(Received for publication, October 31, 1995; and in revised form, November 28, 1995)
From the
The second messenger cAMP stimulates the expression of a number
of target genes via the protein kinase A-mediated phosphorylation of
CREB at Ser-133 (Gonzalez, G. A., and Montminy, M. R.(1989) Cell 59, 675-680). Ser-133 phosphorylation enhances CREB activity
by promoting interaction with a 265-kDa CREB binding protein referred
to as CBP (Arias, J., Alberts, A., Brindle, P., Claret, F., Smeal, T.,
Karin, M., Feramisco, J., and Montminy, M.(1994) Nature 370,
226-228; Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M.,
Montminy, M. R., and Goodman, R. H.(1993) Nature 365,
855-859). The mechanism by which CBP in turn mediates induction
of cAMP-responsive genes is unknown but is thought to involve
recruitment of basal transcription factors to the promoter. Here we
demonstrate that CBP associates specifically with RNA polymerase II in
HeLa nuclear extracts. This association in turn permits RNA polymerase
II to be recruited to CREB in a phospho-(Ser-133)-dependent manner. As
anti-CBP antiserum, which inhibits recruitment of CBP and RNA
polymerase II to phospho-(Ser-133) CREB, attenuates transcriptional
induction by protein kinase A in vitro, our results
demonstrate that the CBPRNA polymerase II complex is critical for
expression of cAMP-responsive genes.
A number of hormones and growth factors stimulate the expression of target genes by inducing the reversible phosphorylation of specific transcription factors(4) . Although phosphorylation has been shown to regulate a number of nuclear factors by inducing their nuclear targeting or DNA binding activities, the cAMP-responsive transcription factor CREB belongs to a group whose transactivation potential is affected(1, 5) . In this regard, Chrivia et al.(3) have characterized a nuclear CREB binding protein, termed CBP, which binds to CREB in a phospho-(Ser-133)-dependent manner. The requirement for CBP in mediating cAMP-dependent transcription has been demonstrated by cellular microinjection experiments in which CBP antisera blocked transcriptional induction by cAMP (2) and by transient transfection experiments in which overexpression of CBP could potentiate CREB activity in response to agonist(2, 6) . Here we examine the mechanism by which CBP interacts with the transcriptional apparatus to induce target gene expression in response to hormonal stimulation. Our results suggest that CBP is constitutively associated with specific components of the transcriptional apparatus and that this association in turn permits recruitment of certain basal factors to promoters of cAMP-responsive genes.
Preliminary evidence suggesting that CBP migrates as a high molecular mass complex of 2000 kDa during gel filtration chromatography (not shown) prompted us to examine whether CBP might stimulate cAMP-responsive genes by virtue of its association with specific basal transcription factors. When purified from HeLa nuclear extracts by phosphocellulose chromatography (Fig. 1, top), CBP was detected predominantly in the 0.3 M KCl ``B'' fraction, which also contained RNA polymerase II. Following subsequent fractionation over a Mono-S ion exchange resin (Fig. 1, bottom), CBP again eluted with peak fractions of RNA polymerase II. In contrast to the relatively sharp elution profile for CBP, however, RNA polymerase II appeared to be more broadly distributed, indicating that only a fraction of RNA polymerase II may be associated with CBP. By contrast with RNA polymerase II, CBP did not co-elute from the Mono-S column with TFIIB, a basal factor that has been reported to interact with CBP in GST pull-down assays(6) .
Figure 1: The CREB co-activator CBP fractionates with RNA polymerase II during purification from HeLa nuclear extracts. Top, Western blot analysis of HeLa nuclear extract following fractionation over P11 phosphocellulose resin. A-D correspond to HeLa protein fractions eluted from the P11 column with 100 mM, 300 mM, 500 mM, and 1.0 M KCl, respectively. Analysis of CBP, RNA polymerase II (RNA Pol II) in individual fractions as indicated on left. Bottom, Western blot analysis of the 300 mM KCl HeLa ``B'' fraction (top) following fractionation over Mono-S ion exchange resin. Elution profile for CBP and RNA polymerase II as indicated on left. FT, flow-through.
