that AIB1 also regulates ERa turnover through recruit- ment of components of the ubiquitin-proteasome path- way. Suppression of AIB1 leads to ERa stabilization in the presence of estradiol (E2) and subsequent reduction of ERa transcriptional activity (15). The activity of AIB1 is modulated post-transcriptionally by phosphorylation which results in increased AIB1 degradation (16,17). Recently, atypical protein kinase C (aPKC) has been shown to stabilize AIB1 protein levels in cancer cells (10), supporting the existence of an equilibrium of diﬀer- ent kinases implicated in the promotion and prevention of AIB1 degradation, so called dual kinase regulation. Casein kinase 1 (CK1), a highly conserved Ser/Thr pro- tein kinase family, is ubiquitously expressed in all eukary- otic organisms (18,19) and alterations in the expression and/or activity of CK1 have been observed in breast car- cinomas (19,20). Among CK1 isoforms, CK1d is able to phosphorylate a diversity of substrates, modulating their activity and subcellular localization (19,21). As changes in the activity of CK1d, or mutations of CK1d-speciﬁc phos- phorylation sites within it’s substrates, contribute to dys- regulation of various signaling pathways (19,22–25), we wished to establish the role of CK1d in the regulation of ERa activity. Here, we observe that CK1d can interact with and phos- phorylate both AIB1 and ERa in vitro. Furthermore, overexpression of CK1d protein levels in MCF7 cells enhanced the interactions between ERa and AIB1. CK1d silencing results in decreased ERa transcriptional activity even though paradoxically ERa levels appear increased. Moreover, these results demonstrate that the eﬀects of CK1d silencing on AIB1 can be rescued by pro- teasomal inhibition, suggesting that phosphorylation of AIB1 by CK1d protects it from degradation. Finally we identify S601 in AIB1, a hitherto unidentiﬁed site, as the main phosphorylation aa targeted by CK1d, which is required for the activity of AIB1. These data herein, and recent evidence regarding dual kinase regulation (10), sug- gest a general mechanism by which the characteristics of the AIB1–ERa partnership can be modulated by CK1d-mediated phosphorylation. MATERIALS AND METHODS Chemicals and reagents E2 was obtained from Sigma (Gillingham, UK) and dissolved in ethanol; Charcoal-dextran stripped serum (DSS) was obtained from Gemini (Bolnet, UK). The cell-permeable proteasome inhibitor MG132 and the kinase inhibitor IC261 were obtained from Merck (Nottingham, UK). Rabbit polyclonal CK1d antibody was from Santa Cruz (Heidelberg, Germany); mouse monoclonal ERa, rabbit monoclonal anti-phosphoERa– S118 and anti-b-actin mouse monoclonal antibodies were purchased from Abcam (Cambridge, UK) and Millipore (Southampton, UK) respectively. HRP (horse- radish peroxidase)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibodies were from GE Healthcare (Slough, UK). Nucleic Acids Research, 2009, Vol. 37, No. 9 3111 Plasmids The expression plasmid for yellow ﬂuorescent protein (EYFP)–CK1d was constructed as follows. PCR ampliﬁ- cation was carried out using mouse testis cDNA as a template and the CK1d primers: 50-GGATCCATGGAG CTGAGGGTCGGGACA-30, 30-GGATCCTCAGTAGG TGGTACGTCGTGG-50, which contain a BamH1 restriction site (underlined). The PCR product was then cloned into the multiple cloning site of pcDNA3.1/V5-His- TOPO vector (Invitrogen, Paisley, UK) and then sub- cloned into the BamH1 site of pEYFP vector (BD Biosciences Clontech, Heidelberg, Germany). The expres- sion plasmid for human Flag-tagged AIB1 (pCMV- Flag-AIB1) was generated as described previously (26). The expression plasmids: (i) pSG5-ERa (27) and (ii) pGEX4T–AIB1 (encompassing amino acids 582–800) (26) were kind gifts from Professors Simak Ali and Bert W. O’Malley respectively. Site-directed mutagenesis The expression plasmid pCMV-Flag-AIB1 baring a muta- tion at aa S601A was generated using the QuikChange site-directed mutagenesis kit according to manufacturer’s instructions (Stratagene). The plasmid pCMV-Flag-AIB1 served as a template and the complementary primers used were: 50-GACAAAGAAAGTAAGGAGGCCAGTGTT GAGGGGGCAGAG-30 (sense) and 50-CTCTGCCCCC TCAACACTGGCCTCCTTACTTTCTTTGTC-30 (anti- sense). The plasmids pGEX4T–AIB1 aa 582–800 contain- ing mutations at aas: S601A, S664A, T714A, S715A and S794 of AIB1, were constructed using the plasmid pGEX4T–AIB1 582–800 as a template and the following complimentary primers: (i) S601A: 50-GACAAAGAA AGTAAGGAGGCCAGTGTTGAGGGGGCAGAG-30 (sense) and 50-CTCTGCCCCCTCAACACTGGCCTCC TTACTTTCTTTGTC-30 (antisense), (ii) S664A: 50-GT CTCCTCCTCTACAGCTGG AGGAGTATCCTC-30 (sense) and 50-GAGGATACTCCTCCAGCTGTAGAG GAGGAGAC-30 (antisense), (iii) T714A: 50-GACA CC AGCAGTATAGCTTCTTGTGGGACGG-30 (sense) and 50-CCGTCCCCACAAGAAGCTATACTGCTGGT GTC-30 (antisense), (iv) S715A: 50-CCAGCAGTATAAC TGCTTGTGGGGACGGAAATG-30 (sense) and 50-CAT TTCCGTCCCCACAAGCAGTTATACTGCTGG-30 (antisense), (v) S794A: 50-GACAAGTGAAGAGGGAG CTGGAGACTTGGATAATC-30 (sense) and 50- GATT ATCCAAGTCTCCAGCTCCCTCTTCACTTGTC-30 (antisense). Cell culture and transfections MCF7, T47D, MELN (28) and COS-1 cells were main- tained in DMEM supplemented with 10% DSS, 1% peni- cillin/streptomycin and 2% glutamine. HeLa cell line expressing wild-type ERa has been described elsewhere (29) (a kind gift from Dr Shapiro). All cells were incubated at 378C in a humidiﬁed 5% CO2 atmosphere. MCF7 and MELN cells were transfected with 5 nM siRNA using Hiperfect according to the manufacturer’s instructions (Qiagen, Crawley, UK). Transient transfections of COS-1