EGF stimulates gastrin promoter through activation of Sp1
kinase activity
Sergey
Chupreta1,
Ming
Du1,
Andrea
Todisco1, and
Juanita L.
Merchant1,2,3
Departments of 1 Internal Medicine and
2 Physiology and 3 Howard Hughes
Medical Institute, University of Michigan, Ann Arbor, Michigan 48109
 |
ABSTRACT |
Epidermal
growth factor (EGF) receptor activation stimulates gastrin gene
expression through a GC-rich element called gastrin EGF response
element (gERE). This element is bound by Sp1 family members and is a
target of the ras-extracellular signal-regulated kinase (Erk)
signal transduction cascade. This raised the possibility that Sp1 may
be phosphorylated by kinases of this signaling pathway. Erk is capable
of phosphorylating other mitogen-inducible transcription factors, e.g.,
Elk and Sap, suggesting that Erk may also mediate EGF-dependent
phosphorylation of Sp1. This possibility was tested by studying
Sp1-dependent kinase activity in extracts prepared from EGF-activated
AGS cells by use of solid-phase kinase assays and immunoprecipitation
of metabolically labeled Sp1. The results revealed that Sp1 kinase
activity (like gastrin promoter activation) is inhibited by PD-98059
and, therefore, is dependent on mitogen-activated protein kinase kinase
1 (Mek 1). However, EGF-dependent activation of endogenous Erk did not
account for most of the Sp1 kinase activity, since Erk and additional
Sp1 kinase activity analyzed in a solid-phase kinase assay eluted from
an ion-exchange column in different fractions. Phosphoamino acid
analysis of in vivo radiolabeled Sp1 demonstrated that the kinase
phosphorylates Sp1 on Ser and Thr in response to EGF. Therefore, most
EGF-stimulated Sp1 kinase activity is Mek 1 dependent and distinct from Erk.
extracellular signal-regulated kinase; signal transduction; gene
expression; PD-98059; phosphorylation; epidermal growth factor
 |
INTRODUCTION |
THE GENE ENCODING THE PEPTIDE hormone gastrin is
produced by neuroendocrine cells of the adult stomach and is positively
regulated by epidermal growth factor (EGF) receptor activation through
an Sp1 binding site (16, 33). This raised the possibility that Sp1
transactivation may be modulated through inducible phosphorylation by
the ras-extracellular signal-regulated kinase (Erk) pathway, as
demonstrated for many other EGF-regulated promoters (43). Ras
activation of the gastrin promoter is clinically relevant because of
the elevated levels of gastrin mRNA in colon cancers containing an
activating mutation of K-ras (41). Thus understanding how the
ras-Erk pathway stimulates gastrin gene expression may further
our understanding of gastrointestinal transformation. Consistent with
the role of activated Ras in colonic transformation and gastrin gene
expression, we recently showed that activation of the gastrin promoter
through the GC-rich gastrin EGF response element (gERE) is
ras-Erk dependent (34). Moreover, purified Erk 2 phosphorylates
Sp1 and stimulates increased Sp1 binding and affinity for DNA. We also
found that overexpression of Erk stimulates gastrin promoter activation
and that the Mek 1 inhibitor PD-98059 prevents Sp1-dependent
transactivation of the gastrin promoter (34).
Initial understanding of the role of Sp1 focused on its ability to
regulate constitutively active genes (11) that mediate interaction with
the basal transcription apparatus (45). However, recent evidence has
emerged to indicate that the activity of Sp1 is modulated by cell
growth and differentiation (5, 12, 37, 40, 47, 48). It appears that
growth regulation of Sp1-dependent transcription correlates with a
change in the state of Sp1 phosphorylation. Moreover, the kinases
phosphorylating Sp1 as well as activated signaling pathways appear to
be agonist, cell type, and promoter dependent. Thus Sp1 no longer
represents a transcription factor unaffected by extracellular signals
but exhibits complexities as intricate as other mitogen-inducible
transcription factors, e.g., signal tranducers and activators of
transcription (STAT), Elk, and Sap1A. This may be surprising, since
many promoters contain GC-rich sites capable of binding Sp1. Yet, in
many instances, these sites are bound not only by Sp1 and Sp1 family
members but by other zinc finger transcription factors that may compete
or cooperate with Sp1 (26, 27, 29, 35, 49, 50).
The focus of the present study was to determine whether Sp1 is a
substrate for endogenously activated Erk alone or whether the "Sp1
kinase" activity represents additional EGF-activated kinases. We
found that the major Ser/Thr kinase activity phosphorylating Sp1
targeted its NH2-terminal domain and was distinct from Erk.
 |
EXPERIMENTAL PROCEDURES |
Plasmids.
pGEX1-Sp1FLU or pGEX1N-Sp1ABC containing full-length human Sp1 or amino
acids 1-530 of Sp1 cDNA (NH2-ter1-530
Sp1) were gifts from J. Horowitz (Duke University, Durham, NC) (51).
Cell culture.
AGS cells (derived from a human gastric adenocarcinoma) (4) were
purchased from American Type Culture Collection and cultured in DMEM
(GIBCO-BRL) containing 10% FCS, 100 µg/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 5%
CO2-95% air in 35-mm six-well culture dishes at 37°C.
The cells were stably transfected with the 240 gastrin-luciferase
reporter construct (240 GasLuc) or the 240 GasLuc construct containing
a 4-bp mutation of the EGF response element (m240 GasLuc), selected in
G418 (GIBCO-BRL), and pooled as previously described (33). The 240 GasLuc construct contained 240 bp of the human gastrin promoter, and
the first exon ligated upstream of the luciferase reporter in pGL2
basic (Promega). Calcium coprecipitation (5 Prime-3 Prime) was
used to create stable transfections into AGS cells. The cells were incubated for 48 h in Ham's F-12 nutrient mixture containing 100 µg/ml penicillin and 100 µg/ml streptomycin (GIBCO-BRL)
without serum before treatment with 10 nM EGF.
Immunoblots.
Whole cell extracts prepared from AGS cells were lysed in 10 mM HEPES
at pH 7.7, 30 mM NaCl, 2% glycerol, 0.5% Triton X-100, 3 mM
MgCl2, 0.2 mM EDTA, 0.2 mM Na3VO4,
2 mM NaF, 2 mM sodium pyrophosphate, and one Complete tablet per 10 ml
(Boehringer-Mannheim), which contained a cocktail of protease
inhibitors. The extracts were heat denatured in Laemmli sample buffer,
resolved on a 7.5 or 10% Laemmli gel, and then electroblotted onto
polyvinylidene difluoride membrane (Bio-Rad). After the sample was
blocked for 1 h in 100 mM Tris · HCl, pH 7.5, 0.9%
NaCl, and 0.05% Tween 20 containing 5% nonfat dry milk (TTBS), the
blot was exposed to antibody diluted in TTBS for 1 h and then rinsed
three times in TTBS. The blot was then incubated for 1 h in
anti-goat IgG-horseradish peroxidase (1:1,000 dilution). After three
additional 15-min rinses in TTBS, the resulting protein-antibody
complexes were detected by chemiluminescence (SuperSignal, Pierce
Biochemicals). Sp1 and Erk antibodies were purchased from Santa Cruz
Biotechnology; phospho-Erk antibody was purchased from New England
BioLabs. Rsk, p38, DNA-dependent protein kinase, and JNK antibodies
were purchased from Santa Cruz Biotechnology.
