In this study, we used anti-acetylated lysine 310 antibodies to demonstrate that RelA is acetylated on lysine 310 both in vitro by p300 and in vivo in response to TNF-α stimulation. We further find that phosphorylation of RelA at either serine 276 or serine 536 facilitates the acetylation of lysine 310, likely by enhancing the binding of RelA to p300. Finally, the importance of these sequential posttranslational modifications of RelA for full transcriptional activation of an endogenous NF-κB target gene is shown.
Although p300 acetylates RelA in vivo (4
), recombinant p300 fails to acetylate RelA in vitro despite employing conditions where the acetylation of p53 and histones by recombinant p300 is readily detected (22
). This result raises the possibility that a p300 cofactor may be required for RelA acetylation. Consistent with this notion, RelA is readily acetylated in vitro when p300 immunoprecipitates from 293T cells are used as the acetyltransferase (Fig. ). It has been reported that p300 requires human immunodeficiency virus Tat protein for the in vitro acetylation of another subunit of NF-κB, p50 (11
). Although certainly not a viral factor, p300 appears to require an additional cellular factor for the effective in vitro acetylation of RelA. The nature of this putative cofactor is currently under study. p300/CBP is not the only HAT that mediates acetylation of RelA. PCAF displays such activity, and other HATs have been implicated as coactivators of NF-κB (11
). It is possible that different HATs selectively target different lysine residues and that other NF-κB coactivators with HAT activity, such as SRC-1 and SRC-3, play a role, independently or in conjunction with p300/CBP, in the acetylation of different sites in RelA.
Our studies of RelA acetylation have been facilitated by the use of specific anti-acetylated lysine 310 antibodies. Using this antibody, we now demonstrate that lysine 310 in the endogenous RelA protein is acetylated in vivo in a stimulus-coupled manner. Although the exact kinetics and stoichiometry of this acetylation reaction are not precisely known, the acetylation of lysine 310 is temporarily delayed relative to the phosphorylation of RelA. Specifically, acetylation of lysine 310 occurs 10 min after TNF-α stimulation in 293T cells (Fig. ), while phosphorylation occurs as early as 5 min after stimulation (23
; data not shown). In addition, RelA is phosphorylated in both the cytoplasm and the nucleus (23
), whereas acetylation appears restricted to the nucleus, consistent with the nuclear localization of p300/CBP (14
). In vitro assays of RelA acetylation further support a contingent relationship between acetylation and phosphorylation. Although recombinant RelA can be forcibly acetylated by p300 in vitro in the absence of phosphorylation (Fig. ), prior phosphorylation of RelA markedly enhances its acetylation at lysine 310 in vitro. TNF-α-induced acetylation of RelA is also severely compromised in RelA-deficient cells reconstituted with RelA S276A or RelA S536A (Fig. ). Of note, TNF-α-induced phosphorylation of RelA at serines 276 and 536 remains unchanged in RelA-deficient cells reconstituted with RelA-K310R compared with cells reconstituted with wild-type RelA (see Fig. S1 in the supplemental material). Together, these various lines of evidence strongly support a model where the acetylation of RelA at lysine 310 is enhanced by prior phosphorylation of RelA at serine 276 or serine 536. It should be noted that RelA is also acetylated at other sites (5
). Unfortunately, site-specific anti-acetylated lysine antibodies are not yet available for their study. It will be interesting to determine whether these other acetylation sites similarly undergo signal-dependent acetylation that is regulated by prior phosphorylation.
In ChIP assays, we demonstrated that RelA acetylated on lysine 310 is effectively recruited to the endogenous IL-8 promoter in vivo in a stimulus-coupled manner (Fig. ). Of note, acetylation of lysine 221 improves DNA binding of RelA, while acetylation of lysine 310 does not. However, due to the lack of the appropriate site-specific antibody, we were unable to evaluate the status of lysine 221 acetylation in these ChIP assays. Nevertheless, these findings further strengthen the notion that acetylation of lysine 310 in RelA plays important roles in modeling the overall transcriptional response elicited by NF-κB. However, how acetylation of lysine 310 regulates the overall transcriptional response of NF-κB is not yet clear. It is possible that acetylation of lysine 310 might recruit an unidentified factor which is required for the full transactivation of NF-κB.
In ChIP assays, we also detected the recruitment of phospho-276 RelA to the κB enhancer of the IL-8 promoter after TNF-α stimulation (Fig. ). We suspect that some of the bound RelA proteins contain both phosphorylated and acetylated forms of RelA, although sequential ChIP assays will be required to confirm this supposition. We also suspect that phospho-536 RelA is effectively recruited to endogenous κB enhancers after TNF-α stimulation, but this has been technically challenging to demonstrate because, in our hands, the currently available anti-phospho-536 RelA antibody generates a very high nonspecific background signal in the ChIP assays.
