Functional Interactions, Phosphorylation, and Levels of 3',5'-Cyclic Adenosine Monophosphate-Regulatory Element Binding Protein and Steroidogenic Factor-1 Mediate Hormone-Regulated and Constitutive Expression of Aromatase in Gonadal Cells
Diana L. Carlone and
JoAnne S. Richards
Department of Cell Biology Baylor College of Medicine
Houston, Texas 77030
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ABSTRACT
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The proximal promoter of the rat aromatase CYP19
gene contains two functional regions that, by 5'-deletion analyses,
have been shown to confer hormone/cAMP inducibility to chimeric genes
in primary cultures of rat granulosa cells and constitutive expression
in R2C Leydig cells. Promoter region A binds Steroidogenic Factor-1
(SF-1); region B binds cAMP-regulatory element binding protein (CREB)
and two other factors (designated X and Y). Mutations were generated
within the context of the intact promoter to selectively eliminate the
binding of either SF-1, CREB, CREB plus factors X and Y, or all of the
above. When expression vectors that failed to bind either CREB alone or
CREB plus factors X and Y were transfected into granulosa cells,
cAMP-dependent chloramphenicol acetyltransferase (CAT) activity was
reduced 65% indicating that CREB alone, and not factors X and Y,
mediates the cAMP response of this cAMP response element-like domain.
Similarly, cAMP-dependent CAT activity was reduced 50% in constructs
that failed to bind SF-1 and was abolished with vectors that were
unable to bind either factor. In R2C Leydig cells, the absence of
either CREB or SF-1 binding resulted in an almost complete loss in CAT
activity. Both immunoreactive CREB and phosphorylated CREB
(phosphoCREB) were present in extracts and nuclei of R2C cells.
Immunoreactive phosphoCREB was low in granulosa cell extracts and
nuclei but increased rapidly (90 min) in response to FSH/cAMP and was
highest at 48 h, at a time when SF-1 was also phosphorylated and
expression of the endogenous gene was elevated. Although the amount of
CREB and SF-1 remained unchanged in response to FSH, LH mediated a
rapid decrease in the amount of SF-1 (but not CREB) that is coincident
with decreased aromatase mRNA in luteinizing granulosa cells. Taken
together, the data indicate that expression of the aromatase gene is
dependent on the additive interactions of regions A and B of the
aromatase promoter in granulosa cells and the synergistic interactions
of these same regions in R2C cells and that these interactions are
dependent, in turn, on the phosphorylation of CREB and SF-1 and the
content of these factors, as well as the presence of putative
coregulatory molecules.
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INTRODUCTION
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Maturation and differentiation of the ovarian follicle involves
not only the presence of pituitary gonadotropins but also the
activation of specific ovarian genes in response to these peptide
hormones (1, 2). The CYP19 gene encodes the aromatase cytochrome P450
enzyme that converts testosterone to estradiol. Expression of this gene
is regulated throughout the life cycle of the rat follicle and corpus
luteum by the sequential actions of the gonadotropins, FSH and LH, and
by the cytokine PRL (3, 4, 5, 6, 7, 8). In response to FSH, steady state levels of
aromatase mRNA increase, with maximal levels detected in granulosa
cells of the preovulatory follicles (3). The surge of LH that initiates
the onset of ovulation exerts a dramatic negative effect on steady
state levels of aromatase message resulting in a complete loss of
transcript within 6 h (5, 6). During pregnancy, PRL is responsible
for elevated levels of aromatase message and protein in the corpus
luteum (4). These changes, which occur in vivo, can be
mimicked in granulosa cells cultured sequentially in the presence of
FSH and LH (7, 8). In contrast to these sequential changes in aromatase
expression that characterize granulosa cell differentiation and
luteinization, aromatase mRNA is constitutively expressed at high
levels in the rat R2C Leydig cell line (9).
One of the mechanisms by which FSH regulates transcription of the
aromatase gene involves stimulation of the cAMP-signaling cascade that
activates A-kinases and leads to the phosphorylation of key substrates
including specific transcription factors (1, 2). Two regions within the
rat aromatase promoter have been identified to mediate cAMP-induced
expression in granulosa cells (10, 11, 12). These same two regions are also
present in the promoter II of the human aromatase gene (13). The most
proximal element (region A) lies between -90 and -66 bp and binds
Steroidogenic Factor-1 (SF-1) or adrenal binding protein (Ad4BP), an
orphan receptor member of the steroid/thyroid hormone receptor
superfamily (14, 15). The hexameric SF-1-binding site [(C/A) AGGTCA]
is present within a large number of steroidogenic genes (14, 15) and
has been shown to be critical for their transcriptional activation in
adrenal and gonadal cells. Targeted deletion of the SF-1 gene results
in mice that lack gonads and adrenals and exhibit abnormal gonadotrope
development (16), indicating that the expression and activity of SF-1
are critical during early embryonic development and likely involve
SF-1-regulated expression of tissue-specific genes in addition to those
encoding steroidogenic enzymes (17). In the adult rat ovary, the levels
of SF-1 mRNA and protein are relatively constant (10, 11, 18)
suggesting that posttranslational modifications as well as
transcriptional control may be important for mediating the functional
activity of SF-1.
The distal element of the aromatase promoter (region B) lies between
-161 and -138 bp and contains a cAMP-response element (CRE)-like
sequence (TGCACGTCA) (19, 20). This region has been shown to bind
CRE-binding protein (CREB) along with two additional proteins
designated as X and Y (12). Because mutations that prevent binding of
all three proteins result in a decrease in promoter activity (12), we
hypothesized that either CREB alone or that factors X and Y (with
putative functions redundant of CREB) were responsible for the
transactivation of promoter activity at this site. This hypothesis was
based on the known complexity of the CREB/ATF superfamily (21), on the
presence of multiple, alternatively spliced forms of CREB in different
tissues, and on the fertility of the original CREB knockout mice
(21, 22, 23). More recently, Blendy et al. (24) demonstrated
that although the original CREB knockout mice did not express CREB
-
or
-isoforms, the levels of CREB ß as well as the CREB/ATF family
member, CRE modulator, were increased. Therefore, factors X and Y that
bind the aromatase CRE region were considered potential members of the
CREB/activating transcription factor (ATF) family.
