p300

Molecular Cell, Vol. 1, 661–671, April, 1998, Copyright 1998 by Cell Press

Phosphorylation of NF-kB p65 by PKA Stimulates Transcriptional Activity by Promoting a Novel Bivalent Interaction with the Coactivator CBP/p300 Haihong Zhong,* Reinhard E. Voll,* and Sankar Ghosh*† * Section of Immunobiology and Department of Molecular Biophysics and Biochemistry Howard Hughes Medical Institute Yale University School of Medicine New Haven, Connecticut 06520

Summary The transcriptional activity of NF-kB is stimulated upon phosphorylation of its p65 subunit on serine 276 by protein kinase A (PKA). The transcriptional coactivator CBP/p300 associates with NF-kB p65 through two sites, an N-terminal domain that interacts with the C-terminal region of unphosphorylated p65, and a second domain that only interacts with p65 phosphorylated on serine 276. Accessibility to both sites is blocked in unphosphorylated p65 through an intramolecular masking of the N terminus by the C-terminal region of p65. Phosphorylation by PKA both weakens the interaction between the N- and C-terminal regions of p65 and creates an additional site for interaction with CBP/p300. Therefore, PKA regulates the transcriptional activity of NF-kB by modulating its interaction with CBP/p300. Introduction The inducible transcription factor NF-kB (nuclear factor kB) is responsible for regulating the expression of a wide variety of genes (Baldwin, 1996; May and Ghosh, 1997). The most abundant form of the protein is a heterodimer of p50 and p65 subunits, in which the p65 subunit contains the transcriptional activation domain. In uninduced cells, NF-kB remains in the cytoplasm bound to inhibitory proteins known as IkBs (inhibitors of NF-kB). Upon treatment of cells with inducers such as tumor necrosis factor a, interleukin-1, or lipopolysaccharide (LPS), the IkB proteins are phosphorylated, ubiquitinated, and degraded. The released NF-kB then translocates to the nucleus and up-regulates gene expression (Baldwin, 1996; May and Ghosh, 1997). We have recently demonstrated that cytosolic complexes of NF-kB:IkB also contain the cAMP-dependent protein kinase catalytic subunit of PKA, PKAc, whose activity is inhibited by its association with the NF-kB:IkB complex (Zhong et al., 1997). The degradation of IkB proteins during induction of NF-kB leads to the activation of the associated PKAc and the concomitant phosphorylation of the p65 subunit of NF-kB. The phosphorylation of p65 by PKA is required for efficient transcriptional activation by NF-kB; however, the mechanism responsible for the enhancement of transcription remains unclear. Recent studies have suggested that the activity of many inducible transcription factors is regulated through their † To whom correspondence should be addressed (e-mail: sankar.

[email protected]).

interaction with cellular coactivators such as CBP (CREB-binding protein)/p300 (Janknecht and Hunter, 1996a, 1996b). Coactivator molecules are believed to link enhancer-bound transcription factors with the general transcriptional machinery, and CBP/p300 has been linked to transcriptional regulation by several transcription factors such as c-Jun (Bannister et al., 1995), c-Fos (Janknecht and Nordheim, 1996), p53 (Avantaggiati et al., 1997; Lill et al., 1997), c-Myb (Dai et al., 1996; Oelgeschlager et al., 1996), Sap-1 (Janknecht and Nordheim, 1996), MyoD (Yuan et al., 1996), E12/E47 (Eckner et al., 1996), Tax (Kwok et al., 1996), and nuclear hormone receptors (Chakravarti et al., 1996). In addition to these transcription factors, CBP/p300 has also been shown to interact with TBP (TATA box–binding protein) (Swope et al., 1996; Dallas et al., 1997) and TFIIB (Kwok et al., 1994), thereby providing possible targets of interaction for this protein on the general transcriptional apparatus. It has been reported recently that CBP/p300 can be detected associated with NF-kB in cells, and cotransfection of CBP/p300 enhances NF-kB–dependent transcription (Gerritsen et al., 1997; Perkins et al., 1997). However, the regulation of this interaction was not explored, and it remained unclear whether CBP/p300 was an obligatory component in the transcriptionally active, nuclear form of NF-kB or acted to enhance NF-kB– dependent transcriptional activity. We report that association of NF-kB with CBP/p300 is dependent upon phosphorylation of NF-kB p65 by PKA, and that CBP/p300 plays an essential role in NF-kB transcriptional activity. The association between CBP/ p300 and NF-kB p65 occurs through a bivalent interaction that consists of a phosphorylation-independent interaction and a PKA–phosphorylation-dependent interaction. The phosphorylation-dependent interaction involves the KIX region of CBP, which is also responsible for binding to the PKA-phosphorylated transcription factor, CREB (cyclic AMP response element–binding protein) (Parker et al., 1996). Disruption of either the phosphorylation-independent or phosphorylation-dependent interaction through mutagenesis dramatically lowers the efficiency of NF-kB–dependent transcription, demonstrating the requirement of both domains for stable association and consequent transcriptional activity. Access to both CBP-interacting regions in unphosphorylated p65 is blocked by an intramolecular masking of the N-terminal region of p65 by the C-terminal region. Phosphorylation by PKA weakens this intramolecular association and promotes association with CBP/p300 by creating a site for phosphorylation-dependent interaction, as well as making the site for phosphorylationindependent interaction accessible. Thus, our results describe a mechanism that regulates the association of NF-kB p65 and CBP and explains how phosphorylation of p65 by PKA enhances NF-kB–dependent transcription. Results CBP Enhances NF-kB–Dependent Transcription To determine whether phosphorylation by PKA enhanced the transcriptional activity of NF-kB by influencing its

