Growth Factors Signal to Steroid Receptors through
Mitogen-activated Protein Kinase Regulation of p160 Coactivator
Activity*
Gabriela N.
Lopez
,
Christoph W.
Turck§¶,
Fred
Schaufele
,
Michael R.
Stallcup
, and
Peter J.
Kushner
§**
From the
Metabolic Research Unit,
§ Department of Medicine, and ¶ Howard Hughes
Medical Institute, University of California, San Francisco, California
94143 and the
Department of Pathology, University of Southern
California, Los Angeles, California 90033
Received for publication, November 28, 2000, and in revised form, March 26, 2001
 |
ABSTRACT |
Promoter-bound steroid receptors activate gene
expression by recruiting members of the p160 family of coactivators.
Many steroid receptors, most notably the progesterone and estrogen
receptors, are regulated both by cognate hormone and independently by
growth factors. Here we show that epidermal growth factor regulates the activities of the p160 GRIP1 through the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases. ERKs phosphorylate GRIP1 at a specific site, Ser-736, the integrity of which is required for full growth factor induction of GRIP1 transcriptional activation and coactivator function. We propose that
growth factors signal to nuclear receptors in part by targeting the
p160 coactivators.
 |
INTRODUCTION |
Nuclear receptors such as the estrogen receptor
(ER)1 and progesterone
receptor (PR) tether via their DNA binding domain to response elements
in the promoter region of target genes and stimulate transcription. To
do so the receptors must bind to coactivators that they recruit through
transcriptional activation functions, the constitutive AF-1, found in
the amino-terminal receptor domain, and the hormone-activated AF-2 in
the carboxyl ligand binding domain (LBD) (for review, see Ref. 1).
Perhaps the most important of these coactivators is the p160 family,
SRC-1 (N-CoA1), GRIP1 (TIF2/N-CoA2), and ACTR (pCIP/AIB1/RAC3).
These bind to the LBD only in the presence of cognate hormone, and
their binding is blocked by antagonist ligands. The mechanism of
binding is now understood in atomic detail and involves the docking of
coactivator nuclear receptor boxes, which have the motif
LXXLL with a hydrophobic cleft that forms on the surface of
the hormone-bound LBD (2-6). The AF-1 domain of the estrogen,
androgen, and perhaps other receptors also contacts the p160s but does
so through surfaces outside of the nuclear receptor boxes (7, 8).
The p160s are complex proteins with multiple domains (Fig. 1). In
addition to the nuclear receptor boxes they have two intrinsic transcriptional activation domains AD1 and AD2, whose activities may be
monitored when the coactivators are directly tethered on DNA via fusion
to a heterologous DNA binding domain (8, 9). AD1, which is essential
for transcriptional mediation by p160s, is coextensive with the binding
domain for the CBP/p300 family of coactivators. CBP/p300
complexes with the p160s and synergizes in coactivator function (10,
11). In particular, CBP/p300s contain a potent acetyltransferase
activity that can transfer acetate from acetyl-CoA to histones and also
to other proteins in the complex on DNA (12-18). AD2 contributes to
coactivation by p160s in some circumstances and does so in part by
binding CARM1 and other proteins that have histone methyltransferase
activity (19). The coactivators are believed to mediate transcriptional activation by remodeling chromatin through their histone modification activities and also by direct effects on the transcriptional complex.
In addition to regulating AF-2, hormones regulate steroid receptors
(but not other nuclear receptors) in part by releasing the receptors
from inhibitory complexes with heat shock proteins. The activity of
steroid receptors is not regulated solely by hormones, however. Growth
factors, such as EGF and insulin-like growth factor 1, can have
profound and surprising effects on steroid receptors, even in the
absence of cognate hormone (for review, see Ref. 20). In the most
dramatic examples, EGF activates progesterone receptors almost as well
as does hormone (21) and will partly activate ER in the absence of
hormone and enhance activity in the presence of hormone (22). This
later activity has been traced in part to EGF initiation of a cascade
through the ERK family of MAP kinases that ultimately phosphorylates
the ER at Ser-118 in the AF-1 region (23-26). Phosphorylation of
Ser-118 leads to increased AF-1 activity by increasing binding of a p68
RNA helicase that is or becomes bound to CBP. This
Ser-118-dependent link of AF-1 to CBP is in addition to the
Ser-118-independent link with GRIP1 and thereby CBP (27).