Electrophoretic mobility shift assay.
AGS whole cell extracts were prepared as described above, snap frozen,
and stored at
80°C until use. A double-stranded
oligonucleotide probe corresponding to the gERE GGGGCGGGGTGGGGGG was
end-labeled using Klenow Exo-enzyme (New England BioLabs) and
[
-32P]dCTP (Amersham). The probe was
purified using Quick Spin columns (Boehringer Mannheim). Ten micrograms
of whole cell extract were incubated in a final volume of 20 µl
containing 10 mM Tris · HCl, pH 7.9, 1 mM
ZnCl2, 100 mM KCl, 1 mM EDTA, 300 ng
dI · dC, 1 mM dithiothreitol (DTT), 5 mM
MgCl2, 10% glycerol, and radiolabeled probe (30,000 cpm/0.01 ng) at 25°C and then resolved on a 4% nondenaturing polyacrylamide gel containing 45 mM Tris base, 45 mM boric acid, and 1 mM EDTA. Antibodies to Sp1, Sp3, and Sp4 were purchased from Santa Cruz
Biotechnology. Whole cell extracts were pretreated with 1-5 units
of calf intestinal phosphatase (CIP) for 20 min at 30°C
in electrophoretic mobility shift assay (EMSA) buffer before the probe
was added.
Expression of fusion proteins.
A 300-ml starter culture of transformed Escherichia coli strain
BL21 (DE3) protease-deficient bacteria (Novagen) was grown overnight at
25°C and then used to inoculate 1 liter of fresh Luria broth
containing 50 µg/ml ampicillin. The 1-liter culture was grown until
log phase reached a density of A600 = 0.4 optical density unit at 25°C, where A600 is absorbance
at 600 nm. Protein expression was initiated by the addition of a final
concentration of 0.4 mM isopropyl-
-thiogalactopyranoside. After
2-3 h the cells were collected by centrifugation for 10 min at
8,000 rpm in a Beckman JA-10 rotor, resuspended in 15 ml of lysis
buffer (1% Triton X-100, 1 mM DTT, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 0.1 mM phenylmethylsulfonyl
fluoride), and then sonicated twice for 15 s at 30% output power at
4°C. Bacterial debris was removed by centrifugation at 15,000 rpm
for 30 min in a Beckman SW28 rotor. The fusion protein was isolated
from the supernatant by batch chromatography with use of
glutathione-Sepharose 4B beads at 25°C according to the
manufacturer's instructions (Pharmacia). After the beads were washed
three times in lysis buffer with protease inhibitors, the bound protein
was stored in PBS with 0.02% sodium azide at 4°C until use (within
1 wk). The concentration of protein on the beads was determined by the method of Bradford (8), and the purity was confirmed by SDS gel
electrophoresis and Coomassie blue staining. Glutathione
S-transferase (GST) protein alone was prepared by expressing
the pGEX vector (Pharmacia).
Column purification of extracts.
Whole cell AGS extracts were prepared by detergent lysis in 0.5%
Triton X-100, 50 mM
-glycerophosphate, pH 7.2, 0.1 mM sodium orthovanadate, 2 mM MgCl2, 1 mM EGTA, 10 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM DTT, snap
frozen, and stored at
80°C until use. MonoQ anion-exchange
resin (1 ml; Pharmacia) was preequilibrated in column buffer consisting
of 50 mM
-glycerophosphate, pH 7.2, 0.1 mM sodium orthovanadate,
1 mM EGTA, and 1 mM DTT. The extracts were thawed, mixed, and
loaded onto a preequilibrated MonoQ anion-exchange column three times.
The column was washed three times in "column buffer" before
elution of 1-ml fractions with an NaCl gradient of 180-560 mM. The
fractions were collected in tubes containing 50 ml of 2 mM sodium
orthovanadate and one Complete tablet per 2 ml of buffer. The extracts
were analyzed by solid-phase kinase assays or by Western blots.
Solid-phase kinase assays.
To study phosphorylation of Sp1, AGS cells grown to ~70% confluency
were placed under serum-free conditions (Ham's F-12 nutrient mixture)
for 48 h before the start of the experiment. Cells were treated for 1 h
with 50 µM Mek 1 kinase inhibitor PD-98059 (BioMol Research Labs)
before addition of 10 nM EGF. Whole cell extracts were prepared as
previously described, and the protein concentration was determined by
the method of Bradford (8). Thirty micrograms of GST-Sp1 fusion protein
immobilized on beads or GST alone expressed from the empty pGEX vector
were incubated with 200 µg of whole cell extracts with agitation for
3 h. Alternatively, 0.1 µg of recombinant GST-Erk 2 enzyme (Upstate
Biotechnology) was incubated with 30 µg of GST-Sp1. Nonadherent
protein was removed by centrifugation in an Eppendorf refrigerated
microfuge for 5 min at 12,000 rpm and then washed three times in HEPES
binding buffer (20 mM HEPES, pH 7.7, 50 mM NaCl, 2.5 mM
MgCl2, 0.1 mM EDTA, 0.05% Triton X-100). The beads were
resuspended in 30 µl of kinase buffer (20 mM HEPES, pH 7.7, 20 mM
MgCl2, 20 mM p-nitrophenyl phosphate, 0.1 mM
Na3VO4, 2 mM DTT, 20 mM ATP, 5 µCi of
[
-32P]ATP) for 30 min at 30°C. The
reaction was terminated on ice, and the beads were washed 3 times with
20 times the volume of kinase buffer without radiolabel. The protein
was eluted in 30 µl of Laemmli sample buffer at 90°C for 2 min
and resolved on a 10% SDS-polyacrylamide gel before autoradiography.
Analysis of phosphate incorporation was determined on a Molecular
Dynamics PhosphorImager. The gel was stained with Coomassie blue dye to verify loading.
In vivo phosphate labeling and immunoprecipitation of Sp1.
AGS cells were cultured on 100-mm dishes, then preincubated in
phosphate-free DMEM (GIBCO) for 1 h before the addition of labeling
medium. The cells were incubated for 3 h in 3 ml of labeling medium
consisting of phosphate-free DMEM with 100 µCi/ml
[32P]orthophosphate (Amersham) before the
addition of PD-98059 for 1 h and then 10 nM EGF for 30 min. The cells
were lysed on ice in 1 ml of RIPA buffer (1% Nonidet P-40, 1% sodium
deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM sodium phosphate, pH 7.2, 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodium orthovanadate, 100 U/ml
aprotinin). Sp1 protein was immunoprecipitated from cell extracts by
use of rabbit polyclonal anti-Sp1 antibody and protein A-agarose (Santa Cruz). The immune complex was washed four times with RIPA buffer, then
boiled for 3 min in 30 µl of 2× Laemmli sample buffer
and resolved on a 10% SDS-polyacrylamide gel. Phosphorylated Sp1 was detected by autoradiography and analyzed on a PhosphorImager.