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mutant p65 in cotransfection assays indicated that CBP was now ineffective in stimulating transcription, and this result along with the demonstration of a costimulatory role for PKA suggested that the effect of CBP on NFkB–mediated transcription was dependent on phosphorylation of NF-kB p65 by PKA. The dependence of CBP-mediated stimulation of p65 transcriptional activity on phosphorylation raises the question of why transfection of CBP in the absence of PKA stimulates p65-dependent transcription (Figure 1A, lane 2 versus lane 4). A possible explanation is the presence of low-level, basal PKA activity in cells grown in culture (Zhong et al., 1997) that would phosphorylate a portion of the transfected p65. To determine whether basal PKA activity was responsible for the observed effect of CBP on p65, we carried out transfection experiments similar to those in Figure 1A except for the addition of either H-89, a specific inhibitor of PKA, or ML-7, a related compound that inhibits the activity of myosin light chain kinase, but not PKA, to the transfected cells. CBP was able to stimulate p65-mediated transcription in ML-7–treated cells but not H-89–treated cells (Figure 1B). This experiment indicates that phosphorylation of p65 is required for CBP to stimulate NF-kB–dependent transcription.

Figure 1. Stimulation of NF-kB–Dependent Transcription by CBP Is Dependent upon Phosphorylation of p65 by PKA (A) Jurkat cells were transfected with the NF-kB–dependent reporter plasmid pBIIx-Luc and the indicated constructs, using lipofectamine (GIBCO). The p65 and PKA mutants have been described previously (Zhong et al., 1997). The cells were harvested 36 hr after transfection, and the extracts were used to assay for luciferase activity. (B) Hela cells were transfected with pBIIx-Luc, p65, and CBP as indicated, and the cells were treated 6 hr before harvesting with indicated concentrations of H-89 or ML-7 (Zhong et al., 1997). Luciferase activity in the extracts was measured as in (A).

interaction with CBP/p300, we carried out cotransfection experiments in Jurkat T cells using a NF-kB–dependent reporter construct. We verified that cotransfection of CBP increased NF-kB p65-dependent transcription from reporter constructs (Figure 1A, lane 4) (Gerritsen et al., 1997; Perkins et al., 1997); however, we found that inclusion of PKAc further stimulated transcription driven by NF-kB p65 and CBP (Figure 1A, lane 5), and this stimulation was not observed when a catalytically inactive form of PKAc was used (Figure 1A, lane 10). In agreement with our previous results (Zhong et al., 1997), we found that the stimulatory effect of PKA on NF-kB– driven transcription was due to phosphorylation at Ser276, since a site-specific mutant of p65, in which Ser276 was changed to alanine (S276A), was unaffected by cotransfection with PKA (Figure 1A, lane 8). Use of this

Association of NF-kB and CBP/p300 In Vivo Is Influenced by PKA The enhancement of transcription by CBP/p300 is mediated by its physical association with transcription factors (Janknecht and Hunter, 1996a, 1996b). Therefore, the stimulation of NF-kB–dependent transcription by CBP suggests that PKA phosphorylation probably influences association of CBP with NF-kB. To test this hypothesis, we examined the association of CBP with NFkB in vivo in stimulated cells. We stimulated 70Z/3 cells with LPS for different lengths of time and immunoprecipitated NF-kB complexes from nuclear extracts using antibodies directed against the p65 subunit. The immunoprecipitates were fractionated on SDS-PAGE (polyacrylamide gel electrophoresis) and immunoblotted with either antibodies against CBP (Figure 2A, left) or antibodies against p300 (Figure 2A, right). In both cases NFkB from stimulated cells was associated with CBP or p300. Since both CBP and p300 appeared to be functioning in an equivalent manner, to simplify further analysis we carried out the remainder of the study using CBP. To determine whether the association of p65 and CBP in vivo was influenced by PKA, as suggested by the results of the experiment shown in Figure 1, we transfected CBP and p65 into COS cells with or without PKA. Cotransfection of PKA significantly increased the amount of CBP that was coprecipitated with p65 (Figure 2B, lane 2 versus lane 3), thus indicating that phosphorylation by PKA enhances the association of NF-kB with CBP. To test the effect of mutating the serine 276 in p65 on this association, we transfected flu-tagged wild-type (WT) and S276A mutant p65 with CBP and PKA (Figure 2C). We then immunoprecipitated CBP and the presence of flu-p65 in the immunoprecipitates was tested using immunoblots (Figure 2C, left). We also carried out the reciprocal experiment in which the flu-p65 was immunoprecipitated and the presence of CBP in the immunoprecipitates tested by immunoblotting with anti-CBP

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Figure 2. Association of CBP/p300 with NFkB Is Dependent upon Phosphorylation of p65 by PKA (A) The association of p65 with CBP/p300 in vivo was examined by stimulating 70Z/3 cells with LPS. Nuclear extracts from cells stimulated for the indicated periods of time were then immunoprecipitated with antibodies against p65. The immunoprecipitates were fractionated on SDS-PAGE and analyzed by immunoblotting with antibodies against CBP (left) or p300 (right). (B) Association of p65 and CBP is influenced by PKA. The CBP and p65 constructs were transfected into COS cells as indicated. The cells were harvested 24 hr after transfection, and immunoprecipitations were carried out on extracts using antibodies against p65. The immunoprecipitates were analyzed by immunoblotting with antibodies against CBP. (C) The association of p65 and CBP depends upon phosphorylation of p65 at serine 276. Flu-tagged p65 constructs, either WT or mutants, were transfected into COS cells with CBP and PKA. The extracts were subjected to immunoprecipitation with antibodies against CBP (left) or flu epitope (right) 24 hr after transfection. The immunoprecipitates were fractionated on SDS-PAGE and immunoblotted with anti-flu monoclonal antibodies (left) or anti-CBP antibodies (right).

antibody (Figure 2C, right). In both experiments, the association of CBP with p65 containing the mutated PKA phosphorylation site (S276A) was drastically reduced (Figure 2C, lanes 4 and 5 [left] and lanes 1 and 2 [right]), while the association of the flu-tagged WT p65 with CBP was dramatically enhanced upon the inclusion of PKA (Figure 2C, lanes 6 and 7 [left] and lanes 3 and 4 [right]).