Although EGF-mediated phosphorylation of steroid receptors underlies
part of EGF enhancement, it cannot underlie all of it. In particular,
mutation of Ser-118 to glutamate in the ER AF-1 domain, while blocking
phosphorylation, nonetheless preserves EGF enhancement of ER action
(25). Furthermore, careful mutation of sites of phosphorylation in the
progesterone receptor again reveals a potent action of EGF in the
absence of direct receptor phosphorylation (21). These studies suggest
the existence of a pathway of EGF action that targets a nonreceptor
protein. We have explored the possibility that this unknown target is
one of the p160 coactivators, particularly GRIP1. We present evidence that at least part of the signal from EGF to steroid receptor is
conducted through the p160 coactivators.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture, Transient Transfection, and Luciferase Assay
HeLa cells were transfected by electroporation as previously
described (28). Generally transfections included 5 µg of
luciferase reporter plasmid, 1.0 µg of Gal-GRIP expression
vectors, and 1.0 µg of
-actin-
-galactosidase expression vector
for internal control. After electroporation, the cells were treated
with vehicle, EGF (25 ng/ml), or TGF-
(15 ng/ml). Luciferase and
chloramphenicol acetyltransferase values are the means and standard
deviations of triplicate treatments from a single experiment,
representative of at least three independent experiments.
In Vitro Kinase Assay
GST-GRIP fragments were expressed in bacteria (HB101) and
partially purified by glutathione-Sepharose affinity column. Beads bearing the fusion proteins (1-3 µg of total protein) were subjected to in vitro phosphorylation by activated ERK2 according to
the instructions of the supplier (Stratagene, La Jolla, CA). The
phosphorylated products were extracted from the beads, resolved by 10%
polyacrylamide gel electrophoresis, stained with Coomassie Blue to
monitor expression, and subjected to autoradiography.
Phosphorylation Site Mapping
Approximately 8.5 µg of total protein on beads were labeled in
a 50-µl final reaction volume, extracted from the beads, and purified
by 10% SDS-polyacrylamide gel electrophoresis. The gel was fixed,
dried, and exposed to x-ray film to visualize the radiolabeled proteins. In-gel digestion of the protein with trypsin or
endoproteinase Glu-C (Roche Diagnostics, Indianapolis, IN) was carried
out as described previously (29). The resultant peptides were separated using reversed phase HPLC on a microbore C8 column (Vydac,
Hesperia, CA), and the collected fractions were subjected to
scintillation counting. Individual peptides were subjected to covalent
Edman degradation on a Sequelon AA membrane (PerSeptive Biosystems, Cambridge, MA) with a protein sequencer (model 492; Applied Biosystems, Foster City, CA). The anilinothiazolínoneamino acids were
extracted from the filter with neat trifluoroacetic acid and
scintillation counted. Radioactive profiles for each sequencing run
were compared with theoretical peptide sequences derived from the protein.
