From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208
Received for publication, February 13, 2001, and in revised form, April 9, 2001
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ABSTRACT |
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Protein-tyrosine phosphatase 1B (PTP1B) is an
important regulator of protein-tyrosine kinase-dependent
signaling pathways. Changes in expression and activity of PTP1B have
been associated with various human diseases; however, the mechanisms by
which PTP1B expression is regulated have yet to be characterized.
Previously, we have shown that the expression of PTP1B is enhanced by
p210 Bcr-Abl and that PTP1B is a specific antagonist of transformation induced by this oncoprotein protein-tyrosine kinase. Here we have characterized the PTP1B promoter and demonstrate that a
motif with features of a stress-response element acts as a
p210 Bcr-Abl-responsive sequence,
termed PRS. We have shown that three C2H2 zinc
finger proteins, namely Sp1, Sp3, and Egr-1, bind to PRS. Whereas
binding of either Sp1 or Sp3 induced promoter function, Egr-1 repressed Sp3-mediated PTP1B promoter activation. The binding of
Egr-1 to PRS is suppressed by p210 Bcr-Abl due to the inhibition of
Egr-1 expression, resulting in the enhancement of PTP1B
promoter activity. Our data indicate that Egr-1 and Sp family proteins
play a reciprocal role in the control of expression from the
PTP1B promoter.
The actions of protein-tyrosine phosphatases
(PTPs)1 and kinases (PTKs)
are coordinated in vivo to regulate the reversible phosphorylation of tyrosine residues in proteins and thus control such
fundamental physiological processes as growth and proliferation, differentiation, migration, metabolism, and cytoskeletal function. Furthermore, disturbance of the delicate balance between the actions of
PTKs and PTPs has been shown to be the cause of human diseases, including cancer, diabetes, and inflammation. The PTPs are now known to
consist of a large family of receptor-like and nontransmembrane enzymes
that rival the PTKs in structural diversity and complexity (1).
Furthermore, PTPs can both antagonize PTK-induced signaling events and
cooperate with PTKs to promote signal transduction processes in
vivo, thus highlighting further their importance as an integral
component of the cellular response to environmental cues. With this
realization that PTPs are critical regulators of cellular signaling
events, they have become a focus of attention for their potential to
identify novel targets for therapeutic intervention in human disease.
The prototypic member of the PTP family is PTP1B. It was first purified
from human placenta as a catalytic domain of 37 kDa (2) and was
subsequently shown to occur in vivo as a full-length protein
of ~50 kDa (3-5). PTP1B is composed of an N-terminal catalytic
domain fused to a non-catalytic, C-terminal segment that serves a
regulatory function, targeting the protein to the cytoplasmic face of
membranes of the endoplasmic reticulum (6). Recently, a number of
insights have been gained into the physiological function of PTP1B that
have emphasized its potential importance in various human disease
states. For example, PTP1B has been reported to act as a negative
regulator of signaling events initiated by several growth
factor/hormone receptor PTKs, including the epidermal growth factor and
insulin receptors, as well as signaling events induced by cytokines
(1). Several reports noted enhanced expression levels of PTP1B in
diabetic and insulin-resistant patients and animal models that may
affect insulin signaling (7-9). Furthermore, it was also reported that
there might be a negative feedback loop, governed by insulin and
insulin-like growth factor stimulation, which could lead to
up-regulation of PTP1B gene expression (10, 11). Of
particular interest has been the demonstration that PTP1B knockout mice
display hypersensitivity to insulin and resistance to diet-induced
obesity (12, 13). Recent structural studies have defined how PTP1B
recognizes the activation loop of the insulin receptor as a substrate
and thus can modulate signaling in response to stimulation by the
hormone (14). These observations suggest the exciting possibility that
an inhibitor of PTP1B may be of therapeutic use in the treatment of
diabetes and obesity (15). Furthermore, changes in the levels of PTP1B
have been noted in several human diseases, particularly those
associated with disruption of the normal patterns of tyrosine
phosphorylation. Work from our own laboratory (16) has shown that the
expression of PTP1B is induced specifically by the p210 Bcr-Abl
oncoprotein, a PTK that is directly responsible for the initial
manifestations of chronic myelogenous leukemia. We have also observed
that PTP1B, but not TC-PTP, its closest relative, suppresses
p210 Bcr-Abl-mediated signaling and transformation (17). Therefore,
PTP1B may also function as an antagonist of the p210 Bcr-Abl
oncoprotein PTK in vivo. In addition, changes in PTP1B
levels have been associated with other human cancers associated with
oncoprotein PTKs (18, 19). However, the exact molecular mechanism by
which PTP1B gene expression is regulated in these contexts
remains to be determined.
