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(Received for publication, January 9, 1997, and in revised form, May 21, 1997)
From the Georgetown University Medical Center, Department of
Pharmacology and the Lombardi Cancer Center,
Washington, D.C. 20007
ABSTRACT Transcription factor Sp1 is a phosphoprotein
whose level and DNA binding activity are markedly increased in
doxorubicin-resistant HL-60 (HL-60/AR) leukemia cells. The
trans-activating and DNA binding properties of Sp1 in
HL-60/AR cells are stimulated by cAMP-dependent protein
kinase (PKA) and PKA agonists and inhibited by PKA antagonists as well
as by the PKA regulatory subunit. Reporter gene activity under the
control of the Sp1-dependent SV40 promoter is stimulated in
insect cells transiently expressing Sp1 and PKA, and the DNA binding
activity of recombinant Sp1 is activated by exogenous PKA in
vitro. These results indicate that Sp1 is a cAMP-responsive transcription factor and that Sp1-dependent genes may be
modulated through a cAMP-dependent signaling pathway.
INTRODUCTION Sp1 is a ubiquitous transcription factor that binds to the
consensus sequence (G/A)(G/A)GGCG(G/T)(G/A)(G/A)(G/T) or GC box (1). It
was initially identified as a HeLa cell-derived factor that activated
six tandem Sp1 sites in the SV40 early promoter (2-4). Sp1 elements of
varying affinity have been characterized in the
HIV-11 (5), herpes simplex
virus thymidine kinase (6), metallothionein IIA (7), and
MDR1 (8) promoter regions, among others.
Sp1 is glycosylated (9) and is phosphorylated at its N terminus by
DNA-dependent protein kinase, a nuclear Ser/Thr kinase that
is stimulated by 3 Type I cAMP-dependent protein kinase (PKA) is a tetrameric
holoenzyme consisting of two regulatory cAMP-binding (R-I) subunits and
two catalytic (C) subunits that dissociate upon binding cAMP (14).
Dissociation of PKA results in its activation and the translocation of
the catalytic subunit to the nucleus (15). Nuclear localization of PKA
is essential to mediate induction of cAMP-regulated genes (16, 17) via
phosphorylation of the cAMP response element (CRE)-binding protein,
CREB (18, 19). Recently, another CRE, termed CRS, has been
characterized in the promoter region of members of the CYP
(20-22) and ferredoxin (23) genes. The CRS binds a transcription
factor similar to Sp1 in size and sequence specificity, and the binding
of this factor to the CRS is inhibited by an Sp1 consensus
double-stranded oligonucleotide (24). The CRS confers high basal levels
of transcription in adrenocortical tumor cells, and gene expression
through the CRS is stimulated by forskolin, an activator of adenyl
cyclase (24).
HL-60/AR leukemia cells (25) exhibit a multidrug-resistant (MDR)
phenotype with constitutively high Sp1 and CREB DNA binding activities
(11, 26). Reversion of drug resistance by the type I PKA antagonist
8-Cl-cAMP results in the down-regulation of CREB DNA binding activity
but not in the levels of CREB and other cAMP-regulated transcription
factors (26), suggesting that the presence of PKA-dependent
transcription factors in these cells may be a prerequisite for
maintenance of the MDR phenotype.
In the present study, we investigated whether the
cAMP-dependent signaling pathway could modulate the DNA
binding and trans-activating properties of Sp1. Our results
indicate for the first time that Sp1 is activated by PKA and that a
clearly defined cAMP signaling pathway may be responsible for
up-regulating the DNA binding and trans-activating
properties of this transcription factor.
EXPERIMENTAL PROCEDURES HL-60 and HL-60/AR cells (25) were obtained
from the American Type Culture Collection and Dr. James E. Gervasoni,
Jr. (Columbia University), respectively. HL-60 cells were grown in RPMI
1640 medium supplemented with 10% heat-inactivated fetal calf serum (Biofluids, Inc.), 40 mM Hepes (pH 7.4), and 50 µg/ml
gentamicin. HL-60/AR cells were maintained in the same medium with 1 µM doxorubicin but were diluted 10-fold with
doxorubicin-free medium prior to use.
8-Cl-cAMP and doxorubicin were obtained from the Natural Products
Branch, Developmental Therapeutics Program, National Cancer Institute.
RpcAMP[S], SpcAMP[S], and Rp-8-Cl-cAMP[S] were obtained from
Biolog Life Science (Bremen, Germany).
Nuclear extracts were prepared from
5 × 106 cells as described (27). Nuclear extracts (7 µg of protein) were incubated for 30 min at room temperature with 2 fmol (680 Ci/mmol) of a double-stranded Sp1 consensus
oligodeoxynucleotide (Stratagene) that was end-labeled with
[ When Sp1 DNA binding activity was measured in the presence of PKA, 0.5 ng of purified Sp1 (99% purity, Promega) was incubated with 40 units
of the PKA catalytic subunit (Sigma) in the presence of 10 mM MgCl2, 20 mM Tris (pH 7.4), and
40 µM ATP.
Nuclear extracts (50 µg of protein) were
separated by SDS-polyacrylamide gel electrophoresis and blotted onto
nitrocellulose as described previously (28). Sp1 was detected with a
rabbit polyclonal antibody (generously provided by Dr. Robert Tjian, University of California at Berkeley) diluted 1:1,000 in 1% dry milk,
10 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 0.05%
Tween 20. Alkaline phosphatase-conjugated goat anti-rabbit IgG served
as the secondary antibody, and CSPD® served as the chemiluminescent
substrate (Tropix Inc.). Autoradiography was performed by exposure of
the blot to Fuji RX film.
HL-60 and HL-60/AR cells were suspended at a
concentration of 1.5 × 107 cells/0.5 ml RPMI 1640 containing 40 mM Hepes (pH 7.4). The cell suspension was
incubated for 15 min in a 0.4-cm electroporation cuvette with either 20 µg of pSV-CAT (Promega) or p Sf9 insect cells were transfected by lipofection (Lipofectin, Life
Technologies, Inc.). Cells were grown in serum-free SF900 medium (Life
Technologies, Inc.) under air at 27 °C and transfected with 10 µg
of pSV40-CAT, 2 µg of pPacSp1, and 10 µg of
pPacC Cells were harvested by
centrifugation at 500 × g for 5 min, washed once with
cold phosphate-buffered saline, suspended in 90 µl of 250 mM Tris (pH 7.8) containing 0.5 mM
phenylmethylsulfonyl fluoride, and sonicated with ten 1-s bursts at
4 °C. Cell debris was removed by centrifugation at 10,000 × g for 10 min at 4 °C. 60 µl of extract were heated at
70 °C for 10 min and centrifuged at 10,000 × g for
10 min, and the supernatant was used for measuring CAT activity (30).
Cell lysate (50 µl) was incubated in a reaction mixture containing:
100 mM Tris (pH 7.8), 6 mM MgCl2,
75 mM KCl, 0.5 mM sodium acetate, 0.5 mM coenzyme A, 3.75 mM ATP, 50 µM
chloramphenicol, and 0.25 µCi of [14C] chloramphenicol
(40-60 mCi/mmol, New England Nuclear) for 4-20 h at 37 °C.
