CSN5/Jab1, one of the subunits of the CSN complex, has been described as a nuclear receptor coactivator (6
). One essential function of CSN could be in the cellular response to the environment (8
) as a mediator between kinase signaling and protein degradation (13
). This complex plays a key role in sustaining the activity of SCF and other cullin-based ubiquitin ligases. These ligases are activated by neddylation of the cullin subunit. CSN could be involved in its deneddylation, a role attributed at least in part to CSN5/Jab1, which contains an NEDD8 isopeptidase active site.
Here, we show that CSN5/Jab1 coimmunoprecipitates with ERα, indicating that they interact directly or indirectly and that the increase in hormone-induced transcription resulting from increased amounts of CSN5/Jab1 is accompanied by an increase in hormone-induced ERα degradation. These results, taken together with the role of CSN on cullin-based ubiquitin ligases, support the recent observation that disruption of the NEDD8 pathway impairs ligand-ERα degradation (9
). In addition, we show that the amount of coimmunoprecipitated ERα and CSN5/Jab1 is increased in the presence of curcumin, suggesting a stabilization of the interaction by this inhibitor of a kinase activity associated with CSN (4
). Curcumin also blocks ligand-dependent ERα phosphorylation and ligand-dependent ERα degradation, suggesting that, as described for p53, CSN-dependent phosphorylation of ERα could target it for degradation. The effect of kinases involved in cell signaling was recently investigated to address the question of the link between ERα phosphorylation and degradation. An earlier study (19
) described an enhanced degradation of ERα by PKC, regardless of its ligand binding status. In contrast, PKA, mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase impeded the degradation of ERα, whether ligand free or complexed to estradiol. No correlation between the capacity of kinase inhibitors to affect ERα stability and their effect on basal or estradiol-induced transcription was found (19
). However, there is as yet no clear evidence that ERα phosphorylation is involved in regulating its ligand-induced degradation (16
ERα is phosphorylated on multiple amino acids, mainly Ser residues located within its N-terminal activation domain. Ser-118, the site predominantly phosphorylated in vivo, is phosphorylated in response to the activation of the MAPK pathway and to estradiol. The identity of the kinases responsible for Ser-118 phosphorylation in response to estradiol binding is controversial. One possible candidate is the TFIIH cyclin-dependent kinase (7
). Ser-167 is also phosphorylated in response to the activation of the MAPK pathway. In addition, Ser-167 is phosphorylated in vitro by CKII and AKT (2
). To address a possible role of ERα phosphorylation in its degradation and/or cycling on estrogen-responsive promoters, we investigated the effect of kinase inhibitors on ligand-induced ERα degradation. H7, which inhibits a wide spectrum of kinases (PKA, PKG, and PKC), had no effect on ERα stability. We also investigated the effect of DRB, an inhibitor of CDK7, which blocks transcription by RNA Pol II, and again we found no effect on ERα stability. These results, obtained for short treatment times with DRB, agreed with data obtained with pituitary cells (1
). However, for long treatment times with DRB in the presence of trichostatin A, ERα degradation was prevented (25
; unpublished data). This suggests that the effect of DRB on ERα degradation is indirect and it demonstrates that transcription is not required for ligand-dependent ERα degradation.
Recently, it was demonstrated that to activate ERα-dependent transcription, SRC3/AIB1, a coactivator of ERα, must be phosphorylated (39
). In addition, it was shown that in presence of estradiol, SRC3/AIB1 is necessary for ERα binding on the TFF1/pS2 promoter and for ERα degradation by the proteasome (29
). SRC3/AIB1 itself is degraded in a estradiol-dependent fashion. The effect of curcumin on ERα degradation may result from phosphorylation inhibition of ERα and/or of a coactivator such as SRC3/AIB1. In the presence of ICI, SRC3/AIB1 does not interact with ERα whereas curcumin blocks the ICI-induced degradation of ERα. Three kinases, inositol 1,3,4-triphosphate 5/6 kinase (31
), CKII, and protein kinase D (35
), have been described as being associated with CSN. Of these, CKII is a good candidate for ERα phosphorylation, since as mentioned above CKII was found to be capable of phosphorylating ERα on Ser-167 (2
). These three kinases have not been proposed as candidate kinases for SRC3/AIB1 phosphorylation. Taken together with the observation that curcumin inhibits E2-dependent phosphorylation, these data suggest that the target of the curcumin-sensitive kinase would be ERα itself rather than a coactivator.
