(Received for publication, October 27, 1994; and in revised form, April 5, 1995)
From the
cAMP response element-binding protein (CREB) participates in
both constitutive and cAMP-induced transcription of cAMP-responsive
genes. CREB-mediated constitutive transcription requires only
CREB-binding sites and a minimal promoter region (containing the TATA
through start sequences), indicating that CREB interacts directly with
components of the general transcription machinery. In this study, a
coimmunoprecipitation assay was used to test for interaction of CREB
with the general transcription factors (TF) TFIIB and TFIID and the
core component of TFIID, TATA-binding protein (TBP). Human TFIIB and
TBP, tagged with distinct epitopes (eTFIIB and eTBP), were expressed in
and purified from Escherichia coli, and holo-eTFIID,
containing eTBP, was obtained from the HeLa cell line LTR
cAMP response element-binding protein (CREB)
CREB
contains independent domains that mediate constitutive and
kinase-inducible transcription(5) . The constitutive activation
domain (CAD) of CREB maps to the C-terminal portion of the protein
(amino acids 165-255). Deletion of the CREB CAD abolishes
constitutive transcription without impairing inducibility by PKA, and
fusion of the CAD to the GAL4 DNA-binding domain restores constitutive
activity to the level observed for wild-type CRG. Expression of either
the constitutive or PKA-inducible CREB activities required only GAL4
sites to bind CRG ligated to a minimal promoter encompassing the TATA
footprint through the transcription start site of either the
phosphoenolpyruvate carboxykinase or E1b gene(5) . This result
indicates that CREB need not interact with other promoter-bound
regulators outside of the TATA-based initiation complex. Together,
these data suggest that the CREB CAD stimulates basal or constitutive
transcription through interaction with one or more general
transcription factors bound to the minimal promoter at the TATA box.
The general transcription factors are defined as those required for
accurate initiation of in vitro transcription and are named
TFIIA, TFIIB, etc. based on their chromatographic properties (7, 8, 9) . Regulatory factors, such as CREB,
bound to upstream sites in the promoter are thought to stimulate
transcription through interactions with the general transcription
factors, assembled with RNA polymerase II into a preinitiation complex
at the start site of
transcription(9, 10, 11) . ATF/CREB family
members qualitatively alter the binding of TFIID and associated
proteins to a target promoter, facilitating subsequent interaction with
TFIIB and other general transcription factors and increasing protection
downstream of the TATA element(12, 13) . The
activation domains of both VP16 (14) and the E1A protein (15, 16) interact directly with TBP. In addition, the
VP16 activation domain (14) and others (14, 17, 18) interact directly with TFIIB.
VP16 also has been shown to play a role in recruiting TFIIB to the
initiation complex(19, 20) .
The minimal factor
requirement to support transcription from a promoter is (i) TBP, (ii)
TFIIB, and (iii) the RNA polymerase II-TFIIF complex (9, 21) . In at least one case, the initiator
recognizing element YY1 can substitute for TBP and requires only TFIIB
and polymerase II for basal transcription(22) . Moreover, the
TFIIF requirement has been further refined to only the RAP30 component
of TFIIF(23) . However, many transcription activators require
TFIID rather than TBP to mediate regulated, as opposed to basal,
transcription(10, 24, 25) . Given that CREB
interacts with general transcription factors to promote constitutive
activation and that TBP/TFIID and TFIIB are indispensable for RNA
polymerase II-mediated transcription, we tested the ability of CREB to
interact with TBP, TFIID, and TFIIB in this study. To determine which
targets or combinations of targets would interact with CREB in a
coimmunoprecipitation assay, we used TBP and TFIIB tagged with distinct
epitopes and the eTFIID fraction from a HeLa cell line (LTR
Figure 1:
Assay for interaction of TBP with E1A,
luciferase, and CREB. Plasmids containing cDNAs for E1A, luciferase (LUC), nothing, or CREB were used to program a coupled in
vitro transcription-translation system. Equimolar amounts of
Figure 2:
Interaction assay for CREB with eTFIIB.
[
Figure 3:
Interaction of CREB with holo-eTFIID. A, [
Figure 4:
Effect of addition of TFIIB on CREB
binding to eTBP. A,
Figure 5:
Effect of immunodepletion of TFIIB on CREB
interaction with TFIID.
Figure 6:
Effect of mutation of the PKA
phosphorylation site in CREB on interaction with eTFIIB or holo-eTFIID.