Acetylated chloramphenicol was recovered by three extractions with
xylene, and the amount of acetylated [14C]chloramphenicol
was determined by liquid scintillation spectrometry (31).
The human Sp1 cDNA was
cloned into the NotI and SmaI site in baculovirus
expression vector pVL1392 under the control of the polyhedrin promoter
(32). The recombinant virus was plaque purified and used to infect Sf9
cells as described previously (33). Sf9 cells (4 × 106) were infected with 5 plaque-forming units/cell of
recombinant virus for 1 h, and cells were harvested 60 h
after infection. Cells were washed once in ice-cold phosphate-buffered
saline and suspended in 4 ml of buffer D containing 20 mM
Hepes (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 10 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mg/ml aprotinin and homogenized three times with 20 strokes of a Dounce homogenizer at 10-min intervals. Cell homogenates
were centrifuged at 100,000 × g for 1 h at
4 °C. The resulting supernatant was applied to a radial DEAE column
(MemSep1010, Millipore) attached to a Pharmacia FPLC. Elution was
carried out at 4 °C at a flow rate of 2 ml/min with a linear
gradient of 0-1.0 M NaCl in buffer D, and 2-ml fractions
were collected. Fractions containing Sp1 were determined by Western
blotting and were pooled, diluted 3-fold with buffer D, and applied to
a Mono Q HR 5/5 column (Pharmacia). Elution was carried out with a
linear gradient of 0-0.4 M NaCl in buffer D at a flow rate
of 1.0 ml/min. Sp1 was approximately 25% pure after Mono Q
chromatography as determined by Rapid Coomassie Stain (Diversified
Biotech) of 10% SDS-polyacrylamide gels.
For determining Sp1 DNA binding activity, the fractions obtained from
the Mono Q chromatography step were incubated with PKA catalytic
subunit for 10 min at 30 °C in a 50-µl reaction mixture containing
20 mM Tris-HCl (pH 7.4), 10 mM
MgCl2, and 40 µM ATP (28), and gel shift
assays were carried out as described above.
Cell extracts were prepared from
Sp1-expressing Sf9 cells as described above and incubated for 20 min at
30 °C with a buffer containing 20 mM Tris-HCl (pH 7.4),
10 mM MgCl2, and 4 µCi of [ For gel shift assays, 30 ng (0.5 footprint unit) of purified Sp1
(Promega) were incubated for 20 min in the presence or the absence of
20 units of purified bovine heart PKA catalytic subunit RESULTS 8-Cl-cAMP specifically targets the high affinity cAMP
binding sites in the regulatory R-I subunit of PKA (35), which results in the sustained activation and proteolysis of type I PKA and the
down-regulation of cAMP-dependent transcription factors
(26, 28). To determine if Sp1 DNA binding activity is regulated in a
similar fashion, HL-60/AR cells were treated for 48 h with 2.5 µM 8-Cl-cAMP, a 10% inhibitory concentration, and DNA
binding activity was measured by mobility shift analysis (Fig.
1A). Nuclear extracts from
HL-60/AR cells (Fig. 1A, lanes 3 and
4) exhibited increased basal Sp1 DNA binding activity
compared with wild type cells (Fig. 1A, lanes 1 and 2). Treatment of HL-60/AR cells with 8-Cl-cAMP markedly
reduced Sp1 DNA binding activity (Fig. 1A, lanes
5 and 6). Because the MDR phenotype in HL-60/AR cells
is maintained by exposure to doxorubicin, Sp1 DNA binding activity was
measured in the absence of selective pressure by maintenance of
HL-60/AR cells in drug-free medium (Fig. 1B). Growth of
cells in doxorubicin-free medium for 2 weeks resulted in the virtual disappearance of Sp1 activity (Fig. 1B, lane 1)
that was dramatically induced upon re-exposure to 1 µM
doxorubicin for either 24 or 48 h (Fig. 1B, lanes
2 and 3). In contrast, maintenance of HL-60/AR cells in
1 µM doxorubicin plus the addition of either the
nonhydrolyzable PKA antagonists RpcAMP[S] and Rp-8-Cl-cAMP[S] (36)
or 8-Cl-cAMP counteracted the up-regulation of Sp1 activity by
doxorubicin (Fig. 1B, lanes 4-6). The levels of
Sp1 as determined by Western blotting were not affected under these
conditions (results not shown).
To
determine whether the increased Sp1 DNA binding activity in HL-60/AR
cells correlated with Sp1-dependent transcriptional activation, HL-60/AR cells were transfected with a plasmid containing the CAT reporter gene under the control of the SV40 promoter, which
contains six tandem Sp1 response elements (2) (Fig.
2). CAT activity was increased 27-fold in
HL-60/AR cells compared with wild type cells, and transcriptional
activation correlated closely with DNA binding activity (Fig. 1). In
comparison, transcription from p
We next determined if endogenous PKA is required for Sp1
trans-activation. In these experiments, HL-60/AR cells were
cotransfected with pSV40-CAT and pOT1521R-I containing the R-I subunit
of PKA (38) (Fig. 3). In the absence of
R-I overexpression, treatment of HL-60/AR cells with the
nonhydrolyzable and membrane permeable PKA agonist, SpcAMP[S] (36),
stimulated CAT activity approximately 2-fold, whereas RpcAMP[S]
attenuated CAT activity by 20%. Transfection of HL-60/AR cells with
increasing amounts of R-I produced a progressive inhibition of CAT
activity in the absence of drug treatment, and upon treatment with
RpcAMP[S], reporter gene activity was blocked completely. In
contrast, the inhibitory effect of R-I was reversed with SpcAMP[S].
Transfection with the empty vector, pOT1521, did not inhibit CAT
activity (results not shown).
To further assess the requirement for PKA in Sp1-dependent
trans-activation, Sf9 insect cells, which lack endogenous
Sp1 and have low PKA activity, were cotransfected with pSV40-CAT and
pPacSp1 in the presence or the absence of
pPacC
Because
these results strongly suggested that PKA was necessary for the
activation of Sp1 trans-activating and DNA binding activities, Sp1 was expressed in Sf9 insect cells using a recombinant baculovirus, and DNA binding activity was determined in the absence and
the presence of PKA (Fig. 5A).
Sp1 was partially purified by DEAE and Mono Q anion-exchange
chromatography that resulted in the elution of approximately equal Sp1
levels in fractions 17-20 as determined by immunoblotting (Fig.
5B). Fractions 17 and 18 were devoid of basal DNA binding
activity, but the addition of exogenous PKA resulted in a marked
stimulation of activity (Fig. 5A). On the other hand,
fractions 19 and 20 did exhibit DNA binding activity that was further
stimulated by PKA. Sp1 phosphorylation was also determined in
vitro with cell lysates from Sp1-expressing insect cells (Fig.