Plasmids
Expression Vectors--
To construct GalDBD-GRIP1, the GalDBD
coding fragment from pGBT9·GRIPFL (32) was removed with
HindIII-EcoRI and ligated to
SmaI-EcoRI-cut pBS (Stratagene) giving rise to
pBS·Gal4DBD. An EcoRI fragment of GRIP1 (amino acids
5-1462) from pGBT9 vector was then inserted into pBS·Gal4DBD. The
entire Gal4 DBD-GRIP1 coding segment was then removed with
XbaI-EcoRV and subcloned into the
NheI-EcoRV site of commercially available pCMV
vector (Stratagene). GalDBD-CBP has been described by Swope et
al. (30). GST-GRIP1-479, GST-GRIP1-766, GST-GRIP184-766 and
GST-GRIP766-1462 vectors for bacterial expression have been described
by Webb et al. (7). The mutants GST-GRIPS736A and
GST-GRIPS554A for mammalian expression were generated by polymerase
chain reaction from parental vector GST-GRIP184-766 incorporating a
mutagenic primer with Pfu polymerase (QuickChange
site-directed mutagenesis kit, Stratagene). MPK-1 (CL100) expression
vector was a gift of D. Stokoe (Cancer Center, University of
California, San Francisco). MEKK97R expression vector has been
described by Mansour et al. (31). pSG5-GRIPS736A was
constructed from pSG5-GRIPFL (32) using mutagenic primer incorporation
by polymerase chain reaction as described above. Gal-GRIPS736A and
Gal-GRIPS554A are derivatives of Gal-GRIPFL vector and were mutated as
indicated above. All the mutants were confirmed by sequencing, and
generally, functional assays of two clones were carried out. pGFP-GRIP
has green fluorescent protein fused to the amino terminus of GRIP
expression vector and was a gift from Yihong Wan (University of
Colorado, Health Sciences Center), and pGFP-GRIPS736A was constructed
by removing a 1612-nucleotide BstXI-BspEI
fragment from pSG5-GRIPS736A and inserting it into the
BstXI-BspEI sites of pGFP-GRIP. The point
mutation was confirmed by sequencing. The pSG5ER S118A mutant is a
derivative of HEO (33) and is described by Webb et
al. (7).
Reporters--
GalRE-luc contains five Gal4 response elements
upstream of a minimal adenovirus E1b promoter and has been previously
described (34). It is devoid of the pUC AP1 site as described by Webb et al. (36). ERE-luc contains the 45-mer estrogen response
element from the Xenopus vitellogenin A2 promoter driving
expression of the luciferase gene cloned in a pUC vector devoid of the
vector AP1 site (35) and has been previously described (36). The p
TAT3-luc reporter has three glucocorticoid response elements from the regulatory region of tyrosine aminotransferase gene
placed upstream of the minimal Drosophila distal alcohol
dehydrogenase promoter (
33 to +55) driving luciferase expression
(37).
Western Blot
20 million HeLa cells were transfected with 20 µg of
GFP-GRIPwt or GFP-GRIPS736A expression vector, incubated overnight at 37 °C, exposed to EGF for 2 h, and harvested in lysis buffer
(10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 20 mM
-glycerophosphate, 0.1% Triton X-100), protease inhibitors, and
Ser/Thr and Tyr phosphatase inhibitors (Sigma). The whole cell extract
(50 µg) was resolved by 4-15% gradient polyacrylamide gel
electrophoresis, transferred to a polyvinylidene difluoride membrane
(Bio-Rad), and probed with anti-green fluorescent protein (GFP)
antibodies (Convance, Berkeley, CA) or GRIP1 antibodies (Affinity
BioReagents, Inc., Golden, CO) followed by the corresponding
peroxidase-conjugated anti-IgG. Signals were visualized by the ECL
detection kit (Amersham Pharmacia Biotech).
Fluorescence Microscopy
After transfections cells to be analyzed by microscopy were
distributed on cover glass-containing wells, and a duplicate group of
cells was treated for regular luciferase activity. Images were collected on a Zeiss Axioplan epifluorescence microscope using a 63×,
1.35 numerical aperture oil immersion objective and fluorescein isothiocyanate excitation and emission filters (Chroma Corp., Brattleboro, VT). Digital images were collected using a Xillix CCD
camera and integration times from 50 to 150 ms. All the images presented in Fig. 6 were collected at the same integration times and
processed identically.
 |
RESULTS |
A recent report suggests that growth factor stimulation of the
transcriptional activation functions of CBP (38, 39) requires the
domain of CBP that mediates p160 binding (10). We thus examined in
transfected cells whether the p160 GRIP1 tethered to a reporter gene
promoter by fusion to the heterologous Gal4 DNA binding domain (Gal-GRIP1, Fig. 1) could
activate transcription in response to growth factors. EGF and TGF-
had no effect on reporter gene expression in the absence of GRIP1 (Fig.