CREB S133A contains alanine in place of serine at the PKA
phosphorylation site.
Figure 7:
Interaction of the CREB constitutive
activation domain with TFIIB and TFIID. The CREB CAD vector encodes the
constitutive activation domain of CREB (amino acids 165-252)
fused to the 8 amino-terminal residues in CREB that provide peptide
initiation. The
We previously demonstrated that CREB mediates both
constitutive and kinase-inducible activities through distinct,
independently acting domains(5) . Given that these activities
of CREB require only binding sites for CREB and minimal promoters
containing the TATA region and start site, we suggested that CREB
interacts directly with one or more components of the general
transcription machinery. Others have shown that the general
transcription factors TBP/TFIID (or YY1), TFIIB, and RNA polymerase
II/RAP30 are sufficient to initiate in vitro transcription(9, 21, 22, 23) .
We now show that CREB interacts directly with both TFIIB and TFIID, but
not with TBP. The CREB-TFIID interaction is presumably mediated through
one or more TAFs in the TFIID complex. CREB interacts independently
with TFIIB and TFIID. These interactions are not affected by mutation
of the PKA phosphorylation site in CREB. Finally, the constitutive
activation domain of CREB is sufficient for interaction with either
TFIIB or TFIID.
The first step in the assembly of the preinitiation
complex is the binding of TFIID to the TATA
element(35, 36) . TFIID is composed of TBP and
TAFs(37, 38) . TBP alone is capable of binding to the
TATA sequence and mediating basal transcription in the absence of
regulators(9) . However, TBP alone cannot mediate activated
transcription regulated by SP1 and other
factors(11, 25, 39, 40, 41) .
Regulated transcription requires the TAFs in the TFIID fraction in
addition to TBP(25, 40, 41) . Different
activators interact with different TAFs of the multisubunit TFIID
complex, contributing to the specificity of gene
regulation(41, 42) . We show here that CREB does not
interact with TBP, but that CREB does interact with holo-TFIID. The
interaction between CREB and TFIID does not require TFIIB and can be
mediated by the constitutive activation domain alone. Although we
cannot rigorously exclude a requirement for other factors associated
with TFIID, our data are consistent with a requirement for the TAFs to
mediate CREB interaction with TFIID. While this study was in its final
stages, Ferreri et al.(43) also showed that CREB does
not interact directly with TBP and provided evidence for an interaction
between CREB and TAF110 mediated by the CREB CAD. Our data are
consistent with theirs, but neither set of data addresses the
possibility that CREB interacts with more than one TAF to form a stable
complex with TFIID. In the study of Ferreri et al., only
TAF110 was examined, whereas in this study, the holo-TFIID fraction
used consisted of many different TAFs associated with TBP. Thus, the
possibility that more than one TAF or other coactivators are involved
in CREB binding to TFIID cannot be ignored.
Following the binding of
TBP or TFIID to the TATA box, other general transcription factors
associate with TFIID to form a preinitiation
complex(9, 44) . The most crucial of these appears to
be TFIIB because TBP, TFIIB, and RNA polymerase II are the minimal
factors required to initiate transcription in
vitro(9, 21) . TFIIB interacts with several
transcription regulatory proteins (14, 17, 18) and with the RNA polymerase
II-TFIIF complex(44) . The most thoroughly studied activator,
herpes simplex virus transcription factor VP16, has been shown to
directly bind to TFIIB and to increase its stable assembly into a
preinitiation complex(19, 20) . We show here that CREB
can interact directly with recombinant TFIIB, purified essentially to
homogeneity from bacterial extracts. This interaction is independent of
phosphorylation of the PKA phosphorylation site (Ser-133) in CREB and
can be mediated by the constitutive activation domain alone. It is not
clear at present whether CREB can affect recruitment of TFIIB to the
promoter. It was demonstrated recently that an adaptor, CBP,
specifically binds to CREB phosphorylated at Ser-133 (45) and
is required for cAMP-induced gene transcription, at least in some cells (46) . CBP augments the effects of CREB phosphorylation and
also binds to TFIIB(47) . Thus, CREB may interact with this
crucial general transcription factor (TFIIB) directly to mediate
constitutive transcription and indirectly through CBP to mediate
kinase-induced transcription.