We have observed that the effects of p210 Bcr-Abl on expression of the
PTP1B gene are manifested at the transcriptional level. In
order to define the mechanism by which PTP1B gene expression is regulated, we cloned the 5'-flanking region of the human gene. We
found two elements that are important for expression from the human
PTP1B promoter. A sequence motif that possesses features of
a site of interaction with GATA-binding proteins was identified at
Isolation and Cloning of the 5'-Flanking Region of the Human
PTP1B Gene and Plasmid Construction--
The 5'-flanking segment of
the human PTP1B gene was isolated by PCR using the
GenomeWalker kit (CLONTECH). Two gene-specific primers (GSPs), 5'-GATAATGGCCGCCCAGCTCCCGGACTTGTC-3' as GSP1 (31-60 bp
relative to the translation start site) and
5'-CCCAAGCTTGAGATCTCTCGAGGATCTGCTCGAACTCCTTTTCCATCTCCAT-3' as GSP2 (1-30 bp relative to the translation start site and including an additional HindIII site), were selected on the basis of
the sequence of the human PTP1B cDNA reported by Brown-Shimer
et al. (5). Primary PCR and secondary nested PCR were
performed on adapter-ligated genomic libraries using the GSP1 and GSP2
gene-specific primers and the AP1 and AP2 adapter sequence primers. The
resulting 2-kb PCR fragment was subcloned into the TA cloning vector
pCR2.1-TOPO (Invitrogen) and sequenced.
A series of 5'-deletion mutants of the PTP1B 5'-flanking region were
made by PCR. The primers used for making of deletion mutants
were as follows: for Determination of 5'-Untranslated Region (UTR) Sequence--
In
order to identify the transcriptional start site, we used the CapSite
5' end cDNA library (Eurogenetec) as a template for PCR, using two
5' adapter sequences (RC1 and RC2) as 5' primers together with
an internal sequence in PTP1B (241-270 bp from the translation
start site, 5'-GTTAGGCAAAGGGCCCTGGGTAAGAATGTA-3') as the 3' primer. The
other internal sequence in PTP1B (1-30 bp relative to the translation
start site, 5'-ATGGAGATGGAAAAGGAGTTCGAGCAGATC-3') was used as the
5' control primer.
Cell Culture--
Parental Rat1 fibroblasts and Rat1 fibroblasts
expressing p210 Bcr-Abl (Rat1 p210), a catalytically inactive mutant of
p210 Bcr-Abl (Rat1 p210 kinase negative) and v-Abl (Rat1 v-Abl) were cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. Drosophila SL2 cells were maintained in Schneider medium supplemented with 10% fetal bovine serum at 25 °C.
Transient Transfection--
Cells were transfected using
LipofectAMINE Reagent (Life Technologies, Inc.) according to the
supplier's protocols. Typically, we used 1 µg of the reporter
plasmid for expression of firefly luciferase and 1 µg of pRL-TK
(Promega), an expression vector containing cDNA encoding
Renilla luciferase, as an internal control of transfection
efficiency. For Drosophila SL2 cells, 2.0 × 106 cells were used for each transfection with
LipofectAMINE Reagent (Life Technologies, Inc.). One µg of the
reporter plasmid for expression of firefly luciferase was used.
Expression plasmids for either human Sp1 (pPac Sp1), Sp3 (pPacU Sp3),
or control plasmid without insert (pPac) were cotransfected with
different amounts of Egr-1 expression plasmid (pPac Egr-1) in the
amounts indicated in the legend to Fig. 6. Cells were incubated with
DNA-lipid complex for 24 h and washed with phosphate-buffered
saline, and luciferase activity was assayed using the Dual-Luciferase
Reporter Assay System (Promega).
DNA "Pull-down" Assay--
Nuclear extracts were prepared
from either Rat1, Rat1 p210 Bcr-Abl, or Rat1 p210 Bcr-Abl kinase
negative cells according to the method of Sadowski and Gilman (24).
Nuclear extract (100 µg) was incubated in a final volume of 1 ml of
20 mM HEPES, pH 7.9, 20 mM NaF, 1 mM Na3VO4, 1 mM
Na4P2O7, 0.125 µM
okadaic acid, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml
pepstatin, 0.2% Nonidet P-40, 100 mM NaCl, 5% glycerol,
together with each biotinylated DNA probe (200 pmol) and 20 µl of
streptavidin-agarose (Oncogene) at 4 °C for 6 h. The
protein-DNA complexes were subjected to SDS-PAGE followed by
immunoblotting with either anti-Egr-1, anti-Sp1, or anti-Sp3
antibodies. Bound proteins were visualized by ECL (Amersham Pharmacia
Biotech).
The following biotinylated, double-stranded DNA probes were used: for
PRS, biotin-5'-CTGCTTCAGGGGCGGAGCCCCTGGCAG-3' as sense and
5'-CTGCCAGGGGCTCCGCCCCTGAAGCAG-3' as antisense; for mutated PRS probe
(PRSM), biotin-5'-CTGCTTCAGAAACGGAGCCCCTGGCAG-3' as a sense
DNA and 5'-CTGCCAGGGGCTCCGTTTCTGAAGCAG-3' as antisense (in
which the underlined bases represent the mutation site).
Immnoblotting--
Nuclear extract (20 µg) from each sample
was resolved by SDS-PAGE and transferred to a PVDF membrane. Membranes
were hybridized with antibodies against Egr-1, Sp1, or Sp3 and
subjected to ECL (Amersham Pharmacia Biotech). Antibodies to Egr-1
(C-19), Sp1 (PEP2), and Sp3 (D-20) were purchased from Santa Cruz Biotechnology.