Mapping the Regions Responsible for Phosphorylation-Independent and Phosphorylation-Dependent Interaction between p65 and CBP To further understand the regulation of NF-kB transcriptional activity by CBP/p300, we proceeded to map the regions of interaction between the two proteins. Two recent reports have demonstrated that the N-terminal region of CBP (amino acids 1–500) can interact with the C terminus of p65 (amino acids 286–550) (Gerritsen et al., 1997; Perkins et al., 1997). To further narrow down the regions in both proteins that are responsible for such phosphorylation-independent interaction and to examine the effect of phosphorylation of p65 by PKA, we generated truncated forms of CBP by in vitro translation

(Figures 3A and 3B, lanes 1–7) and tested their ability to interact with GST-fusion proteins containing the N-terminal (amino acids 1–313) and C-terminal (amino acids 314–550) regions of NF-kB p65 (Figures 3A and 3B). The results showed that in the absence of phosphorylation by PKA only the GST-p65 C-terminal fusion protein interacted with the N-terminal region of CBP (Figure 3B, lanes 8–14). The minimal region of interaction was limited to the N-terminal 450 amino acids of CBP (Figure 3B, lane 14). In contrast, the GST-p65 N-terminal fusion protein (amino acids 1–313) did not interact with CBP (Figure 3B, lanes 15–21). However, phosphorylation of the GST-p65N fusion protein with PKA (the PKA phosphorylation site in p65 is at amino acid 276, and hence lies on the GST-p65N fusion protein) led to efficient association with CBP (Figure 3B, lanes 22–27). The region of CBP that interacted with the phosphorylated GST-p65N lay minimally between amino acids 451 and 593, since deletion of sequences downstream of amino acid 450 abolished interaction completely (Figure 3B, lane 28). This region of CBP, known as the KIX region, is also responsible for interacting with PKA-phosphorylated CREB. To prove that phosphorylation of p65 at serine 276 was necessary for this interaction, we produced a GST-p65N fusion protein in which serine 276

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Figure 3. Phosphorylation-Independent and Phosphorylation-Dependent Bivalent Interaction between NF-kB p65 and CBP (A) Schematic outlines of CBP and p65 with different sites of truncations or mutations in the various contructs used in this and subsequent experiments are indicated. (B) Truncated forms of CBP, generated by restriction enzyme digestions (A), were translated in vitro using rabbit reticulocyte lysates. The 35Slabeled proteins produced were analyzed on SDS-PAGE (lanes 1–7) and helped determine the amount of protein used as input. 2 ml of the in vitro translated proteins were mixed with GST proteins fused to the C-terminal portion (amino acids 314–550) (lanes 8–14), the N-terminal portion (amino acids 1–313) of WT p65 (lanes 15-21 and 22-28), or S276A mutant p65 (lanes 29–35). The N-terminal fusion protein was either used directly (lanes 15-21) or phosphorylated in vitro with PKA and ATP before use (lanes 22-35). Following a 10 min incubation at room temperature, the GST proteins were precipitated with glutathione–agarose beads, and the bound proteins were analyzed by SDS-PAGE followed by fluorography. (C) In vitro translated fragments of CBP, CBP-N (amino acids 1–450), or CBP-KIX (amino acids 451–679) were mixed with GST-p65N (with and without prior phosphorylation by PKA) and GST-p65C and precipitated with glutathione–agarose beads. The precipitated proteins were analyzed as in (B). (D) Comparison of sequences surrounding serine 276 in NF-kB p65 and serine 133 of CREB.

was altered to an alanine. This mutant p65 protein (GSTp65N mt) did not interact with CBP in the presence of PKA, thus establishing the phosphorylation of serine 276 as a critical requirement for the association of this region of p65 with the KIX region of CBP (Figure 3B, lanes 29–35). To further examine this interaction, we subcloned the N-terminal (amino acids 1–450) and KIX regions (amino acids 451–679) of CBP and translated them in vitro (Figure 3C, lanes 1 and 5). We then examined the ability of GST-p65N (amino acids 1–313) (1/2 PKA) and GST-p65C (amino acids 314–550) to interact with

these portions of CBP. As seen in the earlier experiment, the GST-p65C protein interacts with the N-terminal region of CBP (Figure 3C, lane 4) but not the CBP-KIX region (Figure 3C, lane 8). In contrast, only the phosphorylated GST-p65N protein interacts with the KIX region of CBP (Figure 3C, lane 6 versus lanes 2, 3, and 7). The results of the GST-fusion protein pull-down assays indicate that NF-kB p65 and CBP can form two distinct interactions: a phosphorylation-independent interaction that involves the C-terminal region of p65 (amino acids 314–550) and the N-terminal region of CBP

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(amino acids 1–450), and a phosphorylation-dependent interaction that requires phosphorylation of serine 276 of p65 and the KIX region of CBP (amino acids 451–661). As mentioned earlier, the KIX region of CBP has been characterized as being the domain responsible for interacting with the transcription factor CREB, phosphorylated on serine 133. Since both phosphorylated p65 and CREB were recognized by the same domain of CBP, we compared the sequences of CREB and NF-kB p65. Although these proteins belong to distinct families of transcription factors, the regions that include the site of PKA phosphorylation (serine 133 in CREB and serine 276 in p65) bear significant similarity to each other (44% similarity) (Figure 3D), although the postulated sites for phosphorylation are at different ends of the region of homology. Such homology suggests that recognition of both phosphorylated CREB and p65 by the KIX region of CBP might involve a similar set of protein–protein interactions.