Some activators interact with both
TFIIB and TBP or TFIID(18, 30) , and both the TFIIB
and TBP proteins interact with VP16 through the same interaction
domain(19, 48) . In addition, direct association of
TBP with TFIIB has been demonstrated(9, 30) . Factors
bound to the ATF-binding site have been shown to extend the footprint
formed over the TATA box to include the initiation site and downstream
sequences (12) . In addition, ATF-bound factors enhance the
recruitment of TFIIB, TFIIE, and RNA polymerase II to the preinitiation
complex(13) . The nature of the ATF-bound factors has not been
characterized, but they presumably include CREB. Given that TBP and
TFIID interact with each other and that CREB interacts with TFIIB but
not TBP, we considered the possibility that a stable ternary complex
(CREB
In summary, CREB specifically binds
to both TFIID and TFIIB. These interactions are independent of
kinase-induced phosphorylation of Ser-133 in CREB, and the pairwise
interactions between CREB and TFIIB and TFIID occur independently of
the third protein. We have not examined directly whether CREB can
affect recruitment of TFIIB to the promoter, but this is likely in
light of data in other experimental systems. Thus, CREB may promote
basal transcription initiation both through direct interaction with the
TFIID complex and by recruiting TFIIB to the promoter. The exact nature
of the contacts between CREB and these targets remains to be
elucidated, both in terms of which TAFs and other cofactors are
involved and which domains in CREB are responsible for interaction with
different components of the basal transcription complex.
We thank Lorraine Altland for excellent technical
assistance, Drs. J. Hopper and D. Spector for critical discussions of
the work, A. Berk for providing expression vectors for eTBP and E1A and
the HeLa and LTR
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
3.
S-Labeled CREB, synthesized in vitro and
incubated with eTFIIB, was coimmunoprecipitated with antibody
recognizing eTFIIB, indicating that CREB specifically binds to TFIIB.
S-CREB was coimmunoprecipitated with antibody against
eTBP, but only when incubated with the holo-eTFIID complex, not with
eTBP alone. TFIIB interacted with TBP, but CREB was not coprecipitated
with the eTBP antibody when incubated with eTBP plus TFIIB, so CREB did
not form a stable ternary complex with TFIIB and TBP. Conversely,
depletion of TFIIB from the holo-TFIID preparation did not diminish the
level of interaction between CREB and TFIID. Thus, CREB interacts
independently with TFIIB and TFIID, but not directly with TBP. A
protein kinase A phosphorylation site mutant of CREB and wild-type CREB
exhibited equivalent interaction with TFIIB, indicating that this
phosphorylation is not required. Consistent with the role of CREB in
promoting constitutive or basal transcription, the constitutive
activation domain of CREB was sufficient for interaction with both
TFIIB and TFIID.
(
)is a transcription activator that binds to the cAMP
response element within a variety of cAMP-responsive promoters to
regulate gene expression(1, 2, 3) .
Hormone-induced accumulation of cAMP activates protein kinase A (PKA),
which phosphorylates CREB at serine 133 and further enhances
transcription activation. Mutation of the cAMP response element reduces
cAMP-mediated transcription and diminishes constitutive transcription
as well, indicating that the cAMP response element functions both as an
inducible enhancer and a general promoter element(4) . A
protein consisting of the CREB activation domain fused to the GAL4
DNA-binding domain (CRG) can restore both constitutive and
PKA-inducible transcription to a promoter containing a GAL4 site in
place of the cAMP response element, and the constitutive activity of
CRG is unaffected by mutation of the PKA phosphorylation site or by
inhibition of PKA activity by PKI(5, 6) .
3)
expressing eTBP. We show here that CREB interacts directly with TFIIB
and TFIID, but not with TBP.
Plasmids
pRT-CR and its mutants have been
described elsewhere(5) . For in vitro transcription-translation, the wild-type CREB cDNA or CREB S133A,
in which the PKA phosphorylation site is mutated, was cloned into pET3d
(Novagen) between the NcoI and BamHI sites, creating
pET-CR and pET-CR-S133A, respectively. The pET-CAD vector contains
amino acids 1-8 of CREB (to provide peptide initiation) fused to
amino acids 165-243, encoding the CAD, followed by the 6-amino
acid linker region of CRG and an in-frame stop codon.
Human eTFIIB
The human TFIIB expression plasmid
was obtained from Dr. Kent W. Wilcox (Medical College of Wisconsin,
Milwaukee, WI). This plasmid vector contains a T7 phage gene 10 epitope
tag and a 10-histidine tag at the N terminus of the human TFIIB cDNA.