Phosphoamino acid analysis.
Phosphorylated NH2-ter1-530 Sp1 fusion
protein or in vivo radiolabeled immunoprecipitated Sp1 was separated on
a 10% SDS-polyacrylamide gel and transferred to polyvinylidene
difluoride membrane. The bands of interest were excised for analysis by
two-dimensional electrophoresis, as described previously (7). The
migration of phosphoserine, phosphothreonine, and phosphotyrosine was
determined using standard mixtures.
 |
RESULTS |
EGF stimulates the gastrin promoter through an Sp1 binding site.
We previously showed that EGF stimulation of a human gastric cell line
(AGS) increases gastrin promoter activity (16). Furthermore, EGF
induction is mediated by a GC-rich element that binds several zinc
finger factors, including Sp1 (16, 36). To study the time course of the
induction, two lines of pooled stable transformants expressing the 240 gastrin reporter construct (240 GasLuc) and the same construct with a
4-bp mutation in the gERE element were treated over 6 h with 10 nM EGF.
We found that significant EGF induction of the gastrin
promoter was observed by 1 h and was maximal (~6-fold) within 4 h
(Fig. 1). A 4-bp mutation that abolishes Sp1 binding was not induced by EGF over the same time period (16). We
showed previously that the Mek 1 kinase inhibitor PD-98059 blocks EGF
induction of the gastrin promoter by ~50% but has no effect on basal
promoter activity (34).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Epidermal growth factor (EGF) induction of gastrin promoter maps to a
GC-rich element. AGS cells were stably transfected with 240 gastrin-luciferase (GasLuc) construct or m240 GasLuc, which contains a
4-bp mutation within gastrin EGF response element (gERE). Pooled
transformants were treated with 10 nM EGF for up to 6 h. Values (means ± SE for 3 independent experiments performed in triplicate) are
expressed as order of magnitude induction of luciferase activity
normalized to protein.
|
|
EMSA demonstrates Sp1 and Sp3 binding to gERE.
We showed previously that Sp1 comprises the upper portion of the
slower-migrating complex and that Sp3 and ZBP-89 comprise the lower two
complexes (28, 34). To determine whether Sp1 binding to gERE increases
with EGF treatment, whole cell extracts were prepared from AGS cells
after treatment with EGF for various time periods. The results shown in
Fig. 2 indicate that Sp1 and Sp3 binding
increases within 5 min and is maximal by 30 min. However, only Sp1
mediates transactivation of the gastrin promoter (34). Therefore, to
establish whether Sp1 binding is attenuated by pretreatment of the
cells with PD-98059, AGS cells were pretreated with 50 µM PD-98059
before stimulation with EGF. The results show that PD-98059 effectively
prevented Sp1 binding (Fig. 2A), suggesting that Sp1 binding is
in part regulated by Mek 1 kinase activity, consistent with gastrin
promoter induction requiring Mek 1. Furthermore, the results suggest
that Sp1 phosphorylation occurs downstream of Mek 1 kinase activation.
To confirm that the increase in binding was due to Sp1, antibody was
incubated with the extracts before addition of the probe. The results
show that the increase in binding was due primarily to Sp1 and Sp3 and
not to the recruitment of novel transcription factors (Fig.
2B). An immunoblot using Sp1 antibody showed that Sp1 protein
also increases with EGF stimulation within 30 min and is maximal by 4 h
(Fig. 3). This increase was specific, since
Erk 2 protein levels used as a control remained unchanged. Thus protein
abundance may contribute to the sustained increase in Sp1 binding,
whereas the initial increase in binding may be due to increased
phosphorylation.

View larger version (95K):
[in this window]
[in a new window]
|
Fig. 2.
EGF stimulates Sp1 binding to gERE. Whole cell extracts were prepared
from AGS cells treated with 10 nM EGF for up to 6 h. A:
extracts from 48-h serum-starved cells (lane 1) or from cells
treated with EGF for 5, 15, and 30 min and 1, 2, 3, 4, 5, and 6 h
(lanes 2, 3, 4, 5, 6, 7, 8, 9, and 10) were resolved on
a nondenaturing gel. Lanes 11-13, cells pretreated with
PD-98059 (PD) and then treated with EGF for 1-3 h. B:
untreated extracts (lane 1) or extracts from cells treated for
1-4 h (lanes 2-5) were compared with complexes
generated with extracts incubated for 0-3 h with 1 µl of Sp1
antibody (lanes 6-9). Complexes were resolved on a
nondenaturing gel electrophoresed for an extended period of time to
resolve individual bands. *, Supershifted Sp1 protein. All extracts
were incubated with radiolabeled gERE probe. Results are representative
of duplicate experiments.
|
|

View larger version (68K):
[in this window]
[in a new window]
|
Fig. 3.
EGF stimulates an increase in Sp1 protein abundance. Whole cell
extracts were prepared from AGS cells treated with 10 nM EGF for up to
6 h. Lane 1, extracts from 48-h serum-starved cells. Lanes
2, 3, 4, 5, 6, 7, and 8, extracts from cells treated with
EGF for 15 and 30 min and 1, 2, 3, 4, and 6 h. Same blot was probed
first with a 1:250 dilution of Sp1 antibody (top) and then with
a 1:250 dilution of extracellular signal-regulated kinase (Erk) 2 antibody as a control for loading (bottom). Results are
representative of 3 experiments.
|
|
Sp1 binding regulated by phosphorylation.
We focused on the phosphorylation of Sp1, since Sp3 coexpression with
the gastrin promoter in Drosophila Schneider cells has little
effect on activation of the promoter, whereas Sp1 cotransfection stimulates the promoter nearly threefold (34). To examine whether phosphorylation contributed to Sp1 binding, whole cell extracts were
treated with CIP before EMSAs were carried out. The results demonstrate
that CIP treatment decreases binding without affecting the total amount
of Sp1 protein (Fig. 4). Together these
studies indicate that EGF stimulates Sp1 complex binding by two
mechanisms: increased Sp1 abundance and increased binding due to
phosphorylation.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 4.
Phosphorylation regulates Sp1 binding. Whole cell extracts were treated
with increasing amounts of calf intestinal phosphatase (CIP). Same
extract was used in immunoblot, in which Sp1 antibody (1:250) was used
(top), and in an electrophoretic mobility shift assay
(bottom). Lane 1, 30 µg of whole cell extract;
lane 2, 1 U of CIP; lane 3, 3 U of CIP; lane 4,
5 U of CIP.
|
|
Endogenous Erk activity.
Because EGF stimulates gastrin promoter activity and Sp1 binding and
PD-98059 modulates these effects (34), the time course of endogenous
Erk activation was examined. Phospho-Erk antibodies were used to probe
blots containing whole cell extracts prepared from AGS cells after EGF
treatment. The results demonstrated rapid autophosphorylation of Erk
kinases that was inhibited by PD-98059 (Fig.