5C). Assays were conducted with uninfected Sf9 cell extract
in the presence of exogenous purified PKA (lane 1), and in
Sp1-containing extracts in the presence (lane 2) and the
absence (lane 3) of the PKA inhibitor, PKI, as well as in the presence of exogenous PKA (lane 4). These results
indicate that Sp1 is phosphorylated by both endogenous and exogenous
PKA, and that PKA can stimulate Sp1 DNA binding activity. These results also suggest that the presence of partially activated Sp1 in fractions 19 and 20 (Fig. 5A) is a result of its partial
phosphorylation by endogenous PKA.
The phosphorylation of Sp1 by PKA was also determined using purified
Sp1 (Fig. 6, A and
B). Phosphorylated Sp1 was readily detected before (Fig.
6A) and after (Fig. 6B) immunoprecipitation with
an Sp1 polyclonal antibody. It is interesting that PKA coprecipitated with Sp1, suggesting high affinity between these proteins. Sp1 incubated in the presence of PKA exhibited greater DNA binding activity
than in its absence (Fig. 6C), and incubation of PKA phosphorylated Sp1 with PP2A markedly reduced its DNA binding activity
(Fig. 6D).
DISCUSSION The present study provides strong evidence that Sp1
trans-activating and DNA binding activities are modulated
through a cAMP/PKA signaling pathway. Momoi et al. (24) was
the first to show that a CRS with homology to the Sp1 response element
was involved in the cAMP-dependent transcriptional
activation of the bovine CYP 11A and human CYP
21B genes. Our data are consistent with these results and
demonstrate further that PKA can stimulate transcription from an
Sp1-dependent promoter in intact cells as well as activate the DNA binding activity of Sp1 in vitro. In previous
studies, Sp1 was shown to be phosphorylated at multiple sites in HeLa
cell nuclear extracts and to be a substrate in vitro for
DNA-dependent protein kinase; however,
DNA-dependent protein kinase did not affect the extent and
specificity of DNA binding or Sp1-dependent transcription
(40). In contrast, our data indicate that not only is Sp1
phosphorylated by PKA but that the DNA binding and trans-activating activities are stimulated as well. One
mechanism that may account for these results is that phosphorylation of the trans-activation domain of Sp1 results in increased Sp1
multimerization. Sp1 domain B confers high affinity DNA binding (41)
and contains a PKA consensus phosphorylation site at
Thr366. Although still speculative, PKA phosphorylation may
induce conformational changes similar to those that occur by
multimerization through this domain (42, 43). The tight association
between Sp1 and PKA is similar to the complex formed between PKA
catalytic subunit and NF- PKA activity remained unchanged in HL-60/AR cells regardless of whether
cells were maintained in doxorubicin (26), suggesting that
drug-mediated Sp1 activation may require an additional signaling mechanism. One possibility is that protein dephosphorylation is also
regulated by PKA and that this process may be a rate-limiting factor in
Sp1 deactivation. Maximal activation of Sp1 may require the
inactivation of PP1 and PP2A by the PKA-mediated activation of a
nuclear inhibitory protein such as NIPP-1 (45). This mechanism is
analogous to the attenuation of CREB by PP1 and PP2A (46, 47). Phorbol
esters also mediate inactivation of Sp1 DNA binding in HL-60/AR cells
(11), and although PKC does not phosphorylate Sp1 in vitro
(40), it does activate PP2A (48). These results are consistent with the
inhibitory effect of phorbol esters on CRS-dependent
transcription (49). Therefore, the mechanism of Sp1 inactivation may
comprise multiple signaling pathways involving phosphorylation and
dephosphorylation.
Sp1 activity was up-regulated by doxorubicin treatment, suggesting that
MDR-associated drugs can directly influence trans-activation and the MDR phenotype. Because the promoter region in the Sp1 gene
contains five Sp1 sites with three additional elements in the first
intron (50), exposure of cells to doxorubicin may create a positive
feedback loop that results in its autoregulation. This mechanism may
also account for the increased expression of type I PKA, because the
promoter of the R-I gene contains multiple Sp1 response elements (51).
In addition, the MRP drug transporter gene, which is
expressed in HL-60/AR cells, also contains multiple Sp1 elements (52),
and therefore, there may be a close association between selective
pressure and Sp1 activation as a mediator of resistance.
FOOTNOTES ACKNOWLEDGEMENTS We are indebted to Dr. Robert Tjian,
University of California at Berkeley, for providing the Sp1 antibody
and plasmids pPacSp1 and pADH- REFERENCES
Volume 272, Number 34,
Issue of August 22, 1997
pp. 21137-21141
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-termini in DNA (10). Nevertheless, phosphorylation
of Sp1 by DNA-dependent protein kinase does not affect
either its trans-activating or DNA binding activities (10). However, dephosphorylation of Sp1 has been suggested to enhance Sp1 DNA
binding activity (11-13), and thus, the roles of Sp1 phosphorylation and the target protein kinase involved in this process still remain unclear.
-32P]ATP and T4 polynucleotide kinase. Mobility shift
assays were performed as described previously (11). Incubation was
carried out in 20 µl of binding buffer containing: 10 mM
Tris-HCl (pH 7.5), 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 12.5% glycerol, and 2 µg of
poly(dI-dC)·poly(dI-dC). Reaction mixtures with recombinant Sp1 also
contained 10 µM ZnSO4. After incubation, the
reaction mixture was loaded directly onto a 4% polyacrylamide gel and
separated by electrophoresis at 100 V for 4 h at 4 °C (28).
Autoradiography was performed by exposure of the dried gel to Fuji-RX
film.
(
71) CAT, 2 µg pRSV-
Gal (to
normalize for transfection efficiency), and 50 µg DEAE-Dextran
(Promega). Electroporation was carried out at room temperature using a
Gene Pulser (Bio-Rad) set at 300 V and 960 microfarad. Following
electroporation, cells were diluted with 12 ml of RPMI 1640 medium
containing 10% fetal bovine serum, and after 2 h, either 100 µM RpcAMP[S] or 100 µM SpcAMP[S] was added, and cells were incubated for 24 h. In some experiments, HL-60/AR cells were cotransfected in the presence of 2 µM
CdCl2 with control plasmid pOT1521 or pOT1521R-I
(generously provided by Dr. Yoon Sang Cho-Chung, National Cancer
Institute) containing the regulatory subunit of type I PKA under the
control of the mouse metallothionein I promoter (29).
. CAT activity was normalized for transfection
efficiency by cotransfecting cells with 2 µg of pADH-Gal
containing the lacZ gene under the control of the
Drosophila melanogaster alcohol dehydrogenase promoter.
-Galactosidase activity was measured by incubating 30 µl of cell
lysate in 100 µl of reaction mixture containing: 2.5 mg/ml
o-nitrophenyl--D-galactopyranoside in 0.1 M sodium phosphate (pH 7.5), 1 mM
MgCl2, and 45 mM -mercaptoethanol. The
reactions were carried out in 96-well plates for 20-45 min, and
absorbance was read at 410 nm in a microplate reader (Series 750, Cambridge Technology, Inc.).