The functional activity of CREB is dependent upon the phosphorylation
of serine 133 (25) that occurs primarily by activation of A-kinase.
CREB phosphorylation occurs rapidly (within minutes) after stimulation
of cells with hormones or cAMP (25), and the phosphorylation site is
antigenic (26). Furthermore, the ability of phosphoCREB to
transactivate gene expression involves binding to the coactivator CBP
(CREB-binding protein; Refs. 2729). Although recent reports have also
shown that SF-1 can be phosphorylated by A-kinase (30), it is not yet
known whether this phosphorylation is obligatory for transcriptional
activity. As indicated above, regulation of the aromatase gene in cells
is complex. It can be induced in granulosa cells by low levels of
FSH/cAMP, rapidly and dramatically down-regulated in these same cells
by high levels of LH and cAMP, or expressed at elevated,
cAMP-independent levels in an R2C Leydig cell line. These distinct
patterns of aromatase expression indicated that several steps and
signaling pathways might regulate either the content or activity of
factors, such as CREB and SF-1, that bind to regions A and B of the
aromatase gene promoter.
Based on the foregoing observations this study was undertaken to
delineate whether specific interactions of factors binding to region A
and B mediate cAMP-responsive and constitutive activation of the rat
aromatase promoter in granulosa cells and R2C Leydig cells,
respectively. To accomplish this goal, mutations of region A and region
B were generated within the context of an intact promoter. Mutants
within the CRE-like sequence were generated to determine the functional
role of CREB and whether factors X and Y exerted redundant activities.
Mutations within the hexameric SF-1 site were generated to determine
whether SF-1 interacted with CREB for transactivation of the aromatase
promoter. To determine whether changes in the content and
phosphorylation of CREB and SF-1 were related to the activation of the
aromatase gene, Western blot analyses, immunocytochemical localization,
immunoprecipitation, and electrophorectic mobility shift assays (EMSAs)
were performed. Results of these studies indicate that regions A and B
interact in an additive manner in granulosa cells and in a synergistic
manner in R2C Leydig cells, that levels of phosphoCREB are related to
cAMP induction in granulosa cells and constitutive expression in R2C
Leydig cells, and that loss of aromatase mRNA is associated with a
marked decrease in SF-1 in response to the ovulatory LH surge. Thus,
changes in either the phosphorylation state of CREB or the content of
SF-1 appear to exert a major impact on transcriptional activation of
the endogenous gene, further underscoring the importance of the
functional interactions of regions A and B in mediating aromatase gene
expression.
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RESULTS
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Differential Binding of CREB and Additional Proteins to Region B in
the Rat Aromatase Promoter
Region B of the rat aromatase promoter forms three distinct
DNA/protein complexes, one of which has been shown to be CREB by
competitive binding and antibody supershift analyses (Fig. 1
; Ref.12). To elucidate which nucleotides within the
CRE-like region are responsible for the binding of CREB and factors X
and Y, mutant oligonucleotides were synthesized (Fig. 1B
) and tested by
EMSAs using nuclear extracts from granulosa cells of preovulatory
follicles (HEF; Fig. 2
), in which expression of
aromatase has been shown to be the highest (7). As in previous studies
(12), three protein/DNA complexes corresponding to CREB, X, and Y were
formed when the intact -161 oligonucleotide was used as a labeled
probe (Fig. 2
, A-C; lane 2). All three complexes were competed by the
addition of cold competitor -161 oligonucleotide, whereas only the
upper band was competed by an authentic unlabeled CRE oligonucleotide
(Ref. 12; Fig. 2A
; lanes 3 and 4). The faster migrating bands are
nonspecific. The binding of all three complexes was also inhibited by
the addition of unlabeled -158/-157 mutant (-158 M)
oligonucleotides (Fig. 2
B; lane 4). The -156/-155 mutant
oligonucleotide (-156M) failed to compete for the binding of CREB but
did compete for the binding of the other two factors, X and Y (Fig. 2B
;
lane 5). Mutations of bases -155/-154 or -154/-153 (-155M
and -154M, respectively) rendered these oligonucleotides incapable of
competing for binding of CREB, X, or Y (Fig. 2B
; lanes 6 and 7). EMSAs
were also performed using whole cell extracts from R2C Leydig cells and
yielded similar results (data not shown). When increasing
concentrations (50-, 100-, and 200-fold excess) of the competitor
oligonucleotides were added to the reactions, as little as 50-fold
excess of -161, -158M, and -156M competed for the binding of factors
X and Y (Fig. 2C
). The binding of CREB was more effectively reduced by
-158M than -161 and was not altered by -156M. Collectively, these
data show that the binding of CREB can be functionally separated from
that of factors X and Y.

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Figure 1. Mutations in Region B of the Rat Aromatase Promoter
A, Schematic of the regulatory elements of the rat aromatase promoter.
Region A (-90/-66 bp) binds SF-1 (hatched rectangle).
Region B (-161/-138 bp) binds three factors, CREB (solid
rectangle), X (open circle) and Y
(speckled square). B, Mutant oligonucleotides generated
within the CRE-like sequence of region B. -161 represents the intact
region B with the CRE-like sequence underlined. -158M,
-156M, -155M, and -154M designate the mutations
(underlined) at positions -158 and -157, -156 and
-155, -155 and -154, -154 and -153, respectively. The -154M has
previously been described (12).
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Figure 2. Mutations within Region B Generate Distinct Binding
Patterns
A, EMSAs were performed using nuclear extracts (5 µg) prepared
from preovulatory granulosa cells of hormonally primed (HEF) rats and
the -161 (-161/-138) oligonucleotide as probe (200 pg, 13 fmol).
Three distinct protein/DNA complexes were formed (CREB, X, and Y). The
identity of CREB was determined using 200-fold excess of authentic CRE
as shown previously (12). B, EMSAs were performed as described in panel
A using 200-fold excess of unlabeled oligonucleotides (described in
Fig. 1 ) to compete for protein binding. C, EMSAs were performed as
described in panel A except that an increasing amount of unlabeled
oligonucleotide (50-, 100-, and 200- fold excess) was used to
demonstrate specificity of binding. HEF: H, hypophysectomized; E,
estradiol; F, FSH.