5). Serum starvation was sufficient to
inactivate endogenous Erk kinases (Fig. 5, lane 2). Unlike
reports of transient activation of Erk in fibroblasts and PC-12 cells
by EGF (15, 32, 39), Erk kinases in AGS cells remained phosphorylated
for an extended period of time after EGF treatment. Apparently, in some
cells, Erk activation is prolonged if growth factors stimulate the
cells while they are adherent (46).

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 5.
Immunoblot of endogenous Erk 2 in AGS cells. Whole cell extracts
prepared from AGS cells were pretreated with PD-98059 and then treated
for 15 min with 10 nM EGF (lane 1). Lane 2, extracts
from 48-h serum-starved cells; lanes 3, 4, 5, 6, 7, 8, and
9, extracts from cells treated with EGF for 15 and 30 min and 1, 2, 3, 4, and 6 h. Same blot was probed first with a 1:250 dilution of
phospho-Erk (Erk-PO4) antibody (top) and then with
a 1:250 dilution of Erk 2 antibody (bottom).
|
|
Endogenous Sp1 kinase activity is downstream of Mek 1.
To determine whether Sp1 is a target of EGF-activated kinases, a series
of solid-phase kinase assays (20) were performed with extracts from
EGF-treated AGS cells. Solid-phase kinase assays with whole cell
lysates demonstrated that endogenous kinases activated by EGF
phosphorylate the full-length form of Sp1 (Fig.
6A) and the
NH2-terminal truncated form containing amino acids
1-530 (NH2-ter1-530 Sp1) without the
COOH-terminal zinc finger domain (Fig. 6B). Recombinant Erk 2 enzyme was used as a control for Sp1 phosphorylation in this assay
system. Activation of Erk kinases may stimulate downstream kinases (6)
or directly phosphorylate transcription factors, e.g., Elk and Sap
(21). The other phosphoproteins migrating at ~200, 60, and 30 kDa
represented autophosphorylation of endogenous AGS proteins present in
the crude extract and not phosphorylation of residual bacterial
proteins, since these phosphoproteins were not present in Fig. 6B,
lane 11, which also contained bacterially expressed recombinant Sp1
without cell extract. The gel was stained with Coomassie blue to
demonstrate equivalent loading of Sp1 in each lane. Sp1 phosphorylation
was observed with the full-length and truncated forms, indicating
EGF-induced phosphorylation of the NH2 terminus. Within 30 min after EGF induction, a burst of kinase activity hyperphosphorylated
Sp1 up to fourfold over basal levels, with prolonged phosphorylation
averaging 2- to 2.5-fold over the 6-h time course (Fig. 6C).
This follows a time course that parallels Sp1 binding (Fig. 2) but was
somewhat delayed compared with the time course of endogenous Erk kinase
activation (Fig. 5). Moreover, Sp1 was phosphorylated even in
serum-free conditions when Erk kinases were minimally active.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 6.
EGF stimulates Sp1 kinase activity. Full-length Sp1 (A) or
NH2-terminal acids 1-530
(NH2-ter1-530) form (B) was
immobilized on glutathione-agarose beads, incubated with 200 µg of
whole cell extracts from EGF-treated AGS cells, and then incubated with
5 µCi of [ -32P]ATP for 30 min at 30°C.
Phosphorylated Sp1 (Sp1-PO4) was resolved on a 10%
SDS-polyacrylamide gel. Lane 1, cells incubated for 48 h in
serum-free conditions; lanes 2, 3, 4, 5, 6, 7, 8, 9, and
10, cells treated for 5, 15, and 30 min and 1, 2, 3, 4, 5, and 6 h.
Alternatively, 0.1 µg of active recombinant Erk enzyme (rErk 2) was
incubated with glutathione S-transferase (GST)-Sp1 immobilized
on glutathione-agarose beads (lane 11). Top:
autoradiogram; bottom: same gel stained with Coomassie blue dye
(rSp1). C: specific activity of phosphorylated Sp1 (in
B) quantified on a PhosphorImager and plotted as a function of
time for 3 independent experiments.
|
|
To examine the relationship between Mek 1 and the Sp1 kinase activity,
AGS cells were pretreated for 1 h with 50 µM PD-98059 before EGF
treatment and preparation of whole cell extracts. PD-98059 reduced Sp1
phosphorylation by ~60% in solid-phase kinase assays, consistent
with Sp1 hyperphosphorylation resulting from Mek 1-dependent kinases
(Fig. 7, A and B). Because
PD-98059 has an inhibitory effect on the EGF-mediated
hyperphosphorylation of Sp1 in vitro, we investigated whether this
treatment also inhibited Sp1 phosphorylation in vivo. AGS cells were
labeled with [32P]orthophosphate and pretreated
for 1 h with PD-98059 before EGF stimulation. After stimulation, the
cells were lysed and Sp1 was immunoprecipitated with Sp1 antibody. The
results shown in Fig. 7, C and D, demonstrate that
PD-98059 treatment inhibits Sp1 phosphorylation similarly in vivo and
in vitro after EGF stimulation compared with untreated cells. We showed
previously that some EGF-dependent gastrin promoter activity is Mek 1 independent.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
EGF-induced Sp1 phosphorylation inhibited by PD-98059. Solid-phase
kinase assays were performed on extracts from cells pretreated with
PD-98059 and then treated with 10 nM EGF for 1 h. A:
lanes 1-4 contain recombinant Sp1 incubated with whole
cell extracts, and lanes 2 and 4 contain extracts
pretreated with PD-98059 (*). Top: autoradiogram of
Coomassie blue-stained gel (bottom). B:
phosphate-labeled Sp1 was quantified on a PhosphorImager. Values (means ± SE for 4 independent experiments) represent percent inhibition by
PD-98059 at 1 h. ** P < 0.001. C: in vivo
radiolabeled Sp1 immunoprecipitated from untreated cells (lane
1), cells treated with PD-98059 alone (lane 2), cells
treated with EGF alone for 30 min (lane 3), and cells treated
with EGF after pretreatment with PD-98059 for 1 h (lane 4).
Top: autoradiogram of SDS-polyacrylamide gel; bottom:
Coomassie blue-stained gel. D: amount of Pi
incorporated into immunoprecipitated Sp1 quantified on a
PhosphorImager. Results are representative of 2 experiments.
|
|
To determine whether Erk 2 was the only Sp1 kinase
activity, we examined whether all Sp1 kinase activity copurified with
Erk 2. AGS extracts before and after EGF treatment for 30 min were fractionated on a MonoQ anion-exchange column. After the column was
washed with buffer, protein was eluted in an NaCl gradient of
180-540 mM. As predicted from prior studies (14), active Erk
enzyme eluted from the column between 200 and 300 mM NaCl (Fig.
8A). The elution profile of Erk
protein was confirmed using Erk antibody to probe blots containing
protein resolved from each fraction. Untreated and EGF-treated extracts
exhibited the same elution profile, and other fractions did not contain
Erk protein. Phospho-Erk antibody was used to demonstrate the presence
of activated Erk in the EGF-stimulated extracts. The fractions
containing peak Erk activity did not exhibit maximal Sp1 kinase
activity in solid-phase assays. Rather, fractions eluting from the
column at salt concentrations >340 mM contained maximal Sp1 kinase
activity (Fig. 8B). Recombinant Erk 2 enzyme was used as a
positive control. The doublet migrating at 70 kDa below phospho-Sp1
represented autophosphorylation of the GST-Erk 2 enzyme and associated
proteolytic fragments.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 8.