The C-Terminal Region of p65 Interacts with the N-Terminal Region of CBP To further localize the regions responsible for phosphorylation-independent interaction between CBP and p65, we generated smaller subfragments from the N terminus of CBP (amino acids 1–679) and produced them by in vitro translation (Figure 4A, lanes 1–5). The ability of the GST-p65C protein (amino acids 314–550) to interact with these fragments of CBP was assessed in a standard pull-down assay. The results of the pull-down experiment indicated that sequences from amino acids 313– 450 of CBP were able to interact efficiently with GSTp65C (Figure 4A, lane 9). The complementary region in the C terminus of p65 was mapped in finer detail using progressive deletions of p65 from the C terminus (Figure 4B, lanes 1–5). The C-terminal truncations of p65 were translated in vitro and assayed for their ability to interact with GST-CBP (amino acids 313–450). Removal of the last 46 amino acids of p65 had negligible effect on association with CBP (Figure 4B, lane 7), but further removal of amino acids 477–504 almost completely abolished their interaction (Figure 4B, lane 8). Therefore, the minimal sequences responsible for phosphorylation-independent interaction between CBP and p65 are amino acids 313–450 in CBP and amino acids 477–504 in p65. Previous studies that examined the transactivation properties of p65 concluded that the critical elements for transcription were located between amino acids 435 and 550. Within this region, three subdomains were identified as possessing transactivation ability: TA1 (amino acids 521–550), TA19 (amino acids 460–480), and TA2 (amino acids 435–520, hence including TA19) (Schmitz et al., 1994; Schmitz et al., 1995a, 1995b). The transactivation property of these domains was determined by fusing them with GAL4 and measuring the ability of the fusion proteins to stimulate transcription from a GAL4 reporter construct. Since the C-terminal end of p65 (i.e., TA1) is dispensable for CBP binding, we wanted to examine the effect of removing this domain on the transactivating property of p65 (and not as a fusion with GAL4). We therefore generated progressive C-terminal truncations of p65 at amino acids 520, 460, and 435 and tested the ability of these smaller forms of p65 to

Figure 4. Bivalent Interaction between p65 and CBP Is Required for Efficient Transcription by NF-kB (A) Different fragments of CBP were subcloned into pcDNA3 and translated in vitro. The amounts in lanes 1–5 represent approximately 25% of the input used for the pull-down experiment using GST-p65C (lanes 6–10). The proteins precipitated with the GST proteins were analyzed as before. (B) Progressive truncations of p65 from the C terminus were generated using restriction enzyme digestions and produced by translation in vitro. The 35S-labeled p65 proteins were mixed with GSTCBP (amino acids 313–450) fusion protein and precipitated using glutathione-agarose. The amounts in lanes 1–5 represent approximately 25% of the input used for the pull-down experiments (lanes 6–10). (C) C-terminally truncated forms of flu-tagged p65 (which remove the site for phosphorylation-independent interaction with CBP) and serine 276 to alanine mutants of p65 (which removes the site for phosphorylation-dependent interaction with CBP) were tested by cotransfection into Jurkat cells along with a pBIIx-luciferase reporter construct.

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Figure 5. Squelching of NF-kB–Dependent Transcription by p65-Interacting Domains of CBP (A) The indicated fragments of CBP and the pBIIX-luc reporter plasmid were transfected into Jurkat cells. Approximately 24 hr after transfection, the cells were stimulated with P/P for 4 hr, harvested, and assayed for luciferase activity. (B) The different flu-tagged fragments of CBP (A) were transfected along with p65 and PKAC into COS cells. As a control, one transfection was carried out with flu-IkB-b and p65. Extracts were prepared and immunoprecipitated with the anti-flu antibody 24 hr after transfection, followed by immunoblotting with the p65 antibody.

transactivate a NF-kB reporter construct. In multiple experiments we consistently observed little effect on p65-mediated transcription when the last 30 amino acids were deleted (i.e., up to amino acid 520). In contrast, further C-terminal deletion up to amino acid 460 dramatically lowered the amount of p65-dependent transcription (Figure 4C). Since deletion of these last 90 amino acids would also remove the domain of p65 responsible for mediating phosphorylation-independent interaction with CBP, our results suggest that interaction with CBP is important, if not obligatory, for p65 to manifest its transcriptional activity. Hence, the C-terminal region of p65 most likely provides an interaction surface for the cellular coactivator CBP instead of contacting the basal transcriptional complex directly (Kerr et al., 1993; Schmitz et al., 1995b). Mutation of serine 276 of p65 to alanine (p65mt ), which abolishes the PKA phosphorylation–dependent interaction between p65 and

CBP, also dramatically lowers the transactivating potential of p65 (Figure 4C), indicating the importance of both phosphorylation-independent and phosphorylation-dependent interactions with CBP for optimal NF-kB transcriptional activity. Squelching of NF-kB–Dependent Transcription by Fragments of CBP To help provide additional evidence that both phosphorylation-independent and phosphorylation-dependent interactions between CBP and p65 are critical for efficient transcription by NF-kB, we carried out a squelching experiment using fragments of CBP that are responsible for phosphorylation-independent (amino acids 1–450) or phosphorylation-dependent (amino acids 451– 679) interactions with p65 (Figure 4D). As a control we used a fragment of CBP (amino acids 1100–1620) that does not associate with p65. The fragments were

