From the Dipartimento di Scienze Biochimiche, viale
Morgagni 50, 50134 Firenze, Italy and § Istituto di Medicina
Interna, Università di Firenze, viale Morgagni 85, 50134, Firenze, Italy
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ABSTRACT |
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The low molecular weight phosphotyrosine-protein
phosphatase (LMW-PTP) is a cytosolic phosphotyrosine-protein
phosphatase specifically interacting with the activated
platelet-derived growth factor (PDGF) receptor through its active site.
Overexpression of the LMW-PTP results in modulation of
PDGF-dependent mitogenesis. In this study we investigated
the effects of this tyrosine phosphatase on the signaling pathways
relevant for PDGF-dependent DNA synthesis. NIH 3T3 cells
were stably transfected with active or dominant negative LMW-PTP. The
effects of LMW-PTP were essentially restricted to the
G1 phase of the cell cycle. Upon stimulation with
PDGF, cells transfected with the dominant negative LMW-PTP showed an increased activation of Src, whereas the active LMW-PTP induced a
reduced activation of this proto-oncogene. We observe that c-Src binding to PDGF receptor upon stimulation is prevented by
overexpression of LMW-PTP. These effects were associated with parallel
changes in myc expression. Moreover, wild-type and dominant
negative LMW-PTP differentially regulated STAT1 and STAT3 activation
and tyrosine phosphorylation, whereas they did not modify extracellular
signal-regulated kinase activity. However, these modifications were
associated with changes in fos expression despite the lack
of any effect on extracellular signal-regulated kinase activation.
Other independent pathways involved in PDGF-induced mitogenesis, such
as phosphatidylinositol 3-kinase and phospholipase C-1, were not
affected by LMW-PTP. These data indicate that this phosphatase
selectively interferes with the Src and the STATs pathways in PDGF
downstream signaling. The resulting changes in myc and
fos proto-oncogene expression are likely to mediate the
modifications observed in the G1 phase of the cell
cycle.
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INTRODUCTION |
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Protein-tyrosine phosphorylation plays a key role in the regulation of many cellular processes in eukaryotes such as cellular metabolism, proliferation, differentiation, and oncogenic transformation (1). Accumulating evidence indicates that the contribution of phosphotyrosine-protein phosphatases (PTPs)1 to the control of phosphorylation state is as relevant as that of phosphotyrosine-protein kinases. PTPs activity is carefully regulated and appears to be, in most cases, highly specific. The PTPs consist of a family of over 40 enzymes often classified into three groups: 1) receptor-like PTPs; 2) intracellular PTPs; and 3) dual specificity PTPs (2). All PTPs share the signature active site motif CXXXXXR, in which the catalytic cysteine residue is involved in formation of a phosphoenzyme reaction intermediate (3). The low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) is a cytosolic enzyme without extensive sequence homology with other PTPs family members, but it contains the CXXXXXR motif and shares the same catalytic mechanism of classical PTPs (4, 5). Furthermore, the LMW-PTP crystal structure (6) revealed a tridimensionally folded phosphate binding loop that is structurally identical to that contained in the human placenta PTP1B and Yersinia PTP (7, 8).
We have previously demonstrated that the mutation of the cysteine residue in the signature motif to serine (C12S), causes the complete loss of catalytic activity (9). Nevertheless, this dominant negative mutant (dnLMW-PTP) is still able to bind to specific substrates (10). Overexpression of the active phosphatase (wtLMW-PTP) causes a reduction of cell proliferation (11), whereas dnLMW-PTP induces a remarkable increase in DNA synthesis, indicating that this latter molecule behaves as a dominant negative in vivo (12). In addition we have demonstrated that this phenotype is associated with a specific and direct interaction between the active site of dnLMW-PTP and the activated platelet-derived growth factor receptor (PDGF-R) (13). These data indicate that the PDGF-R is a specific substrate of the LMW-PTP and that this phosphatase may be involved in the control of one or more signaling pathways triggered by PDGF-R activation.
Binding of PDGF induces receptor dimerization and autophosphorylation.