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CREB Binding Required for cAMP-Induced and Constitutive
Transactivation of Aromatase CAT Reporter Constructs
The differential binding of CREB and factors X and Y to the
various oligonucleotides provided us with a means of testing the
ability of X and Y to transactivate the aromatase promoter in the
absence of CREB. Therefore, specific mutants of aromatase
promoter-reporter CAT constructs were generated and tested in transient
transfection assays using granulosa cells and R2C Leydig cells (Fig. 3
). When granulosa cells were transfected with the -161
arom CAT or -158M arom CAT constructs (which bind all three proteins)
a 6- to 8-fold increase in CAT activity was observed in the presence of
forskolin (Fig. 3A
). The -156M arom CAT vector that binds factors X
and Y but not CREB exhibited 65% less forskolin-inducible activity
than the -161 arom CAT (Fig. 3A
). Furthermore, the activity of the
-156M arom CAT construct was similar to that of constructs that do not
bind any of the factors, namely, the -154M arom CAT (Fig. 3A
) and
-155M arom CAT (data not shown). The results of these transfection
assays indicate that CREB specifically transactivates cAMP inducibility
within region B of the promoter. In the absence of forskolin, all
constructs tested yielded CAT activity levels comparable to the
negative control construct (pCAT Basic), which contains no promoter
sequence.

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Figure 3. Functional Activity of CREB, X, and Y in Both cAMP
Induction and Constitutive Expression of the Transgene
A, Histogram of the CAT activity (% conversion) in granulosa cells
transfected with 15 µg of various promoter-reporter constructs and
cultured for 24 h. Twenty micrograms of protein were assayed for
18 h to determine CAT activity. The data presented are the mean
value of duplicate samples in two separate experiments. The error bars
represent ± SD. B, Histogram of the CAT activity (%
conversion) in R2C Leydig cells transfected with the same constructs
and cultured for 48 h. Ten micrograms of protein were assayed for
2 h to determine CAT activity. The data presented are the mean
value of duplicate samples in two separate experiments. The error bars
represent ± SD. C, pCAT control; B, pCAT basic.
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Constitutive expression of aromatase by R2C Leydig cells also appears
to depend upon the binding of CREB to a functional CRE (Fig. 3B
). The
-161 and -158M constructs exhibited high CAT activity (in the absence
of forskolin); whereas, constructs lacking CREB binding (-154M and
-156M arom CAT) had minimal CAT activity (7% of -161 arom CAT).
These results indicate that CREB binding to region B is also required
for high constitutive expression of aromatase in R2C Leydig cells.
Cooperative Interactions Occur between CREB and SF-1 in Both
cAMP-Induced and Constitutive Expression of the Aromatase Promoter
SF-1 has previously been shown to be the factor binding to region
A of the rat aromatase promoter (Refs. 10 and 11; see Fig. 8
). To
determine whether SF-1 binding in region A affected either cAMP-induced
or constitutive expression of aromatase, additional aromatase
promoter-CAT constructs were generated. The SF-1-binding site was
mutated either in the context of the intact -161 arom promoter that
contains the functional CRE or in the -95 arom CAT promoter that lacks
the CRE (Fig. 4
). EMSAs were performed to verify that
the mutated hexameric motif (AGGTCA to ATTTCA) was unable to bind SF-1
(data not shown).

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Figure 8. The LH Surge Decreases SF-1 Binding and Protein
Content
A, EMSAs were performed using nuclear extracts (1.5 µg)
prepared from granulosa cells of preantral (HE), preovulatory (HEF),
and ovulatory (HEF plus either 2-h or 10-h hCG) follicles, whole cell
extracts from R2C Leydig cells (7.5 µg), and the SF-1 hexameric
oligonucleotide (-90/-66) as probe (200 pg, 13 fmol). A single
protein/DNA complex corresponding to SF-1 is shown
(arrow; Ref. 12). B, The same nuclear extracts (HEF, HEF
plus 2- or 10-h hCG; 10 µg protein) and whole cell extracts (R2C
Leydig; 50 µg protein) were fractionated on a 10% SDS-PAGE and
transferred to nitrocellulose. Blots were incubated with anti-SF-1
(1:10,000). Immunoreactive proteins were visualized using
[125I]protein A and exposed to x-ray film. C, EMSA was
performed as in panel A using the CRE-like region B of the aromatase
promoter as the labeled probe.
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Figure 4. Schematic of Mutant Aromatase Promoter-Reporter
Constructs
Mutant SF-1-binding constructs were generated in either the context of
the intact promoter (-161 vectors) or a deleted promoter containing
only region A (-95 vectors). The -161, -156M, and -154M were
described in Fig. 1 . The -161M, -156DM, and -154DM were generated by
ligating a mutant region A oligonucleotide into the intact promoter
without or with CRE mutant sites. The -95 and -95M were generated by
ligating either the normal or mutant region A oligonucleotide to the
minimal 50 promoter-reporter construct. Binding of CREB, X, Y, and SF-1
are illustrated by the following symbols: CREB, solid
rectangle; X, open circle; Y, speckled
square; SF-1, hatched square.
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These various constructs were transiently transfected into primary
granulosa cells and tested in the absence or presence of forskolin
(Fig. 5A
). Maximal activity (35% conversion) was
induced by forskolin in cells transfected with the intact -161 arom
CAT construct. Mutation of the SF-1-binding site (-161M arom CAT)
resulted in a 50% reduction in forskolin-induced CAT activity (Fig. 5A
). The level of activity was comparable to that of the -156
M and -154M arom CAT constructs, both of which fail to
bind CREB. No induction of transgene activity by forskolin was observed
in vectors with double mutations, -156DM arom CAT and -154DM arom
CAT, which fail to bind CREB and SF-1. The promoter construct
containing the SF-1-binding site alone (-95 arom CAT) did exhibit a
slight but significant 2-fold induction of CAT activity in response to
forskolin. Mutation of the SF-1-binding site (-95M arom CAT) resulted
in complete loss of forskolin-induced CAT activity. These results
indicate that a cooperative or additive interaction between SF-1 and
CREB is required for cAMP activation of aromatase promoter in granulosa
cells.