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Figure 6. Phosphorylation of NF-kB p65 by PKA Relieves Intramolecular Masking of the p65 N Terminus by the C Terminus and Allows Accessibility to CBP (A) DNA binding by full-length and C-terminally truncated forms of p65 produced by in vitro translation was tested in an electrophoretic mobility shift assay (EMSA). (B) Summary of results using yeast two-hybrid assay system. The p65 C-terminal region fused to the GAL4 DNA binding domain could not be tested as it gave a strong positive signal by itself. (C) The abilty of full-length WT and S276A mutant p65 produced in bacteria to bind DNA was tested with and without prior phosphorylation by PKA. (D) In vitro translated p65C (amino acids 314–550) was mixed together with GST-p65N (amino acids 1–313) fusion protein and precipitated with glutathione–agarose beads. The GST-p65N protein was used by itself or after phosphylation in vitro by PKA. (E) Full-length p65 was produced in bacteria and tested for association with in vitro translated, full-length CBP. After allowing the two proteins to mix with each other, the p65 protein was immunoprecipitated and the immunoprecipitates were analyzed by SDS-PAGE.

cloned with an flu epitope tag at the N terminus to facilitate subsequent analysis. Upon transfection into Jurkat cells, all of the CBP fragments inhibited phorbol myristate acetate/phytohemagglutinin (P/P)–induced transcription from a NF-kB reporter construct with the exception of the fragment that does not interact with p65 (Figure 5A). To verify that the association of these CBP fragments correlated with their ability to inhibit NF-kBdependent transcription, we transfected COS cells (to

ensure that sufficient amounts of the transfected proteins were produced) with WT and S276A mutant p65 along with PKA and the different flu-tagged fragments of CBP. Immunoblot analysis indicated that the different constructs were expressed in nearly equal amounts (data not shown). Immunoprecipitations were carried out from transfected cells using antibodies against the flu epitope, and the immunoprecipitates were fractionated on SDS-PAGE and immunoblotted with the p65

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antibody. All of the CBP fragments that inhibit NF-kB– dependent transcription were found to associate with p65 (Figure 5B, lanes 1–4), as did a flu-tagged IkB-b protein that served as a control (Figure 5B, lane 6). In contrast, the flu-tagged CBP-fragment from amino acids 1100–1620, which did not inhibit NF-kB–dependent transcription, also failed to interact with p65 (Figure 5B, lane 5). Finally, to establish that the association of the CBP fragment amino acids 450–679 with p65 required serine 276, we transfected the flu-tagged CBP-fragments and p65 S276A mutant (Figure 5B, lanes 7–10). Immunoprecipitation of the CBP fragments followed by immunoblotting for p65 indicated that the CBP-KIX region (amino acids 451–679) failed to interact with the S276A mutant p65. These experiments demonstrated the importance of a bivalent interaction between CBP and p65 for efficient transcription by NF-kB.

Phosphorylation of p65 at Ser-276 Inhibits Interaction between the N- and C-Terminal Domains of p65 The association between p65 and CBP through two domains raised the question of whether the two interactions were dependent on each other. The demonstration that both interactions were required for stimulation of NF-kB transcription by CBP suggested that phosphorylation of p65 by PKA influenced the ability of CBP to associate with the C-terminal region of p65, which can occur in the absence of any phosphorylation. The first hint that accessibility of the two regions in p65 (serine 276 and amino acids 460–504) might be linked came from studies where the ability of full-length p65 to bind DNA was examined (Figure 6A) (Nolan et al., 1991). Whereas the full-length p65 homodimer did not bind efficiently to DNA (Figure 6A, lane 3), a shorter version lacking sequences C-terminal to the Rel-homology domain bound to kB sites with high affinity (Figure 6A, lane 6) (Nolan et al., 1991). The inhibitory effect on DNA binding of the C-terminal portion of p65 was not seen in a p50:p65 heterodimer (Figure 6A, lane 1), and one possible explanation for these results was that the C-terminal tail of p65 folded back upon the N-terminal region of the protein, thus interfering with DNA binding. The effect is more pronounced in the p65 homodimer (Figure 6A, lane 3) because of the presence of two p65 C-terminal tails, whereas in the p50:p65 heterodimer the lack of a C-terminal tail in p50 results in less interference on DNA binding (Figure 6A, lane 1). To determine whether the C-terminal portion of p65 (amino acids 314–550) exhibited any affinity for sequences from the N-terminal region of p65 (amino acids 1–313), we set up a yeast two-hybrid interaction assay using p65N (amino acids 1–313) fused to the GAL4 DNAbinding domain as “bait” and p65C (amino acids 314– 550) fused to the GAL4 activation domain as “target.” The C-terminal region of p65 interacted with the N-terminal region of the protein in yeast cells, thus demonstrating an intramolecular association between the N and C termini of p65 (Figure 6B). To narrow down the regions involved in the intramolecular interaction, we reduced the length of the interacting proteins using amino acids 1–194 of p65 as bait and amino acids 436–550 as the