Recombinant eTFIIB was purified from Escherichia coli (BL21(DE3)) as follows. eTFIIB-transformed cells were grown at 37
°C in super broth containing 200 µg/ml ampicillin until A = 0.6 was reached.
Isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 0.4 mM, and the cells were grown at 30
°C for an additional 3 h. Cell lysates from 50 ml of culture were
incubated for 1 h at 4 °C with 1 ml of Ni
-agarose
resin (QIAGEN, Inc.) in 2 ml of buffer A (20 mM Tris-HCl (pH
7.9), 0.1 M KCl, 20% glycerol, 0.1 mM EDTA, 0.1
mM phenylmethylsulfonyl fluoride) containing 1 M ammonium sulfate. The matrix was washed with 10 column volumes of
buffer A containing 25 mM imidazole and with 10 column volumes
of buffer A containing 37.3 mM imidazole. Bound eTFIIB was
eluted in 2 column volumes of the same buffer containing 125
mM imidazole(26) .
Human eTBP
A human eTBP expression plasmid,
containing an epitope of 9 amino acids from the hemagglutinin antigen
of influenza virus fused in frame to the amino terminus of human TBP,
was a generous gift of Dr. A. Berk (UCLA)(15) .
pET-eTBP-transformed cells (BL21(DE3)) were grown at 37 °C in ZY
medium (1% Bactotryptone, 0.5% yeast extract, 0.086 M NaCl)
containing 0.4% glucose in the presence of 100 µg of ampicillin/ml
until A = 0.8 was reached.
Isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 0.5 mM, and the cells were harvested 3
h later. Fractionation of the cell lysate was performed essentially as
described previously(27, 28) .
Holo-eTFIID
The eTFIID fraction of the human
eTBP-expressing LTR3 cell line was isolated by chromatography on
phosphocellulose as described previously(24) . The LTR
3
cell line, in which 80% of the TBP is present as eTBP, was a generous
gift of Dr. A. Berk. Nuclear extracts were prepared by lysis of the
cells with 0.5% Nonidet P-40, collection of the nuclei, and extraction
of the nuclei with 420 mM NaCl as described by Hurst et
al.(29) . TFIIB-depleted LTR
3 nuclear extracts were
prepared by incubation of nuclear extract with a polyclonal anti-TFIIB
antibody for 1 h at 4 °C, followed by addition of protein
A/G-agarose beads and further incubation for 1 h at 4 °C. The beads
were collected by centrifugation to remove antibody-bound TFIIB from
the extracts, and the supernatant nuclear extract was used for
interaction assays. Complete and TFIIB-depleted nuclear extracts were
analyzed for TFIIB by Western blotting with primary antibody against
TFIIB and secondary antibody coupled to horseradish peroxidase. Blots
were developed with chemiluminescence reagents (DuPont NEN).
Coimmunoprecipitation
S-Labeled
proteins were synthesized in a coupled in vitro transcription-translation system using rabbit reticulocyte lysate
(Promega) in the presence of [
S]methionine.
Equimolar amounts of
S-labeled in vitro translated CREB or mutated CREB protein were mixed with 10 pmol of
eTFIIB in 125 µl of buffer D (20 mM Hepes (pH 7.9), 20%
glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM, phenylmethylsulfonyl fluoride)
containing 0.1 M KCl and incubated at 4 °C for 1 h. 2
µg of monoclonal antibody recognizing the T7 Tag epitope on eTFIIB
was added, and the mixture was incubated at 4 °C for 1.5 h. 20
µl of a 50% slurry of protein A/G-agarose beads was added and mixed
for 1 h at 4 °C. Protein complexes immobilized on protein A/G beads
were washed three times with buffer D and suspended in 1
Laemmli sample buffer. One-half of the bound fraction of each sample
was loaded onto 15% SDS-polyacrylamide gels. One-sixtieth of the
original sample (input) or the supernatant (free) was included as
indicated.
S-Labeled proteins were visualized by
autoradiography.