Sp1 kinase activity distinct from Erk. A: whole cell extracts
were prepared from AGS cells treated without or with 10 nM EGF for 30 min. Extracts were fractionated on a MonoQ anion-exchange column and
eluted in 1-ml fractions with 180-360 mM NaCl. Aliquots were
evaluated by immunoblot for Erk activation. Anti-Erk 2 antibody detects
primarily Erk 2 protein; anti-PO4-Erk 1,2 antibody detects
only phospho-Erk proteins. B: autoradiograms of solid-phase
kinase assays performed with eluted fractions with glutathione
S-transferase (GST)-Sp1 as substrate (Sp1-PO4).
|
|
Examination of fractions eluted between 300 and 540 mM NaCl revealed an
increase of at least twofold in the level of Sp1 phosphorylation induced by EGF (Fig. 9A). The level
of Sp1 phosphorylation was not due to phosphorylation of GST, because
GST alone used as a substrate in the kinase assays was not
significantly phosphorylated (Fig. 9B). Moreover,
phosphorylation of Sp1 did not represent autocatalytic activity of an
unidentified kinase, since the assays carried out in the absence of
immobilized Sp1 did not reveal EGF-dependent activity migrating at
~100 kDa for NH2- ter1-530 GST-Sp1 (Fig. 9C). The Sp1 kinase activity in the MonoQ fractions
mapped to the NH2-ter1-530 and was
inhibited by PD-98059 (Fig. 9D). Collectively, these results
indicated that a significant percentage of the Sp1 kinase activity is a
Mek 1-dependent kinase other than Erk.

View larger version (65K):
[in this window]
[in a new window]
|
Fig. 9.
Sp1 kinase activity is novel. A: whole cell extracts were
prepared from AGS cells treated without or with 10 nM EGF for 30 min.
Extracts were fractionated on a MonoQ column, eluted in 1-ml fractions
with 300-540 mM NaCl, and used in solid-phase kinase reactions
with GST-Sp1 as substrate (lanes 1-13). Lane 14, 0.1 µg of recombinant Erk 2 incubated with GST-Sp1. Bottom:
corresponding Coomassie blue-stained gel detecting recombinant Sp1
(rSp1). B: quantification of solid-phase kinase assay from 300 to 540 mM NaCl with GST or GST-Sp1 as substrate. C: whole cell
extracts were prepared from AGS cells treated without or with 10 nM EGF
for 30 min. Extracts were fractionated on a MonoQ column, eluted in
1-ml fractions with 300-540 mM NaCl, and used in solid-phase
kinase reactions with beads but without immobilized recombinant Sp1 as
substrate (lanes 1-13). D: full-length Sp1
(lanes 1-5) or NH2-ter1-530
Sp1 (lanes 6-10) was used in a solid-phase kinase assay
with peak MonoQ fraction isolated from AGS cells before or after
treatment with 10 nM EGF for 10 min and with or without PD-98059
pretreatment for 1 h. Lanes 1 and 6, no treatment;
lanes 2 and 7, treatment with EGF for 10 min; lanes
3 and 8, treatment with EGF for 10 min and pretreatment
with PD-98059; lanes 4 and 9, treatment with PD-98059
alone; lanes 5 and 10, phosphorylation of either form
of Sp1 with Erk 2 enzyme.
|
|
To determine the target residues of this novel Mek 1-dependent kinase,
endogenous Sp1 was immunoprecipitated from radiolabeled AGS cells and
then subjected to phosphoamino acid analysis. As shown in Fig.
10, Sp1 kinase activity in vivo was EGF
dependent and phosphorylated Ser and Thr of Sp1 protein. These results
correlate with the results of phosphoamino acid analysis performed
using recombinant Sp1 protein and activated AGS extracts fractionated on a MonoQ column and eluted with high salt concentration (400-440 mM NaCl; data not shown).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 10.
EGF stimulates phosphorylation of Sp1 on Ser and Thr in vivo.
Immunoprecipitated [32P]orthophosphate-labeled
endogenous Sp1 was resolved on 10% SDS-polyacrylamide gel and then
transferred to polyvinylidene difluoride membrane. Sp1 band was excised
and then submitted for phosphoamino acid analysis. Autoradiography was
performed after 2-dimensional electrophoresis. A: phosphoamino
acid analysis of immunoprecipitated Sp1 from unstimulated cells.
B: analysis after EGF stimulation. C: radioactive spots
containing phosphoserine and phosphothreonine were analyzed on a
PhosphorImager.
|
|
 |
DISCUSSION |
EGF-dependent phosphorylation of Sp1 was studied by partially purifying
distinct Sp1 kinase activity from a human gastric cell line. We showed
recently that EGF stimulates the gastrin promoter through Mek
1-dependent and -independent pathways (34). In addition, overexpression
of Ras and Erk proteins stimulates the gastrin promoter through an Sp1
binding site. The role of Ras activation in the regulation of the
gastrin promoter is significant, since some colon and pancreatic tumors
overexpress gastrin, which correlates with activated ras
mutations. Gastrinomas are malignant pancreatic islet tumors in which
gastrin is overproduced (52). The frequency of ras mutations in
this tumor subtype is unknown; however, 80% of nonislet pancreatic
cancers have K-ras mutations (2). Many of these gastrinomas are
the result of a mutation in the tumor suppresser gene menin (9), which
encodes a corepressor that binds to JunD, a component of activator
protein (AP)-1 (1). It is well known that ras-Erk signaling
targets the Fos component of the activator protein type 1 (AP-1)
complex, and Fos is required for EGF-dependent activation of the
gastrin gene (31). Collectively, these results imply that aberrant
regulation of AP-1 in gastrointestinal tumors modulates gastrin gene
expression. Moreover, K-ras mutations are found frequently in
colon cancers (17) and have recently been correlated with elevated
gastrin gene expression in these tumors (41).
The studies reported here show that gastrin promoter induction by EGF
correlates with an increase in Sp1 binding to gERE, an event that is
partially Mek 1 dependent. Prior studies with purified Erk indicate
that, like other mitogen-inducible transcription factors, e.g., Elk and
Sap (18, 53), Erk is a candidate kinase directly mediating
phosphorylation of Sp1. Indeed, Sp1 contains several consensus Erk
phosphorylation motifs (x-x-S/T-P) (13); there is precedence for Erk
phosphorylation of nuclear proteins, and purified Erk phosphorylates
Sp1 in vitro (this report) (34). However, Erk kinase activity did not
partition on an anion-exchange column with the fractions exhibiting
maximal Sp1 kinase activity. This result may reflect the fact that
phosphorylation of Sp1 by recombinant Erk 2 depends on the level of
enzyme activity and concentration. For example, Erk 1 and Erk 2 can
phosphorylate c-Jun, but not as effectively as Jun
(NH2-terminal) kinase (JNK) activity (20). Regulation of
the gastrin promoter by overexpression of Erk 1 or Erk 2 as expression
vectors represents promoter activation in the presence of
supraphysiological levels (34). Indeed, supraphysiological levels of
Erk may not reflect events in vivo (22). However, expression of
kinase-deficient Erk 1 and Erk 2 prevented gastrin promoter activation,
presumably by abolishing endogenous Erk activity (34). The Sp1 kinase
activity and Erk 2 are Ser/Thr kinases and are Mek 1 dependent.