-32P]ATP (3000 Ci/mmol). In some instances, either 40 units of purified PKA catalytic subunit (Sigma) or 0.5 µg of PKA
inhibitor, PKI (Sigma), was included in the assay. The reaction was
terminated with 5 × SDS-polyacrylamide gel electrophoresis sample
buffer, and samples were separated in a 10% polyacrylamide gel by
SDS-polyacrylamide gel electrophoresis. Autoradiography was performed
by exposing the dried gel to Fuji RX film. Phosphorylation of purified
Sp1 (99% pure, Promega) was carried out in a similar manner.
(Sigma) and
40 µM ATP. For immunoprecipitation, purified Sp1 was
(Promega) was incubated as described for gel shift assays and incubated
overnight at 4 °C with 1 µg of Sp1 polyclonal antibody (Santa Cruz
Biotechnology) as described previously (34).
Fig. 1.
Modulation of Sp1 DNA binding activity in
HL-60/AR cells by doxorubicin and PKA antagonists. Nuclear
extracts were prepared from HL-60/AR cells, and Sp1 DNA binding
activity was measured by mobility shift assay. A, mobility
shift assays were carried out in the absence () and the presence (+)
of a 200-fold molar excess of Sp1 double-stranded competitor. HL-60
cells (lanes 1 and 2); HL-60/AR cells maintained
in 1 µM doxorubicin (lanes 3 and 4)
were treated for 48 h with 2.5 µM 8-Cl-cAMP
(lanes 5 and 6). B, cells were
maintained for 2 weeks in doxorubicin-free medium (lane 1)
and then treated for 24 (lane 2) or 48 h (lane 3) with 1 µM doxorubicin. Cells exposed to 1 µM doxorubicin for 24 h were also cotreated with 100 µM RpcAMP[S] (lane 4), 50 µM Rp-8-Cl-cAMP[S] (lane 5), or 5 µM 8-Cl-cAMP
(lane 6).
[View Larger Version of this Image (26K GIF file)]
(71)-CAT, which contains a single
CRE (37), was enhanced 5-fold in resistant cells (Fig. 2).
Fig. 2.
Sp1- and CREB-dependent reporter
gene expression in HL-60/AR cells. HL-60 and HL-60/AR cells were
co-transfected with 20 µg of pSV40-CAT to assess
Sp1-dependent transcription or p(71)CAT to assess
CRE-dependent transcription and 2 µg of pRSV-Gal to correct for transfection efficiency. CAT activity was measured after
24 h and is normalized per unit (U) of
-galactosidase activity. Each value is the mean ± S.E. of
three experiments.
[View Larger Version of this Image (39K GIF file)]
Fig. 3.
Sp1-dependent reporter gene
expression in HL-60/AR cells is regulated by PKA. HL-60/AR cells
were transfected with pSV40-CAT and varying amounts of pOT1521R-I
containing the PKA regulatory subunit cDNA. Cells were maintained
for 24 h in the absence (
) or the presence of 100 µM RpcAMP[S] (
) or 100 µM SpcAMP[S]
(
) beginning 2 h after electroporation. CAT activity is
normalized to -galactosidase activity and is expressed as a
percentage of control activity in the absence of pOT1521R-I. Each value
is the mean ± S.E. of three experiments.
[View Larger Version of this Image (15K GIF file)]
, which contains the PKA catalytic subunit under
the control of the D. melanogaster actin 5C promoter (39)
(Fig. 4). No CAT activity was observed in
the absence of Sp1 transfection (results not shown), but a small degree of trans-activation was observed when Sp1 alone was
expressed. In contrast, CAT activity was stimulated 9-fold when cells
were transfected with pPacSp1 and pPacC.
Fig. 4.
PKA activates Sp1-dependent
reporter gene expression in Sf9 insect cells. Sf9 cells were
transfected with 10 µg of pSV40-CAT, 2 µg of pPacSp1,
and 10 µg of either pPac0 or pPacC. CAT
activity is normalized to -galactosidase activity that was expressed
by cotransfection with 5 µg of pADH-Gal. Each value is the
mean ± of three experiments.
[View Larger Version of this Image (39K GIF file)]
Fig. 5.
PKA activates baculovirus-expressed Sp1
in vitro. Nuclear extracts were prepared from Sf9
cells 60 h after infection with a recombinant Sp1 baculovirus.
A, Sp1 was eluted from a Mem Sep 1010 radial DEAE column
(Millipore) with a linear gradient of 0-1.0 M NaCl. The
fraction eluting at 0.3 M NaCl contained Sp1 (as detected
by immunoblotting) and was diluted, applied to a MonoQ column, and
eluted with a linear gradient of 0-0.4 M NaCl. Fractions
17-19 were assessed for DNA binding activity by mobility shift assay
in the absence () or the presence (+) of the PKA catalytic subunit.
B, Western blot for Sp1 of fractions 17-20 shown in
A. C, phosphorylation in vitro of Sp1
in cell lysates of Sf9 cells. Lysates were prepared from either
uninfected (lane 1) or Sp1-expressing Sf9 insect cells
(lanes 2-4) and incubated with [-32P]ATP
in the presence (lane 2) or the absence (lane 3)
of the PKA inhibitor, PKI. Extracts were also prepared from Sp1- and PKA-expressing Sf9 cells (lane 4). The upper
arrow denotes phosphorylated Sp1, and the lower arrow
denotes autophosphorylated PKA.
[View Larger Version of this Image (49K GIF file)]
Fig. 6.
In vitro phosphorylation and DNA
binding activity of purified Sp1. A, Sp1 was incubated with
PKA in vitro and separated by SDS-polyacrylamide gel
electrophoresis in a 10% polyacrylamide gel. The upper
arrow denotes Sp1, and the lower arrow denotes the
catalytic subunit of PKA. B, immunoprecipitation of Sp1
incubated as in A with preimmune serum (PIS,
lane 1) or Sp1 antiserum (Sp1, lane
2). The upper arrow denotes Sp1, and the lower
arrow denotes the catalytic subunit of PKA. C, DNA
binding activity of Sp1 in the presence of PKA. Gel shift assays were
as described in Fig. 5. Sp1 was incubated in the absence of exogenous
PKA (lane 1) or in the presence of PKA in the absence
(lane 2) or the presence (lane 3) of a 200-fold
excess of unlabeled Sp1 competitor. Assays were also conducted without
Sp1 in the presence (lane 4) or the absence (lane
5) of PKA. D, incubation of PKA-phosphorylated Sp1 with
PP2A. Gel shift assays were carried out with PKA-phosphorylated Sp1 in
the absence (lane 1) or the presence of 0.5 units PP2A. The
arrow denotes the Sp1-DNA complex.
[View Larger Version of this Image (43K GIF file)]
B (44) and gives further credence to this
mechanism of Sp1 regulation.
Present address: Oxford GlycoSciences, Ltd., 10 The Quadrant,
Abingdon, OX14 3YS, UK.