Northern Blotting--
Total RNA was extracted using the TRIZOL
reagent (Life Technologies, Inc.) according to the manufacturer's
protocols. Total RNA (20 µg) was resolved on a 1%
agarose-formaldehyde gel and transferred to Hybond N+ (Amersham
Pharmacia Biotech) nylon filters. Filters were hybridized with
32P-labeled probe in a buffer comprising 0.5 M
NaPO4, pH 7.0, 0.1 mM EDTA, 7% SDS, 1% bovine
serum albumin at 60 °C for 12-16 h, then washed with 2× SSC, 0.1%
SDS for 10 min and with 0.1× SSC, 0.1% SDS for 30 min at 60 °C,
and then subjected to autoradiography. The cDNA probes used were
rat egr-1 (1.8-kb, EcoRI-BamHI
fragment), human sp1 (2.2- and 1.8-kb, BamHI
fragment) and human sp3 (2.5-kb, NotI fragment).
Definition of Promoter Elements in the Human PTP1B Gene--
In
order to begin to elucidate the molecular mechanisms underlying
regulation of expression of the human PTP1B gene, we
isolated a 2-kb fragment of the 5'-flanking region of the gene. The
sequence of the proximal region up to
A search for potential sites for interaction with DNA-binding proteins
in the 5'-flanking region of the human PTP1B gene, using
TFSEARCH, revealed several candidates. These include potential sites of interaction with the following: the AML-1 DNA-binding protein,
which has been implicated in hematopoietic cell proliferation and
differentiation and is the product of a gene that is disrupted in
certain myelogenous leukemias (25); the AP-1 transcription factor
complex, which is regulated in response to various growth, differentiation, and stress stimuli (26); CHOP, which is induced under
conditions of cellular stress (27); Ets-1, the founding member of a
family of transcription factors that display homology to viral
oncogenes and have been implicated in the control of growth,
differentiation, and development (28); Ikaros 2, from the Ikaros family
of zinc finger transcription factors that have been implicated in the
regulation of lymphocyte development (29); MyoD, the muscle
differentiation factor (30); c-Myb, which is essential for normal
control of hematopoiesis (31); and NF- The Activity of the PTP1B Promoter Is Enhanced by Expression of
p210 Bcr-Abl--
Our laboratory has demonstrated previously (16) that
the levels of PTP1B mRNA and protein are enhanced specifically in
various cells expressing p210 Bcr-Abl and that the PTK activity of the p210 Bcr-Abl oncoprotein is required for this effect. We have now
generated a reporter construct in which expression of luciferase is
driven by potential PTP1B promoter elements. This reporter construct, which contains 5'-flanking sequence from the
PTP1B gene extending from Regulation of PTP1B Promoter Activity by a p210 Bcr-Abl-responsive
Sequence (PRS)--
To define the cis-acting element(s) in the
PTP1B promoter, we made sequential truncations in the
5'-flanking region of the PTP1B gene and incorporated these
deletion mutants into constructs where they drove expression of a
luciferase reporter gene. The promoter activity of these constructs was
compared following transfection into parental and p210
Bcr-Abl-transformed Rat1 fibroblasts. By using this approach, we
identified a transcriptional enhancing element and a potential
p210 Bcr-Abl-responsive sequence,
which we have termed PRS (Fig. 3).
Truncation as far as The C2H2 Zinc Finger Proteins Egr-1, Sp1,
and Sp3 Bind PRS--
Since we had noted that the sequence of the PRS
motif possessed the features of an STRE, we assessed whether mammalian
C2H2 zinc finger proteins would bind to this
motif from the PTP1B promoter. We performed DNA
"pull-down" assays, using biotinylated DNA probes derived from the
sequence of PRS and nuclear extracts of Rat-1 fibroblast lines, to
determine which proteins may bind to this motif. We observed that three
C2H2 zinc finger family transcription factors,
Egr-1, Sp1 and Sp3, from nuclear extracts of parental Rat-1
fibroblasts, associated with PRS probes (Fig.
4A). A mutant PRS probe, in
which STRE-1 was disrupted (PRSM), displayed significantly reduced
affinity for these proteins (Fig. 4A). Furthermore, we have
used nuclear extracts of parental, p210 Bcr-Abl, and catalytically inactive p210 Bcr-Abl-expressing Rat-1 fibroblasts to determine the
effects of expression of p210 Bcr-Abl on the interaction of these
transcription factors with the PRS probe. We observed that expression
of p210 Bcr-Abl led to a decrease in the level of Egr-1 protein (Fig.
4, A and B) and mRNA (Fig. 4C),
compared with that in parental Rat1 cells, resulting in a decrease in
the interaction detected between Egr-1 and the PRS DNA probe (Fig.
4A). In contrast, the presence of p210 Bcr-Abl enhanced the
interaction of Sp1 protein with PRS (Fig. 4A) without
affecting the expression level of this transcription factor (Fig. 4,
B and C). These effects were dependent upon the
catalytic activity of p210 Bcr-Abl. The expression levels of Sp3 and
its association with the PRS probe were unaltered by p210 Bcr-Abl (Fig.
4). These data indicate that PRS, which is a regulator of the activity
of the PTP1B promoter, binds Sp1, Sp3, and Egr-1 and that
expression of p210 Bcr-Abl controls the activity and expression of Sp1
and Egr-1, respectively.