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Figure 5. Functional Interactions of CREB and SF-1-Binding
Sites in Granulosa Cells and R2C Leydig Cells
A, Histogram of the CAT activity (% conversion) in granulosa cells
transfected with 15 µg of various promoter-reporter constructs and
cultured for 24 h. Twenty micrograms of protein were assayed for
18 h to determine CAT activity. The data presented are the mean
value of duplicate samples in two separate experiments. The error bars
represent ± SD. B, Histogram of the CAT activity (%
conversion) in R2C Leydig cells transfected with the same constructs
and cultured for 48 h. Ten micrograms of protein were assayed for
2 h to determine CAT activity. The data presented are a
representative experiment using duplicate samples. The error bars
represent ± SD. C, pCAT control; B, pCAT basic.
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When these same constructs were transfected into R2C cells, different
responses were observed (Fig. 5B
). Whereas the -161 arom CAT exhibited
32% conversion, the -161M arom CAT construct lacking the SF-1-binding
site had only 2% conversion. Thus, mutation of the SF-1-binding site
in the presence of a functional CRE within region B resulted in a
complete loss of CAT activity. Likewise, vectors lacking a functional
CREB-binding site had little activity, and this was not reduced further
in the double mutants (DMs), -156DM arom CAT and -154DM arom CAT. The
-95 arom CAT had low activity that was reduced by mutation of the SF-1
site. These data indicate that both the CREB- and SF-1-binding sites
must be intact and interact in a synergistic manner for transactivation
of the aromatase promoter in R2C cells.
Phosphorylation of CREB and SF-1 in Granulosa Cells and R2C Leydig
Cells
Although CREB homodimers bind DNA at CRE-like sequences,
phosphorylation of CREB at serine 133 is required for it to
transactivate promoter activity (25). Serine 133 resides within a
substrate consensus site for protein kinase A, but other kinases are
also capable of phosphorylating CREB at this same residue (26, 31, 32, 33, 34).
To determine whether CREB was phosphorylated in response to increased
levels of cAMP in granulosa cells and was constitutively phosphorylated
in R2C Leydig cells, Western blot analyses were performed using
specific CREB and anti-phosphoCREB antibodies, the latter of which
recognizes CREB phosphoserine 133 (Ref. 26; Fig. 6
). The
amount of CREB was essentially constant in granulosa cells cultured in
the absence of hormones or in the presence of either forskolin or FSH/T
for 048 h (Fig. 6A
; upper left panel). Similar amounts of
CREB were also detected in R2C cells (Fig. 6A
; upper right
panel). When these same samples were analyzed for the content of
phosphoCREB, the anti-phosphoCREB antibody recognized two distinct
immunoreactive proteins (Fig. 6A
; lower panel). The 43-kDa
phosphoprotein (larger arrow; lower band) comigrated with
CREB and is considered, therefore, to be authentic CREB protein. The
52-kDa immunoreactive phosphoprotein (smaller arrow; upper
band) migrated more slowly than CREB, was not recognized by the
specific CREB antibody, and therefore is not CREB. Because the
anti-phosphoCREB antibody recognizes a phosphorylation region common to
other CREB family members (26), antibodies to other members of the CREB
family (CRE modulator, ATF-1) as well as other CREB antibodies were
tested, but none recognized the higher molecular weight protein (21, 26). Therefore, this protein is either yet another CREB/ATF family
member or is an unrelated protein that has a phosphorylation domain
similar to that present in CREB.

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Figure 6. Phosphorylation of CREB in Response to FSH and
Forskolin
A, Western blot analysis of CREB and phosphoCREB in granulosa cells and
R2C cells. Granulosa cells were cultured for varying times in medium
alone or medium containing either forskolin (Fo; 10 µM)
or FSH (50 ng/ml) and 10 ng/ml testosterone (24 h and 48 h). R2C
cells were cultured for 48 h in medium without hormones. Cell
extracts were prepared, fractionated by 10% SDS-PAGE, and transferred
to nitrocellulose. Blots were incubated with either anti-CREB (1:1000)
or anti-phosphoCREB (1:500) IgG. Immunoreactive proteins (43-kDa CREB,
open arrow; 43 kDa phosphoCREB, large solid
arrow; 52-kDa protein; small solid arrow) were
visualized using [125I]Protein A and exposed to x-ray
film. B, Immature granulosa cells were cultured on coverglass slips in
the presence (+) or absence(-) of forskolin (Fo) for 48 h as
described in panel A. R2C Leydig cells were also cultured for 48
h. Cells were then fixed in 4% paraformaldehyde and incubated
overnight with either anti-CREB (top panel) or
anti-phosphoCREB (bottom panel) IgG. Localization of
these proteins was visualized by fluorescein-labeled anti-rabbit IgG.
Original magnification is 63x.
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In the absence of either forskolin or FSH, the immunoreactive 43-kDa
protein identified as phoshoCREB was barely detectable (Fig. 6A
, lower panel; lane 1, large arrow). The addition
of forskolin caused an increase in immunoreactive phosphoCREB at 90 min
that decreased at 6 h but was again increased to a higher amount
at 48 h. Similar results were observed in response to FSH with the
exception that the secondary increase in phosphoCREB occurred as early
as 24 h and was maintained at 48 h. Because alterations in
the amount of phosphoCREB were not due to changes in the concentration
of CREB protein (Fig. 6A
; upper panel), these results
indicate that the stimulation of cAMP production in granulosa cells
exerts a biphasic response with respect to CREB phosphorylation and
subsequent activation. Immunocytochemical localization studies showed
that high levels of CREB were present in nuclei of granulosa cells
cultured in the presence or absence of forksolin for 48 h (Fig. 6B
; top panel) corresponding to Western analysis data (Fig. 6A
; upper panel). However, granulosa cells cultured for
48 h in the presence of forskolin (+) expressed a higher level of
nuclear phosphoCREB as compared with those cultured without forskolin
(-) (Fig. 6B
; lower panel). CREB and phosphoCREB were also
detected in cell extracts and nuclei of R2C Leydig cells (Fig. 6
).
Interestingly, the highest levels of phosphoCREB, as detected by
Western blotting and indirect immunofluorescent labeling, were observed
in cells expressing the greatest amounts of aromatase mRNA (7, 9).