1A), but these ligands of the EGF receptor each activated
transcription 5-10-fold when GRIP1 was bound to the promoter. The
GRIP1 response to EGF and TGF-
required the action of the ERK family
of MAP kinases because it was abolished by overexpression of the MAP
kinase phosphatase CL100, by a dominant negative MEK that specifically
prevents activation of ERKs, and by PD98059, a specific inhibitor of
MEK activation (Fig. 1, B and C). In control
experiments EGF failed to stimulate reporter gene transcription
mediated by a Gal4 fusion to the herpesvirus VP16 protein, and as
expected, neither PD98059 nor CL100 nor dominant negative MEK was
inhibitory (data not shown). Thus GRIP1 contains an EGF-regulated
transcriptional activation function, and ERKs are a component of the
pathway of activation.

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Fig. 1.
GRIP1 contains a growth factor-regulated and
ERK kinase-mediated transcriptional activation function.
A, activity of a luciferase reporter gene regulated by Gal4
response elements (GalRE-luc) in cells expressing yeast Gal4 DNA
binding domain by itself (Gal-DBD) or fused to GRIP1
(Gal-GRIP1) and treated with growth factors as indicated.
B and C, EGF activation of GRIP1 is abolished by
ERK inhibitors. Activity of GalRE-luc with expression vectors for
Gal-GRIP1, dominant negative MEK (MEK(K97R)), the
dual specificity phosphatase CL100 1B, or the MEK inhibitor PD98059.
TNF, tumor necrosis factor; IGF, immunoglobulin
growth factor.
|
|
To examine whether GRIP1 might serve as a direct substrate for ERKs, we
prepared recombinant GRIP1 in Escherichia coli and incubated it with activated ERK2 in vitro. Among
fragments that represent the entire protein, two fragments from amino
acids 1-766 and 184-766 were strongly phosphorylated by MAP kinase
(Fig. 2A). Fragment 1-479 was
unreactive, and fragment 766-1462 was barely reactive. Thus GRIP1
serves as a MAP kinase substrate in vitro, and the major
site(s) of phosphorylation lies within amino acids 184-766, most
likely between 480 and 766.

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Fig. 2.
ERK phosphorylates GRIP1 on Ser-736 in
vitro. A, domain map of GRIP1 with the
location of the nuclear receptor (NR) boxes, Ser-736 and Ser-554, and
phosphorylation of E. coli-expressed fragments of GRIP1 by
purified and activated ERK2. Products of the reaction with
radiophosphate are shown after gel separation. A major phosphorylation
site is located in the fragment spanning amino acids 184-766.
B, mapping of the site of ERK phosphorylation on GRIP1.
Edman analysis of the major peaks of radiophosphate on GRIP1 after
incubation with ERK2 and digestion with either trypsin or
endoproteinase Glu-C is shown. Both analyses predict Ser-736 as
the major site of phosphorylation. C, confirmation that
S736A is a major site of phosphorylation by ERK. Wild type
(wt) GST-GRIP1-(184-766) and mutants S554A and S736A were
incubated with ERK2 (left panel) or, as a control, with JNK1
(right panel), and the products were autoradiographed after
gel separation.
|
|
The precise site of action of ERK MAP kinase was determined by using
enzymatic protein digests and Edman analysis of the phosphate-labeled GRIP1-(184-766). Each of these digests yielded a single major labeled peptide on HPLC, and the phosphate label was on the fifth amino
acid of the tryptic peptide and the ninth of the V8 peptide (Fig.