Lack of Interaction of CREB with TBP
The
observation of Zhou et al.(24) that E1A interacts
directly with TBP prompted us to investigate the possibility that CREB
may interact directly with TBP. CREB, E1A, and luciferase proteins were
synthesized in vitro and examined, together with a control
lysate, for binding to eTBP. As in the studies of Zhou et al.,
E1A interacted strongly with eTBP, and luciferase did not (Fig. 1). Control lysate, to which no template was added, did
not synthesize any significant amount of protein, and none bound to
eTBP. CREB protein was synthesized, but did not bind to eTBP,
indicating that there is no direct interaction between CREB and TBP.
S-labeled proteins were incubated with eTBP in buffer D
for 1 h at 4 °C and then incubated with antibody against the HA
epitope of eTBP (12CA5) for 1.5 h at 4 °C. Protein A/G-agarose was
used to collect the immunoprecipitates, which were washed three times
with binding buffer. Fractions of the input (I; one-sixtieth),
free (F; one-sixtieth), and bound (B; one-half)
proteins were resolved by SDS-PAGE and visualized by
autoradiography.
Interaction of CREB with TFIIB
We next examined
the possibility that CREB interacted directly with the general
transcription factor TFIIB. S-Labeled in vitro translated CREB was incubated without or with eTFIIB protein and
then with a monoclonal antibody that recognizes the T7 Tag epitope on
eTFIIB (Fig. 2). No binding was observed in the absence of
eTFIIB. In the presence of eTFIIB, CREB was coimmunoprecipitated with
the T7 Tag antibody recognizing eTFIIB. Thus, CREB formed a stable
complex with the TFIIB protein that allowed it to be precipitated with
antibody recognizing the epitope on TFIIB.
S]Methionine-labeled CREB was incubated in the
absence (firstfourlanes) or presence (last four lanes) of 10 pmol of eTFIIB in buffer D for 1 h at
4 °C and immunoprecipitated with a monoclonal antibody (Ab) directed against the T7 Tag on the eTFIIB protein. The
protein-antibody complex was isolated with protein A/G-agarose beads
and washed three times with binding buffer. Fractions of the input (I), free (F), wash (W), and bound (B) proteins were resolved by SDS-PAGE and visualized by
autoradiography. First and fifthlanes,
one-twentieth of the input; second and sixthlanes, one-tenth of the free CREB in the supernatant; third and seventhlanes, one-thousandth of
the wash; fourth and eighthlanes, one-half
of the bound protein.
Interaction of CREB with TFIID
The majority of TBP
is found in complexes with TATA-associated factors (TAFs) in eukaryotic
cells (10, 24) . To determine whether CREB might
interact with TBP indirectly through TAFs, rather than directly with
TBP, we tested the interaction of CREB with a holo-eTFIID fraction, the
eTBP upon which it is based, and eTFIIB. The eTFIID fraction was
purified from LTR3 cells, in which 80% of the TBP present is
eTBP(24) . As shown above, CREB was coimmunoprecipitated with
antibody recognizing eTFIIB (Fig. 3A). Antibody
recognizing eTBP precipitated CREB incubated with eTFIID, but not with
eTBP alone. The composition of the eTFIID fraction precipitated from
LTR
3 cells with the 12CA5 antibody is shown in Fig. 3B. This silver-stained gel shows that, in
addition to eTBP (
39 kDa), bands corresponding to TAFs of
250, 125, 78, and 50 kDa and faint bands at 95 and 30/28 kDa are
coimmunoprecipitated from LTR
3 cells. The band expected at
70
kDa is obscured by residual carrier bovine serum albumin. These
coimmunoprecipitated proteins correspond to the TAFs identified in
LTR
3 cells by Zhou et al.(24) . Thus, our data
suggest that CREB associates with TBP indirectly through interactions
with one or more TAFs, rather than directly interacting with TBP.
S]methionine-labeled CREB was
incubated with eTFIIB, eTBP, or holo-eTFIID (fractionated from the
nuclear extracts of LTR
3 cells stably expressing eTBP) in buffer D
for 1 h at 4 °C and immunoprecipitated with a monoclonal antibody (Ab) directed against the T7 Tag on eTFIIB (first and secondlanes) or the HA epitope tag on eTBP/eTFIID (third through sixthlanes). Antibody-bound
protein complexes were isolated on protein A/G-agarose beads and
washed. The input (I; one-sixtieth of the starting reaction)
and bound (B; one-half of the bound protein) CREB fractions
were resolved by SDS-PAGE and visualized by autoradiography. B, nuclear extracts were prepared from LTR
3 cells
expressing eTBP. The TFIID-containing fraction was obtained by
phosphocellulose chromatography and subjected to immunoprecipitation
with monoclonal antibody against the HA epitope on eTBP under the same
conditions used for interaction assays. The immunoprecipitates were
resolved by SDS-PAGE, and proteins were visualized by silver staining.