Because FSH/cAMP are necessary for expression of the endogenous gene in
granulosa cells (7) and for increased transcription transgenes
containing the SF-1 binding site in region A, we sought to determine
whether SF-1 was also phosphorylated in granulosa cells that express
high levels of aromatase mRNA in response to FSH. Accordingly, in
vivo phosphorylation studies were performed using granulosa cells
cultured for 48 h in the presence (+) or absence (-) of FSH (Fig. 7
). A phosphoprotein of approximately 60 kDa
corresponding in size to SF-1 (see Fig. 8B
) was immunoprecipitated
using an SF-1-specific antibody from cells cultured in the presence of
FSH (Fig. 7
, + lane). A similar band corresponding to SF-1 was not
phosphorylated in cells cultured without FSH (- lane). Thus, SF-1 as
well as CREB appear to be an FSH/cAMP-dependent phosphoprotein.

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Figure 7. Phosphorylation of SF-1 in Response to FSH
SF-1 was immunoprecipitated from cellular extracts (50 µg) prepared
from granulosa cells cultured in the presence (+) or absence (-) of
FSH/t for 48 h and labeled with [32P]orthophosphate
as described in Materials and Methods. A 60-kDa protein
corresponding to SF-1 was detected in the + lane
(arrow).
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LH Reduces SF-1 in Preovulatory Granulosa Cells
Whereas FSH increases aromatase mRNA in granulosa cells, the LH
surge causes a rapid loss of aromatase transcripts in vivo
(3) and in vitro (8). This decrease is presumed to be due,
at least in part, to changes in aromatase transcription rate (8). If
CREB and SF-1 mediate transcription of the endogenous gene, then a
change in the expression, binding, or activation (phosphorylation) of
these factors might be expected to occur in response to high levels of
LH. To investigate these possibilities, two approaches were used.
First, EMSAs were performed by incubating oligonucleotides containing
the hexameric SF-1 binding site with nuclear extracts from granulosa
cells isolated from ovaries of hormonally primed hypophysectomized (H)
rats (Fig. 8A
). A specific SF-1 complex was formed when
oligonucleotides with the hexameric motif were incubated with preantral
(HE) and preovulatory (HEF) granulosa cell extracts (lanes 2 and 3) and
R2C Leydig cell extracts (lane 6). However, when human CG (hCG) was
given to proestrous rats to mimic the endogenous LH surge, the SF-1/DNA
complex was reduced by approximately 50% (lane 4) and 70% (lane 5) at
2 and 10 h, respectively, after treatment. A similar decline in
SF-1 binding was detected when whole cell extracts were prepared from
granulosa cells cultured for 48 h with FSH (preovulatory) and then
exposed to hCG for 2 or 10 h (data not shown). Second, Western
blot analyses were performed to determine whether loss of SF-1 binding
was due to changes in SF-1 protein content. A single immunoreactive
band corresponding to SF-1 at approximately 60 kDa was observed in
nuclear extracts of preovulatory granulosa cells (Fig. 8B
, lane 1).
This immunoreactive SF-1 band was markedly reduced in nuclear extracts
from granulosa cells exposed to hCG for either 2 or 10 h (lanes 2
and 3). SF-1 was also detected in R2C Leydig whole cell extracts (lane
4).
When these same granulosa cell and R2C cell extracts were used in EMSAs
with a labeled aromatase CRE (-161/-138 bp of region A), three
binding complexes were observed with each sample (Fig. 8C
).
Furthermore, no changes in amount of CREB binding (uppermost complex;
see Fig. 2A
) were observed in preovulatory granulosa cells treated with
hCG (LH) (Fig. 8C
). Likewise, the amount of immunoreactive CREB
remained constant during this time period (Ref. 35 and data not shown).
In addition, the phosphorylation state of CREB also did not vary as a
consequence of hCG (data not shown). Taken together, these data
indicate that loss of SF-1 but not CREB is associated with the decrease
in aromatase whereas increased phosphorylation of both factors is
required for induction of the aromatase gene.
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DISCUSSION
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This study documents that different molecular mechanisms mediate
hormone/cAMP induction, down-regulation, and constitutive expression of
the rat aromatase gene by CREB and SF-1 in gonadal cells. Specifically,
the functional interactions of the hexameric (CA)AGGTCA motif that
binds SF-1 (region A) and the CRE that binds CREB and two other factors
(region B) have been analyzed within the context of the intact
promoter. When promoter-reporter constructs were transfected into
granulosa cells and R2C cells, only those vectors that could bind CREB
exhibited functional activity; namely, responsiveness to cAMP in
granulosa cells and high basal activity in R2C cells. These
observations eliminated the possibility that factors X and Y might
provide redundant functions for CREB, a notion that was entertained at
the initiation of these studies because numerous isoforms of CREB had
been identified (21, 22) and because CREB knockout mice exhibited a
normal phenotype devoid of obvious reproductive abnormalities (23).
This phenotype was unexpected given the obligatory roles of FSH and LH
in the growth and function of the ovarian follicle and ovulation (1)
and the presumed preeminence of cAMP and CREB in mediating the actions
of the gonadotropins on specific genes (1, 2, 36, 37). Further analyses
of these mice have provided a different explanation. Blendy et
al. (24) have recently reported that these animals are not
complete CREB knockouts and that CREB ß, which is generally expressed
in extremely low amounts, was present at much higher levels.
One intriguing aspect of the results obtained in these experiments is
that the molecular mechanisms or pathways involved in cAMP activation
of the aromatase promoter in granulosa cells differ from those
controlling constitutive expression in R2C cells. By using selected
mutants of the CREB and SF-1 binding sites within the context of the
intact promoter, we show that CREB and SF-1 interact in an additive
manner to confer cAMP responsiveness in granulosa cells, whereas they
interact synergistically to confer high constitutive activity in R2C
cells. Specifically, within granulosa cells, loss of binding of either
factor in the context of the intact promoter reduced transgene activity
by at least half, whereas complete loss of activity was achieved only
when both CREB and SF-1 binding sites were ablated simultaneously.
Although the involvement of SF-1 in the induction of a cAMP-responsive
gene has been described elsewhere (30), this is the first study to
demonstrate that the hexameric SF-1-binding site is required within the
context of the intact promoter to provide maximal cAMP inducibility. In
R2C Leydig cells that constitutively express high levels of aromatase,
the binding of both CREB and SF-1 are critical for elevated,
cAMP-independent expression of the transgenes. Aromatase promoter-CAT
reporter constructs containing mutations within either CREB- or
SF-1-binding sites or both exhibited little or no activity.