Regulation of PTP1B Promoter Function by the Sp1, Sp3, and Egr-1
Transcription Factors--
In order to test directly whether the
C2H2 zinc finger family transcription factor
Egr-1 may regulate PTP1B promoter activity, we performed
cotransfection experiments in which this protein was expressed together
with the reporter constructs in which expression of the luciferase gene
was driven by the PTP1B promoter. As shown in Fig. 4,
expression of p210 Bcr-Abl in Rat-1 fibroblasts suppressed the levels
of endogenous Egr-1 compared with those observed in the parental cells.
We observed that when we expressed Egr-1 ectopically in p210
Bcr-Abl-transformed Rat-1 fibroblasts, up to levels that approximated
those seen in the parental cells (Fig. 5,
B and C), the luciferase activity driven by the
PTP1B promoter was inhibited in a dose-dependent
manner (Fig. 5A). Similar results were observed with
constructs containing either the full-length promoter or PRS alone
(Fig. 5A). These effects occurred without changes in the
levels of Sp1 or Sp3 (data not shown). Furthermore, inhibition of
promoter function by Egr-1 was not observed in the reporter construct
containing the mutant PRS sequence (PRSM) (Fig. 5A). These
data suggest that PTP1B gene expression may be negatively regulated by the transcription factor Egr-1 acting on the PRS motif in
the promoter.
We also conducted similar luciferase reporter assays in
Drosophila SL2 cells, which lack endogenous Sp factors (39),
to assess the effect of Sp1 and Sp3 proteins on the activity of the human PTP1B promoter. The reporter plasmid containing the
intact PTP1B promoter was cotransfected with expression
vectors for Sp1 and/or Sp3, in the presence or absence of the Egr-1
expression vector. We observed that the intact PTP1B
promoter-induced luciferase activity was up-regulated, in a
dose-dependent manner, by either Sp1 or Sp3, suggesting
that PTP1B gene expression may be positively regulated by
either of these transcription factors (Fig.
6A). Interestingly, whereas
Egr-1 did not affect Sp1-mediated up-regulation of PTP1B promoter
activity, Sp3-mediated transactivation of the PTP1B promoter was
suppressed by Egr-1 in a dose-dependent manner (Fig.
6A). Egr-1 also suppressed PTP1B promoter
transactivation mediated by the expression of both Sp1 and Sp3 in SL2
cells (Fig. 6A). Similar effects were observed with
constructs containing either the full-length promoter or PRS alone
(Fig. 6B). Furthermore, there were no effects of Sp1 or Sp3
on promoter activity in constructs containing the mutated PRS sequence
(PRSM) (Fig. 6C). These data suggest that Sp1 and Sp3 may
act as positive regulators of PTP1B gene expression, whereas
Egr-1 may function to down-regulate the PTP1B promoter by
suppressing Sp3-mediated transactivation and that these effects are
mediated through the PRS motif of the promoter.
In this study, we have cloned 5'-flanking sequences and determined
the transcription initiation site of the human PTP1B gene. In addition, we have identified two promoter elements that are responsible for the regulation of PTP1B expression. A transcriptional enhancing element, which is located between PRS is located between We have demonstrated that three mammalian C2H2
zinc finger proteins, Sp1, Sp3, and Egr-1, are bound to PRS (Fig.
4A). Both Sp1 and Sp3 acted as positive regulators of
PTP1B promoter function (Fig. 6, A and
B), whereas Egr-1 functioned negatively (Fig. 5A) to antagonize Sp3-mediated transactivation of the PTP1B
promoter (Fig. 6, A and B). In parental Rat1
fibroblasts in which promoter activity was low (Fig. 3A),
the PRS element recognized Sp1, Sp3, and Egr-1 (Fig. 4A). In
contrast, expression of p210 Bcr-Abl resulted in decreased levels of
Egr-1 protein and consequently a reduction in PRS-bound Egr-1 (Fig. 4),
whereas there was no detectable effect of the oncoprotein PTK on the
levels of Sp1 or Sp3. Although the levels of Sp1 were unchanged, its
association with PRS was enhanced (Fig. 4A). However, the
mechanism by which Sp1 binding to the PRS element of PTP1B
promoter is regulated remains to be determined. Interestingly, it has
been reported that Sp1 may be phosphorylated in vivo,
raising the possibility that such covalent modification would be of
regulatory significance. Furthermore, enhanced phosphorylation of Sp1
following serum stimulation has been associated with
Sp1-dependent activation of the DHFR promoter
(48), and cAMP-dependent protein kinase phosphorylates Sp1
in vitro, and this phosphorylation leads to increased Sp1
DNA binding (49). Since p210 Bcr-Abl activates multiple signaling
pathways, and its effects on PTP1B expression are dependent upon its
PTK activity, expression of this oncoprotein PTK may induce the
phosphorylation and activation of Sp1 in vivo. It will be
important to define which of the signaling pathways initiated by p210
Bcr-Abl affect both the activity of the Sp family of transcription
factors and the levels of Egr-1.