Another role for CSN is to facilitate the nuclear export of proteins that do not have a nuclear export signal, allowing degradation by the proteasome. Inhibition of nuclear export by LMB on ERα degradation resulted in a block of the estrogen-dependent degradation of ERα, indicating that estradiol-induced ERα-degradation by the proteasome occurs in the cytoplasm. In contrast, ICI-induced ERα degradation was not affected by LMB, indicating that in this case degradation takes place in the nucleus. This conclusion is in agreement with our previous finding that ICI induces the sequestration of ERα in a salt-resistant nuclear compartment in which it is rapidly degraded (12
). Our results are also compatible with data from experiments with fluorescence recovery after photobleaching, which show a decreased mobility of ERα in the nuclei in the presence of ICI (30
). ERα complexed to ICI is degraded by a different pathway than ERα complexed to E2. It is possible that in the presence of ICI, ERα adopts a conformation not recognized by the cell. In this case, the antagonist activity of ICI should result from the rapid degradation of ERα.
An important question is whether ligand-induced ERα degradation and transcription activation are interlinked processes. Our results on the effect of DRB on ligand-induced ERα degradation demonstrate that transcription by itself is not required for ERα degradation. To obtain further insights in this process, we compared, for the same time of treatment, the effect of three types of compounds that inhibit ERα degradation on ERα stability and transcription activation. ALLN, curcumin, and LMB completely prevented ERα degradation, but they had very different effects on transcription. Curcumin totally blocked the expression of a target reporter gene at a dose similar to that inhibiting ERα degradation. ALLN had a minor effect on expression of a reporter gene while LMB increased it slightly. These results differed from previous reports describing a decrease in hormone-induced transcription by treatment of the cells with proteasome inhibitors (17
). The differences in the duration of treatment with the proteasome inhibitor cannot account for this discrepancy. We found that increasing the length of treatment with ALLN increased the expression of the reporter gene (unpublished data). Our results agree with a very recent report demonstrating that proteasome inhibition increases hormone-induced activation (11
). The differences in proteasome inhibitors on hormone-induced transcription probably result from differences in the promoters, as has been suggested elsewhere (11
). All together, these results demonstrate that there is no direct relation between hormone-induced ERα degradation and transcription. Inhibition of ERα nuclear export that results in ERα accumulation in the nucleus is accompanied by increased transcription. Curcumin had a very different effect, blocking both ERα degradation and transcription. ChIP experiments clearly show that the lack of transcription activation is due to a dramatic decrease in ERα binding to its target on an estradiol-regulated promoter. A similar decrease in ERα binding to the TFF1/pS2 promoter was observed when SRC3/AIB1 expression was abolished with a small interfering RNA (29
). One possible mechanism of coactivation by CSN5/Jab1 could be a stabilization of the interaction between ERα and SRC3/AIB1, as was shown for another nuclear receptor and coactivator, the progesterone receptor and SRC1 (6
We propose a model (Fig. ) in which the first event after hormone addition would be ERα and/or coactivator phosphorylation by a curcumin-sensitive kinase associated with the CSN complex (Fig. ). This would allow ERα to interact productively with the TFF1/pS2 promoter (Fig. ). Once the transcription complex is assembled, ERα would be polyubiquitinated (Fig. ) and released from the DNA (D). This complex containing polyubiquitinated ERα would then be exported to the cytoplasm (Fig. ) to be degraded by the proteasome (F). CSN5/Jab1 could also be involved at that step, as has been described for p27Kip1, permitting the export of ERα, which does not have an export signal sequence. Alternatively, ERα could be deubiquitinated and recycled to activate transcription (Fig. ). This hypothesis is supported by the results obtained with LMB, which prevents ERα degradation and increases transcription at the same time. In the presence of ICI, ERα would also be phosphorylated by a kinase (Fig. ), but this does not result in DNA binding. It is targeted to a nuclear subcompartment and is rapidly degraded by a nuclear proteasome (Fig. ). In our experiments, curcumin blocks ERα degradation in the presence of E2 and ICI, impairing its polyubiquitination. According to our model, ERα degradation should not be required to sustain transcription, which as already discussed is indeed slightly enhanced by increased amounts of nuclear ERα. Degradation would be the consequence of one of the dynamic events that lead to transcription initiation rather than of transcription per se.
FIG. 8. Model. (A) In the nucleus, a kinase associated with CSN would phosphorylate ERα complexed to estradiol and/or a coactivator such as SRC3/AIB1, a member of the p160 family. (B) This would result in the binding of the ERα-SRC3/AIB1 complex (more ...)