The observed link between RelA phosphorylation and acetylation is not unique. Specifically, the phosphorylation of both histones and p53 enhances their acetylation. This link also appears due to improved phosphorylation-dependent recruitment of coactivators such as p300 (7
) as is the case for phospho-RelA (Fig. ). Zhong et al. have similarly described that phosphorylation of RelA at serine 276 enhances its assembly with p300 (35
). However, our study now extends this initial finding showing that one important consequence of this improved interaction is the acetylation of a key lysine in RelA that potentiates its overall transcriptional activity.
Phosphorylation of serine 276 also enhances the recruitment of coactivator p300/CBP to and displaces transcriptionally repressive p50-HDAC complexes from the promoter region of several NF-κB target genes, thereby facilitating the acetylation of histones surrounding the gene (29
). As such, the final overall transcriptional output mediated by phosphorylation of serine 276 likely reflects the combined effects produced by modifying RelA, including its increased acetylation and changes in the chromatin structure surrounding the promoter region of NF-κB target genes. Recent studies also identified phosphorylation of serine 276 as essential for protecting the cells from TNF-α-induced cell death (23
). In this regard, deacetylation of lysine 310 by SIRT1 or HDAC3 inhibits the transactivation potential of RelA and similarly augments cellular apoptosis in response to TNF-α (6
). Thus, many of the biological events ascribed to phosphorylation of serine 276 may in fact be linked to acetylation of lysine 310.
Of note, while the introduction of a phosphomimetic S536E mutation in RelA led to enhanced acetylation of lysine 310, the corresponding S276E mutation did not (Fig. ). This finding suggests that mutation of serine 276 may somehow change the overall conformation of the RelA protein “defeating” the potential enhancing effects of the phosphomimetic substitution. Nevertheless, it is clear that phosphorylation of serine 276 enhances the acetylation of lysine 310 (Fig. ) and that the level of acetylation achieved by these mutants closely correlates with their transcriptional activity (Fig. and ).
Consistent with the notion that IKK appears to be the major kinase mediating phosphorylation of serine 536 in response to different stimuli (24
), we find that expression of a kinase-deficient form of both IKK1 and IKK2 inhibits the acetylation of RelA at lysine 310 (Fig. ). Furthermore, phosphorylation of RelA at serine 536 by IKK2 enhances the acetylation of lysine 310 (Fig. ). These results highlight the importance of phosphorylation of serine 536 in the regulation of RelA acetylation. Phosphorylation of serine 536 can be mediated by many kinases in response to many stimuli. For example, IKK1, but not IKK2, phosphorylates serine 536 in response to ligation of the lymphotoxin β receptor (16
) or after stimulation with the human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein (24
). IKK2 plays an essential role in the phosphorylation of RelA on serine 536 induced by lipopolysaccharide or TNF-α (31
). Serine 536 is also phosphorylated by ribosomal subunit kinase 1 after p53 activation and plays a key role in the NF-κB response elicited by tumor suppressor. We suspect that these various kinases in fact play an important role in regulating the overall acetylation status of lysine 310.
While phosphorylation of serine 536 is clearly linked to the transcriptional activation of NF-κB, the exact mechanism underlying this response was not clear. We now demonstrate that phosphorylation of serine 536 stimulates the acetylation of lysine 310 again by enhancing the recruitment of p300 to RelA. Both the N-terminal and C-terminal regions of RelA have been shown to interact with p300 (12
). Phosphorylation at serine 536 may change the conformation of the C-terminal region of RelA promoting its interaction with p300.
In addition to serines 276 and 536, RelA is also phosphorylated at serine 311 by protein kinase C ζ (10
) and at serine 529 by casein kinase II (30
). Whether phosphorylation of RelA at these serines similarly regulates the acetylation of RelA remains unknown. Phosphorylation of serine 311 has been shown to lead to the more effective recruitment of p300/CBP to RelA and to the promoter region of NF-κB target genes, leading to the hyperacetylation of histones and the activation of transcription (10
). It seems likely that phosphorylation of this serine may also influence the acetylation of RelA. Consistent with this hypothesis, we have observed diminished levels of RelA acetylation when serine 311 is substituted with alanine (data not shown).
In summary, our findings indicate that both phosphorylation and acetylation of RelA are required for NF-κB to achieve its full transcriptional activity and that acetylation is largely contingent on prior phosphorylation (Fig. ). Various posttranslational modifications of the histone tails, including phosphorylation and acetylation, contribute to the construction of a specialized “histone code” within localized regions of the chromatin that appears to regulate the association or disassociation of different cofactors. These modifications, alone or in combination, create specific marks defining the actual or potential transcriptional state of the target genes (2
). The different posttranslational modifications of the RelA subunit of NF-κB at specific sites may also create specific marks for the recruitment or divestment of select factors and thus correspond to a “transcription factor code” that dictates specific biological responses of NF-κB in response to different stimuli.
FIG. 6. Schematic model for the role of phosphorylation of serines 276 and 536 in the regulation of RelA acetylation. PKAc- or MSK-1-mediated phosphorylation of serine 276 or IKK-mediated phosphorylation of serine 536 (A) leads to more effective recruitment of (more ...)