Tyrosine phosphorylation sites in the PDGF-R function as high affinity
binding sites for several molecules involved in downstream signal
transduction (14). These proteins bind the phosphorylated receptor
through their Src homology domain 2 or phosphotyrosine binding domains,
and in many cases are themselves substrates of the receptor kinase
activity. These proteins include enzymes such as phospholipase C-1
(PLC
1), phosphatidylinositol 3-kinase (PI3K), Src, or molecular
adapters such as Shc, Grb2, and Nck, leading to different routes that
mediate the biological effects of PDGF. One of the main routes leading
to cell proliferation is the so-called Ras/extracellular
signal-regulated kinase (ERK) pathway. Upon stimulation with growth
factors, Ras activation can be achieved through a variety of signal
transducers like Shc, Grb2, and Sos (15). Alternatively, Syp
phosphatase may function as an adapter molecule for Grb2 and therefore
activate Ras (16). Activated Ras triggers a kinase cascade with
sequential activation of Raf-1, MAP/ERK, and ERK. Upon activation, ERK
translocates to the nucleus where it induces fos
transcriptional activation (17, 18). An alternative route leading to
fos transcription independent of Ras/ERK, is the activation
of the signal transducers and activators of transcription (STAT)
pathway, leading to formation of three transcription factors that bind
to the fos promoter. These transcription factors, known as
sis-inducible factor (SIF) A, B, and C, are STAT-3
homodimers, STAT1/3 heterodimers, and STAT1 homodimers, respectively.
In addition, another Ras-independent pathway relevant for mitogenesis
is the activation of the cytosolic tyrosine kinase Src. Recently,
Barone and Courtneidge (19) have demonstrated that the kinases of the
Src family regulate myc activation, and that myc
is necessary for PDGF-dependent DNA synthesis in NIH 3T3
cells. In fact, myc but not fos rescue the
PDGF-signaling block caused by dominant negative Src. Both the Ras/ERK
and the Src pathways are necessary for DNA synthesis in response to
PDGF, epidermal growth factor, and colony-stimulating factor-1 (19). In
addition, the Src and STAT pathways seems to be linked (20, 21),
although it remains to be established if this connection is directly
mediated by Src kinase. Finally, other pathways relevant for PDGF
mitogenic signaling are PLC
1 and PI3K. PLC
1 catalyzes the
breakdown of phosphatidylinositol bisphosphate, triggering a cytosolic
calcium increase and protein kinase C activation. PI3K is a protein and
lipid kinase that activates the product of the proto-oncogene
akt, protein kinase C
, and other targets, which have been
only partially elucidated.
In this study, we examine the role of the LMW-PTP in signaling pathways originated by PDGF-R activation. We report that LMW-PTP is involved in the regulation of Myc expression through Src activation, and of Fos through an ERK independent pathway mediated by the STAT proteins.
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EXPERIMENTAL PROCEDURES |
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Materials--
Unless specified all reagents were obtained from
Sigma. NIH 3T3 cells were purchased from ATCC; human recombinant
platelet-derived growth factor BB (PDGF-BB) was from Peprotech; RC20
anti-phosphotyrosine antibodies were from Affiniti; enhanced
chemiluminescence kit was from Amersham; c-fos murine
cDNA was from ATCC; human c-myc cDNA was a generous
gift from G. I. Evans; and Src kinase assay kit, anti-Src
antibodies, and PDGF receptor antibodies were from Upstate
Biotechnology, Inc. Agarose-conjugated phosphotyrosine antibodies were
purchased from Oncogene Science. Anti-ERK1, anti-phosphotyrosine, anti-PDGF receptor, and anti-PLC1 antibodies were from Santa Cruz
Biotechnology, and anti-STAT1 and anti-STAT3 antibodies were from
Transduction Laboratories.
Cell Culture and Transfections-- NIH 3T3 cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in 5% CO2 humidified atmosphere. 10 µg of pSVT7PTPC12S (12) or pSVT7PTP (22) and 0.5 µg of pSV2neo, conferring neomycin resistance, were cotransfected in NIH 3T3 cells using the calcium phosphate method. Stable transfected clonal cell lines were isolated by selection with G418 (400 µg/ml). Control cell lines were obtained by transfecting 2 µg of pSV2neo alone. The clonal lines were screened for expression of the transfected genes by a) Northern blot analysis and b) enzyme-linked immunosorbent assay using polyclonal anti-LMW-PTP rabbit antibodies, which do not crossreact with murine endogenous LMW-PTP.
Immunoprecipitations and Western Blot Analysis-- 1 × 106 cells were seeded in 10-cm plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were serum-starved for 24 h before receiving PDGF-BB. Cells were then lysed for 20 min on ice in 500 µl of modified RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 2 mM EGTA, 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Lysates were clarified by centrifugation and immunoprecipitated for 4 h at 4 °C with 5 µg of the specific antibodies. Immune complexes were collected on protein A-Sepharose (Pharmacia Biotech Inc.), separated by SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose (Sartorius). Immunoblots were incubated in 3% bovine serum albumin, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 0.1% Tween-20, for 1 h at room temperature and then probed first with specific antibodies and then with secondary antibodies conjugated with horseradish peroxidase, washed and developed with the enhanced chemiluminescence kit (Amersham).