To test whether CBP in fact associates with RNA polymerase II, we performed co-immunoprecipitation studies with two distinct anti-CBP antisera directed against aa 1-100(5729) and aa 455-679(5614) of the protein (Fig. 2A). Although neither antiserum was capable of recognizing purified RNA polymerase II directly by Western blot or immunoprecipitation assay (not shown), the large subunit of RNA polymerase II was detected in immunoprecipitates of HeLa extracts with both antisera under non-denaturing conditions. In agreement with the broad elution profile of RNA polymerase II following Mono-S chromatography, only a limited fraction (10-20%) of the RNA polymerase II large subunit appeared to be associated with CBP in HeLa extracts. By contrast with RNA polymerase II, other basal transcription factors such as TBP and TFIIB did not appear to associate detectably with CBP in co-fractionation or co-immunoprecipitation assays (Fig. 1), indicating that the CBP-RNA polymerase II interaction was indeed specific. In this regard, RNA polymerase II was found to co-precipitate with CBP even at high concentrations of KCl (0.8 M), suggesting that this complex was also stable.
Figure 2:
CBP is associated with RNA polymerase II
in HeLa nuclear extracts. A, Western blot analysis of
immunoprecipitates (IP) from HeLa nuclear extracts using
anti-CBP antiserum (CBP) and anti-RNA polymerase II
antibody (
RNA POL II). Antisera used for
immunoprecipitation are indicated over each lane. PI,
preimmune antiserum; 5729, rabbit polyclonal
CBP
antiserum raised against CBP polypeptides containing aa 1-117; 5614,
CBP antiserum raised against CBP recombinant CBP
polypeptide extending from aa 455-679; POLII, monoclonal
RNA polymerase II antiserum raised against a C-terminal domain
polypeptide (Promega); ONPUT, crude HeLa nuclear extract prior
to immunoprecipitation. Relative mass, in kilodaltons, indicated
alongside. Asterisks indicate position of CBP and RNA
polymerase II large subunit. Additional CBP-immunoreactive bands in lanes marked 5614 and 5729 correspond to
proteolytic digestion products of CBP, which are generated during
immunoprecipitation with those antisera. B, CBP mediates
recruitment of RNA polymerase II to CREB following PKA-dependent
phosphorylation at Ser-133. Western blot analysis of HeLa nuclear
extracts following affinity chromatography on glutathione-Sepharose
resins containing unphosphorylated (GST
KID) or Ser-133
phosphorylated (GST-(P)-KID) GST-CREB
polypeptide. ONPUT, HeLa nuclear extract (100 µg)
before pull-down assay; MW, relative mass (in kilodaltons).
CBP, anti-CBP antiserum;
RNA POL II,
anti-RNA polymerase II antiserum.
Previous reports showing that CBP interacts with a KID (aa
88-160) in CREB (2, 3) prompted us to examine
whether CBP mediates the PKA-dependent recruitment of RNA polymerase II
to this region (Fig. 2B). Following affinity
chromatography of crude HeLa nuclear extracts over
glutathione-Sepharose resin containing either GSTKID or
GST-phospho-(Ser-133)
KID fusion proteins, CBP was specifically
bound to phospho-(Ser-133)
KID resin. Similarly, RNA polymerase II
was detected on resins containing phospho-(Ser-133)
KID but not
unphosphorylated KID peptide. As purified RNA polymerase II was unable
to bind to phospho-(Ser-133)
KID directly (not shown), these
results indicate that RNA polymerase II is recruited to
phospho-(Ser-133)
KID via CBP.
To test whether PKA simulates
formation of a heteromeric complex consisting of phospho-(Ser-133)
CREBCBP
RNA polymerase II, as predicted by GST affinity
chromatography experiments, we performed immunoprecipitation assays on
crude HeLa nuclear extracts (Fig. 3). Using a CREB antiserum
(253) that can recognize the CREB
CBP complex(8) , we
detected CBP in immunoprecipitates from PKA-treated but not untreated
HeLa nuclear extracts. Similarly, the large subunit of RNA polymerase
II was recovered specifically from immunoprecipitates of PKA-treated
HeLa extracts, demonstrating that PKA induces formation of a
phospho(Ser-133) CREB
CBP
RNA polymerase II complex.