In many promoters containing GC-rich elements, Sp1- and Egr-1-binding
sites have been shown to overlap, and Egr-1 has been shown to modulate
Sp1-mediated transactivation both negatively and positively (45,
50-52). For example, in the case of the platelet-derived growth
factor-A (PDGF-A) and -B (PDGF-B) genes, Egr-1
competes with Sp1 protein for an overlapping region in the promoter and functions as a positive regulator (51, 52). Thus, competition for
promoter-binding sites between Sp and Egr family proteins may be of
regulatory significance. We have observed that ectopic expression of
Egr-1 in p210 Bcr-Abl-transformed Rat-1 fibroblasts was sufficient to
antagonize transcription from the PTP1B promoter (Fig.
5A), consistent with an important role for Egr-1 as a
suppressor of PTP1B expression. Precedent exists for such a function
of Egr-1, which has been shown to act as a negative regulator of
expression from the adenosine deaminase gene promoter (45). The data
from our studies in SL2 cells (Fig. 6) revealed that although
expression of either Sp1 or Sp3 induced PTP1B promoter
activity, this inhibitory effect of Egr-1 appeared to be directed
toward Sp3-mediated transactivation. Thus, the down-regulation of Egr-1
expression, alleviating an inhibitory effect on Sp3-mediated
transactivation, appears to be an important component of the mechanism
of enhancement of PTP1B expression in response to p210 Bcr-Abl.
However, it remains to be determined whether there is competition for
binding sites in the promoter.
It is now becoming apparent that members of the PTP family display
exquisite specificity in their function as regulators of signal
transduction processes in vivo. There are several aspects to
the establishment and maintenance of such specificity. For example,
instances of selectivity in PTP substrate recognition have been
identified and are being characterized at the molecular level (14).
This current study illustrates another aspect of specificity, that of
control of PTP expression. The expression of PTP1B is induced following
aberrant activation of multiple PTK-dependent signaling
pathways in human disease states. For example, PTP1B is induced
specifically in response to p210 Bcr-Abl and functions as an antagonist
of this oncoprotein PTK (16, 17). The results of this study provide the
first mechanistic insights into the control of PTP1B
promoter function and will form the basis for further analyses of the
expression of this important signaling molecule under normal and other
pathophysiological conditions. In particular, although these data
illustrate a reciprocal role of Egr-1 and Sp family members in
controlling PTP1B promoter activity in transfection assays,
it will be important to investigate further which proteins interact
with the promoter under physiological conditions and to examine the
impact of Sp family members on the activity of chromosomal template.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
167 to
151 bp from the transcription start site. Disruption of this site inhibited promoter activity in the presence of p210 Bcr-Abl, but responsiveness to the oncoprotein PTK was maintained. However, a p210 Bcr-Abl-responsive
sequence, termed PRS, which was important for stimulation
of activity in response to the PTK, was identified at
49 to
37 bp
from the transcription start site. The PRS is contained in a sequence
that displays features of a stress-response
element (STRE), a feature originally identified in
Saccharomyces cerevisiae (20-22) and that functions as a
binding site for C2H2 zinc finger proteins
(23). In our study, we have shown that three mammalian
C2H2 zinc finger proteins, Egr-1, Sp1, and Sp3,
bind to PRS. Of these, both of Sp1 and Sp3 function as positive
regulators, whereas Egr-1 represses Sp3-mediated transactivation of the
PTP1B gene. Furthermore, expression of p210 Bcr-Abl results in down-regulation of the levels of Egr-1. Our data illustrate that the
reciprocal actions of the Sp1/Sp3 and Egr-1
C2H2 zinc finger transcription factors are an
important aspect of the regulation of PTP1B expression in response to
the p210 Bcr-Abl oncoprotein.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
838/+145,
5'-ACTATAGGGCACGCGTCTGGGCCCCACCACACCA-3'; for
247/+145,
5'-ACTATAGGGCACGCGTCCGTGGGCGGGGCTCCCGGG-3'; for
167/+145,
5'-ACTATAGGGCACGCGTACGCGCGCTATTAGATATCT-3'; for
151/+145, 5'-ACTATAGGGCACGCGTATCTCGCGGTGCTGGGGCC-3'; for
49/+145,
5'-ACTATAGGGCACGCGTGGGCGGAGCCCCTGGCAGGCGT-3'; for
37/+145,
5'-ACTATAGGGCACGCGTTGGCAGGCGTGATGCGTAGT-3'; for
49(M1)/+145,
5'-ACTATAGGGCACGCGTAAACGGAGCCCCTGGCAGGCGT-3; and for
49(M2)/+145, 5'-ACTATAGGGCACGCGTGGGCGGAGAAACTGGCAGGCGT-3. The underlined bases represent the site of mutation. The following 3'
primer was used for each PCR to exclude ATG translation initiation site: 5'-CCCAAGCTTGACGGGCCAGGGCGGCTGCTGCGCCTCCTT-3'. Amplified PCR products were digested by MluI and
HindIII and inserted into the pGL3 Basic (Promega) reporter
plasmid, in which promoter activity can be detected by expression of
firefly luciferase.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
838 bp from the transcription
initiation site is shown in Fig.