Cell Cycle Analysis--
Growth data were obtained plating
2 × 105 cells for 48 h. Cell number was
estimated by counting in a Burker chamber. Cell doubling times
(cdt) were calculated as: cdt = 48/(b a), where a is
log2 of the cell number at time of plating and b
is log2 of cell number at 48 h of standard exponential
growing conditions.
mRNA Analysis-- 1 × 107 cells were serum-starved for 24 h and than stimulated with 30 ng/ml of PDGF. Total RNA was purified according to a method previously described (24). 10 µg of total RNA were separated onto a denaturing 15% formaldehyde-agarose gel and blotted onto nitrocellulose filters using standard methods. Hybridization was performed overnight in 4× standard saline citrate, 0.1% SDS, 5× Denhart's, 1 mM EDTA, 20 mM sodium phosphate, pH 7.2, at 65 °C using a random priming labeled cDNA with specific activity of about 109 cpm/µg. Washings were performed twice in 0.5× standard saline citrate, 0.1% SDS at 55 °C; the filter was then autoradiographed and the signals quantitated with a densitometer. Normalization was done on the basis of a human actin cDNA probe hybridization.
ERK Activity Assay--
ERK activity was assayed by
phosphorylation of microtubule-associated protein 2 (MAP-2) as
described by Mihasaka (25). Briefly, 3 × 105 cells
were seeded in 6-cm dishes and serum-starved for 24 h. Cells
stimulated with 5-50 ng/ml PDGF were harvested by scraping in 0.2 ml
of lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 2 mM EGTA, 1 mM orthovanadate, 0.1 mM molybdate, 1% Triton X-100, 1 mM
phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). 10 µl of cell lysate (1.5 mg/ml of total protein) were
incubated with 2 µg of MAP-2 for 10 min at 30 °C in a final volume
of 25 µ l, containing 50 mM Tris-HCl, pH 7.4, 2 mM EGTA, 10 mM MgCl2, 40 µM [-32P]ATP (1 µCi, 3000 Ci/mmol).
The reaction was stopped by the addition of 4× Laemmli
SDS-polyacrylamide gel electrophoresis sample buffer. Phosphorylated
MAP-2 was resolved by SDS-polyacrylamide gel electrophoresis (7.5%
acrylamide gel). Coomassie Blue-stained bands containing MAP-2 protein
were excised from the gel, and incorporated radioactivity was measured
by scintillation counting. ERK activity was also measured by an
in vitro kinase assay after immunoprecipitation. 70 µg of
total proteins in modified RIPA lysis buffer were immunoprecipitated with anti-ERK1 polyclonal antibodies and with the use of protein A-Sepharose. After five washings with 1 ml of modified RIPA buffer, the
immunobeads were incubated in 25 µl of a buffer containing 15 mM Tris-HCl, pH 7.4, 15 mM MgCl2,
0.5 mM EGTA, 40 mM ATP, 1 µCi
[
-32P]ATP (3000 Ci/mmol), and 10 µg of a peptide
derived from myelin basic protein (Santa Cruz Biotechnology). After 5 min of incubation at 30 °C the reaction was stopped by adding 20 µl of 40% trichloroacetic acid. 25 µl of the reaction mixture were
spotted onto 2 × 2-cm phosphocellulose disks, washed four times
in 0.75% phosphoric acid and once in acetone. Radioactivity was
evaluated by scintillation counting. The amount of immunoprecipitated
ERK1 from each sample was evaluated by anti-ERK1 immunoblot.
Densitometric analysis of the immunoblot was used for normalization of
the immunokinase assay.
Cell Motility Assay-- Migration of NIH 3T3 cells expressing wtLMW-PTP or dnLMW-PTP was assayed with the Boyden chamber system for chemotaxis (Nucleopore Corp., Pleasanton, CA) equipped with 8-µm pore polyvinylpyrrolidone-free polycarbonate filters (13-mm diameter) (26). Polycarbonate filters were precoated with human type I collagen (20 µg/ml in phosphate-buffered saline, pH 7.4) for 30 min at 37 °C and placed between the chemoattractant (lower chamber) and the upper chamber. The lower chamber was filled with medium supplemented with different concentrations of PDGF-BB. Cells cultured in serum-free Dulbecco's modified Eagle's medium were suspended by trypsinization, and 3 × 104 cells in 200 µl was added to the top wells and incubated at 37 °C in 5% CO2 for 6 h. After incubation the cells that had attached to the upper side but had not migrated through the filter were mechanically removed using cotton swabs. The filters were fixed in 96% methanol and stained with Harris hematoxylin solution. Chemotaxis was quantitated by counting the cells that had migrated to the lower surface of the polycarbonate filters. For each filter the number of cells in six randomly chosen fields was determined, and the counts were averaged (mean ± S.D.).