The asterisks indicate gel artifacts observed in all lanes. BSA, bovine serum albumin.
TFIIB Does Not Permit Interaction of CREB with
TBP
TFIIB has been shown to interact with
TBP(9, 30) . Therefore, the TBPTFIIB complex is
a potential target for interaction with CREB. To determine whether
TFIIB can interact with both CREB and TBP and thus recruit TBP to a
complex with CREB,
S-labeled CREB was incubated with TFIIB
and eTBP, alone or in combination. CREB was immunoprecipitated when
incubated with eTFIIB, but not with TBP (Fig. 4A), as
observed above (Fig. 3). Preincubation of eTBP with an excess of
TFIIB still did not permit immunoprecipitation of CREB with antibody
against eTBP (Fig. 4A), indicating that the
TBP
TFIIB complex is not a strong target for CREB interaction. To
verify that eTFIIB interacted stably with eTBP under our experimental
conditions,
S-labeled eTBP was incubated with eTFIIB and
precipitated with antibody against eTFIIB. Interaction between eTBP and
eTFIIB was observed (Fig. 4B) and was more pronounced
than that seen between CREB and TFIIB. A similar degree of interaction
was observed when eTBP precipitates were electrophoresed and probed
with the TFIIB antibody (data not shown). Thus, TFIIB and TBP interact
under these conditions, but the TBP
TFIIB complex does not bind
CREB.
S-labeled CREB was incubated
with the indicated target proteins, eTFIIB and eTBP, alone or together,
and then immunoprecipitated with antibody (Ab) against the
appropriate epitopes, indicated at the top. Protein A/G-agarose was
used to collect the immunoprecipitates, which were washed three times
with binding buffer. Fractions of the input (I; one-sixtieth)
and bound (B; one-half) proteins were resolved by SDS-PAGE and
visualized by autoradiography. B, a cDNA encoding eTBP was
used to program in vitro transcription-translation, and
S-labeled eTBP was incubated with eTFIIB in buffer D for 1
h at 4 °C and then immunoprecipitated with antibody recognizing the
T7 Tag epitope on eTFIIB. Protein A/G-agarose was used to collect the
immunoprecipitates, which were washed three times with binding buffer.
Fractions of the input (one-sixtieth) and bound (one-half) proteins
were resolved by SDS-PAGE and visualized by
autoradiography.
CREB Interacts with TFIID Immunodepleted of
TFIIB
It is possible that the interaction observed between the
TFIID fraction and CREB was due to association of TFIIB with eTFIID.
The TFIID fraction used in the above studies contained readily
detectable TFIIB, as judged by Western blotting with antibody
recognizing native TFIIB (data not shown). To determine whether TFIIB
influenced the interaction of CREB with TFIID, we prepared nuclear
lysates containing eTFIID from LTR3 cells, in which 80% of the TBP
is eTBP. Half of the extract was depleted of TFIIB by incubation with
antibody recognizing native TFIIB and protein A/G-agarose beads and by
removal of the beads by centrifugation. Both the complete nuclear
extract and the TFIIB-depleted nuclear extract were examined for
interaction with CREB (Fig. 5A, NE and NE-TFIIB, respectively). Depletion of TFIIB did not alter CREB
interaction with eTFIID in the nuclear extract, indicating that TFIIB
is not required for interaction between CREB and TFIID. Complete and
immunodepleted nuclear extracts (Fig. 5B, NE and NE-TFIIB, respectively) were subjected to Western
blotting with a polyclonal TFIIB antibody, and the bands were detected
by enhanced chemiluminescence. Immunodepletion appeared to have been
effective (<0.3 fmol). We estimate that 0.3-0.6 fmol of CREB
is bound to TFIIB or TFIID in these assays, based on
3%
incorporation of 10-20 fmol of input protein. Therefore, it is
unlikely that residual TFIIB is responsible for the interaction of CREB
with eTFIID.
