RESULTS CK1d silencing modulates ERa transcriptional activity and decreases E2-induced expression of ERa regulated genes To investigate the involvement of CK1d in E2-dependent transcriptional activation of ERa, MELN cells (MCF7 cells, stably transfected with a luciferase reporter gene under the control of an estrogen response element using the b-globin promoter) were transfected with negative control siRNA (CT siRNA) or CK1d siRNA (5 nM), trea- ted with 10 nM E2 for 24 h, and luciferase activities mea- sured. Treatment with E2 alone resulted in a 20-fold induction of luciferase activity. There were no eﬀects of CT siRNA on the activity of ERa in this assay. However, in the presence of CK1d siRNA the E2-dependent lucifer- ase activity was decreased 35%, implying an association of CK1d in E2-induced ERa activation (Figure 1A). Quantitative real-time PCR (qRT-PCR) conﬁrmed 80% reduced CK1d mRNA levels after siRNA treat- ment (Figure 1B). We next examined the eﬀects of CK1d silencing on ER-regulated gene expression by performing qRT-PCR for two well-known estrogen-induced genes (pS2 and PR). Treatment of MCF7 cells with CK1d siRNA for 48 h reduced expression of both pS2 and PR, by 55% and 43%, respectively (Figure 1C and D). These results, in conjunction with the luciferase assay performed in MELN cells, conﬁrmed either a direct or indirect involve- ment of CK1d in the regulation of ERa target gene expres- sion. Use of 1mM IC261 (30), a small molecule inhibitor preferentially inhibiting the CK1d and e isoforms, resulted in a higher reduction of pS2 and PR expression (69% and 66%, respectively) (Figure 1E and F). This diﬀerence can be explained by the fact that the use of siRNA knocked down 80% of CK1d; therefore the lesser eﬀect on gene expression after siRNA treatment, compared to IC261 treatment, is due to the residual CK1d. To establish whether downregulation of ERa-targeted genes expression is partly due to CK1d catalytic suppression, we trans- fected MCF7 cells for 24 h with an expression plasmid encoding EYFP-CK1d. Overexpression of CK1d (con- ﬁrmed by western blotting) resulted in a 2-fold increase in E2-induced expression of pS2 (Figure 1G), demonstrat- ing that the presence of CK1d regulates ERa target gene expression. ERa–S118 is not targeted for phosphorylation by CK1d Since we have previously shown that phosphorylation at S118 of ERa is implicated in regulation of transcriptional activation (31,32), we analyzed the eﬀects of CK1d silenc- ing on phosphorylation of ERa. MCF7 cells, untrans- fected or transfected with either CT siRNA or CK1d siRNA, were stimulated for 30 min with 100 nM E2 and then probed for ERa phospho-S118. Short-term E2 treat- ment induced ERa phosphorylation at S118 in both untransfected and control siRNA-transfected cells (Figure 2A). This phosphorylation was not suppressed by CK1d siRNA, but was instead increased (Figure 2A and B). Furthermore, the total ERa protein content was also increased in the CK1d siRNA-treated cells. Nucleic Acids Research, 2009, Vol. 37, No. 9 3113 Quantiﬁcation and comparison of the phospho- S118:ERa ratio in treated and untreated MCF7 cells implicated upregulation of ERa protein levels as cause of the increase in observed ERa phospho-S118 phosphor- ylation levels (Figure 2B). In order to further validate that ERa–S118 is not phos- phorylated by CK1d in vivo, we used a stably transfected HeLa cell line expressing wild-type ERa (Figure 2C). Compared with E2 treatment, CK1d siRNA failed to reduce the phosphorylation of S118 in Hela-ERa cells (Figure 2D and E). These results indicate that the involve- ment of CK1d in the regulation of ERa transcriptional activation is not mediated via phosphorylation at S118, but could result from phosphorylation either at: (i) other ERa site(s) or (ii) proteins that interact and modulate ERa activity. CK1d silencing stabilizes ERa protein levels in breast cancer cell lines Next, we investigated protein levels of ERa following inhi- bition of the proteasome in the presence of E2 (100 nM). Addition of MG132 (5 mM), a cell permeable proteasome inhibitor, prevented ERa degradation in E2-treated MCF7 (Figure 3A and B) and T47D breast cancer cells (Figure 3C and D) Moreover, in the presence of E2, both CK1d silen- cing and MG132 treatment elevated ERa levels, while co-treatment resulted in further increases. Similar results were obtained in MCF7 cells, using another independent CK1d siRNA, ensuring that the data do not reﬂect oﬀ- target eﬀects (Supplementary Figure 1A and B). Finally, in order to investigate whether the observed CK1d silenc- ing-related increase of ERa protein levels might be induced by an increase of ERa gene transcription, we performed qRT-PCR and examined the ERa mRNA expression levels upon CK1d-siRNA treatment. Our results did not reveal any signiﬁcant alteration of ERa mRNA following CK1d RNA interference (data not shown). Taken together, these data suggest that CK1d is implicated in the stabiliza- tion of ERa in E2-stimulated cells, potentially through direct or indirect phosphorylation events. CK1d silencing decreases AIB1 protein levels in the presence of E2 We have demonstrated that inhibition of CK1d leads to a decrease in ERa transcriptional activity and a reduction of E2-responsive gene expression, whilst stabilizing ERa protein levels. Related ‘contradictory’ or apparently ‘paradoxical’ observations have been reported for AIB1 co-activator suppression (15), where AIB1 is necessary for E2-induced ERa degradation, optimal binding of ERa to target gene promoters and full ERa transcriptional activity. Consequently, we investigated the eﬀects of CK1d silencing on AIB1 protein levels. MCF7 cells were trans- fected either with CT siRNA or CK1d siRNA in the presence or absence of E2 (100 nM), followed by immu- noblotting using a mouse monoclonal AIB1 antibody. Whereas in untreated, E2-treated and control siRNA- treated cells the AIB1 protein levels remained unaﬀected, treatment with CK1d siRNA followed by E2 stimulation resulted in a 60% decrease of AIB1 protein levels