Phosphatidylinositol 3-Kinase Activity--
PI3K assay was
performed as described elsewhere (27, 28). Briefly, serum-starved cells
were incubated with 30 ng/ml of PDGF for 10 min and then lysed in RIPA
buffer. Equal amounts of protein were immunoprecipitated using an
agarose-conjugated anti-phosphotyrosine antibody. After washing, the
immunobeads were resuspended in 50 µl of 20 mM Tris-HCl,
pH 7.5, 100 mM NaCl, and 0.5 mM EGTA. 0.5 µl
of 20 mg/ml phosphatidylinositol was added, mixed, and incubated at
25 °C for 10 min. 1 µl of 1 M MgCl2 and 10 µCi of [-32P]ATP (3000 Ci/mmol) were then added
simultaneously and incubated at 25 °C for an additional 10 min. The
reaction was stopped by the addition of 150 µl of chloroform,
methanol, 37% HCl, 10:20:0.2. The samples were extracted with
chloroform and dried. Radioactive lipids were separated by thin-layer
chromatography using chloroform, methanol, 30% ammonium hydroxide,
water 46:41:5:8. After drying, the plates were autoradiographed.
Identity of the 3-OH phosphorylated lipids after separation by
thin-layer chromatography has been previously confirmed using
high-pressure liquid chromatography (28). The radioactive spots
corresponding to phosphatidylinositol phosphate were scraped and
counted in a liquid scintillation counter.
Gel Mobility Shift Assay--
This assay has been carried out as
described previously (29). Briefly, double-stranded synthetic
oligonucleotides corresponding to the high affinity m67 oligonucleotide
5'-CAGTTCCCGTCAATC (30) were synthesized using a 329 DNA/RNA
synthesizer (Applied Biosystems). The single-stranded oligonucleotides
were annealed and labeled using T4 polynucleotide kinase and
[-32P]ATP (3000 Ci/mmol). Labeled DNA was separated
from the unincorporated radioactivity using an ion-exchange column
(Schleicher & Schuell). 10 µg of total cell extracts were incubated
in binding buffer (35 mM HEPES, pH 7.8, 0.5 mM
EDTA, 0.5 mM dithiothreitol, 10% glycerol, and 250 mM spermidine) containing 10 µg/ml of poly(dI·dC) (Pharmacia) and 50,000-100,000 cpm of radiolabeled DNA for 30 min at
25 °C.
Src Kinase Assay--
1.5 × 106 cells were
seeded in 10-cm dishes, serum-starved for 24 h and then stimulated
with 50 ng/ml of PDGF for 5 min at 37 °C. Cell were lysed in
modified RIPA buffer (50 mM Tris-HCl, pH 7.5, 2 mM EGTA, 1 mM orthovanadate, 1% Nonidet P-40,
0.25% sodium deoxycholate, 1 mM phenylmethanesulfonyl
fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Lysates were
clarified by centrifugation and assayed for protein content. 5 µg of
anti-Src antibodies were added to 500 µg of total proteins. The
reaction mixtures were incubated overnight at 4 °C. Immune complexes
were collected using 50 µl of protein A-Sepharose resin that had been
preincubated with 5 µg of rabbit anti-mouse IgG. The beads were
incubated in the reaction mixture for 1 h at 4 °C. The beads
were washed three times with 500 µl of modified RIPA buffer and once
with 1 ml of cold phosphate-buffered saline. The beads were then
incubated for 15 min at 30 °C in 40 µl kinase buffer (25 mM Tris-HCl, pH 7.2, 30 mM MgCl2, 6 mM MnCl2, 0.5 mM EGTA, 0.1 mM orthovanadate, 0.5 mM dithiothreitol, 125 µM [-32P]ATP, corresponding to 1 µCi)
containing 10 µg of a Src specific peptide. The reaction was stopped
by adding 20 µl of 40% trichloroacetic acid. After centrifugation 25 µl of supernatant were spotted onto 2 × 2-cm phosphocellulose
paper, that was subsequently washed in a 0.75% phosphoric acid
solution and then in pure acetone. Radioactivity was quantitated in a
scintillation counter. The amount of immunoprecipitated c-Src from each
sample was evaluated by anti-c-Src immunoblot. Densitometric analysis
of the immunoblot was used for normalization of the immunokinase
assay.