target. There was significant association between the amino acids 436–550 portion and the amino acids 1–194 portion, suggesting that the p65 protein could fold as a result of the affinity between the two ends. Such an association between the two halves of p65 could therefore be responsible for interference with DNA binding by full-length p65. To determine whether phosphorylation of p65 by PKA might relieve the intramolecular masking of the p65 N-terminal region by the C terminus, we examined the effect of phosphorylation on DNA binding by p65. We therefore used a full-length p65 protein produced in bacteria (with a ten-histidine tag at the N terminus from pET 19 vector) and tested its ability to bind to a kB site containing DNA probe in an electrophoretic mobility shift assay (EMSA) (Figure 6C). As seen with the in vitro translated p65 protein (Figure 6A, lane 3), the full-length p65 protein binds inefficiently to DNA (Figure 6C, lane 1). Phosphorylation by PKA, however, significantly enhances DNA binding by this protein (Figure 6C, lane 2), and inclusion of the specific PKA inhibitor PKI in the in vitro phosphorylation reaction blocks this stimulation of DNA binding (Figure 6C, lane 3). To further prove that phosphorylation at serine 276 was responsible for the phosphorylation-mediated enhancement of DNA binding, we produced a S276A mutant p65 protein and tested its DNA binding properties in a similar assay. As expected, DNA binding by the p65 mutant was not significantly affected by PKA (Figure 6C, lanes 4 and 5). To test whether the interaction between the two halves of p65, as revealed in the experiment presented above, was sufficient to allow for a stable physical association, we used a GST-p65N (amino acids 1–313) fusion protein to pull down an in vitro translated p65C portion (amino acids 314–550) (Figure 6D, lane 3). In the absence of PKA-mediated phosphorylation, the GST-p65N protein associated, albeit weakly, with the p65C protein (Figure 6D, lane 4); however, phosphorylation by PKA abolished this interaction (Figure 6D, lane 5). If, instead of WT p65N, the GST p65N S276A mutant was used, addition of PKA had no effect on its association with the p65C protein (Figure 6D, lanes 6 and 7). To further test the hypothesis that accessibility to both CBP-binding domains in p65 was blocked in the full-length unphosphorylated p65 protein, we used full-length, bacterially expressed p65 WT and S276A mutant proteins and examined their ability to associate with full-length in vitro translated CBP. Full-length p65 interacted only very weakly with CBP (Figure 6E, lane 1); prior phosphorylation by PKA significantly increased the efficiency of interaction of WT p65 (lane 2) with CBP, but not that of the S276A mutant (lanes 3 and 4) with CBP. Discussion The activity of transcription factors is regulated at multiple levels: at the level of synthesis, subcellular localization, or posttranslational modification. In the case of NF-kB, its sequestration in the cytoplasm through association with IkB proteins led to most attention being focused on mechanisms that regulate the degradation of IkB and the subsequent nuclear translocation of NFkB. This aspect of NF-kB regulation has also garnered

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Figure 7. Model Depicting How Phosphorylation of p65 by PKA Stimulates the Transcriptional Activity of NF-kB The inactive, cytoplasmic complex of NF-kB:IkB:PKAc responds to external signals such as interleukin-1, tumor necrosis factor a, or LPS by phosphorylation and degradation of the IkB protein. The removal of IkB activates PKAc, which phosphorylates the NF-kB p65 subunit on serine 276. The phosphorylation of p65 weakens the interaction of the p65 N-terminal region with the C-terminal region, unmasking the phosphorylation-independent, CBP-interaction domain in p65. The phosphorylated p65 then associates with CBP through a bivalent interaction, CBP-KIX (amino acids 450–679) with phospho-serine 276, and CBP amino acids 313–450 with p65C (amino acids 477–504).

interest because the pathways leading to the specific phosphorylation, ubiquitination, and degradation of IkB proteins involve novel mechanisms that are highly regulated. However, most other inducible transcription factors, including AP-1, STAT, and CREB are regulated through direct phosphorylation of the transcription factor itself. We have recently shown that activation of NFkB is also associated with phosphorylation of the p65 subunit by PKA. The PKA responsible for p65 phosphorylation is activated by a mechanism that does not involve changes in cAMP levels (Zhong et al., 1997). Cytoplasmic NF-kB:IkB complexes contain the catalytic subunit of PKA associated with both p65 and IkB, in a manner that blocks its catalytic activity. Upon degradation of IkB, the bound PKA is activated and phosphorylates the p65 subunit on serine 276 (Figure 7) (Zhong et al., 1997). We show that the effect of this phosphorylation is to allow association with the cellular coactivator CBP/p300. The association of CBP/p300 with NF-kB is bivalent and involves a phosphorylation-independent

and phosphorylation-dependent interaction. However, the phosphorylation of p65 by PKA regulates the overall association, because in the absence of phosphorylation both sites of interaction in p65 for CBP are masked. Phosphorylation at serine 276 of p65 by PKA induces a conformational change that unmasks both of the domains for interaction with CBP. Thus, phosphorylation of serine 276 in p65 serves two purposes: it weakens the intramolecular interaction between the N- and C-terminal portions of p65, allowing CBP to access the phosphorylation-independent site of interaction in p65, and it creates a site for phosphorylation-dependent interaction with the KIX region of CBP (Figure 7). The net result is the establishment of a stable interaction between phosphorylated p65 and CBP, which is a crucial prerequisite for efficient transcription by NF-kB. PKA regulates the activity of another widely studied inducible transcription factor, CREB. Activation of PKA leads to the phosphorylation of CREB at serine 133, and the phosphorylated protein then binds to the KIX region of CBP. Binding of CBP to CREB is essential for transcriptional activity, and therefore, phosphorylation by PKA is the key regulatory step in the activation of CREB. CREB is a member of the larger activating transcription factor/jun family of proteins, and there is little overlap in either sequence or regulation between these proteins and those belonging to the Rel/NF-kB family of proteins. It was therefore remarkable that phosphorylation by PKA stimulates the activity of both transcription factors through a similar mechanism, the recruitment of CBP. However, the use of a bivalent interaction between p65 and CBP apparently indicates a more stringent control in the recruitment of CBP to nuclear NF-kB complexes. The homology in sequence between the region of p65 and CREB that is responsible for associating with the KIX region of CBP indicates the evolutionary conservation of this interaction. The position of the site of phosphorylation (serine 133 in CREB and serine 276 in p65) at opposite ends in the region of homology suggests that recognition of the phosphorylated proteins by CBPKIX involves that domain of the protein rather than just the phosphorylated amino acid. It also highlights the uniqueness of RelB, because among the mammalian Rel proteins RelB is the only one that lacks a PKA phosphorylation site in the Rel domain and is also quite divergent in sequence in that region of the Rel domain. Since our results suggest that recruitment of CBP to p65 (and possibly c-Rel)-containing NF-kB complexes is essential for transcriptional activity, it raises the question of how RelB-containing NF-kB complexes activate transcription. In this regard the presence of the N-terminal leucine zipper and transactivation domain in RelB is probably of significance, since they could provide an alternate means for RelB to interact with the basal transcriptional machinery. However, the prediction that nuclear RelB-containing transcriptional complexes may function without CBP remains to be proven. The use of the same domain in CBP for interaction with both CREB and p65 suggests an explanation for a well-characterized but unexplained effect of cAMP on NF-kB activity in T cells. It has been reported that elevation of cAMP by agents such as forskolin leads to the inhibition of NF-kB–dependent transcription (Chen and