Figure 3:
PKA stimulates formation of a
phospho-(Ser-133) CREB-CBP-RNA polymerase II (POLII)
heteromeric complex in HeLa nuclear extracts. Western blot analysis (CBP,
RNA POLII) of immunoprecipitates (IP)
prepared from control(-) or PKA (+) treated HeLa nuclear
extract with various antisera under non-denaturing conditions. ONPUT, crude HeLa nuclear extract prior to
immunoprecipitation; 253, polyclonal anti-CREB antiserum
raised against the full-length recombinant CREB protein (aa
1-341); 5729, CBP antiserum raised against a recombinant
CBP polypeptide extending from aa 1-100; PI, preimmune
serum.
In
order to determine whether recruitment of the CBPpolymerase II
complex to phospho-(Ser-133) CREB is critical for transcriptional
induction by PKA, we performed in vitro transcription assays
on crude HeLa nuclear extracts (Fig. 4). Addition of PKA to
nuclear extracts induced transcription from a cAMP-responsive template
containing three consensus cAMP-responsive elements (3
CRE)
approximately 4-fold. But PKA treatment had no effect on an internal
control adenovirus major late promoter template lacking CRE sites.
Addition of affinity-purified CBP antiserum, which blocks recruitment
of the CBP
RNA polymerase II complex to CREB(2) ,
specifically inhibited PKA-inducible transcription from the 3
CRE template. Unrelated antiserum (anti-corticotropin-releasing factor
binding protein) had no effect on PKA induction of the 3
CRE
template, however, demonstrating that the inhibition by anti-CBP
antiserum was indeed specific.
Figure 4:
The
CBP-RNA polymerase II complex is required for transcriptional induction
by PKA in vitro. Primer extension analysis of in vitro transcription reactions performed with HeLa nuclear extracts in
the presence or absence of purified PKA catalytic subunit, as indicated
above each lane (-, +). 3XCRE,
transcription template containing three tandemly repeated somatostatin
CRE sites inserted upstream of the adenovirus major late promoter. MLP, adenovirus major late promoter construct lacking CRE
sequences. Transcription reactions containing affinity-purified CBP antiserum or control anti-corticotropin-releasing
factor binding protein (
CRFBP) antiserum as indicated
over corresponding lanes.
Our observation that CBP is found in
a high molecular weight complex is supported by recent findings of
Maldonado et al.,()suggesting that CBP is
contained within purified preparations of a mammalian RNA polymerase II
holoenzyme. In contrast to results of Kwok et al.(6) ,
TFIIB did not appear to associate detectably with CBP in HeLa extracts.
These results would suggest that TFIIB may interact with CBP only after
being recruited to the promoter.
Our results do not address whether
CBP interacts directly with RNA polymerase II. In preliminary GST
pull-down assays, purified recombinant CBP polypeptides are unable to
associate directly with the large subunit of RNA polymerase II, ()suggesting that the interaction between CBP and RNA
polymerase II may either require post-translational modification (i.e. phosphorylation) or may involve other proteins within
the RNA polymerase II holoenzyme complex.
In a previous study, we found that the activity of purified phospho-(Ser-133) CREB in cell-free transcription assays was indistinguishable from that of unphosphorylated CREB(9) . In this report, we found the addition of PKA to HeLa nuclear extracts during the transcription reaction was critical for Ser-133 phosphorylation-dependent activity. These results are consistent with recent findings that other PKA-dependent events in addition to CREB phosphorylation are required for transcriptional induction by cAMP in vivo(8) . In this regard, CBP contains a consensus PKA phosphorylation site at Ser-1772, and it is tempting to speculate that the interaction between CBP and RNA polymerase II may itself be regulated by CBP phosphorylation. In yeast, interaction between an upstream activator (GAL4) and a component of the yeast RNA polymerase II holoenzyme complex (GAL11) is sufficient for transcriptional induction(10) . The importance of CBP in mediating not only cAMP but also mitogen-inducible transcription (2) indicates that CBP may similarly provide contact points for recruitment of mammalian RNA polymerase II holoenzyme by multiple signal-dependent activators.