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RESULTS |
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LMW-PTP Expression-- NIH 3T3 cell line was chosen to assess the role of LMW-PTP in PDGF signaling. Expression plasmids containing the coding sequence for either wtLMW-PTP and dnLMW-PTP, in which the critical active site cysteine residue had been mutated to serine (C12S), were transfected into the NIH 3T3 cell line. Clonal selection of neomycin-resistant cells resulted in the establishment of cell lines overexpressing active LMW-PTP or the dominant negative form of LMW-PTP, respectively. To verify the overexpression of LMW-PTP and to choose clones with comparable LMW-PTP expression for subsequent studies, we used polyclonal anti-LMW-PTP antibodies in an enzyme-linked immunosorbent assay analysis of whole cell lysates from each of the derivative cell lines in comparison to neomycin cells. Enzyme-linked immunosorbent assay quantification data of LMW-PTP overexpression are presented in Fig. 1A. For further analysis, we chose four NIH 3T3-transfected clones with comparable LMW-PTP expression levels (wtLMW-PTP cl.2 and cl.12 and dnLMW-PTP cl.6 and cl.14, respectively). We previously demonstrated that overexpression of wtLMW-PTP and dnLMW-PTP in NIH 3T3 cells resulted in an association between the phosphatase and the PDGF receptor, leading, in the case of wtLMW-PTP, to receptor dephosphorylation (12, 13). We analyzed the PDGF receptor tyrosine phosphorylation of both cl.2 and cl.12 overexpressing wtLMW-PTP and cl.6 and cl.14 overexpressing dnLMW-PTP, respectively. PDGF receptor was immunoprecipitated from cells stimulated with 30 ng/ml PDGF-BB for 5' and then its tyrosine phosphorylation was analyzed by anti-phosphotyrosine immunoblotting. Fig. 1B presents the results obtained. Scanning densitometry of these data revealed that both dnLMW-PTP-overexpressing clones showed an increased tyrosine phosphorylation of PDGF receptor (lanes 2 and 3) with respect to control (lane 1). In contrast, clones overexpressing the active phosphatase (lanes 4 and 5) showed the opposite phenotype, displaying a decreased level of receptor phosphorylation. As already published, overexpression of the dnLMW-PTP in NIH 3T3 cells causes an increased mitogenic response to PDGF, whereas overexpression of the wtLMW-PTP has an opposite effect (11, 12). Moreover, we observed that cell morphology, viability, and protein content were not affected by LMW-PTP overexpression. In any case, no difference among clones overexpressing either wtLMW-PTP or dnLMW-PTP was ever observed. For these reasons, clones 2 and 14 were randomly chosen for further analysis.
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LMW-PTP Acts Specifically in G1 Phase-- To investigate the role of LMW-PTP in the downstream signaling events originating from PDGF-R, we analyzed the possible variations in cell cycle phases distribution in response to LMW-PTP overexpression. The cellular doubling time during exponential growth in complete medium was 20.7 ± 0.4 h (mean ± S.D.) for neomycin cells (expressing neomycin resistance alone), 17.6 ± 0.4 and 29.9 ± 0.7 h for NIH 3T3 cells overexpressing dnLMW-PTP or wtLMW-PTP, respectively. These data are in agreement with our previous results clearly showing an opposite phenotypic effect of dnLMW-PTP with respect to wtLMW-PTP. The effects on cell cycle distribution was analyzed by flow cytometry, and results are presented in Table I. dnLMW-PTP-overexpressing cells showed a shorter G1 phase in comparison to neomycin cells, whereas the duration of the other phases remained almost identical. In contrast, cells transfected with wtLMW-PTP showed a marked increase in the duration of G1 phase with respect to neomycin cells but also presented modifications in S and G2/M phases, suggesting the existence of possible additional effects of wtLMW-PTP overexpression.
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LMW-PTP Modulates Src Activity and Myc Expression-- The cytosolic tyrosine kinase Src is recruited and activated by phosphorylated PDGF-R. To investigate the possible interaction of LMW-PTP with this pathway, we evaluated the effects of phosphatase overexpression in NIH 3T3 cells on Src activity in response to PDGF stimulation. Overexpression of the dnLMW-PTP (Fig. 2A) greatly increased Src activity upon stimulation with PDGF in comparison to neomycin cells (about 60% increase), whereas overexpression of wtLMW-PTP resulted in a dramatic decrease of Src kinase activity (less than 30% with respect to neomycin cells). No significant change was observed in the Src activity of serum-starved unstimulated cells.
