Western blot analysis with
anti-phosphotyrosine antibody. The positions of PTP1C and the 95-kDa (p95) and 100-kDa (p100) proteins are
indicated.
In our analysis of the PTP1C gene, we have recently shown that the expression of PTP1C in hematopoietic cells versus non-hematopoietic cells is regulated by two tissue-specific promoters. The hematopoietic form of the PTP1C transcript is initiated exclusively from a specific promoter (P2), whereas in non-hematopoietic cells, an upstream promoter (P1) is transcriptionally activated(33) . Indeed, PTP1C has been detected in a variety of non-hematopoietic cells(5, 12, 30) , from which PTP1C was initially identified(5) . Recent studies have further established that in hematopoietic cells, PTP1C negatively regulates a number of major signaling pathways. For instance, PTP1C inhibited B cell signaling and turned off erythropoietin-stimulated hematopoiesis(27, 28) . However, the function of PTP1C in non-hematopoietic cells is largely unknown. Since the EGF receptor is expressed in many non-hematopoietic cells, but is not found in hematopoietic cells, we used EGF as a model system to investigate the effect of PTP1C on mitogenic pathways in 293 cells.
The function of PTP1C in EGF- and serum-activated mitogenic pathways was investigated by expression of wild-type PTP1C and its inactive mutant form in 293 cells. Overexpression of a wild-type PTP1C slightly enhanced EGF- and serum-activated mitogenesis as compared with the control cells. The positive effect of PTP1C on cell growth and DNA synthesis was more apparent at low concentrations of serum and EGF (see Fig. 2and Fig. 3). It is possible that at high concentrations of the mitogens, other elements may have been activated in multiple signal transduction pathways that might mask the positive function of PTP1C. Most important, overexpression of a catalytically inactive mutant of PTP1C strongly suppressed all mitogen-activated pathways, as measured by cell growth, DNA synthesis, early gene transcription, MAP kinase phosphorylation, and MAP kinase kinase activity. Taken together, the results suggest that, in contrast with its negative regulatory roles observed in hematopoietic cells, the intrinsic phosphatase activity of PTP1C in 293 cells likely functions as a positive regulator in EGF- or serum-stimulated mitogenesis. Interestingly, the catalytically inactive mutant of PTP2C, a structural homologue of PTP1C, displayed a similar positive effect on mitogenesis activated by several growth factors in non-hematopoietic cells(18, 19, 20, 21) . However, although PTP1C and PTP2C share a great deal of sequence homology, two lines of evidence suggested that the positive effect of overexpressed PTP1C on mitogen-activated pathways in 293 cells was not due to competition with PTP2C for the same phosphotyrosine-binding site(s) on the target. First, in similar experiments, the catalytically inactive mutant of PTP2C in 293 cells bound to a 47-kDa protein(21) , rather than to the 95-kDa protein shown to bind to the inactive PTP1C here, indicating that the two SH2 domain-containing phosphatases target different molecules. Second, the inhibitory function of the overexpressed catalytically inactive mutant of PTP1C in EGF-stimulated SRE-Luc activity was nearly completely abolished by wild-type PTP1C. In this experiment, PTP2C caused an increase in response to EGF nearly identical to that observed in the absence of the dominant negative form of PTP1C(21) . Together, these results suggest that in 293 cells, the positive effects of overexpressed PTP1C and PTP2C on EGF-stimulated mitogenesis arise through different signaling pathways.
Although our data suggest that PTP1C, like PTP2C, functions as a
positive regulator in the mitogen-activated Ras-Raf-MAP kinase pathway
in 293 cells, the mechanism by which the inactive mutant PTP1C
attenuated the EGF- and serum-activated signaling is unknown. It is
known that PMA, a potent activator of protein kinase C, induces MAP
kinase activation in a Ras-independent manner(34) . Since
mutant PTP1C strongly suppressed the serum- and EGF-stimulated activity
of the SRE-Luc reporter gene, but did not interfere with the PMA
activation of the gene, it is very likely that PTP1C C455S targets an
upstream molecule in the Ras-MAP kinase pathway, rather MAP kinase
itself or its downstream molecules. This molecule may link Ras-Raf
activation as suggested for the positive role of PTP2C in
insulin-stimulated signaling(19, 20) . Alternatively,
tyrosine-phosphorylated PTP1C, as with PTP2C(35) , can serve as
a docking site for Grb2 binding(24) . Formation of the
PTP1C-Grb2 complex may activate the Ras-Raf-MAP kinase pathway.
However, expression of the catalytically inactive mutant PTP1C C455S
appeared to have no significant effect on complex formation between
Grb2 and the EGF receptor, and site-directed mutagenesis experiments
also showed that eliminating the Grb2-binding site on PTP1C did not
reduce the mitogenic responses. ()Ideally, as a positive
inducer in signaling, PTP1C (or PTP2C) may dephosphorylate a molecule
on its phosphotyrosine site(s) that negatively regulate its function,
such as the kinases of the Src family. In searching for potential
targets for PTP1C, we have found a tyrosine-hyperphosphorylated 95-kDa
protein in cells expressing the inactive mutant PTP1C. This protein was
co-immunoprecipitated with mutant PTP1C C455S, but not with the active
phosphatase. Since the 95-kDa protein bound to the glutathione S-transferase-SH2 fusion protein of PTP1C in vitro,
it is likely that PTP1C associates with the 95-kDa protein through its
SH2 domain(s). These results suggest that this tyrosine-phosphorylated
95-kDa protein might be one of the downstream targets of PTP1C in
signaling.
The demonstration of the positive regulatory function for PTP1C in mitogenesis as described here suggests a more complicated role for these SH2-containing protein-tyrosine phosphatases than previously considered, such as the view that PTP1C plays only a negative role in hematopoietic cells and, conversely, that PTP2C, a mammalian homologue of corkscrew, the Drosophila csw gene product, functions as a positive regulator. In fact, we have observed that PTP2C, in contrast with its positive function in mitogen-activated pathways, plays a negative role in membrane ruffling signaling(36) . The negative function of PTP2C in regulating EGF-dependent cell growth was also recently reported(37) . A positive regulatory function for PTP1C has also been suggested for the thrombin-activated mitogen pathway in blood platelets(26) . On the other hand, PTP1C was found to down-regulate the proliferation of v-Src-transformed rat fibroblast cells(38) . Thus, the results presented here, together with previous data, imply that PTP1C participates in complicated and multiple signaling pathways, where, depending on cell types, its compartmentalization, and which molecules it interacts with, the enzyme can play either a positive or negative role in regulating individual signal transduction pathways.