3116 Nucleic Acids Research, 2009, Vol. 37, No. 9 Figure 3. CK1d silencing stabilizes ERa protein levels. (A) MCF7 or, (C) T47D cells were untransfected or transfected with 5 nM CK1d siRNA for 72 h. MG132 (5 mM) was added for 6 h and following cells were treated with E2 (100 nM) for 45 min. Cell extracts were immunoblotted. Immunoblot showing silencing of CK1d. Quantitative analysis of ERa protein levels with or without MG132 pretreatment in the presence of E2 in (B) MCF7 or, (D) T47D cells. All data are representative of results from two independent experiments. Error bars represent SD of two separate experiments in triplicate. The eﬀects of CK1d silencing on AIB1 protein levels were also investigated by IF, where MCF7 cells, untransfected or transfected with CK1d siRNA, were stimulated with E2, and the subcellular localization of AIB1 was visualised using the AIB1 antibody. Treatment with CK1d siRNA resulted in a signiﬁcantly decrease in nuclear AIB1 com- pared to untreated cells, in the presence of E2 (Figure 4E). CK1d associates with human ERa and AIB1 and regulates their interactions In order to clarify whether ERa and AIB1 are physiological substrates for CK1d, we examined whether these proteins can physically associate in vivo. COS-1 cells were transi- ently transfected for 24 h with expression plasmids encod- ing for either: (i) ERa and CK1d or, (ii) AIB1 and CK1d, following treatment with or without 100 nM E2 for 30 min. Subsequently, the potential interactions between: (i) ERa– CK1d and, (ii) AIB1-CK1d were assessed by immunopre- cipitation of CK1d followed by analyses of the immune complexes for the presence of ERa and AIB1 respectively. An association of CK1d with ERa as well as with AIB1 in the presence or absence of E2 was observed, although the interaction was reduced in the absence of E2 (Figure 5A). In addition, CK1d was able to co-precipitate with ERa phosphorylated at S118, suggesting that it is able to interact with this form of ERa (Figure 5A). The ability of CK1d to interact with the ERa–AIB1 com- plex, led us to examine whether CK1d is able to inﬂuence the interactions between ERa and AIB1. To address this issue, we overexpressed CK1d in MCF7 cells for 24 h, and performed co-immunoprecipitation experi- ments following treatment with 100 nM E2 for 30 min. Our results showed that increased CK1d protein levels resulted in an increased ERa–AIB1 association, compared to untransfected cells, in the presence of E2 (Figure 5B). Overall, these data demonstrate that CK1d is involved in the regulation of ERa–AIB1 interactions, critical for the modulation of ERa transcriptional activity. CK1d phosphorylates ERa in the AF1-DBD domain and AIB1 at S601 in vitro Since the phosphorylation status of ERa and AIB1 play essential roles in their functions, we examined the ability of CK1d to phosphorylate these proteins. We performed in vitro kinase assays using the puriﬁed catalytic subunit of CK1 as source of enzyme activity. As ERa-substrates we