1A. In order to identify the
transcriptional start site, we used the CapSite 5' end cDNA library
(Eurogenetec) as a template for PCR, using two 5' adapter sequences
(RC1 and RC2) as 5' primers together with an internal sequence in PTP1B (241-270 bp from the translation start site) as the 3' primer. Another
internal sequence in PTP1B (1-30 bp relative to the translation start
site) was used as the 5' control primer. By this approach, we cloned a
0.45-kb fragment (Fig. 1B), sequenced it, and identified 145 bp as the 5'-UTR in human PTP1B mRNA (Fig. 1A).
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Fig. 1.
A, nucleotide sequence of the promoter
region of the human PTP1B gene, highlighting potential
recognition motifs for DNA-binding proteins. B,
determination of 5'-UTR of human PTP1B gene. The DNA
sequence of the 5'-UTR upstream from the translation start site of
human PTP1B was determined by PCR using CapSite cDNA
(Eurogentec) as a template. Primary PCR and secondary nested PCR were
performed using the 1RC and 2RC primers corresponding to the adapter
sequences and adapter-ligated CapSite cDNA derived from human
placenta. The 3' primer consists of 241-270 bp from the translation
start site of human PTP1B and the control 3' primer consists
of 1-30 bp from the translation start site. The resulting 0.45-kb PCR
fragment (Product 1) was subcloned and sequenced. The 5'-UTR
is shown as a gray box in A.
B, which is a critical
regulator of immune and inflammatory gene expression in cell
proliferation and apoptosis (32). In addition, there are putative
binding sites for GATA-binding proteins, which are important regulators
of diverse cell differentiation pathways during development (33-36)
and clusters of GC-rich regions, consistent with sites of interaction
with C2H2 zinc finger proteins, such as the
specificity protein (Sp) family (37). Finally, we observed that the
human PTP1B gene lacks a TATA box, an initiator, or a downstream promoter element (38). It has been reported that promoters
that lack either a TATA box or an initiator element often contain
multiple binding sites for Sp1, as we have noted here for the human
PTP1B gene. However, unlike many such genes that are often
associated with "housekeeping" functions, PTP1B expression varies
in response to a variety of signals, and the enzyme serves a critical
function as a regulator of tyrosine
phosphorylation-dependent signaling pathways (1).
2 kb to +145 bp relative to the
transcription initiation site, was transfected into various Rat-1
fibroblast lines, and production of firefly luciferase was monitored as
a measure of promoter activity. Transfection efficiency was
standardized according to expression of Renilla luciferase
from a control plasmid. We observed 4-fold higher promoter activity in
p210 Bcr-Abl-transformed Rat-1 cells (Rat1 p210 Bcr-Abl) compared with
the parental Rat1 cells (Fig. 2). In
addition, we observed that optimal expression from the PTP1B
promoter required the kinase activity of p210 Bcr-Abl. Furthermore,
expression of v-Abl, which shares the same catalytic domain as p210
Bcr-Abl, stimulated promoter activity only weakly (Fig. 2), in
agreement with our previous observations of the effects of these PTKs
on the levels of PTP1B protein (16). These data suggest that p210
Bcr-Abl triggers signaling events that culminate specifically in
stimulation of the PTP1B promoter.
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Fig. 2.
The activity of the human PTP1B
promoter is enhanced by p210 Bcr-Abl in Rat1 fibroblasts. We
constructed a luciferase reporter plasmid containing the intact
PTP1B promoter. Reporter activity was assessed in parental
Rat1 fibroblasts (Rat1) and Rat1 fibroblasts expressing p210
Bcr-Abl (Rat1 p210), a catalytically inactive mutant form of
p210 Bcr-Abl (Rat1 p210KN) and v-Abl (Rat1
v-Abl). The cells were transiently transfected with 1 µg of the
intact human PTP1B promoter luciferase construct, containing
the 5'-flanking region of the human PTP1B gene,
(pGL3-Basic: 2k/+145), control plasmid, which
contains SV40 promoter and enhancer sequence,
(pGL3-Control), or empty vector (pGL3-Basic), and
1 µg of pRL-TK, to normalize the transfection efficiency. Cells were
harvested, lysed, and assayed for luciferase activity. Values were
normalized for transfection efficiency and represent the means ± S.D. of three separate experiments.
167 bp from transcription initiation site did
not affect total promoter activity or responsiveness to p210 Bcr-Abl.
However, we identified a transcriptional enhancing element, between
167 and
151 bp upstream from the transcription start site (Fig.
3A), that displays features of a binding site for GATA
family binding proteins (33). Deletion of this element by truncation to
151 bp suppressed promoter activity by ~60%, but the
responsiveness of promoter activity to p210 Bcr-Abl was retained (Fig.
3A). Truncation to
49 bp upstream from the transcription
start site resulted in no further alteration in promoter activity,
whereas promoter function was severely attenuated by truncation to
37
bp. Importantly, the residual activity was no longer responsive to p210
Bcr-Abl (Fig. 3A). This latter element, spanning
49 to
37 bp upstream from the transcription start site, is termed PRS and
corresponds to a segment possessing features of an STRE (Fig.