§
To whom correspondence should be addressed. Tel.: 202-687-8324;
Fax: 202-687-8324; E-mail: glazerr{at}gunet.georgetown.edu.
1
The abbreviations used are: HIV-1, human
immunodeficiency virus, type 1; PKA, cAMP-dependent protein
kinase; C, PKA catalytic subunit; R-I, type I cAMP regulatory
subunit; MDR, multidrug-resistant; PP1, protein phosphatase 1; PP2A,
protein phosphatase 2A; CAT, chloramphenicol acetyltransferase.
Gal and to Dr. Yoon Sang
Cho-Chung, National Cancer Institute, for providing pOT1521R-I,
p(71)CAT, and the C cDNA.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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 P. R. Kiela, N. Kuscuoglu, A. J. Midura, M. T. Midura-Kiela, C. B. Larmonier, M. Lipko, and F. K. Ghishan Molecular mechanism of rat NHE3 gene promoter regulation by sodium butyrate Am J Physiol Cell Physiol, July 1, 2007; 293(1): C64 - C74. [Abstract] [Full Text] [PDF]
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B.-K. Chen, C.-C. Huang, W.-C. Chang, Y.-J. Chen, U. Kikkawa, K.-i. Nakahama, I. Morita, and W.-C. Chang PP2B-mediated Dephosphorylation of c-Jun C Terminus Regulates Phorbol Ester-induced c-Jun/Sp1 Interaction in A431 Cells Mol. Biol. Cell, March 1, 2007; 18(3): 1118 - 1127. [Abstract] [Full Text] [PDF]
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Md. R. Amin, J. Malakooti, R. Sandoval, P. K. Dudeja, and K. Ramaswamy IFN-{gamma} and TNF-{alpha} regulate human NHE3 gene expression by modulating the Sp family transcription factors in human intestinal epithelial cell line C2BBe1 Am J Physiol Cell Physiol, November 1, 2006; 291(5): C887 - C896. [Abstract] [Full Text] [PDF]
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N. Pore, A. K. Gupta, G. J. Cerniglia, Z. Jiang, E. J. Bernhard, S. M. Evans, C. J. Koch, S. M. Hahn, and A. Maity Nelfinavir Down-regulates Hypoxia-Inducible Factor 1{alpha} and VEGF Expression and Increases Tumor Oxygenation: Implications for Radiotherapy. Cancer Res., September 15, 2006; 66(18): 9252 - 9259. [Abstract] [Full Text] [PDF]
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 K. Hasegawa, S. Wakino, T. Tanaka, M. Kimoto, S. Tatematsu, T. Kanda, K. Yoshioka, K. Homma, N. Sugano, M. Kurabayashi, et al. Dimethylarginine Dimethylaminohydrolase 2 Increases Vascular Endothelial Growth Factor Expression Through Sp1 Transcription Factor in Endothelial Cells Arterioscler. Thromb. Vasc. Biol., July 1, 2006; 26(7): 1488 - 1494. [Abstract] [Full Text] [PDF]
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N. Pore, Z. Jiang, A. Gupta, G. Cerniglia, G. D. Kao, and A. Maity EGFR Tyrosine Kinase Inhibitors Decrease VEGF Expression by Both Hypoxia-Inducible Factor (HIF)-1-Independent and HIF-1-Dependent Mechanisms. Cancer Res., March 15, 2006; 66(6): 3197 - 3204. [Abstract] [Full Text] [PDF]
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 M. L. Spengler and M. G. Brattain Sumoylation Inhibits Cleavage of Sp1 N-terminal Negative Regulatory Domain and Inhibits Sp1-dependent Transcription J. Biol. Chem., March 3, 2006; 281(9): 5567 - 5574. [Abstract] [Full Text] [PDF]
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 M. Stark and Y. G. Assaraf Loss of Sp1 function via inhibitory phosphorylation in antifolate-resistant human leukemia cells with down-regulation of the reduced folate carrier Blood, January 15, 2006; 107(2): 708 - 715. [Abstract] [Full Text] [PDF]
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 Z. Wu, H.-P. Kim, H.-H. Xue, H. Liu, K. Zhao, and W. J. Leonard Interleukin-21 Receptor Gene Induction in Human T Cells Is Mediated by T-Cell Receptor-Induced Sp1 Activity Mol. Cell. Biol., November 15, 2005; 25(22): 9741 - 9752. [Abstract] [Full Text] [PDF]
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J.-J. Hung, C.-Y. Wu, P.-C. Liao, and W.-C. Chang Hsp90{alpha} Recruited by Sp1 Is Important for Transcription of 12(S)-Lipoxygenase in A431 Cells J. Biol. Chem., October 28, 2005; 280(43): 36283 - 36292. [Abstract] [Full Text] [PDF]
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B. L. Kelly, R. Vassar, and A. Ferreira {beta}-Amyloid-induced Dynamin 1 Depletion in Hippocampal Neurons: A POTENTIAL MECHANISM FOR EARLY COGNITIVE DECLINE IN ALZHEIMER DISEASE J. Biol. Chem., September 9, 2005; 280(36): 31746 - 31753. [Abstract] [Full Text] [PDF]
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 R. J. Phillips, A. J. Tyson-Capper (nee Pollard), J. Bailey, S. C. Robson, and G. N. Europe-Finner Regulation of Expression of the Chorionic Gonadotropin/Luteinizing Hormone Receptor Gene in the Human Myometrium: Involvement of Specificity Protein-1 (Sp1), Sp3, Sp4, Sp-Like Proteins, and Histone Deacetylases J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3479 - 3490. [Abstract] [Full Text] [PDF]
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X. Chen, H.-Y. Zhang, K. Pavlish, and J. N. Benoit Effects of chronic portal hypertension on small heat-shock proteins in mesenteric arteries Am J Physiol Gastrointest Liver Physiol, April 1, 2005; 288(4): G616 - G620. [Abstract] [Full Text] [PDF]
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 G. Pages and J. Pouyssegur Transcriptional regulation of the Vascular Endothelial Growth Factor gene-a concert of activating factors Cardiovasc Res, February 15, 2005; 65(3): 564 - 573. [Abstract] [Full Text] [PDF]
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 J. Ye, D. Shedd, and G. Miller An Sp1 Response Element in the Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50 Promoter Mediates Lytic Cycle Induction by Butyrate J. Virol., February 1, 2005; 79(3): 1397 - 1408. [Abstract] [Full Text] [PDF]
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S. Prudhomme, G. Oriol, and F. Mallet A Retroviral Promoter and a Cellular Enhancer Define a Bipartite Element Which Controls env ERVWE1 Placental Expression J. Virol., November 15, 2004; 78(22): 12157 - 12168. [Abstract] [Full Text] [PDF]
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 S. Dunzendorfer, H.-K. Lee, and P. S. Tobias Flow-Dependent Regulation of Endothelial Toll-Like Receptor 2 Expression Through Inhibition of SP1 Activity Circ. Res., October 1, 2004; 95(7): 684 - 691. [Abstract] [Full Text] [PDF]