Molecular Cell 670

Rothenberg, 1994; Neumann et al., 1995). Based on our current results, it is possible to postulate that elevation of cAMP would lead to the generation of phosphorylated CREB and this phosphorylated CREB could sequester CBP, particularly if phospho-CREB has a higher affinity for CBP than phospho-p65, thus inhibiting NF-kBdependent transcription. Therefore, if the amount of CBP is limiting in cells as suggested by the stimulation of transcription observed upon transfecting CBP, such a model would predict a hierarchy in CBP-dependent transcription factors where the transcription factors with the greatest affinity for CBP would be able to suppress the transcription by factors with lower affinity for CBP. A surprising finding that is revealed from our studies is the obligatory role for CBP in NF-kB–dependent transcription. The analysis of the transcriptional activity of NF-kB p65 in transfected cells revealed that the last 45 amino acids of p65 were dispensable for effective transcription; however, any further deletion resulted in a loss of transcriptional activity (Figure 4C). Examination of earlier reports on the transactivation domains in p65 revealed that the majority of the experiments were carried out using fusions between small domains of p65 and GAL4 (Schmitz et al., 1994; Schmitz et al., 1995a, 1995b). Therefore, although these subregions in p65, TA1, TA19, and TA2 can interact with TFIIB and TBP, what appears more important in context of the fulllength p65 is its ability to associate stably with CBP. However, despite our results that implicate a direct association between CBP and p65, it remains possible that in certain instances the interaction between CBP and p65 is linked through TBP and TFIIB. It has been reported that the N-terminal domain of CBP, i.e., the region responsible for phosphorylation-independent binding to p65, can bind to TBP (Swope et al., 1996). Since the C-terminal region of p65 can also bind to CBP, the possibility exists that on certain promoters, TBP helps to recruit both CBP and NF-kB p65. Existence of such complexes in the nucleus, however, remains to be established. Why does the NF-kB system use CBP? The recent finding that CBP/p300, in addition to their ability to provide a bridge to the basal transcriptional machinery, have endogenous histone acetylation properties provides a possible explanation (Bannister and Kouzarides, 1996; Ogryzko et al., 1996). The inhibition of NF-kB– dependent transcription by adenovirus E1a protein (Parker et al., 1997), which blocks the ability of CBP to associate with the histone acetylase P/CAF (Yang et al., 1996), suggests that histone acetylation plays an important role in transcription through NF-kB. However, unlike p53, another transcription factor regulated by CBP, p65 is not directly acetylated by CBP (data not shown) (Gu and Roeder, 1997). Acetylation of p53 has been proposed to weaken an intramolecular interaction in the protein (Gu and Roeder, 1997), similar to the situation in p65 except that phosphorylation instead of acetylation is utilized. Another transcription factor that interacts with CBP, c-myb (Dai et al., 1996; Oelgeschlager et al., 1996), has also been shown to be regulated by an intramolecular interaction between the N and C termini of the protein (Dash et al., 1996), although the mechanism responsible for disruption of this intramolecular

