Nucleic Acids Research, 2009, Vol. 37, No. 9 3121 Figure 7. Functional role of CK1d-dependent phosphorylation of S601 in ERa-dependent transcription. MCF7 cells (2 105) were plated in 6-well plates in phenol red-free DMEM containing 10% DSS. (A) Cells were co-transfected either with 1mg of: (i) pCMV-Flag-AIB1 and (EYFP)–CK1d plasmids or, (ii) pCMV-Flag-AIB1/S601A and (EYFP)–CK1d plasmids for 24 h. (B) Cells were transfected with 1mg of: (i) pCMV-Flag-AIB1 and, (ii) pCMV-Flag-AIB1/S601A plasmid for 24 h, followed by treatment with 1 mM IC261 for 3 h. Subsequently, all cells were treated or not (vehicle) with E2 (10 nM) for 24 h. Cells were harvested and total RNA was extracted and used to synthesize cDNA by reverse transcription, as described in ‘Materials and Methods’ section. Gene expression of pS2 was measured by qRT-PCR. Error bars represent SD of two separate experiments, each in triplicate (P < 0.05). Figure 8. A model for the regulation of ERa transcriptional activity, involving CK1d and AIB1. In the presence of E2, CK1d interacts with and phosphorylates AIB1 predominantly at S601 and probably ERa at other site(s), inducing the formation of a complex between them that is required for the complete transcriptional activity of ERa. Inhibition of CK1d-mediated phosphorylation, potentially alters the conformation of AIB1 and promotes its proteasome-mediated degradation; subsequently, ERa protein levels are stabilized and ERa activity is decreased. with its known functional domains. bHLS/PAS: basic helix-loop-helix/Per-Arnt-Sim domain; RID: receptor interacting domain; CID: CBP/p300 interacting domain; HAT: histone acetyltransferase domain. The positions of all identiﬁed phosphorylation amino acids are indicated. (D) In vitro kinase assays were performed using recombinant human CK1d as source of enzyme and as substrates: (i) puriﬁed full-length AIB1 and (ii) GST- recombinant human AIB1 (aa 582–800). (E) CK1 phosphorylates one major residue of AIB1. GST–AIB1 aa 582–800 was phosphorylated in vitro by the catalytic subunit of CK1 for the indicated times. Up to 1.6 mol phosphate/mol protein were incorporated into the GST–AIB1 aa 582–800 fusion protein. (F) Phosphorylation of GST–AIB1 fusion proteins by CK1d. In vitro kinase assays were performed using recombinant human CK1d as the source of enzyme and: (i) GST–AIB1 aa 582–800 (wt), (ii) GST–AIB1 aa 582–800 (S664A), (iii) GST–AIB1 aa 582–800 (T714A), (iv) GST–AIB1 aa 582–800 (S715A), (v) GST–AIB1 aa 582–800 (S794A) and (vi) GST–AIB1 aa 582–800 (S601A), as substrates. Proteins were separated by SDS-PAGE and the phosphorylated proteins were detected by autoradiography. Quantiﬁcation of phosphate incorporation of phosphorylated proteins was measured by Cerenkov counting. Error bars represent SD of two experiments, each in triplicate. C: Coomasie. A: Autoradiogram.