©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Phosphotyrosine Phosphatase PTP1D, but not PTP1C, Is an Essential Mediator of Fibroblast Proliferation Induced by Tyrosine Kinase and G Protein-coupled Receptors (*)

Nathalie Rivard (§) , Fergus R. McKenzie , Jean-Marc Brondello , Jacques Pouysségur

From the (1) Centre de Biochimie, CNRS-UMR134, Parc Valrose, 06108 Nice, Cédex 2, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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.


INTRODUCTION

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- and pp60. This specific association then permits activation of their respective signaling pathways (3) .

Several phosphotyrosine phosphatases (PTPs)() 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.

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 (-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) .

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 -thrombin on early gene transcription and DNA synthesis. In light of these findings, the importance of PTP1C and PTP1D to mitogenic signaling is discussed.


EXPERIMENTAL PROCEDURES

Materials Highly purified human -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.


RESULTS

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).

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, -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).




DISCUSSION

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 (-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.

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 -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.

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, -thrombin, and PDGF are strong stimulators of MAP kinase activity in CCL39 cells, whereas TPA and insulin are much less potent (35, 36) .

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, -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.

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 -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.

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 -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.

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 -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.

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.


FOOTNOTES

*
This work was supported by grants from CNRS, INSERM, and Association pour la Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Holds a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. To whom correspondence should be addressed. Tel.: 33-93-52-99-25; Fax: 33-93-52-99-17.

The abbreviations used are: PTP, protein-tyrosine phosphatase; PDGF, platelet-derived growth factor; VSVG, vesicular stomatitus virus glycoprotein; SRE, serum-responsive element; DMEM, Dulbecco's modified Eagle's medium; pNPP, para-nitrophenyl phosphate; MAP, mitogen activated protein; CS, cysteine to serine mutation; WT, wild type; TPA, 12- O-tetradecanoylphorbol-13-acetate; FCS, fetal calf serum.


ACKNOWLEDGEMENTS

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.


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