Not only are interactions of regions A and B of the aromatase promoter
cell specific, but these interactions are associated with specific
changes in the phosphorylation and amount of factors that regulate
aromatase induction in the ovary by FSH, its down-regulation by LH, and
its constitutive expression in R2C Leydig cells. Phosphorylation of
CREB was associated with both cAMP induction of aromatase transgenes in
granulosa cells and with their constitutive expression in R2C Leydig
cells. Although there is abundant evidence that cAMP can activate
A-kinase in granulosa cells (38) and transactivate genes containing CRE
sites (36, 37), the kinase(s) responsible for the phosphorylation of
CREB in the R2C Leydig cells have not yet been identified. Because
forskolin (cAMP) does not regulate expression of the endogenous
aromatase gene or the transgenes in R2C Leydig cells (9, 10), it is
possible that A-kinase is maximally activated by factors present in the
serum in which the cells are cultured. Alternatively, other kinases
(e.g. C-kinase, calmodulin (CAM) kinase, RSK-2) that are
capable of phosphorylating CREB at the serine 133 residue (31, 32, 33, 34), the
site obligatory for transactivation (25) and antibody recognition (26),
may be activated in R2C cells. Thus, it is possible that cellular
signaling pathways other than, or in addition to, A-kinase
phosphorylate and activate CREB in granulosa cells and R2C cells.
Phosphorylation of CREB is also dependent on cell differentiation. One
of the novel observations of these studies is that although the amount
of CREB remained constant during granulosa cell differentiation (Fig. 6A
; Refs. 12 and 35), the phosphorylation of CREB was markedly elevated
in cells cultured in the presence of FSH or forskolin for 48 h.
Thus, the phosphorylation of CREB is not only rapid and transient (25)
but is subsequently increased and maintained as the cells
differentiate. As a consequence of this biphasic pattern of
phosphorylation in response to FSH, the highest levels of phosphoCREB
in granulosa cells were coincident with the maximal induction of the
endogenous aromatase gene (7) and the acquisition of the preovulatory
phenotype (1). Several factors may account for the greater
phosphorylation of CREB in the differentiated cells. First, although
stimulation of cAMP production by FSH and forskolin is greatest at 60
min in these cultured cells, the nucleotide continues to be synthesized
at elevated levels for 2448 h (7, 39). Second, total A-kinase
activity may be increased. Previous studies have shown that the
concentrations of the regulatory type IIß (RIIß) subunit of
A-kinase is increased dramatically in preovulatory granulosa cells
in vivo and in vitro (8, 39, 40, 41). Although we
originally thought that the increase in this regulatory subunit might
serve as a sponge to reduce the sensitivity of granulosa cells to high
cAMP and thereby prevent premature luteinization, it is also possible
that these high levels of RIIß reflect a transient increase in total
kinase holoenzyme with the rapid dissociation of the subunits and the
transport of the activated C subunit to the nucleus. Such a hypothesis
would support the observations of McKnight and colleagues (42), who
have shown that overexpression of regulatory subunits leads to more
holoenzyme. Alternatively, the activity of a specific phosphatase may
be decreased as granulosa cells differentiate, thereby leading to
increased phosphorylation of CREB as well as other granulosa cell
proteins (43). These include RIIß (39, 44), SF-1 (Fig. 7
), and a
protein that is antigenically distinct from CREB but which is
recognized by the phosphoCREB antibody (Fig. 6A
). Whether this protein
is a member of the CREB family or an unrelated protein with a similar
antigenic phosphorylation site remains to be determined.
SF-1 is known to play a key role in ovarian cell function based on
targeted disruption of the SF-1 gene in transgenic mice (16), altered
function of granulosa cells cultured in the presence of antisense
oligonucleotides (45), and the regulation of transcription of specific
transgenes containing the hexameric SF-1-binding site (10, 18, 30).
However, little is yet know about the phosphorylation of SF-1 and
whether phosphorylation of this factor regulates its transcriptional
activity. In the adult ovary and developing follicles, SF-1 mRNA and
protein levels appear to be relatively constant (10, 11, 18),
suggesting that activation of this orphan receptor occurs through a
posttranslational modification. We show herein that granulosa cells
cultured with FSH for 48 h contain similar levels of SF-1 but
greater amounts of phosphorylated SF-1 than those cultured in the
absence of FSH. It is not yet known whether this phosphorylation occurs
as a consequence of A-kinase or whether it alters the activity of SF-1
in granulosa cells. However, Zhang and Mellon (30) have recently
demonstrated that A-kinase can phosphorylate recombinant SF-1 in
vitro on serine and threonine residues, thereby indicating that
SF-1 is a putative substrate for A-kinase. Although the levels of SF-1
protein do not exhibit major changes during follicular growth or in
response to low levels of FSH in culture (10), elevated levels of LH
(high cAMP?) acting on differentiated, preovulatory granulosa cells
cause a dramatic decrease in SF-1 protein. This loss of SF-1 protein is
associated with the rapid loss of aromatase mRNA in granulosa cells of
ovulating follicles. Because the amount of CREB and phosphoCREB did not
change during this time (data not shown), the down-regulation of
aromatase appears to be associated more with the loss of SF-1 than to
any change in CREB. These results provide a physiological basis to
support the pharmacological actions of antisense oligonucleotides that
decrease SF-1 and aromatase in cultured cells (45).
In summary, these studies provide evidence that the additive
interactions of regions A and B of the aromatase promoter in granulosa
cells and the synergistic interactions of these same regions in R2C
cells are dependent on the phosphorylation of CREB and SF-1, the
amounts of these factors, as well as the presence (activation) of
additional coregulatory molecules (46, 47, 48), one of which is likely to
be such CREB binding protein, CBP.
 |
MATERIALS AND METHODS
|
---|
Animals
Immature (day 22 of age) Holtzman Sprague-Dawley female rats
(Harlan, Indianapolis, IN) were housed on a 16-h light, 8-h dark
regimen in the Center for Comparative Medicine at Baylor College of
Medicine and provided food and water ad libitum. Animals
were treated in accordance with the principles and procedures outlined
in the NIH Guide for the Care and Use of Laboratory Animals. All
procedures were approved by the Institutional Committee on Animal Care
and Use, Baylor College of Medicine (Houston, TX).