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C. Banchio, L. M. Schang, and D. E. Vance Phosphorylation of Sp1 by Cyclin-dependent Kinase 2 Modulates the Role of Sp1 in CTP:Phosphocholine Cytidylyltransferase {alpha} Regulation during the S Phase of the Cell Cycle J. Biol. Chem., September 17, 2004; 279(38): 40220 - 40226. [Abstract] [Full Text] [PDF]
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N. C. Browner, H. Sellak, and T. M. Lincoln Downregulation of cGMP-dependent protein kinase expression by inflammatory cytokines in vascular smooth muscle cells Am J Physiol Cell Physiol, July 1, 2004; 287(1): C88 - C96. [Abstract] [Full Text] [PDF]
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L. Zhang and P. A. Insel The Pro-apoptotic Protein Bim Is a Convergence Point for cAMP/Protein Kinase A- and Glucocorticoid-promoted Apoptosis of Lymphoid Cells J. Biol. Chem., May 14, 2004; 279(20): 20858 - 20865. [Abstract] [Full Text] [PDF]
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D. Trisciuoglio, A. Iervolino, A. Candiloro, G. Fibbi, M. Fanciulli, U. Zangemeister-Wittke, G. Zupi, and D. Del Bufalo bcl-2 Induction of Urokinase Plasminogen Activator Receptor Expression in Human Cancer Cells through Sp1 Activation: INVOLVEMENT OF ERK1/ERK2 ACTIVITY J. Biol. Chem., February 20, 2004; 279(8): 6737 - 6745. [Abstract] [Full Text] [PDF]
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G. Wang, A. B. Leiter, E. W. Englander, and G. H. Greeley Jr. Insulin-Like Growth Factor I Increases Rat Peptide YY Promoter Activity through Sp1 Binding Sites Endocrinology, February 1, 2004; 145(2): 659 - 666. [Abstract] [Full Text] [PDF]
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 P. Grimaldi, F. Capolunghi, R. Geremia, and P. Rossi Cyclic Adenosine Monophosphate (cAMP) Stimulation of the Kit Ligand Promoter in Sertoli Cells Requires an Sp1-Binding Region, a Canonical TATA Box, and a cAMP-Induced Factor Binding to an Immediately Downstream GC-Rich Element Biol Reprod, December 1, 2003; 69(6): 1979 - 1988. [Abstract] [Full Text] [PDF]
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 J. B. Griffin, R. Rodriguez-Melendez, and J. Zempleni The Nuclear Abundance of Transcription Factors Sp1 and Sp3 Depends on Biotin in Jurkat Cells J. Nutr., November 1, 2003; 133(11): 3409 - 3415. [Abstract] [Full Text] [PDF]
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J. Zhang, S. Wang, R. A. Wesley, and R. L. Danner Adjacent Sequence Controls the Response Polarity of Nitric Oxide-sensitive Sp Factor Binding Sites J. Biol. Chem., August 1, 2003; 278(31): 29192 - 29200. [Abstract] [Full Text] [PDF]
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Q. Wei, W. K. Miskimins, and R. Miskimins The Sp1 Family of Transcription Factors Is Involved in p27Kip1-Mediated Activation of Myelin Basic Protein Gene Expression Mol. Cell. Biol., June 15, 2003; 23(12): 4035 - 4045. [Abstract] [Full Text] [PDF]
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A. Ray, P. Ray, N. Guthrie, A. Shakya, D. Kumar, and B. K. Ray Protein Kinase A Signaling Pathway Regulates Transcriptional Activity of SAF-1 by Unmasking Its DNA-binding Domains J. Biol. Chem., June 13, 2003; 278(25): 22586 - 22595. [Abstract] [Full Text] [PDF]
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I. J. Arinze and Y. Kawai Sp Family of Transcription Factors Is Involved in Valproic Acid-induced Expression of Galpha i2 J. Biol. Chem., May 9, 2003; 278(20): 17785 - 17791. [Abstract] [Full Text] [PDF]
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 H. Ryu, J. Lee, K. Zaman, J. Kubilis, R. J. Ferrante, B. D. Ross, R. Neve, and R. R. Ratan Sp1 and Sp3 Are Oxidative Stress-Inducible, Antideath Transcription Factors in Cortical Neurons J. Neurosci., May 1, 2003; 23(9): 3597 - 3606. [Abstract] [Full Text] [PDF]
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 D. L. Russell, K. M. H. Doyle, I. Gonzales-Robayna, C. Pipaon, and J. S. Richards Egr-1 Induction in Rat Granulosa Cells by Follicle-Stimulating Hormone and Luteinizing Hormone: Combinatorial Regulation By Transcription Factors Cyclic Adenosine 3',5'-Monophosphate Regulatory Element Binding Protein, Serum Response Factor, Sp1, and Early Growth Response Factor-1 Mol. Endocrinol., April 1, 2003; 17(4): 520 - 533. [Abstract] [Full Text] [PDF]
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R. J. Phillips, J. Bailey, S. C. Robson, and G. N. Europe-Finner Differential Expression of the Adenylyl Cyclase-Stimulatory Guanosine Triphosphate-Binding Protein Gs{alpha} in the Human Myometrium during Pregnancy and Labor Involves Transcriptional Regulation by Cyclic Adenosine 3',5'-Monophosphate and Binding of Phosphorylated Nuclear Proteins to Multiple GC Boxes within the Promoter J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5675 - 5685. [Abstract] [Full Text] [PDF]
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C. Uchida, T. Oda, T. Sugiyama, S. Otani, M. Kitagawa, and A. Ichiyama The Role of Sp1 and AP-2 in Basal and Protein Kinase A-induced Expression of Mitochondrial Serine:Pyruvate Aminotransferase in Hepatocytes J. Biol. Chem., October 11, 2002; 277(42): 39082 - 39092. [Abstract] [Full Text] [PDF]
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S. Yun, A. Dardik, M. Haga, A. Yamashita, S. Yamaguchi, Y. Koh, J. A. Madri, and B. E. Sumpio Transcription Factor Sp1 Phosphorylation Induced by Shear Stress Inhibits Membrane Type 1-Matrix Metalloproteinase Expression in Endothelium J. Biol. Chem., September 13, 2002; 277(38): 34808 - 34814. [Abstract] [Full Text] [PDF]
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H. J. Goldberg, C. I. Whiteside, and I. G. Fantus The Hexosamine Pathway Regulates the Plasminogen Activator Inhibitor-1 Gene Promoter and Sp1 Transcriptional Activation through Protein Kinase C-beta I and -delta J. Biol. Chem., September 6, 2002; 277(37): 33833 - 33841. [Abstract] [Full Text] [PDF]
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X. Ye and S. F. Liu Lipopolysaccharide Down-regulates Sp1 Binding Activity by Promoting Sp1 Protein Dephosphorylation and Degradation J. Biol. Chem., August 23, 2002; 277(35): 31863 - 31870. [Abstract] [Full Text] [PDF]