2B). Each of these assays predicts that the major site of phosphorylation of GRIP1 is serine 736 (Fig. 2B), which is
an ERK consensus site. To confirm the identity of the site, the
phosphorylation reactions were repeated with wild type GRIP1-(184-766)
and mutants in which Ser-736 or Ser-554, chosen because it resembles a
MAP kinase site, was mutated to alanine. Phosphorylation was diminished on GRIP1 mutated on Ser-736 (Fig. 2C). The S554A mutant was
phosphorylated as efficiently as wild type. In control experiments,
JNK1, a MAP kinase with different specificity, phosphorylated all three
substrates equally. We concluded that Ser-736 is a major target for
phosphorylation of GRIP1 by ERK but not JNK in vitro.
To examine the role of Ser-736 in vivo, we repeated our
studies of EGF activation of tethered GRIP1 using both wild type and mutants. EGF potentiated transcriptional activation mediated by wild
type GRIP1 or the S554A mutant but was consistently weaker on the S736A
mutant (Fig. 3A, upper
panel). Despite this diminution of response, the
Gal-GRIPS736A mutant was well expressed as detected by Western blots
(not shown) and by its ability to serve as "bait" in a mammalian
two-hybrid assay (Fig. 3A, lower panel). These results indicate that GRIP1 bound directly to the promoter requires Ser-736 for full activation by EGF.

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Fig. 3.
Mutation of Ser-736 to Ala decreases
EGF-induced transcriptional activation by Gal-GRIP1 and diminishes
EGF-induced coactivator functions. A, top
panel, expression of a GalRE-luc reporter gene activated by
Gal-GRIP1 wild type or the indicated mutant and induced with EGF. *,
p < 0.028 as compared with EGF-induced level of
Gal-GRIPwt by standardized Student's t test. Bottom
panel, comparison of the ability of Gal-GRIPwt and Gal-GRIPS736A
to act as bait in a mammalian two-hybrid assay in which they are
tethered on a GalRE-luc reporter gene and activated with an expression
vector for the ligand binding domain of estrogen receptor fused to the
VP16 activation domain (ER-LBDVP16) in the absence or
presence of EGF or E2. B, expression of a PRE-luciferase
reporter gene (TAT3-luc) activated by PR, EGF, or the
progestin RU5020 as indicated and potentiated by wild type or S736A
mutant of GRIP1. Values correspond to the mean ± %cv of
triplicate determinations from a representative experiment that was
performed separately at least three times. **, p < 0.003 as compared with the RU5020-induced coactivation by GRIPwt; *,
p < 0.01 for EGF-induced coactivation by mutant
GRIPS736A compared with EGF-induced levels by GRIPwt.
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|
We then tested the requirement for Ser-736 when GRIP1 functions as a
coactivator for the PR activated by EGF signaling. We used a reporter
gene with three response elements from the tyrosine aminotransferase
promoter (TAT3-luc), which the PR activates 3-fold in the presence of
EGF (Fig. 3B). Wild type GRIP1 as well as the S554A mutant
potentiated PR-mediated transcription of the reporter gene
approximately 4-fold after activation with EGF. Both the hormone-liganded and constitutive activity of the PR were potentiated, as is frequently observed for nuclear receptors with overexpressed coactivators (data not shown). The S736A mutant was only half as
effective as wild type in potentiating PR action. In control experiments with hormone-activated glucocorticoid receptor, which does
not respond to EGF, both wild type and S736A GRIP1 potentiated receptor
action to similar extents (data not shown), indicating that the Ser-736
mutant of GRIP1 retains full function in some contexts. S736A GRIP1
was, however, less able than wild type GRIP1 to potentiate
progestin-activated PR. Some of this deficit may reflect a role for low
level activation of ERK or some other kinase on the inducible functions
of GRIP1 on progestin-PR, even in the absence of deliberate EGF
stimulation (data not shown). For other potential explanations see
Lange et al. (40). Thus S736A GRIP1 retains full ability to
serve as a coactivator in some circumstances but is deficient in
EGF-regulated coactivator function on the PR.