Collectively, these studies suggest that the Sp1 kinase activity lies
downstream of Erk 2, although other protein targets of Erk that bind to
upstream elements cannot be ruled out (Fig.
11). The Rsk 1-3 kinases are known
to reside downstream of Erk and phosphorylate transcription factors (6,
54). However, we found that Rsk 1-3 kinases, p38, JNK,
DNA-dependent kinase, as well as Erk 1 and Erk 2, also do
not copurify with the Sp1 kinase activity (data not shown). Another
Erk-independent kinase has been reported that mediates the feedback
regulation of the guanyl nucleotide exchange protein SOS (22). This
novel but unidentified kinase is activated by insulin, is also Mek 1 dependent, and regulates the dissociation of the Grb2-SOS complex.
Therefore, precedence exists for novel Mek 1-dependent subsets of
growth-regulated kinases.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 11.
Proposed role of Sp1 kinase activity in regulation of gastrin gene
expression. Ras and Erk expression stimulate gastrin promoter activity.
Inhibition of mitogen-activated protein kinase kinase (Mek) 1 blocks
Sp1 kinase activity, demonstrating that this activity lies downstream
of Mek. Erk 2 phosphorylates Sp1 and possibly targets elsewhere in
promoter; however, Sp1 kinase activity exists primarily in fractions
devoid of Erk 2. Therefore, primary "Sp1 kinase" activity is Mek
1 dependent and likely Erk dependent. Mek 1-dependent, Erk-independent
Sp1 kinase activity has not been excluded.
|
|
During preparation of this report, Black et al. (5) reported that Sp1
is phosphorylated in vivo during the G0-to-G1
transition in serum-activated fibroblasts. The growth-regulated Sp1
kinase is distinct from previously identified kinases. Phosphorylation occurred exclusively on Ser in the zinc finger domain between amino
acids 612 and 678, which resulted in a decrease in Sp1 binding to a
GC-box within the dihydrofolate promoter. Phosphorylation within the
DNA-binding domain may interfere with binding because of steric
hindrance or charge repulsion (23). Therefore, the observed decrease in
Sp1 binding in response to a growth signal was difficult to reconcile
with the Sp1-dependent stimulation of the dihydrofolate promoter. The
authors suggested that Sp1 may cooperate with other coregulators to
mediate overall transcriptional activation, despite a decrease in Sp1
binding. Although it was not evaluated in their study, another
possibility is that increased phosphorylation directly regulates the
level of transcriptional activity. However, the absence of
phosphorylation in the Ser/Thr-rich transactivation domain makes this
function less likely.
Another unusual feature of the Sp1 kinase activity reported by Black et
al. (5) is the significant delay in Sp1 phosphorylation (maximal at 6 h) compared with increased Sp1 abundance, which was maximal within
2 h. An alternative explanation is that the late burst in
phosphorylation of the zinc finger domain represents a mechanism to
decrease binding and shut off transcription of the dihydrofolate
promoter at the end of the G1 phase. Phosphorylation of Sp1
has been shown to activate proteosomal and nonproteosomal degradation
of Sp1, which inhibits transcription in some instances (19, 30, 38,
44). The Sp1 kinase activity reported here and by Black et al. may also
represent two different enzymes: a kinase activated early that targets
the Sp1 transactivation domain to activate transcription and a kinase
activated late that targets the DNA-binding domain to inhibit
transcription. The Sp1 kinase activity described in the present study
correlates with transcriptional activation. We examined early time
points that revealed a rapid increase in phosphorylation that was
maximal within 30 min and was sustained for
6 h. The increase in Sp1 abundance was not significant until 30 min after treatment and was not
maximal until ~4 h, indicating some delay in altering Sp1 abundance
compared with modulating the level of phosphorylation. Moreover, the
two kinase activities reported by each group may be distinct from each
other simply because of differences in cell types, ligands, and promoters.
Other investigators have shown that kinase activity affects Sp1 binding
and, presumably, transactivation. Chun et al. (10) showed that human
immunodeficiency virus type 1 TAT protein forms a complex with Sp1 that
augments DNA-dependent protein kinase phosphorylation of Sp1. Ser-131
within the NH2-terminal domain appears to be a target of
the kinase and required for TAT-dependent transactivation. In
regenerating liver, casein kinase 2 activity phosphorylates the zinc
finger domain of Sp1 (Thr-579) and decreases Sp1 binding (3). In
glucose-responsive promoters, glucose-inducible Sp1 binding is
prevented by casein kinase 2 phosphorylation (55). Apparently,
carbohydrate dynamically regulates the level of O-glycosylation on amino acid hydroxyl groups, which in turn are targets for
phosphorylating enzymes. Dephosphorylation of DNA binding domains and
subsequent O-glycosylation stimulates binding and transcription
and prevents Sp1 proteolysis (19, 25). In contrast, Sp1 transactivation and binding appear to be enhanced if phosphorylation occurs outside the
zinc finger domain. Increased protein kinase A activity in HL-60 cells
stimulates Sp1 phosphorylation upstream of the zinc finger domain and
subsequent binding, possibly through the protein kinase A consensus
site at Thr-366 (47). In contrast, protein kinase C
translocates to
the nucleus in the absence of the von Hippel-Lindau tumor suppressor
gene product, phosphorylating Sp1 in the zinc finger domain and
stimulating the vascular EGF promoter (42). Collectively, these studies
demonstrate that Sp1 is regulated in a dynamic fashion by
phosphorylation at several different protein domains by a wide range of
protein kinases.
The initial report on Sp1 phosphorylation demonstrated that the
modification was mediated by a DNA-dependent kinase that requires Sp1
to be prebound to DNA (24). Growth-regulated kinase phosphorylation of
Sp1 occurs in the presence of ethidium bromide used to disrupt the
protein-DNA interaction (5). Moreover, the signal transduction pathway(s) regulating DNA-dependent kinase activity remains obscure (10). Thus DNA-dependent kinase does not appear to be the major kinase
regulating receptor-mediated induction of Sp1 phosphorylation.
In summary, EGF regulation of the gastrin promoter is mediated in part
through activation of ras and Mek 1 (Fig. 11). A novel Sp1
kinase activated by EGF lies downstream of Mek 1 and, possibly, Erk.