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V. Gavrilyuk, C. Dello Russo, M. T. Heneka, D. Pelligrino, G. Weinberg, and D. L. Feinstein Norepinephrine Increases Ikappa Balpha Expression in Astrocytes J. Biol. Chem., August 9, 2002; 277(33): 29662 - 29668. [Abstract] [Full Text] [PDF]
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 C. Hong, Hyeon.-A. Kim, G. L. Firestone, and L. F. Bjeldanes 3,3'-Diindolylmethane (DIM) induces a G1 cell cycle arrest in human breast cancer cells that is accompanied by Sp1-mediated activation of p21WAF1/CIP1 expression Carcinogenesis, August 1, 2002; 23(8): 1297 - 1305. [Abstract] [Full Text] [PDF]
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D.-B. Kim and N. A. DeLuca Phosphorylation of Transcription Factor Sp1 during Herpes Simplex Virus Type 1 Infection J. Virol., June 5, 2002; 76(13): 6473 - 6479. [Abstract] [Full Text] [PDF]
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J. Milanini-Mongiat, J. Pouyssegur, and G. Pages Identification of Two Sp1 Phosphorylation Sites for p42/p44 Mitogen-activated Protein Kinases. THEIR IMPLICATION IN VASCULAR ENDOTHELIAL GROWTH FACTOR GENE TRANSCRIPTION J. Biol. Chem., May 31, 2002; 277(23): 20631 - 20639. [Abstract] [Full Text] [PDF]
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A. R. Nawrocki, C. E. Goldring, R. M. Kostadinova, F. J. Frey, and B. M. Frey In Vivo Footprinting of the Human 11beta -Hydroxysteroid Dehydrogenase Type 2 Promoter. EVIDENCE FOR CELL-SPECIFIC REGULATION BY Sp1 AND Sp3 J. Biol. Chem., April 19, 2002; 277(17): 14647 - 14656. [Abstract] [Full Text] [PDF]
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M. Tone, Y. Tone, J. M. Babik, C.-Y. Lin, and H. Waldmann The Role of Sp1 and NF-kappa B in Regulating CD40 Gene Expression J. Biol. Chem., March 8, 2002; 277(11): 8890 - 8897. [Abstract] [Full Text] [PDF]
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I. Lacroix, C. Lipcey, J. Imbert, and B. Kahn-Perles Sp1 Transcriptional Activity Is Up-regulated by Phosphatase 2A in Dividing T Lymphocytes J. Biol. Chem., March 8, 2002; 277(11): 9598 - 9605. [Abstract] [Full Text] [PDF]
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H. Sellak, X. Yang, X. Cao, T. Cornwell, G. A. Soff, and T. Lincoln Sp1 Transcription Factor as a Molecular Target for Nitric Oxide- and Cyclic Nucleotide-Mediated Suppression of cGMP-Dependent Protein Kinase-I{alpha} Expression in Vascular Smooth Muscle Cells Circ. Res., March 8, 2002; 90(4): 405 - 412. [Abstract] [Full Text] [PDF]
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S. A. Shell, C. Fix, D. Olejniczak, N. Gram-Humphrey, and W. H. Walker Regulation of Cyclic Adenosine 3',5'-Monophosphate Response Element Binding Protein (CREB) Expression by Sp1 in the Mammalian Testis Biol Reprod, March 1, 2002; 66(3): 659 - 666. [Abstract] [Full Text] [PDF]
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 T. Wang, W. P. Lafuse, and B. S. Zwilling NF{kappa}B and Sp1 Elements Are Necessary for Maximal Transcription of Toll-like Receptor 2 Induced by Mycobacterium avium J. Immunol., December 15, 2001; 167(12): 6924 - 6932. [Abstract] [Full Text] [PDF]
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Y. Ge, L. H. Matherly, and J. W. Taub Transcriptional Regulation of Cell-specific Expression of the Human Cystathionine beta -Synthase Gene by Differential Binding of Sp1/Sp3 to the -1b Promoter J. Biol. Chem., November 16, 2001; 276(47): 43570 - 43579. [Abstract] [Full Text] [PDF]
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P. R. Kiela, E. R. Hines, J. F. Collins, and F. K. Ghishan Regulation of the rat NHE3 gene promoter by sodium butyrate Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G947 - G956. [Abstract] [Full Text] [PDF]
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 T. M. Lincoln, N. Dey, and H. Sellak Signal Transduction in Smooth Muscle: Invited Review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression J Appl Physiol, September 1, 2001; 91(3): 1421 - 1430. [Abstract] [Full Text] [PDF]
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T. Ragoczy and G. Miller Autostimulation of the Epstein-Barr Virus BRLF1 Promoter Is Mediated through Consensus Sp1 and Sp3 Binding Sites J. Virol., June 1, 2001; 75(11): 5240 - 5251. [Abstract] [Full Text]
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C. Aigueperse, P. Val, C. Pacot, C. Darne, E. Lalli, P. Sassone-Corsi, G. Veyssiere, C. Jean, and A. Martinez SF-1 (Steroidogenic Factor-1), C/EBP{beta} (CCAAT/Enhancer Binding Protein), and Ubiquitous Transcription Factors NF1 (Nuclear Factor 1) and Sp1 (Selective Promoter Factor 1) Are Required for Regulation of the Mouse Aldose Reductase-Like Gene (AKR1B7) Expression in Adrenocortical Cells Mol. Endocrinol., January 1, 2001; 15(1): 93 - 111. [Abstract] [Full Text]
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S. Chupreta, M. Du, A. Todisco, and J. L. Merchant EGF stimulates gastrin promoter through activation of Sp1 kinase activity Am J Physiol Cell Physiol, April 1, 2000; 278(4): C697 - C708. [Abstract] [Full Text] [PDF]
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 R. Margana, K. Berhane, M. N. Alam, and V. Boggaram Identification of functional TTF-1 and Sp1/Sp3 sites in the upstream promoter region of rabbit SP-B gene Am J Physiol Lung Cell Mol Physiol, March 1, 2000; 278(3): L477 - L484. [Abstract] [Full Text] [PDF]
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S. Wang, W. Wang, R. A. Wesley, and R. L. Danner A Sp1 Binding Site of the Tumor Necrosis Factor alpha Promoter Functions as a Nitric Oxide Response Element J. Biol. Chem., November 19, 1999; 274(47): 33190 - 33193. [Abstract] [Full Text] [PDF]
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M. Algarte, H. Kwon, P. Genin, and J. Hiscott Identification by In Vivo Genomic Footprinting of a Transcriptional Switch Containing NF-kappa B and Sp1 That Regulates the Ikappa Balpha Promoter Mol. Cell. Biol., September 1, 1999; 19(9): 6140 - 6153. [Abstract] [Full Text] [PDF]
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H.-C. Liu, J.-T. Shen, L. B. Augustin, J. L. Ko, and H. H. Loh Transcriptional Regulation of Mouse delta -Opioid Receptor Gene J. Biol. Chem., August 13, 1999; 274(33): 23617 - 23626. [Abstract] [Full Text] [PDF]