Estrogen receptor activation of transcription from a reporter gene
regulated by a consensus ERE is potentiated by EGF to varying extents
depending on cells and culture conditions. We tested the ability of an
ER S118A, mutated in the major site of EGF-dependent phosphorylation, for response to EGF with GRIP1. Without elevated GRIP1, EGF had little or no effect on transcriptional activation by ER
S118A either in the absence or presence of estrogen (Fig. 4A, E2). With
elevated GRIP1, EGF potentiated both unliganded and estrogen-liganded
ER S118A action 2-fold. Interestingly, the S736A GRIP1 mutant was
unable to support an EGF response. The isolated ER AF-2 function, which
can be tested as a fusion of the ER LBD to the yeast Gal4 DNA
binding domain tethered on a Gal4 response element, does not respond to
EGF (Fig. 4B). The S736A mutant was undiminished in its
ability to function as a coactivator for isolated ER AF-2. Thus, S736A
retains full coactivator function with the isolated ER LBD but is
deficient in coactivator function for full-length ER S118A.

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Fig. 4.
A, effect of GRIP1 wild type and S736A
on the expression of an ERE-luciferase reporter gene
(ERE-luc) activated by ER-S118A and estrogen (E2)
or EGF as indicated. B, effect of GRIP1 wild type and S736A
on expression of a GalRE-luciferase reporter gene
(GalRE-luc) activated by a Gal4-ER LBD fusion protein,
estrogen, and EGF as indicated.
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As noted above, GRIP1 is also bound to and synergizes with CBP/p300
(10, 11, 41). It is possible that EGF regulates GRIP1 coactivator
activities by affecting the efficiency of this interaction. Indeed, it
was recently reported that activated MEK increased the association of
CBP and the p160 AIB1 (42). Although expression of activated MEK led to
phosphorylation of AIB1 the site(s) was not analyzed. We therefore
examined the ability of wild type and S736A GRIP1 to potentiate
transcription mediated by CBP. We tethered CBP directly to a promoter
by fusion to a heterologous Gal4 DNA binding domain. In the absence of
overexpressed GRIP1 the CBP domain weakly activates transcription (Fig.
5). Coexpression of wild type GRIP1 or
the S554A mutant markedly potentiates transcription, which becomes
sensitive to further induction by EGF. The S736A mutant of GRIP1 is,
however, compromised in its ability to activate tethered CBP, and the
EGF response is decreased.

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Fig. 5.
Mutation of Ser-736 to Ala impairs the
ability of GRIP1 to serve as an EGF-inducible coactivator for
Gal-CBP. Expression of the GalRE-luc reporter gene when activated
by a fusion of the p160 binding domain of CBP to yeast Gal4 DNA binding
domain (Gal-CBP), wild type or mutant versions of GRIP1, and
EGF is as indicated.
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To confirm that the defects of the S736A mutant are not due to loss of
stability or nuclear localization ability, we fused both wild type and
mutant GRIP1 to GFP and monitored GRIP1 expression levels and nuclear
localization. Both wild type and Ser-736 were well expressed and both
localized to the nucleus (Fig. 6,
top panel). Western blots indicated equal expression of wild
type and mutant (Fig. 6, middle panel). When fused to GFP
wild type GRIP1 had full coactivator action with tethered CBP and EGF,
but the S736A mutant was diminished (Fig. 6, bottom panel).
Thus the S736A mutation does not affect GRIP1 expression or
localization yet has a specific effect on EGF-responsive coactivator
function.

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Fig. 6.
Expression and localization of GRIP1 is
unaffected by the S736A mutation. Top
panels, fluorescence from fusions of GFP to GRIP1 wild type
or S736A expressed in HeLa cells either untreated or EGF-treated as
indicated. Middle panel, Western blot of the transfected
cell extracts probed with anti-GFP. Lower panel, effect of
GFP-GRIP1 wild type or S736A on expression of a GalRE-luc reporter gene
activated with Gal-CBP and EGF as indicated.
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 |
DISCUSSION |
The observations above indicate that EGF-activated ERK MAP kinase
potentiates the transcriptional activation functions of GRIP1.