Promoter Constructs
Construction of the -161 aromatase-CAT reporter construct has
been previously described (12). The mutant CRE aromatase promoter-CAT
reporter constructs (-158M, -156M, -155M, -154M) were generated by
chemical synthesis of oligonucleotides (Genosys, The Woodlands, TX)
containing the designated mutations within the -161/-138 region (Fig. 1
). Annealed oligonucleotides were ligated into the -138 aromatase CAT
vector (digested with HindIII and XhoI),
previously described (12). Constructs containing the SF-1-binding site
mutation within the context of the intact region (-161M) or within the
CRE mutant constructs [DMs designated -156DM and -154DM] were
generated by chemical synthesis (Genosys) of a mutant oligonucleotide
corresponding to region A (-95/-50 bp) in which the SF-1-binding site
was mutated from AGGTCA to ATTTCA. The annealed mutant SF-1
oligonucleotide was ligated into the -161, -156M, or -154M aromatase
CAT vectors at the DRAII and APOI sites. To generate the -95 and SF-1
mutant (-95M) aromatase CAT vectors, the normal or mutant
oligonucleotide (-95/-50 bp) was ligated to the -50/+13 bp fragment
of the aromatase promoter and inserted into the pCAT basic vector. The
sequence and orientation of each mutant were confirmed by sequence
analyses and purified by a CsCl gradient. The pCAT Control vector
(Promega, Madison, WI), which contains the Simian Virus-40 promoter and
enhancer sequences, served as a positive control for transfection
reproducibility. The pCAT basic (Promega), which lacks eukaryotic
promoter and enhancer sequences, was used as control for basal vector
activity.
Electrophoretic Mobility Shift Assays
To test the ability of factors to bind these mutant
oligonucleotides, EMSAs were performed as previously described (48, 49). Briefly, nuclear extracts (NE) were prepared from granulosa cells
of hypophysectomized (H) rats treated sequentially with estradiol (HE;
preantral), FSH (HEF; preovulatory), and an ovulatory dose of hCG as
previously described (12). Oligonucleotides were labeled with
[P32]deoxy-CTP (3000 Ci/mmol, ICN, La Mesa, CA) and DNA
Polymerase-l (Klenow enyzme; large fragment, Promega, Madison, WI) to a
specific activity of 108. For studies investigating the
protein/DNA interactions at the CRE (region B) site, 5 µg NE protein
from preovulatory (HEF) granulosa cells were incubated with labeled
-161/-138 fragment (200 pg, 13 fmol) in the presence of 5.8 µg
poly(deoxyinosinic-deoxycytidylic)acid for 30 min at room temperature.
Specificity was analyzed by adding increasing concentrations of
unlabeled competitor DNA, either the aromatase CRE or a commercial
consensus CRE (Promega) as previously described (12). To determine SF-1
binding, 1.5 µg NE protein from granulosa cells of preantral (HE),
preovulatory (HEF), and ovulatory (hCG, 2 and 10 h) rats were
incubated with the labeled hexameric (-90/-66) motif (10).
Protein/DNA complexes were resolved on 5% polyacrylamide gels in 0.5x
TBE (0.089 M Tris borate, pH 8.3, and 2 mM
EDTA) at 150 V at 22 C after 30 min prerun. The gels were dried and
exposed to Kodak X-AR film at -80 C. Quantification of SF-1 binding
was determined using a Betascope 603 Blot Analyzer (Betagen Corp.,
Waltham, MA).
Transfection and CAT Assays
Granulosa cells were isolated from immature female rats (day 23
of age) (36, 39). Cells were cultured in 35-mm multiwell plates in 3 ml
DMEM-F-12 media (1:1, GIBCO/BRL, Gaithersburg, MD) supplemented with 20
mM HEPES (Curtain Matheson Scientific, Inc., Houston, TX),
100 IU/ml penicillin, 100 mg/ml streptomycin (Sigma, St. Louis, MO),
and 1% FBS (Hyclone, Logan, UT). The cells were transiently
transfected 18 h later using 15 µg plasmid DNA and the calcium
phosphate precipitation method (50). Four hours later the DNA was
removed, the cells were washed with HBSS
-Mg-Cl (GIBCO/BRL) and cultured in the
presence or absence of 10 µM forskolin (dissolved in
ethanol, Calbiochem, San Diego, CA). After 24 h, the cells were
lysed by freeze-thaw procedure, and the protein concentrations were
determined by the method of Bradford using the Bio-Rad (Richmond, CA)
protein assay. CAT activity was analyzed using 20 µg protein for
18 h according to a standard protocol (51). The amount of
radioactivity in the substrate and acetylated products was quantified
using the Betascope 603 Blot Analyzer.
R2C Leydig cells [American Type Cell Collection (ATCC), Rockville,
MD] were grown in Hams F-10 (GIBCO/BRL) supplemented with 12.5%
equine serum (Hyclone), 2.5% FBS (GIBCO/BRL), and 100 IU/ml
penicillin, 100 µg/ml streptomycin. Cells were plated overnight at a
density of 500,000 cells per 35-mm multiwell plate and transiently
transfected in a manner similar to granulosa cells, with the exception
that 4 h after DNA addition, cells were glycerol shocked (20% in
HBSS +Mg+Cl, GIBCO/BRL) for 2 min at 37 C,
washed with HBSS -Mg-Cl (GIBCO/BRL), and
cultured for 48 h in the R2C medium mentioned above. CAT assays
were performed using 10 µg protein for 2 h.
Western Blot Analysis
Western blot analyses of CREB and phosphorylated CREB
(phosphoCREB) were performed as described previously (26) using
specific antibodies that recognized CREB and phosphoCREB (serine 133).