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LMW-PTP Affects PDGF-stimulated Chemotaxis-- Recent evidences indicates that the Src tyrosine kinase directly phosphorylates the PDGF-R in Tyr-934 (31). This leads to a negative modulation of the signal transduction pathway leading to motility response and shifts the response to increased mitogenicity. Since LMW-PTP appears to modulate the Src kinase activity, dnLMW-PTP or wtLMW-PTP-overexpressing cells were analyzed for their ability to migrate against a gradient of PDGF-BB, employing the leading front assay in a modified Boyden chamber. Measurements were done also in comparison with NIH 3T3 overexpressing c-Src. Cells at a density of 1.5 × 105/ml were resuspended after trypsinization and centrifugation in Dulbecco's modified Eagle's medium and seeded in the upper part of the modified Boyden chamber, while medium containing PDGF-BB was added below the 150-µm thick micropore filter. Compared with neomycin cells (Fig. 4), dnLMW-PTP-overexpressing cells displayed decreased PDGF-stimulated chemotaxis, whereas the wtLMW-PTP showed a clear extension in chemotactic response. As expected, c-Src-overexpressing cells showed a clear decrease in cell mobility as previously reported by Hansen (31). These data suggest that LMW-PTP influences the PDGF receptor-regulated chemotaxis. LMW-PTP probably modulates a signal transduction pathway leading to increased motility response and to a decreased mitogenic response.
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LMW-PTP Is Not Involved in the Regulation of the Ras/ERK Pathway-- The kinase cascade originating by activation of Ras results in ERK activation, which migrates to the nucleus and phosphorylates different transcription factors. The Ras/ERK pathway has been shown to be necessary for growth factor-dependent DNA synthesis. Therefore, we evaluated whether the observed effects of LMW-PTP on cell proliferation could be mediated by actions on this signaling pathway. Three different methods were used to address this issue. First, we assayed ERK activity by measuring the ability of total cell lysates to phosphorylate MAP-2, a specific ERK substrate. As shown in Fig. 5A, PDGF rapidly stimulated the enzyme activity in all cell lines, reaching a maximum within 5 min and slowly declining thereafter. No significant differences were observed in MAP-2 phosphorylation comparing neomycin cells with dnLMW-PTP- or wtLMW-PTP-overexpressing cells.
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LMW-PTP Modulates Fos Expression via STAT1 and STAT3
Activation--
Several soluble mediators activate the so called STATs
pathway with differing specificity with respect to the activated
kinases and the STAT protein complexes formed (33). PDGF and epidermal growth factor induce activation of STAT1 and STAT3 through tyrosine phosphorylation, which binds to the promoter of fos as homo-
and heterodimers known as SIF-A, SIF-B, and SIF-C (34). We analyzed STAT1 and STAT3 tyrosine phosphorylation after PDGF stimulation for 15 min. (Fig. 6). In
dnLMW-PTP-overexpressing cells both STAT1 and STAT3 showed an increased
tyrosine phosphorylation with respect to mock transfected cells,
whereas in wtLMW-PTP-overexpressing cells a decrease of STAT1 and STAT3
tyrosine phosphorylation was observed. In addition, we analyzed STAT1
and STAT3 activation in a gel mobility shift assay using a high
affinity mutated oligonucleotide from the fos promoter (m67)
according to Wagner (30). Formation of the three DNA binding complexes
in response to PDGF stimulation (Fig.
7A) was markedly increased in
cells expressing the dnLMW-PTP (lane 6), whereas it was
dramatically reduced in those transfected with wtLMW-PTP (lane
5). As expected, no complex was formed in the absence of PDGF
stimulation (lanes 1 to 3). Competition with an
excess of unlabeled m67 oligonucleotide resulted in the disappearance of the three complexes (lanes 10-12 and 16),
whereas addition of an unrelated oligonucleotide (specific for NFB,
lane 17) did not affect the DNA binding. The identity of the
proteins involved in DNA binding was confirmed by preincubating the
cell lysates with anti-STAT1 (lanes 7-9) or STAT3
antibodies (lane 14). Anti-STAT1 antibodies resulted in a
further reduction ("supershift") of the electrophoretic mobility of
SIF-B and SIF-C, the two complexes which contain STAT1. Anti-STAT3
antibodies caused the disappearance of the two slower migrating
complexes corresponding to SIF-A and SIF-B, which contain STAT3. As a
control, non-immune mouse IgG did not affect DNA binding of any of the
SIF complexes.
