Figure 5. CK1d associates with ERa and AIB1 in vivo and regulates their interactions. (A) Lysates from untreated or E2-stimulated COS-1 cells were immunoprecipitated with CK1d or non-immune IgG fol- lowed by immunoblotting with anti-ERa, anti-pS118/ERa and anti- AIB1. (B) MCF7 cells (i) untransfected or (ii) transfected with a (EYFP)-CK1d plasmid were stimulated with 100 nM E2 for 30 min before precipitation with anti-AIB1 antibody. The interaction between AIB1 and ERa was visualised by western blotting using an anti-ERa antibody. Equal amounts of protein were added in both lanes. Since phosphorylation of ERa S118 is correlated with ERa activity, we next examined phospho-S118 levels in MCF7 cells following CK1d silencing. Our results demon- strate increased phospho-S118 levels that paradoxically followed a CK1d siRNA-induced dependent increase in total ERa. These data suggest that S118 is not phosphory- lated by CK1d implicating other unidentiﬁed ERa site(s) as targets for CK1d, which eventually could modulate the activity of ERa. However, CK1d appears to be involved in the regulation of ERa protein levels, as demonstrated in two diﬀerent breast cancer cell lines (MCF7 and T47D) after treatment with CK1d siRNA resulting in ERa stabi- lization. It is well known that ERa protein turnover occurs through the proteasome in a cyclic manner, dependent on ligand binding, which ensures a stable and balanced cellu- lar level of the receptor (6,37). Moreover, it has been reported that the diﬀerential transcriptional activity of ERa produced upon binding of diﬀerent synthetic ligands (SERMs), correlates with the degradation rate of ERa (38), with higher activities corresponding to faster degradation. Various kinases and multiple agents that activate or inhibit phosphorylation have been implicated in modifying the proteasome-mediated stability of ERa and consequently its activity (8,39–41). Taken together, our data suggest that CK1 represents a new protein kinase involved in the stabilization of ERa. Additionally, several observations have linked the recruitment of ERa co-factors with ligand-dependent deg- radation of ERa. Particularly in the case of AIB1, it has been shown that siRNA-mediated silencing of AIB1 results in ERa stabilization, but a loss in ERa activity. This stabilization correlates with a reduction in Nucleic Acids Research, 2009, Vol. 37, No. 9 3119 recruitment of components of the ubiquitin proteasome machinery (15). Therefore, we have also examined the eﬀects of CK1d silencing on AIB1 protein levels. CK1d siRNA treatment in the presence of E2 produced a 60% decrease in AIB1 protein levels, an eﬀect that was con- ﬁrmed by IF, where the overall ﬂuorescent signal of AIB1 was markedly weaker when cells were treated with CK1d siRNA. The decrease in AIB1 levels was rescued by proteasome inhibition, implicating CK1d in the regulation of AIB1 proteasome-mediated degradation. Our in vitro kinase assays demonstrated that AIB1, in addition to ERa, can be a substrate for CK1d phosphor- ylation. Use of an AIB1 GST-fusion protein revealed that the prospective phosphorylation site(s) of AIB1 targeted by CK1d are located within amino acids 582–800, which encompass the RID domain of AIB1, required for inter- actions with NRs. Time-course experiments revealed that there is one major phosphorylation site for CK1 within aa 582–800 of AIB1. In vitro kinase assays using various GST–AIB1 fusion proteins as substrates revealed that CK1 mainly phosphorylates S601 in AIB1, a hitherto uni- dentiﬁed phosphosite. The presence of a serine residue at position n 3 of S601 (K596ESKES601) resembles the opti- mal consensus recognition motif for CK1 (42), thereby favouring S601 as the main phosphorylation site targeted by CK1. Examination of the role of S601 revealed a phys- iological importance of this site in the activity of AIB1 in breast cancer cells, after demonstrating that mutation of this aa negatively inﬂuenced the expression levels of the E2-dependent pS2 genes. However, additional experi- ments are required in order to elucidate the exact molec- ular mechanism of the involvement of S601 in the regulation of AIB1’s co-activation function. Our data herein, reveal an association of CK1d with ERa and AIB1, which can result in a complex formation that enables CK1d to interact, phosphorylate and exert its eﬀects by regulating the interaction between ERa and AIB1 and thereby modulate the functions of these two proteins. AIB1 is a target for multiple signaling pathways and its phosphorylation state is a determinant of its functionality including degradation and interactions with other proteins (17). Phosphorylation of AIB1 by diﬀerent kinases has been associated with increased AIB1 degradation (16,17); however, a recent report (10) identiﬁed aPKC as the ﬁrst kinase able to stabilize AIB1 in cancer cells and thereby enhance its activity. Based on our results, we pro- pose a model where CK1d protects AIB1 from proteolysis, as part of a balanced equilibrium in which kinases are required to maintain a consistent protein level of AIB1 (Figure 8). However, aberrations in this balance towards CK1d, as occurs in breast cancer, would be expected to increase AIB1 levels, and therefore also the expression of monoclonal antibody. Samples were probed for equal loading using a b-actin speciﬁc monoclonal antibody. (B) Quantitative analysis of AIB1 protein levels is given as fold of control. (C) MCF7 cells were untransfected or transfected with 5 nM CT siRNA or with 5 nM CK1d siRNA for 72 h. Where indicated, cells were incubated with MG132 (5mM) for 6 h and and then treated with E2 (100 nM) for 45 min. Cell extracts were immunoblotted with AIB1 antibody. (D) Quantitative analysis of AIB1 protein levels with or without MG132 pre-treatment in the presence of E2. All data are repre- sentative results from two independent experiments. (E) Fluorescence microscopy was performed on either untransfected MCF7 cells or cells transfected with 5 nM CK1d siRNA for 72 h followed by the addition of E2 (100 nM) for 45 min. All cells were grown on coverslips, then ﬁxed and stained as described in ‘Materials and Methods’ section.