used: (i) full-length recombinant human ERa, (ii) GST- recombinant human ERa fragment encompassing the AF1 transactivation domain and the DNA-binding domain (aa 1–280) and (iii) GST-recombinant human ERa fragment encompassing the LBD (aa 283–595) (Figure 6A and B). As AIB1-substrates we used: (i) pur- iﬁed full-length AIB1 and (ii) GST-recombinant human AIB1 fragment encompassing the RID region, responsible for ligand-dependent interaction with NRs (aa 582–800) (Figure 6C and D). We demonstrated that CK1d can phosphorylate ERa in vitro, and its prospective phosphorylation target(s) are located within the AF1-DBD area of ERa (Figure 6B). Moreover, CK1d was able to phosphorylate full-length AIB1 within aa 582–800 (Figure 6D). Use of additional GST-fusion proteins containing fragments derived from AIB1 (containing aa 1–321, aa 321–581 and aa 841–1081) were not phosphorylated by CK1d (our data not shown and also shown by others) (26). Bioinformatic sequence examination (http://scansite. mit.edu) revealed several potential CK1d phosphorylation sites in the AF1-DBD domain of ERa and in the region between aa 582 and 800 of AIB1. In order to determine the stoichiometry of AIB1 phos- phorylation by CK1d, we performed in vitro time course kinase assays using the GST-AIB1 fusion protein encom- passing aa 582–800 as substrate and the puriﬁed catalytic subunit of CK1 as enzyme. Our results demonstrated that 1.6 mol of phosphate were incorporated per mol of pro- tein, implying the existence of at least one major aa in AIB1 (within the region of aa 582–800) phosphorylated by CK1 in vitro (Figure 6E). Subsequently, to identify the exact phosphorylation site(s) of AIB1, we generated dif- ferent GST-AIB1 aa 582–800 fusion proteins baring muta- tions from S or T to A at various prospective aas (S601, S664, T714, S715, S794) that could be phosphorylated by CK1d. Our in vitro kinase assays clearly revealed that the AIB1 residues (i) S664, (ii) T714 and (iii) S715 were not targeted for phosphorylation by CK1 (Figure 6F). Mutation of S794 to A resulted in a 10% reduction of phosphorylation compared to the wild-type GST-AIB1 aa 582–800, whilst the degree of phosphorylation of GST-AIB1 aa 582–800 S601A was less then 40% com- pared with that of wild type GST-AIB1 aa 582–800 (Figure 6F). These results strongly suggest that the major site of AIB1 phosphorylated by CK1d is S601. Phosphorylation of S601 by CK1d in vivo affects the co-activation function of AIB1 In order to examine the importance of S601 in relation to CK1d phosphorylation in vivo, an AIB1 expression plas- mid baring a mutation at S601A was generated (pCMV- Flag-AIB1/S601A). Following this, the eﬀects of S601A mutant on the ability of AIB1 to function as a transcrip- tional co-activator was assessed by measuring the estro- gen-dependent induction of endogenous pS2 mRNA levels in MCF7 cells, after increasing or decreasing CK1d pro- tein levels, respectively. In the presence of E2, transient co-transfections of wt AIB1 and CK1d resulted in an increase ( 60%) of pS2 gene expression levels, compared Nucleic Acids Research, 2009, Vol. 37, No. 9 3117 to untransfected cells (Figure 7A), while a small increase ( 10%) of pS2 mRNA levels was observed after over- expression of the AIB1/S601A mutant along with CK1d (Figure 7A). To further establish that the observed changes in the co-activation functions of AIB1 can be attributed to phosphorylation of S601 by CK1d, MCF7 cells were treated with 1mM IC261 and pS2 mRNA levels were quantiﬁed. In accordance with ‘our hypothesis’, in the presence of E2 inhibition of CK1d led to decreased activity of wt AIB1 to basal pS2 mRNA levels (Figure 7B) compared to over-expression of both wt AIB1 and CK1d. As expected, use of the IC261 inhibitor did not alter the activity of AIB1/S601A mutant (Figure 7B). We conclude that these functional data indicate that phospho-S601 res- idue within the RID domain is necessary and suﬃcient for AIB1 co-activation functions. DISCUSSION Protein phosphorylation, an essential post-translational modiﬁcation, regulates protein functions including activ- ity, stability, subcellular localization and interactions with other proteins and substrates (33,34). Kinases are there- fore considered key regulatory proteins and elucidation of their roles in signaling pathways is essential (35). In the present study, we have identiﬁed ERa and AIB1 as novel substrates for CK1d in vitro that are able to interact in a cellular context, proposing an involvement of CK1d in regulating their interactions and functions. In addition, we have identiﬁed S601 within AIB1 as a novel phosphor- ylation site targeted by CK1d in vitro, while AIB1-S601A mutant protein negatively inﬂuenced AIB1’s co-activation function in ERa-dependent transcription. Furthermore, using CK1d siRNA in MCF7 breast cancer cells, we demonstrated that CK1d is required for full ERa tran- scriptional activity and is involved in the regulation of ERa protein levels in the presence of E2. Finally, we have shown that suppression of CK1d results in protea- some-mediated degradation of AIB1, in the presence of E2, implying that CK1d phosphorylation protects AIB1 from proteolysis. We examined the eﬀects of CK1d silencing on the tran- scriptional activity of ERa and observed a 35% decrease in the E2 response of an integrated ERE reporter gene. Moreover, the eﬀects of CK1d silencing on the expression levels of two endogenous estrogen-responsive genes, pS2 and PR, resulted in reductions of 55% and 43% respec- tively when CK1d siRNA was used, while the reductions were even higher (69% and 66%) in the presence of a CK1d inhibitor (IC261). In contrast, overexpression of CK1d resulted in a 2-fold increase in pS2 mRNA levels. In vitro kinase assays using the CK1d as enzyme and dif- ferent ERa fragments as substrates showed that CK1d predominantly phosphorylates the AF1 domain of ERa. Thus far, various kinases have been identiﬁed that can phosphorylate the AF1 domain and thereby regulate ERa activity (3,36). In this report, we identify a novel involvement of CK1 as a direct and/or indirect regulator of ERa transcriptional activity.