3B). STREs were originally identified in the promoter of the
DDR2 and CTT1 gene in S. cerevisiae
and shown to bind C2H2 zinc finger proteins
following stimulation by heat or osmotic stress (23). We introduced
nucleotide substitution mutations to assess the importance of the
putative STREs in PRS, and we observed that disruption of STRE-1
abolished p210 Bcr-Abl responsiveness, whereas mutation of STRE-2 was
without effect (Fig. 3A). These data suggest that PRS may
have a critical role in p210 Bcr-Abl-mediated up-regulation of PTP1B
expression.
View larger version (26K):
[in a new window]
Fig. 3.
Identification of a p210 Bcr-Abl response
element in the human PTP1B promoter.
A, luciferase constructs bearing various deletions within
the sequences upstream, on the 5' side, of the human PTP1B
gene were generated. Rat1 (stippled bar) and Rat1
p210 Bcr-Abl-transformed cells (black bar) were transiently
transfected with 1 µg of human PTP1B promoter luciferase
construct containing segments of the human PTP1B promoter
including PRS-mutated sequences (pGL3-Basic:-49(M1)/+145 for
STRE1 and pGL3-Basic:-49(M2)/+145 for STRE2), control
plasmid, which contains SV40 promoter and enhancer sequence,
(pGL3-Control), empty vector (pGL3-Basic), and 1 µg of pRL-TK to normalize transfection efficiency. Cells were
harvested, lysed, and assayed for luciferase activity. Values were
normalized to transfection efficiency and represent the means ± S.D. of three separate experiments. B, schematic profile of
human PTP1B promoter. Two elements were identified as
important for the human PTP1B promoter-driven luciferase
activity in response to p210 Bcr-Abl. A p210 Bcr-Abl-responsive
sequence (PRS), which possesses features of an STRE that
interacts with C2H2 zinc finger proteins, was
identified at 49 to
37 bp from the transcription start site. In
addition, an element possessing a conserved sequence motif for
recognition of GATA family proteins, such as GATA-1, GATA-2, was
identified at
167 to
151 bp from the transcription start site and
functioned as a transcriptional enhancing element.
View larger version (24K):
[in a new window]
Fig. 4.
Characterization of proteins that associate
with the PRS element in the human PTP1B promoter.
A, effect of p210 Bcr-Abl on the association of Egr-1, Sp1,
and Sp3 with the PRS element in the PTP1B promoter. Nuclear
extracts were prepared from parental (Rat1), p210
Bcr-Abl-transformed (Rat1 p210), and p210 Bcr-Abl-inactive
mutant (Rat1 p210KN)-expressing Rat1 cells. The extracts
were incubated with biotinylated PRS or mutated PRS (PRSM)
double-stranded DNA probe and streptavidin-agarose for 6 h at
4 °C. The protein-DNA complexes were subjected to SDS-PAGE followed
by immunoblotting with either anti-Egr-1, Sp1, or Sp3 antibodies. The
level of each transcription factor was assessed by immunoprecipitation
(IP) and immunoblotting (IB) using the
appropriate antibodies, followed by visualization by ECL. B,
the expression level of Egr-1 was suppressed by p210 Bcr-Abl. Nuclear
extract (20 µg) of each sample was resolved by SDS-PAGE, transferred
to PVDF membrane, and immunoblotted with antibodies to Egr-1, Sp1, or
Sp3 to assess changes in the expression levels of the transcription factors. The blots were visualized by
ECL. C, the expression of egr-1 mRNA was
suppressed by p210 Bcr-Abl. Total RNA (20 µg) from each sample was
resolved by electrophoresis and transferred to nylon membrane.
Membranes were hybridized with 32P-labeled cDNA probe
and subjected to autoradiography. The probes used were rat
egr-1 and human sp1 and sp3. Equal
loading and integrity of the RNA samples was confirmed by ethidium
bromide staining of 28 S and 18 S rRNAs in the gel.
View larger version (27K):
[in a new window]
Fig. 5.
Ectopic expression of Egr-1 represses the
activity of the human PTP1B promoter in p210
Bcr-Abl-transformed Rat1 fibroblasts. A, luciferase
reporter constructs, bearing either intact 5'upstream sequence of the
human PTP1B gene (pGL3: 2k/+145),
5'-deleted sequence (pGL3:
49/+145), PRS-mutated
sequence (pGL3:
49(M1)/+145), or a control
plasmid (pGL3-Basic) were generated. These reporters were
cotransfected with the indicated quantities of Egr-1 expression plasmid
or control plasmid lacking insert (Empty vector) in Rat1
p210 Bcr-Abl-expressing cells (black bar) or parental Rat1
cells (open bar). Luciferase activity was normalized to
transfection efficiency and is presented as the mean ± S.D. of
three separate experiments. B, immunoblot analysis of Egr-1
protein. Nuclear extract (20 µg) from each sample in Fig.
6A was separated by SDS-PAGE, transferred to PVDF membrane,
and immunoblotted with antibodies to Egr-1. The blots were visualized
by ECL. C, ectopically expressed Egr-1 binds to the PRS
element of the PTP1B promoter. Parental (Rat1) and p210
Bcr-Abl-transformed (Rat1 p210) Rat1 cells were
cotransfected with Egr-1 expression plasmid (2 or 5 µg) and control
empty vector to a total of 5 µg of DNA. Nuclear extracts were
prepared and DNA binding assays performed using the PRS-containing
probe. The levels of PRS-bound Egr-1 and total Egr-1 in the extract
were assessed by immunoblotting (IB) with anti-Egr-1
antibodies and visualized by ECL.