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PI3K or PLC1 Are not Targets of the LMW-PTP--
PI3K and
PLC
1 have been shown to contribute in the transduction of the
mitogenic signal originating from PDGF-R activation (14). To
investigate if LMW-PTP overexpression caused perturbations in these
pathways, we analyzed PI3K and PLC
1 activity. PDGF induced a
dramatic up-regulation of PI3K activity (Fig.
8A), but this variation was
not significantly influenced by the overexpression of either dnLMW-PTP
or wtLMW-PTP (lanes 5 and 6) in comparison to
neomycin cells (lane 4). Furthermore, we evaluated the
amount of receptor-recruited PLC
1. Immunoprecipitates obtained with anti-PDGF receptor antibodies were analyzed on Western blot using anti-PLC
1 antibodies (Fig. 8B). No marked difference was
present in the three samples.
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DISCUSSION |
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Recent data from our laboratory have provided evidence for a role of LMW-PTP in the regulation of PDGF-dependent mitogenesis. In addition, we have reported that the PDGF receptor is a specific in vivo substrate of the LMW-PTP in NIH 3T3 cells and that this phosphatase physically associates with the activated receptor (12). However, no data were available regarding the molecular mechanisms by which the LMW-PTP can shut off the mitogenic signals initiated by PDGF. In this report, we first established that the overexpression of dnLMW-PTP induces a significant shortening of G1 cell cycle phase whereas S and G2/M remain almost unchanged, indicating that the LMW-PTP acts specifically in G1 phase. This is in agreement with the hypothesis that LMW-PTP acts on the activated PDGF receptor (11, 12), which is a specific effector of G1 progression. We have also observed that overexpression of the wtLMW-PTP markedly prolongs the doubling time although the changes are not uniquely restricted to the G1 phase. It is possible that overexpression of the active enzyme can artifactually interact with other cellular districts and/or functions, whereas the dnLMW-PTP would cause fewer and more specific perturbations of the physiology of the cells. However, actions on phases of the cell cycle other than G1 cannot be completely ruled out by the present data.
The main finding of this study is that in cells transfected with the
LMW-PTP not all the pathways originating from the activated PDGF
receptor are affected. Rather, this phosphatase appears to be highly
specific in modulating the Src and the STATs pathways without
interacting with other important routes such as Ras/ERK, PI3K, and
PLC1. The effects on ERK activation are particularly striking since
many extracellular and intracellular effectors may converge at this
level. Along these lines, the lack of changes in ERK activity further
confirms that neither PI3K nor PLC
1 are involved in mediating the
effects of the LMW-PTP. In fact, PI3K has been shown to signal through
routes that are at least in part dependent on Ras (35), and activation
of PLC
1 results in the generation of signals that ultimately lead to
ERK activation via Ras-dependent and -independent pathways
(36, 37). Overexpression of the active and inactive forms of the
LMW-PTP determine a reduced or increased activation of both Src and
STATs pathways. Furthermore, LMW-PTP prevents the direct association of
Src with the tyrosine-phosphorylated PDGF receptor. These modifications
are associated with similar changes in the nuclear targets of these
signaling pathways, namely myc and fos.
Therefore, not only LMW-PTP modulate post-receptor signaling, but the
resulting modifications are sufficient to affect activation of specific
transcription factors.
Src kinase transduces PDGF signaling in a ERK-independent manner (19). Recent results indicate that Src is the starting point of a route that leads to Myc expression, whereas it is not necessary for Ras activation. This so called "Src pathway" is in turn independent of Ras and appears to be required for PDGF-induced DNA synthesis. In fact, microinjection of a dominant negative Myc (Myc In 373) completely inhibits the mitogenic response to PDGF (19). Furthermore, microinjection of antibodies directed against all three Src kinase family proteins blocks both PDGF-induced Myc expression and entry of NIH 3T3 cells into S phase (38). Hence, these data strongly support the hypothesis that myc transcription is under the control of Src family kinases and is required for mitogenesis. Data presented herein indicate that the LMW-PTP is involved in the modulation of the Src pathway activated by the PDGF-R since Src recruitment by the phosphorylated PDGF-R is modified by LMW-PTP overexpression. Src kinase receptor association directly up-regulates its tyrosine kinase activity. It is likely that the modulation in the Src kinase activity observed in LMW-PTP-overexpressing cells is mediated by the LMW-PTP prevention of binding of Src to the receptor. Recent data reported by our group indicate that LMW-PTP is a target of v-Src in vitro (39). However, we failed to observe a physical interaction between c-Src and either dnLMW-PTP or wtLMW-PTP in vivo. A recent paper reports that the recruitment of the Src tyrosine kinase by PDGF-R leads to decreased PDGF-stimulated chemotaxis directly mediated by PDGF-R Tyr-934 phosphorylation by Src kinase (31). Our data on the modulation of the PDGF chemotactic response by LMW-PTP are in agreement with a specific action of LMW-PTP on the Src-mediated pathways leading to increased mitogenicity through Myc activation and to decreased PDGF-stimulated chemotaxis.