Granulosa cells were isolated and cultured for varying amounts of time
(see Fig. 6
legend) in the presence or absence of ovine FSH (50 ng/ml,
NIH oFSH-16; National Hormone and Pituitary Program, Baltimore, MD) or
forskolin (10 µM) together with testosterone (10 ng/ml,
Steraloids, Keene, NH). R2C Leydig cells were cultured as described
above. After culture, cells were washed with PBS and lysed with boiling
SDS sample buffer [100 mM Tris (pH 6.8), 2% SDS, 20%
glycerol, 10% ß-mercaptoethanol, and bromophenol blue, 100 C]. Cell
extracts were then boiled (100 C) for 5 min. Samples were
electrophoresed on a 10% SDS-polyacrylamide gel by standard methods
(52). Proteins were transferred to a nitrocellulose membrane
(Schleicher & Schuell, Keene, NH) in a buffer containing 25
mM Tris, 192 mM glycine, and 20% methanol (53)
at 12 V for 18 h at 4 C. Blots were rinsed in Tris-buffered
saline-0.05% Tween-20 (TBS-T) and blocked with 4% BSA (BSA, Fraction
V, United States Biochemical Corp., Cleveland, OH) for 1 h at 22
C. The blots were incubated with rabbit anti-phosphoCREB IgG (1:5,000;
generously provided by Dr. David Ginty, Johns Hopkins University,
Baltimore, MD) in 4% BSA in TBS-T for 18 h at 4 C, with shaking.
To detect CREB, the blots were first blocked with 5% nonfat milk in
TBS-T for 1 h at 22 C, shaking. Blots were then incubated with
rabbit anti-CREB IgG (1:1000 in TBS-T, Ab 244 generously provided by
Dr. Marc Montminy, Salk Institute, La Jolla, CA) at 4 C for 18 h,
shaking. After several TBS-T washes, the presence of CREB or
phosphoCREB was detected using I125 protein A (1:1000
vol/vol in TBS-T, specific activity 1 mCi/ml, ICN) for 3 h at 22 C
with shaking. The blots were washed with TBS-T and exposed to x-ray
film.
Western blot analyses of SF-1 were performed as described previously
(54). Briefly, nuclear extracts (10 µg) from granulosa cells of
preovulatory (HEF) and ovulatory (HEF treated for 2 and 10 h with
hCG) follicles, as well as 50 µg of whole cell extracts from R2C
Leydig cells, were boiled (100 C) in an SDS-PAGE loading buffer [0.05
M cyclohexylaminoethane sulfonic acid, 2% SDS, 10%
glycerol, 2% ß-mercaptoethanol, and bromophenol blue, pH 9.5] and
electrophoresed on a 10% SDS-polyacrylamide gel by standard methods
(52). Proteins were transferred to nitrocellulose membrane (Schleicher
& Schuell) in a buffer containing 25 mM Tris, 192
mM glycine, and 20% methanol (53) at 12 V for 18 h at
4 C. The blots were rinsed in 140 mM NaCl, 10
mM KPO4, pH 7.5, 10 mg/ml BSA for 6 h at
22 C with shaking. The blots were then incubated with rabbit anti-Ad4BP
(SF-1) IgG (1:10,000; generously provided by Dr. Ken-ichirou Morohashi,
Kyushu University, Fukuoka, Japan) in 140 mM NaCl, 10
mM KPO4, pH 7.5, 10 mg/ml BSA, 0.1% Triton
X-100, 0.02% SDS for 18 h at 4 C. The blots were washed in 140
mM NaCl, 10 mM KPO4, pH 7.5, 0.1%
Triton X-100, 0.02% SDS at 22 C. After the washes, SF-1 was detected
using [125I]Protein A (1:1000 in wash buffer; specific
activity, 1 mCi/ml) for 3 h at 22 C with shaking. The blots were
washed and exposed to x-ray film.
Immunocytochemistry
Granulosa and R2C Leydig cells were cultured as above on glass
coverslips for varying times in the presence or absence of FSH or
forskolin. Cells were fixed in 4% paraformaldehyde (Electron
Microscopy Sciences, Fort Washington, PA) in PBS for 30 min at 22 C,
washed in 10 mM glycine in PBS, and treated with 0.5%
NP-40 in PBS. The cells were blocked with 3% BSA in PBS and incubated
with anti-phosphoCREB IgG (1:1000) or anti-CREB IgG (1:500) in 3% BSA
in PBS at 4 C for 18 h. After several PBS washes, cells were
incubated with fluorescein-labeled goat anti-rabbit IgG (1:20, Pierce,
Rockford, IL) in 3% BSA in PBS for 1 h at 22 C. CREB and
phosphoCREB were visualized on a Zeiss Axiophot microscope.
Phosphorylation and Immunoprecipitation of SF-1
Phosphorylation of granulosa cell proteins was performed as
previously described (39). Briefly, granulosa cells were cultured in
serum free DMEM:F12 in the presence or absence of FSH and testosterone
for 48 h. Cells were then washed and preincubated for 2 h in
phosphate-free DMEM (GIBCO). Fresh medium with FSH and testosterone and
2.5 mCi/ml of [32P]orthophosphate (ICN) were added for
2 h. The cells were then washed with PBS and resuspended in whole
cell extract buffer containing phosphatase inhibitors (55). The cells
were lysed by freeze-thaw procedure, and the protein concentrations
were determined by the method of Bradford using the Bio-Rad protein
assay. Immunoprecipitations were performed as follows. Protein A
Sepharose CL-4B beads (Sigma) were preswollen in PBS, 0.1% SDS, 0.2%
Triton X-100, and incubated with equal volumes of anti-Ad4BP IgG (1:100
in the above buffer) for 7 h at 4 C, with shaking. Unbound
antibody was removed by excess washing of the Sepharose beads. An equal
volume of the 32P-labeled whole cell extract protein (50
µg) was then incubated with the beads for 18 h at 4 C, with
shaking. Beads were washed with excess buffer to remove unbound labeled
proteins. Bound 32P-labeled proteins were eluted from the
beads by addition of an equal volume of SDS-PAGE loading buffer and
boiled at 100 C for 10 min. Samples were electrophoresed on a 10%
SDS-polyacrylamide gel by standard methods (52), dried, and exposed to
x-ray film.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Kristen Williams for her technical
assistance and Dr. Marc Montminy (Salk Institute, La Jolla Ca), Dr.
David Ginty (Johns Hopkins University, Baltimore MD), and Dr.
Ken-ichirou Morohashi (Kyushu University, Japan) for their generous
gifts of the CREB, phosphoCREB, and Ad4BP antibodies, respectively.
 |
FOOTNOTES
|
---|
Address requests for reprints to: JoAnne S. Richards, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.
This work was supported by NIH Grants 16272 (to J.S.R.), HD-07165, and
NRSA-HD-07991 (to D.L.C.).
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