Furthermore, activated ERKs phosphorylate GRIP1 on serine 736 in
vitro. Mutation of Ser-736 to alanine substantially reduces the
ability of GRIP1 to enhance transcription of the EGF-activated PR and
of the estrogen- and EGF-potentiated ER. Because the mutant GRIP1 is
fully able to function as a coactivator for the GR and for the isolated
ER LBD AF-2, receptors that do not respond to EGF, it appears that the
defect of S736A is limited and may indeed be specific for GRIP1
coactivator function with EGF-responsive steroid receptors or other
transcription factors. In sum these observations suggest that
EGF-activated ERKs increase selected coactivator functions of GRIP1
through a pathway requiring the integrity and most likely the
phosphorylation of Ser-736.
While this study was in preparation, it was reported that AIB1, another
member of the p160 family, contained a transcriptional function that
was activated by transfection with constitutive MEK, a potent activator
of ERKs (42). Activation by MEK was independent of the D2 domain of
AIB1, suggesting that AD1 was responsible. Indeed fragments of AIB1
that contain AD1 and the nuclear receptor boxes are phosphorylated in
MEK-transfected cells, suggesting that the target of MEK activation is
in this region and may require AD1 for an output. Interestingly, a
deletion of AD1 of GRIP1 compromises the EGF response with ER and PR
(data not shown).
Activation by MEK was also reported to increase the binding of CBP with
AIB1 fragments that contain the AD1 domain. Our studies complement this
observation in that we observed an EGF-induced increase in the
functional interaction between the p160 binding domain of CBP tethered
to DNA by fusion to the Gal4 DNA binding domain and GRIP1. The
interaction is compromised by the Ser-736 mutation, suggesting a direct
role for this site in the interaction. Sequence comparison of AIB1 and
GRIP1 indicates that the Ser-736 residue is not conserved. Therefore
mechanistic comparison must await the identification of the
MEK-phosphorylated residues in AIB1 and their mutational analysis.
The EGF pathway to GRIP1 and thence to transcription factors that
recruit GRIP1 may provide a biological means to coordinate EGF effects
at many different transcription factors and their target genes. It has
been well established that EGF initiates a signal transduction
phosphorylation cascade that leads to transcription factors such as
Elk-1, ER, and PR. Direct phosphorylation of the transcription factors
is required for wild type Elk-1 and ER response to EGF. Nonetheless,
there are hints that in the absence of direct phosphorylation, such as
when the ER Ser-118 is mutated to glutamate, an EGF response persists.
Thus, we suggest that for an EGF-induced signal cascade to be fully
effective the cascade may need to target both the transcription factor
and the recruited coactivator. This double requirement may prevent
random biological noise from activation of the EGF response.
 |
ACKNOWLEDGEMENTS |
We thank Paul Webb, Branka Kovacic, and David
Stokoe for plasmids and helpful discussion.
 |
FOOTNOTES |
*
This work was supported by United States Public Health
Service Grants R01 CA 80210 and R01 DK51083 from the National
Institutes of Health and Grant DAMD17-99-1-9110 from the Army Breast
Cancer Research Program (to P. J. K.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
Director of, consultant to, and has significant financial interests
in KaroBio AB, a Swedish pharmaceutical development company with
interests in this area of research. To whom correspondence should be
addressed. Tel.: 415-476-6790; E-mail: kushner@itsa.ucsf.edu.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M010718200
 |
ABBREVIATIONS |
The abbreviations used are:
ER, estrogen
receptor;
PR, progesterone receptor;
LBD, ligand binding domain;
EGF, epidermal growth factor;
ERK, extracellular signal-regulated kinase;
MAP, mitogen-activated protein;
HPLC, high pressure liquid
chromatography;
GFP, green fluorescent protein;
MEK, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase;
JNK, c-Jun NH2-terminal kinase;
GST, glutathione
S-transferase;
luc, luciferase;
DBD, DNA binding domain;
ERE, estrogen response element;
PRE, progesterone response element;
wt, wild type;
TGF, transforming growth factor;
CBP, CREB-binding protein;
GRIP1, glucocorticoid receptor interacting protein 1.
 |
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