S-Labeled CREB was incubated with
nuclear extract from LTR
3 cells (NE) or with LTR
3
nuclear extract immunodepleted of TFIIB (NE-TFIIB) and then
with antibody (Ab) against the HA epitope in eTFIID. Protein
A/G-agarose was used to collect the immunoprecipitates, which were
washed three times with binding buffer. Fractions of the input (I; one-sixtieth) and bound (B; one-half) proteins
were resolved by SDS-PAGE and visualized by autoradiography. B, nuclear extracts from LTR
3 cells were treated with
protein A/G-agarose alone (left lane) or with a polyclonal
anti-TFIIB antibody plus protein A/G-agarose (right lane).
Following removal of the agarose beads, proteins were separated by
SDS-PAGE, transferred to nitrocellulose, and probed with a polyclonal
TFIIB antibody.
Effect of Phosphorylation of CREB by PKA on Its
Interaction with TFIIB or TFIID
Transcription activation by CREB
is enhanced by cAMP-dependent PKA-mediated phosphorylation of Ser-133,
and reticulocyte lysates are known to have endogenous PKA(31) .
To determine whether phosphorylation of CREB by PKA in the lysate
contributed to the interaction of CREB with TFIIB or TFIID, we used a
PKA site mutant of CREB, CREB S133A, in which Ser-133 is changed to
alanine. This mutation completely abolishes phosphorylation by PKA (2, 32) and PKA inducibility in transfection assays,
but has no effect on constitutive transcription activity in these
assays(5, 6) . Mutation of the CREB phosphorylation
site in CREB S133A had no effect on its ability to interact with TFIIB (Fig. 6) or TFIID (data not shown) in vitro in the
coimmunoprecipitation assay. The lack of effect of mutation of the PKA
phosphorylation site demonstrates that phosphorylation by PKA is not
crucial for the interaction of CREB with either eTFIIB or holo-eTFIID.
S-Labeled wild-type (WT)
CREB or mutant CREB S133A was incubated with eTFIIB, immunoprecipitated
with T7 Tag antibody and protein A/G-agarose beads, and washed three
times in binding buffer. Fractions of the input (I;
one-sixtieth) and bound (B; one-half) proteins were resolved
by SDS-PAGE and visualized by
autoradiography.
Binding of the CAD of CREB to TFIIB and
TFIID
Constitutive activation and kinase-inducible activation by
CREB are mediated by independent domains in the protein. The CAD
located between amino acids 165 and 243 of CREB provides the majority
of constitutive activity(5, 33, 34) . The CAD
was fused to the 8 amino-terminal amino acids of CREB to permit
initiation of protein synthesis. The S-labeled CREB CAD
protein was incubated with TFIIB and TFIID to determine whether the
CREB CAD can mediate binding to these proteins. The CREB CAD bound to
both TFIIB and TFIID (Fig. 7), indicating that the CAD is
sufficient for interaction with these general transcription factors, as
is consistent with the role of the CAD in promoting transcription
activation in the absence of phosphorylation of CREB by PKA.
S-labeled CREB CAD was incubated with
eTFIIB or eTFID and then immunoprecipitated with antibody directed
against either the T7 Tag (for eTFIIB) or the HA epitope (for eTFIID)
and protein A/G-agarose beads and washed three times in binding buffer.
Fractions of the input (I; one-sixtieth) and bound (B; one-half) proteins were resolved by SDS-PAGE and
visualized by autoradiography.
TFIIB
TBP) might be formed, allowing the
immunoprecipitation of CREB by antibody recognizing epitope-tagged TBP
in the presence of TFIIB. We found no evidence for such a complex.
Although both CREB and TBP interact with TFIIB, they do not appear to
form a stable ternary complex, at least under our experimental
conditions. One possibility is that CREB and TBP have similar or
overlapping recognition sites on TFIIB such that binding is mutually
exclusive. In that case, CREB might interact with TFIIB and recruit it
to the initiation complex, after which TFIIB would form a stable
complex with TBP to facilitate transcription initiation. Consistent
with this notion is the demonstration by Hai et al.(13) that ATF factors facilitate formation of the
initiation complex, but are not required for maintenance of this
complex. Thus, CREB may play a role in recruiting TFIIB to the
initiation complex. Studies to determine whether CREB can recruit TFIIB
are underway in our laboratory.
3 cell lines, K. W. Wilcox for the eTFIIB
expression vector, R. Maurer for providing the PKAc expression plasmid,
and M. Ptashne for the antibody to the GAL4 DNA-binding domain.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.