Fos activation appears to be strongly implicated in PDGF mitogenic signaling (17, 18). Activation of fos transcription by PDGF is mediated by the serum response element (SRE) and the sis-inducible element (SIE) of the promoter. The SRE interacts with the ternary complex factor, whose components Elk-1 and SAP-1 are direct targets of ERK (18). Conversely, SIE interacts with proteins of the STAT family, STAT1 and STAT3, which form three transcription factors, namely SIF-A, -B, or -C (30). We find that the LMW-PTP modulates Fos expression, which is greater in cells transfected with the dnLMW-PTP and reduced in those expressing wtLMW-PTP. These changes are associated with a modulation of both tyrosine phosphorylation and DNA binding activity of STAT1 and STAT3. On the other hand, ERK activity, measured by three independent methods, is unaffected by the overexpression of the LMW-PTP. The SRE and the SIE are both able to drive fos transcription in studies using deletion mutants of the promoter and a reporter gene (30). However, the relative contribution of the SIE to fos transcription is controversial (40). Data from the present study provide evidence that Fos expression can be modulated by the STATs pathway in vivo in the absence of any changes in the ERK/SRE pathway, even though the possible contribution of other factors cannot be completely ruled out. Therefore, the effects of the LMW-PTP suggest that both the SRE and the SIE are necessary to achieve optimal fos transcription.
The mechanisms involved in the reduced activation of STAT proteins by the LMW-PTP remain to be clarified. Three different Jak kinases, Jak-1, Jak-2, and Tyk-2 have been shown to interact with the PDGF receptor (36). However, none of these enzymes is necessary per se to obtain STATs phosphorylation. Recent data indicate that v-Src transformed cells show greater activation of STAT3 (20, 21), indicating a possible cross-talk between the Src and the STATs pathways. In addition, we have observed that in PDGF-stimulated NIH 3T3 cells c-Src overexpression leads to an up-regulation of both STAT1 and STAT3 (41).
In this study we present evidences showing that the activation of both STAT1 and STAT3 are clearly affected by the LMW-PTP, a finding that could be explained by the concomitant action of LMW-PTP on Src activity, thus suggesting that the STATs and Src pathways could be related. However, the direct LMW-PTP action on a Src-independent STATs pathway cannot be excluded.
In summary, the results of this study indicate that LMW-PTP decreases PDGF mitogenic signaling by selectively interacting with the Src and the STATs pathway, resulting in modulated expression of myc and fos, two proto-oncogenes crucial for G1 progression. Whether the LMW-PTP affects PDGF receptor signal transduction pathways through the dephosphorylation of different receptor tyrosines remains to be established. On the other hand, this phosphatase may directly and specifically interact with only one phosphorylated tyrosine, leading to pleiotropic effects. The available data indicate that LMW-PTP may act as a physiological modulator of the effects of PDGF on cell growth.
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FOOTNOTES |
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* This work was supported by the Italian Association for Cancer Research (AIRC) and in part by the Ministero della Università e Ricerca Scientifica e Tecnologica (MURST) and Consiglio Nazionale delle Ricerche (CNR).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.
§ Supported by grants from MURST (Liver cirrhosis and viral hepatitis project) and from the Italian Liver Foundation.
To whom correspondence should be addressed. Tel.:
39-55-413765; Fax: 39-55-4222725; E-mail:
raugei{at}cesit1.unifi.it.
1
The abbreviations used are: PTP,
phosphotyrosine-protein phosphatase; LMW-PTP, low molecular weight PTP;
dnLMW-PTP, dominant negative LMW-PTP; ERK, extracellular
signal-regulated kinase; MAP-2, microtubule-associated protein 2; PDGF,
platelet-derived growth factor; PDGF-R, PDGF receptor; PI3K,
phosphatidylinositol 3-kinase; PLC1, phospholipase C
1; SIE,
sis-inducible element; SIF, sis-inducible factor;
SRE, serum response element; STAT, signal transducer and activator of
transcription; wtLMW-PTP, wild-type LMW-PTP.
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