3110–3123 Nucleic Acids Research, 2009, Vol. 37, No. 9 doi:10.1093/nar/gkp136 Published online 1 April 2009 CK1d modulates the transcriptional activity of ERa via AIB1 in an estrogen-dependent manner and regulates ERa–AIB1 interactions Georgios Giamas1, Leandro Castellano 1 , Qin Feng 2 , Uwe Knippschild3, Jimmy Jacob 1 , Ross S. Thomas1, R. Charles Coombes1, Carolyn L. Smith2, Long R. Jiao1 and Justin Stebbing1,* 1Department of Medical Oncology, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 ONN, UK, 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA and 3Department of General-, Visceral- and Transplantation Surgery, University of Ulm, D 89075 Ulm, Germany Received January 19, 2009; Revised February 13, 2009; Accepted February 18, 2009 ABSTRACT Oncogenesis in breast cancer often requires the overexpression of the nuclear receptor coactivator AIB1/SRC-3 acting in conjunction with estrogen receptor-a (ERa). Phosphorylation of both ERa and AIB1 has been shown to have profound effects on their functions. In addition, proteasome-mediated degradation plays a major role by regulating their stability and activity. CK1d, a member of the ubiqui- tous casein kinase-1 family, is implicated in the pro- gression of breast cancer. In this study, we show that both ERa and AIB1 are substrates for CK1d in vitro, and identify a novel AIB1 phosphorylation site (S601) targeted by CK1d, significant for the co-activator function of AIB1. CK1d is able to inter- act with ERa and AIB1 in vivo, while overexpression of CK1d in breast cancer cells results in an increased association of ERa with AIB1 as con- firmed by co-immunoprecipitation assays from cell lysates. Using an siRNA-based approach, luciferase reporter assays and qRT-PCR, we observe that silencing of CK1d leads to reduced ERa transcrip- tional activity, despite increased ERa levels, simi- larly to proteasome inhibition. We provide evidence that AIB1 protein levels are reduced by CK1d silenc- ing, in an estradiol-dependent manner; such desta- bilization can be inhibited by pre-treatment with the proteasome inhibitor MG132. We propose that differing activities adopted by ERa and AIB1 as a consequence of their interactions with and phos- phorylation by CK1d, particularly AIB1 stabilization, influence the transcriptional activity of ERa, and therefore have a role in breast cancer development. INTRODUCTION Estrogen receptor alpha (ERa), a member of the nuclear receptor (NR) superfamily of transcription factors, has been one of the most successful therapeutic targets for breast cancer (1). It contains 595 amino acids with a cen- tral DNA-binding domain (DBD) and transcriptional activation occurs through at least two distinct transactiva- tion domains located in the amino-terminal A/B region (AF-1) and the carboxy terminal E region of the receptor (AF-2). The AF-1 domain is hormone-independent, whereas the AF-2 domain is estrogen-dependent; both AF domains are required for maximal ERa transcrip- tional activity (2). In addition to being activated upon bind- ing estrogen, we and others have shown that ERa can be activated by phosphorylation (3). Activation of ERa is also coupled with its degradation by the ubiquitin- proteasome pathway (4–6). Upon ligand binding, ERa becomes ubiquitinated and is targeted by the 26S protea- some for degradation (7). However, various direct or indi- rect mechanisms have been described that can protect ERa from proteasomal degradation and thereby modulate its transcriptional activity including the involvement of the Ampliﬁed in breast cancer-1 (AIB1) protein (8–10). AIB1 (SRC-3/ACTR/pCIP/RAC3), a p160 co-activat- ing oncogene overexpressed in diﬀerent types of cancer, especially breast tumors (11), associates with and regulates ERa transcriptional activity; AIB1 recruits co-factors that possess histone acetyl-transferase activity and thus increases ERa transcriptional activity through chromatin remodeling (12–14). Interestingly, it has been reported *To whom correspondence should be addressed. Tel: +44 208 7468295; Fax: +44 208 8461433; Email: g.giamas@imperial.ac.uk or j.stebbing@imperial.ac.uk 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.