There are >100 putative Ser and Thr phosphorylation sites within the
NH2-terminal domain of Sp1. These sites will be evaluated once the Sp1 kinase(s) is cloned to determine which of the potential Sp1 phosphorylation motifs are bona fide targets of this kinase and to
determine whether they contribute to Sp1-dependent transcription.
 |
ACKNOWLEDGEMENTS |
The authors thank S. A. Tarlé for expert technical
assistance. Oligonucleotides were synthesized by the University of
Michigan DNA synthesis core facility.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants DK-45729 (to J. L. Merchant) and DK-02336
(to A. Todisco). J. L. Merchant is an investigator of the Howard Hughes
Medical Institute. A. Todisco is a recipient of an American
Gastroenterological Association Industry Research Scholar Award and a
grant from the Charles E. Culpeper Foundation. This study was also
supported in part by National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-34533 to the University of Michigan
Gastrointestinal Peptide Research Center.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. L. Merchant,
1150 West Medical Dr., MSRB I, 3510, Ann Arbor, MI 48109-0650
(E-mail: merchanj{at}umich.edu).
Received 2 July 1999; accepted in final form 25 October 1999.
 |
REFERENCES |
1.
Agarwal, SK,
Guru SC,
Heppner C,
Erdos MR,
Collins RM,
Park SY,
Saggar S,
Chandrasekharappa SC,
Collins FS,
Spiegel AM,
Marx SJ,
and
Burns AL.
Menin interacts with the AP-1 transcription factor Jun D and represses Jun D-activated transcription.
Cell
96:
143-152,
1999[ISI][Medline].
2.
Almoguera, C,
Shibata D,
Forrester K,
Martin J,
Arnheim N,
and
Perucho M.
Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes.
Cell
53:
549-554,
1988[ISI][Medline].
3.
Armstrong, SA,
Barry DA,
Leggett RW,
and
Mueller CR.
Casein kinase II-mediated phosphorylation of the C terminus of Sp1 decreases its DNA binding activity.
J Biol Chem
272:
13489-13495,
1997[Abstract/Free Full Text].
4.
Barranco, SC,
Townsend CM, Jr,
Casartelli C,
Macik BG,
Burger NL,
Boerwinkle WR,
and
Gourley WK.
Establishment and characterization of an in vitro model system for human adenocarcinoma of the stomach.
Cancer Res
43:
1703-1709,
1983[Abstract].
5.
Black, AR,
Jensen D,
Lin SY,
and
Azizkhan JC.
Growth/cell cycle regulation of Sp1 phosphorylation.
J Biol Chem
274:
1207-1215,
1999[Abstract/Free Full Text].
6.
Blenis, J.
Signal transduction via the MAP kinases: proceed at your own RSK.
Proc Natl Acad Sci USA
90:
5889-5892,
1993[Abstract].
7.
Boyle, WJ,
van der Geer P,
and
Hunter T.
Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates.
Methods Enzymol
201:
110-149,
1991[ISI][Medline].
8.
Bradford, MM.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
9.
Chandrasekharappa, SC,
Guru SC,
Manickam P,
Olufemi S-E,
Collins FS,
Emmert-Buck MR,
Debelenko LV,
Zhuang Z,
Lubensky IA,
Liotta LA,
Crabtree JS,
Wang Y,
Roe BA,
Weisemann J,
Boguski MS,
Agarwal SK,
Kester MB,
Kim YS,
Heppner C,
Dong Q,
Spiegel AM,
Burns AL,
and
Marx SJ.
Positional cloning of the gene for multiple endocrine neoplasia type 1.
Science
276:
404-407,
1997[Abstract/Free Full Text].
10.
Chun, RF,
Semmes OJ,
Neuveut C,
and
Jeang KT.
Modulation of Sp1 phosphorylation by human immunodeficiency virus type 1 Tat.
J Virol
72:
2615-2629,
1998[Abstract/Free Full Text].
11.
Courey, AJ,
and
Tjian R.
Mechanisms of transcriptional control as revealed by studies of human transcription factor Sp1.
In: Transcriptional Regulation, edited by McKnight SL,
and Yamamoto KR.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1992, p. 743-789.
12.
Daniel, S,
Zhang S,
DePaoli-Roach AA,
and
Kim K-H.
Dephosphorylation of Sp1 by protein phosphatase 1 is involved in the glucose-mediated activation of the acetyl-CoA carboxylase gene.
J Biol Chem
271:
14692-14697,
1996[Abstract/Free Full Text].
13.
Davis, RJ.
The mitogen-activated protein kinase signal transduction pathway.
J Biol Chem
268:
14553-14556,
1993[Free Full Text].
14.
Dent, P,
Haser W,
Haystead TAJ,
Vincent LA,
Roberts TM,
and
Sturgill TW.
Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro.
Science
257:
1404-1407,
1992[ISI][Medline].
15.
De Vries-Smits, AMM,
Burgering BMT,
Leevers SJ,
Marshall CJ,
and
Bos JL.
Involvement of p21 ras in activation of extracellular signal-regulated kinase 2.
Nature
357:
602-604,
1992[ISI][Medline].
16.
Ford, MG,
Valle JD,
Soroka CJ,
and
Merchant JL.
EGF receptor activation stimulates endogenous gastrin gene expression in canine G cells and human gastric cell cultures.
J Clin Invest
99:
2762-2771,
1997[Abstract/Free Full Text].
17.
Forrester, K,
Almoguera C,
Han K,
Grizzle WE,
and
Perucho M.
Detection of high incidence of K-ras oncogenes during human colon tumorigenesis.
Nature
327:
298-303,
1987[ISI][Medline].
18.
Gille, H,
Sharrocks AD,
and
Shaw PE.
Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter.
Nature
358:
414-417,
1992[ISI][Medline].
19.
Han, I,
and
Kudlow JE.
Reduced O-glycosylation of Sp1 is associated with increased proteasome susceptibility.
Mol Cell Biol
17:
2550-2558,
1997[Abstract].
20.
Hibi, M,
Lin A,
Smeal T,
Minden A,
and
Karin M.
Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain.
Genes Dev
7:
2135-2148,
1993[Abstract].
21.
Hill, CS,
and
Treisman R.
Transcriptional regulation by extracellular signals: mechanisms and specificity.
Cell
80:
199-211,
1995[ISI][Medline].
22.
Holt, KH,
Kasson BG,
and
Pessin JE.
Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.
Mol Cell Biol
16:
577-583,
1996[Abstract].
23.
Hunter, T,
and
Karin M.
The regulation of transcription by phosphorylation.
Cell
70:
375-387,
1992[ISI][Medline].
24.
Jackson, S,
Gottlieb T,
and
Hartley K.
Phosphorylation of the transcription factor Sp1 by the DNA-dependent protein kinase.
In: Advances in Second Messenger and Phosphoprotein Research, edited by Greengard P,
and Robison GA.. New York: Raven, 1993, vol. 28, p. 279-286.
25.
Jackson, SP,
and
Tjian R.
O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation.
Cell
55:
125-133,
1988[ISI][Medline].
26.
Janson, L,
and
Pettersson U.
Cooperative interactions between transcription factors Sp1 and OTF-1.
Proc Natl Acad Sci USA
87:
4732-4736,
1990[Abstract].
27.
Khachigian, LM,
Williams AJ,
and
Collins T.
Interplay of Sp1 and Egr-1 in the proximal platelet-derived growth factor A-chain promoter in cultured vascular endothelial cells.