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I. J. Gonzalez-Robayna, T. N. Alliston, P. Buse, G. L. Firestone, and J. S. Richards Functional and Subcellular Changes in the A-Kinase-Signaling Pathway: Relation to Aromatase and Sgk Expression during the Transition of Granulosa Cells to Luteal Cells Mol. Endocrinol., August 1, 1999; 13(8): 1318 - 1337. [Abstract] [Full Text]
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R. Ahlgren, G. Suske, M. R. Waterman, and J. Lund Role of Sp1 in cAMP-dependent Transcriptional Regulation of the Bovine CYP11A Gene J. Biol. Chem., July 2, 1999; 274(27): 19422 - 19428. [Abstract] [Full Text] [PDF]
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K. Su, M. D. Roos, X. Yang, I. Han, A. J. Paterson, and J. E. Kudlow An N-terminal Region of Sp1 Targets Its Proteasome-dependent Degradation in Vitro J. Biol. Chem., May 21, 1999; 274(21): 15194 - 15202. [Abstract] [Full Text] [PDF]
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C. Li, F. B. Kraemer, T. E. Ahlborn, and J. Liu Induction of Low Density Lipoprotein Receptor (LDLR) Transcription by Oncostatin M Is Mediated by the Extracellular Signal-regulated Kinase Signaling Pathway and the Repeat 3 Element of the LDLR Promoter J. Biol. Chem., March 5, 1999; 274(10): 6747 - 6753. [Abstract] [Full Text] [PDF]
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I. Alroy, L. Soussan, R. Seger, and Y. Yarden Neu Differentiation Factor Stimulates Phosphorylation and Activation of the Sp1 Transcription Factor Mol. Cell. Biol., March 1, 1999; 19(3): 1961 - 1972. [Abstract] [Full Text] [PDF]
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A. R. Black, D. Jensen, S.-Y. Lin, and J. C. Azizkhan Growth/Cell Cycle Regulation of Sp1 Phosphorylation J. Biol. Chem., January 15, 1999; 274(3): 1207 - 1215. [Abstract] [Full Text] [PDF]
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M. Hauses, R. R. Tonjes, and M. Grez The Transcription Factor Sp1 Regulates the Myeloid-specific Expression of the Human Hematopoietic Cell Kinase (HCK) Gene through Binding to Two Adjacent GC Boxes within the HCK Promoter-Proximal Region J. Biol. Chem., November 27, 1998; 273(48): 31844 - 31852. [Abstract] [Full Text] [PDF]
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N. Dumais, B. Barbeau, M. Olivier, and M. J. Tremblay Prostaglandin E2 Up-regulates HIV-1 Long Terminal Repeat-driven Gene Activity in T Cells via NF-kappa B-dependent and -Independent Signaling Pathways J. Biol. Chem., October 16, 1998; 273(42): 27306 - 27314. [Abstract] [Full Text] [PDF]
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R. A. Rajakumar, S. Thamotharan, R. K. Menon, and S. U. Devaskar Sp1 and Sp3 Regulate Transcriptional Activity of the Facilitative Glucose Transporter Isoform-3 Gene in Mammalian Neuroblasts and Trophoblasts J. Biol. Chem., October 16, 1998; 273(42): 27474 - 27483. [Abstract] [Full Text] [PDF]
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Z.-Z. Hu, L. Zhuang, J. Meng, and M. L. Dufau Transcriptional Regulation of the Generic Promoter III of the Rat Prolactin Receptor Gene by C/EBPbeta and Sp1 J. Biol. Chem., October 2, 1998; 273(40): 26225 - 26235. [Abstract] [Full Text] [PDF]
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J. B. Redell and B. L Tempel Multiple Promoter Elements Interact to Control the Transcription of the Potassium Channel Gene, KCNJ2 J. Biol. Chem., August 28, 1998; 273(35): 22807 - 22818. [Abstract] [Full Text] [PDF]
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K. Yamada, T. Tanaka, K. Miyamoto, and T. Noguchi Sp Family Members and Nuclear Factor-Y Cooperatively Stimulate Transcription from the Rat Pyruvate Kinase M Gene Distal Promoter Region via Their Direct Interactions J. Biol. Chem., June 9, 2000; 275(24): 18129 - 18137. [Abstract] [Full Text] [PDF]
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S. Horie, H. Ishii, F. Matsumoto, M. Kusano, K. Kizaki, J. Matsuda, and M. Kazama Acceleration of Thrombomodulin Gene Transcription by Retinoic Acid. RETINOIC ACID RECEPTORS AND Sp1 REGULATE THE PROMOTER ACTIVITY THROUGH INTERACTIONS WITH TWO DIFFERENT SEQUENCES IN THE 5'-FLANKING REGION OF HUMAN GENE J. Biol. Chem., January 19, 2001; 276(4): 2440 - 2450. [Abstract] [Full Text] [PDF]
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A.-C. Poncelet and H. W. Schnaper Sp1 and Smad Proteins Cooperate to Mediate Transforming Growth Factor-beta 1-induced alpha 2(I) Collagen Expression in Human Glomerular Mesangial Cells J. Biol. Chem., March 2, 2001; 276(10): 6983 - 6992. [Abstract] [Full Text] [PDF]
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Y. Yang, C. K. Hwang, E. Junn, G. Lee, and M. M. Mouradian ZIC2 and Sp3 Repress Sp1-induced Activation of the Human D1ADopamine Receptor Gene J. Biol. Chem., December 1, 2000; 275(49): 38863 - 38869. [Abstract] [Full Text] [PDF]
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K.-Y. Kim, T. Rhim, I. Choi, and S.-S. Kim N-Acetylcysteine Induces Cell Cycle Arrest in Hepatic Stellate Cells through Its Reducing Activity J. Biol. Chem., October 26, 2001; 276(44): 40591 - 40598. [Abstract] [Full Text] [PDF]
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T. Fukada and N. K. Tonks The Reciprocal Role of Egr-1 and Sp Family Proteins in Regulation of the PTP1B Promoter in Response to the p210 Bcr-Abl Oncoprotein-tyrosine Kinase J. Biol. Chem., June 29, 2001; 276(27): 25512 - 25519. [Abstract] [Full Text] [PDF]
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D. N. Douglas, V. W. Dolinsky, R. Lehner, and D. E. Vance A Role for Sp1 in the Transcriptional Regulation of Hepatic Triacylglycerol Hydrolase in the Mouse J. Biol. Chem., June 29, 2001; 276(27): 25621 - 25630. [Abstract] [Full Text] [PDF]
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H. Sellak, X. Yang, X. Cao, T. Cornwell, G. A. Soff, and T. Lincoln Sp1 Transcription Factor as a Molecular Target for Nitric Oxide- and Cyclic Nucleotide-Mediated Suppression of cGMP-Dependent Protein Kinase-I{alpha} Expression in Vascular Smooth Muscle Cells Circ. Res., March 8, 2002; 90(4): 405 - 412. [Abstract] [Full Text] [PDF]
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