interaction in c-myb remains to be characterized. Therefore, it appears that the intramolecular association observed for p65 might be a commonly used regulatory strategy; however, the exact mechanism used to regulate this interaction is different for individual transcription factors. The results in this paper reveal a mechanism by which the transcriptional activity of NF-kB is regulated by the cellular coactivators CBP/p300. Our studies show that recruitment of CBP is obligatory for NF-kB transcription and hence suggest that regulation of CBP could be used to directly influence the transcriptional activity of NFkB. More intriguingly, we have found that the same domain on CBP is utilized for interacting with both NFkB p65 and CREB; therefore, it appears that the two transcription factors would compete for CBP, which is probably limiting in cells. Further studies are clearly necessary to examine whether such regulatory schemes involving CBP recruitment are employed by other transcription factors. Experimental Procedures Antibodies and Reagents The sources of the different antibodies used were as follows: antip65 was from Biomol (SA-171); anti-CBP (A-22) and anti-p300 (N-15) were from Santa Cruz (sc-369, sc-584). Anti-Flu antibody (12CA5) was produced and purified in this laboratory. The cAMP-dependent protein kinase (catalytic subunit) was from Promega. The PKI (amino acids 6–22) amide peptide was from Biomol. Immunoprecipitations and Immunoblotting Immunoprecipitations and immunoblotting were done as described previously (Thompson et al., 1995). 1 ml of anti-p65, 10 ml of anti-CBP, or 10 ml of anti-Flu antibodies were used for immunoprecipations. Recombinant Protein Expression in Bacteria and GST Pull-Down Assays Full-length p65 recombinant protein was obtained from P. Sigler’s laboratory at Yale University. It was purified according to protocols described previously (Thanos and Maniatis, 1992). For full-length p65 mutant (in which Ser-276 is mutated to Ala), the cDNA encoding the p65 mutant was subcloned into the Xpress vector pRSET (Invitrogen), and the plasmid was used to transform E. coli BL21 (DE3) cells. Cells were grown to OD595 of 0.8, and induced with 1 mM IPTG for 3 hr at 288C. After lysing cells by sonication, the recombinant proteins were purified by a nickel affinity column using manufacturer’s protocols (Novagen). For GST fusion proteins, different portions of p65 were cloned into pGEX vectors (Pharmacia). Recombinant proteins were expressed and purified as reported previously (Thompson et al. 1995). In GST pull-down assays, equal amounts of GST fusion proteins were immobilized on glutathione–agarose beads and incubated at 48C for 1 hr with 2–5 ml of 35S-labeled in vitro translated proteins in TNT buffer (20 mM Tris [pH 8.0], 150 mM NaCl, 1% Triton-X, 0.5 mM PMSF). After extensive washing with TNT buffer (53), the bound proteins were eluted in SDS sample buffer and separated on SDSPAGE followed by fluorography. In Vitro PKA Phosphorylation Assays 0.5 mg of GST-p65 (amino acids 1–313) or recombinant full-length p65 proteins were incubated at 308C for 30 min in 30 ml of assay buffer containing 20 mM Tris (pH 7.5), 100 mM NaCl, 12 mM MgCl2 , and 2.5 mM ATP in the presence of 1 ml (50 units) of PKAc (Promega). Yeast Two-Hybrid Assay The expression vectors pAD-GAL4 and pBD-GAL4 were from Stratagene. Different portions of p65 were cloned into either pAD-GAL4 or pBD-GAL4 vector to generate target or bait plasmids as shown

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in Figure 6B. The bait and target plasmids were transformed and coexpressed into the yeast host HF7c by LiAc method as described in Matchmaker Library protocols (Clontech). Colony lift of b-galactosidase filter assays was performed to detect the interaction between the two expressed proteins. Acknowledgments We would like to thank Y. Korkhin and F. T. F. Tsai for providing the full-length p65 protein, D. Chakravarti and T. Kouzarides for CBP constructs, and D. Schatz, M. Solomon, and D. Sengupta for carefully reviewing the manuscript. R. E. V. was supported by a fellowship from the Deutsche Forschungsgemeinschaft. The work in this paper was supported by funding from the Howard Hughes Medical Institute and National Institutes of Health grant RO1 AI 33443. Received December 1, 1997; revised February 3, 1998. References Avantaggiati, M.L., Ogryzko, V., Gardner, K., Giordano, A., Levine, A.S., and Kelly, K. (1997). Recruitment of p300/CBP in p53-dependent signal pathways. Cell 89, 1175–1184. Baldwin, A.S. (1996). The NF-kB and IkB proteins: new discoveries and insights. Annu. Rev. Immunol. 14, 649–681. Bannister, A.J., and Kouzarides, T. (1996). The CBP co-activator is a histone acetyltransferase. Nature 384, 641–643. Bannister, A.J., Oehler, T., Wilhelm, D., Angel, P., and Kouzarides, T. (1995). Stimulation of c-Jun activity by CBP: c-Jun residues Ser63/ 73 are required for CBP-induced stimulation in vivo and CBP binding in vitro. Oncogene 11, 2509–2514. Chakravarti, D., LaMorte, V.J., Nelson, M.C., Nakajima, T., Schulman, I.G., Juguilon, H., Montminy, M., and Evans, R.M. (1996). Role of CBP/p300 in nuclear receptor signaling. Nature 383, 99–103. Chen, D., and Rothenberg, E.V. (1994). Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles. J. Exp. Med. 179, 931–942. Dai, P., Akimaru, H., Tanaka, Y., Hou, D.X., Yasukawa, T., KaneiIshii, C., Takahashi, T., and Ishii, S. (1996). CBP as a transcriptional coactivator of c-Myb. Genes Dev. 10, 528–540. Dallas, P.B., Yaciuk, P., and Moran, E. (1997). Characterization of monoclonal antibodies raised against p300: both p300 and CBP are present in intracellular TBP complexes. J. Virol. 71, 1726–1731. Dash, A.B., Orrico, F.C., and Ness, S.A. (1996). The EVES motif mediates both intermolecular and intramolecular regulation of c-Myb. Genes Dev. 10, 1858–1869. Eckner, R., Yao, T.P., Oldread, E., and Livingston, D.M. (1996). Interaction and functional collaboration of p300/CBP and bHLH proteins in muscle and B-cell differentiation. Genes Dev. 10, 2478–2490. Gerritsen, M.E., Williams, A.J., Neish, A.S., Moore, S., Shi, Y., and Collins, T. (1997). CREB-binding protein/p300 are transcriptional coactivators of p65. Proc. Natl. Acad. Sci. USA 94, 2927–2932. Gu, W., and Roeder, R.G. (1997). Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595–606. Janknecht, R., and Hunter, T. (1996a). A growing coactivator network. Nature 383, 22–23.

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Kwok, R.P., Lundblad, J.R., Chrivia, J.C., Richards, J.P., Bachinger,