View larger version (23K):
[in a new window]
Fig. 6.
Egr-1 suppresses Sp1/Sp3-mediated activation
of human PTP1B promoter. Drosophila
SL2 cells were cotransfected with luciferase reporter constructs
bearing the intact 5' upstream sequence of the human PTP1B gene (pGL3: 2k/+145)
(A), 5' end-deleted sequence (pGL3:
49/+145)
(B), or PRS-mutated sequence (pGL3:
49(M1)/+145)
(C) and indicated quantities of expression plasmid for Sp1
(pPac Sp1), Sp3 (pPacU Sp3), or Egr-1 (pPac
Egr-1). The total quantity of pPac plasmid DNA was made up to 5 µg by inclusion of the control plasmid lacking the insert
(pPac). After incubation with the DNA-lipid complex for
24 h to facilitate transfection, the cells were lysed, and
luciferase activity was assayed. Values were normalized to transfection
efficiency and represent the means ± S.D. of three separate
experiments. White bar, experiments using pPac vector and
Egr-1; diagonal-lined bar, vector and Sp1; heavy
diagonal-lined bar, vector, Sp1, and Egr-1;
cross-hatched-lined bar (white and black background), vector
and Sp3; cross-hatched line (black and white background),
vector, Sp3, and Egr-1; black bar, vector, Sp1, Sp3, and
Egr-1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
167 and
151 bp upstream of the transcription start site, features the consensus sequence for
recognition of GATA-binding proteins. GATA-binding proteins are a
family of transcription factors that recognize the consensus sequence
T/A(GATA)A/G and are regulators of developmental differentiation pathways. GATA-1 is the founder of a family that now comprises six
members. GATA-1, -2, and -3 are crucial for hematopoiesis (33), GATA-2
and -3 have been implicated in the development of the central nervous
system (34, 36), and GATA-4, -5, and -6 are involved in the development
of the heart and viscera (35). However, further studies will be
required to identify and characterize the proteins that bind to this
site in the PTP1B promoter. Although this element is
important for maximal promoter activity (Fig. 3A),
comparison of the promoter sequences in human and mouse revealed that
the highest level of conservation was in the region surrounding the
second element, PRS (40). Therefore, since this sequence similarity is
consistent with a function of PRS as a general regulator of PTP1B
expression, at this time we have focused our attention on this latter motif.
49 and
37 bp upstream of the transcription
start site in the human PTP1B and is composed of a
palindromic sequence (AGGGG and in the reverse orientation CCCCT)
containing STREs. STREs were first identified in S. cerevisiae and shown to bind C2H2 zinc
finger proteins following stress stimuli (23). Several
C2H2 zinc finger transcription factors have
been identified in mammals, including the specificity protein (Sp)
family (Sp1-4), the early growth response family (Egr-1-4), and
Wilm's tumor proteins (WT1) (37, 41). Sp family members recognize
GC-rich promoter elements such as the GC box, which appears frequently
in the regulatory regions of genes that respond to signaling cues (37).
Sp family proteins are structurally conserved but mediate distinct
functions. For example, Sp1 has been implicated in promoting
transcriptional activation, whereas Sp3 functions either as a repressor
(42) or an activator (43, 44). Egr-1 is the prototypic member of the
Egr family of transcription factors, which contain a DNA binding domain
that is homologous to that found in Sp1 and recognize the consensus
GC-rich DNA sequence (45, 46). Similarly to Sp3, Egr-1 behaves both as
a positive (47) and negative (45) regulator of gene transcription,
depending upon the cell type.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. G. Suske for expression plasmids for Sp1 and Sp3; Drs. J. Milbrandt and E. Adamson for expression plasmids of Egr-1; and Drs. L. Klaman and B. Neel for a plasmid containing the 5'-flanking region of the murine PTP1B. We are grateful to Dr. Bill Tansey for constructive criticism of the manuscript.
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FOOTNOTES |
---|
* This work was supported by the Naito Foundation, a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad (to T. F.), Grant CA53840 from the National Institutes of Health (to N. K. T.), and Grant P30CA45508 from the National Institutes of Health.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY029236.
To whom correspondence should be addressed: Cold Spring Harbor
Laboratory, Demerec Bldg., 1 Bungtown Rd., Cold Spring Harbor, NY
11724-2208. Tel.: 516-367-8846; Fax: 516-367-6812; E-mail: tonks@cshl.org.
Published, JBC Papers in Press, April 20, 2001, DOI 10.1074/jbc.M101354200
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ABBREVIATIONS |
---|
The abbreviations used are: PTP, protein-tyrosine phosphatase; PTP1B, protein-tyrosine phosphatase 1B; PTK, protein-tyrosine kinase; STRE, stress-response element; PCR, polymerase chain reaction; GSPs, gene-specific primers; PVDF, polyvinylidene difluoride; kb, kilobase pair; bp, base pair; PAGE, polyacrylamide gel electrophoresis; UTR, untranslated region.
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