J Biol Chem
270:
27679-27686,
1995[Abstract/Free Full Text].
28.
Law, GL,
Itoh H,
Law DJ,
Mize GJ,
Merchant JL,
and
Morris DR.
Transcription factor ZBP-89 regulates the activity of the ornithine decarboxylase promoter.
J Biol Chem
273:
19955-19964,
1998[Abstract/Free Full Text].
29.
Lee, Y-H,
Yano M,
Liu S-Y,
Matsunaga E,
Johnson PR,
and
Gonzalez FJ.
A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP
and an Sp1 factor.
Mol Cell Biol
14:
1383-1394,
1994[Abstract].
30.
Leggett, RW,
Armstrong SA,
Barry D,
and
Mueller CR.
Sp1 is phosphorylated and its DNA binding activity down-regulated upon terminal differentiation of liver.
J Biol Chem
270:
25879-25884,
1995[Abstract/Free Full Text].
31.
Marks, P,
Iyer G,
Cui Y,
and
Merchant JL.
Fos is required for EGF stimulation of the gastrin promoter.
Am J Physiol Gastrointest Liver Physiol
271:
G942-G948,
1996[Abstract/Free Full Text].
32.
Marshall, CJ.
Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.
Cell
80:
179-185,
1995[ISI][Medline].
33.
Merchant, JL,
Demediuk B,
and
Brand SJ.
A GC-rich element confers epidermal growth factor responsiveness to transcription from the gastrin promoter.
Mol Cell Biol
11:
2686-2696,
1991[ISI][Medline].
34.
Merchant, JL,
Du M,
and
Todisco A.
Sp1 phosphorylation by Erk 2 stimulates DNA binding.
Biochem Biophys Res Commun
254:
454-461,
1999[ISI][Medline].
35.
Merchant, JL,
Iyer GR,
Taylor BR,
Kitchen JR,
Mortensen ER,
Wang Z,
Flintoft RJ,
Michel J,
and
Bassel-Duby R.
ZBP-89, a Krüppel-type zinc finger protein, inhibits EGF induction of the gastrin promoter.
Mol Cell Biol
16:
6644-6653,
1996[Abstract].
36.
Merchant, JL,
Shiotani A,
Mortensen E,
Shumaker D,
and
Abraczinskas D.
EGF stimulation of the human gastrin promoter requires Sp1.
J Biol Chem
270:
6314-6319,
1995[Abstract/Free Full Text].
37.
Miltenberger, RJ,
Farnham PJ,
Smith DE,
Stommel JM,
and
Cornwell MM.
v-Raf activates transcription of growth-responsive promoters via GC-rich sequences that bind the transcription factor Sp1.
Cell Growth Differ
6:
549-556,
1995[Abstract].
38.
Mortensen, ER,
Marks PA,
Shiotani A,
and
Merchant JL.
Epidermal growth factor and okadaic acid stimulate Sp1 proteolysis.
J Biol Chem
272:
16540-16547,
1997[Abstract/Free Full Text].
39.
Muroya, K,
Hattori S,
and
Nakamura S.
Nerve growth factor induces rapid accumulation of the GTP-bound form of p21 (ras) in rat pheochromocytoma PC12 cells.
Oncogene
7:
277-281,
1992[ISI][Medline].
40.
Nakano, K,
Mizuno T,
Sowa Y,
Orita T,
Yoshino T,
Okuyama Y,
Fujita T,
Ohtani-Fujita N,
Matsukawa Y,
Tokino T,
Yamagishi H,
Oka T,
Nomura H,
and
Sakai T.
Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative human colon cancer cell line.
J Biol Chem
272:
22199-22206,
1997[Abstract/Free Full Text].
41.
Nakata, H,
Wang SL,
Chung DC,
Westwick JK,
and
Tillotson LG.
Oncogenic ras induces gastrin gene expression in colon cancer.
Gastroenterology
115:
1144-1153,
1998[ISI][Medline].
42.
Pal, S,
Claffey KP,
Cohen HT,
and
Mukhopadhyay D.
Activation of Sp1-mediated vascular permeability factor/vascular endothelial growth factor transcription requires specific interaction with protein kinase C
.
J Biol Chem
273:
26277-26280,
1998[Abstract/Free Full Text].
43.
Pelech, SL,
and
Sanghera JS.
MAP kinases: charting the regulatory pathways.
Science
257:
1355-1356,
1992[ISI][Medline].
44.
Piedrafita, FJ,
and
Pfahl M.
Retinoid-induced apoptosis and Sp1 cleavage occur independently of transcription and require caspase activation.
Mol Cell Biol
17:
6348-6358,
1997[Abstract].
45.
Pugh, BF,
and
Tjian R.
Mechanism of transcriptional activation by Sp1: evidence for coactivators.
Cell
61:
1187-1197,
1990[ISI][Medline].
46.
Renshaw, MW,
Ren XD,
and
Schwartz MA.
Growth factor activation of MAP kinase requires cell adhesion.
EMBO J
16:
5592-5599,
1997[Abstract/Free Full Text].
47.
Rohlff, C,
Ahmad S,
Borellini F,
Lei J,
and
Glazer RI.
Modulation of transcription factor Sp1 by cAMP-dependent protein kinase.
J Biol Chem
272:
21137-21141,
1997[Abstract/Free Full Text].
48.
Saffer, JD,
Jackson SP,
and
Thurston SJ.
SV40 stimulates expression of the trans-acting factor Sp1 at the mRNA level.
Genes Dev
4:
659-666,
1990[Abstract].
49.
Seto, E,
Lewis B,
and
Shenk T.
Interaction between transcription factors Sp1 and YY1.
Nature
365:
462-464,
1993[ISI][Medline].
50.
Tan, S-H,
Gloss B,
and
Bernard H-U.
During negative regulation of the human papillomavirus-16 E6 promoter, the viral E2 protein can displace Sp1 from a proximal promoter element.
Nucleic Acids Res
20:
251-256,
1991[Abstract].
51.
Udvadia, AJ,
Roger KT,
Higgins PD,
Murata Y,
Humphrey PA,
and
Horowitz JM.
Sp-1 binds promoter elements regulated by the RB protein and Sp-1 mediated transcription is stimulated by RB coexpression.
Proc Natl Acad Sci USA
90:
3265-3269,
1993[Abstract].
52.
Walsh, JH.
Role of gastrin as a trophic hormone.
Digestion
47:
11-16,
1990[ISI][Medline].
53.
Whitmarsh, AJ,
Shore P,
Sharrocks AD,
and
Davis RJ.
Integration of MAP kinase signal transduction pathways at the serum response element.
Science
269:
403-407,
1995[ISI][Medline].
54.
Xing, J,
Ginty DD,
and
Greenberg ME.
Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase.
Science
273:
959-963,
1996[Abstract].
55.
Zhang, S,
and
Kim K-H.
Protein kinase CK2 down-regulates glucose-activated expression of the acetyl-CoA carboxylase gene.
Arch Biochem Biophys
338:
227-232,
1997[ISI][Medline].
Am J Physiol Cell Physiol 278(4):C697-C708
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society