3122 Nucleic Acids Research, 2009, Vol. 37, No. 9 ERa-respondent target genes. These genes are critical to breast cancer growth, as evidenced by the successes in the use of anti-estrogens to treat breast cancer, so we spec- ulate here that increases in their expression may be a determinant of cancer progression and/or resistance to anti-estrogens. It is interesting to note here that both the receptor and its co-activator partner are subject to phos- phorylation by the same kinase, introducing hierarchical, sequential or simultaneous phosphorylation possibilities in this dual regulatory pathway. However, using puriﬁed substrates in the in vitro kinase assays suggests that CK1d can interact with and phosphorylate both independently. Our results, identifying a novel AIB1 phosphorylation site (S601) that aﬀects the co-activation function of AIB1, in conjunction with a new report which suggests that tyrosine phosphorylation of AIB1 is also required for its activity (43) demonstrate the importance of post- translational modiﬁcations as an additional level of tran- scriptional regulation. Generation of a phospho-speciﬁc antibody against S601 in AIB1 is currently underway; correlations between phosphorylated levels of AIB1 at S601 in breast cancer samples and clinical outcome para- meters such as time to progression, overall survival and clinical endocrine resistance, remain to be tested. It has already been shown that ERa and AIB1 shuttle between the cytoplasm and the nucleus (44,45). Phosphor- ylation events induced by diﬀerent kinases occur in both compartments inﬂuencing their functions, including stabi- lity, mobility and activity (3,46–49) as per our proposed model (Figure 8). In this report we identify CK1d, a pre- dominantly cytoplasmic protein (50,51), as a novel kinase implicated in the modulation of physiological aspects of both ERa and AIB1. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We are indebted to Professors Bert W. O’Malley and Simak Ali for their advice and critical reviews of this work. We would also like to thank Dr Elena Kulinskaya of the Statistical Advisory Service (Imperial College, London) for helping with the statistical analyses of our data. FUNDING Citrina Foundation, the family of Janet Booker and the Hammersmith Hospitals’ Trustees’ Research Committee. Conﬂict of interest statement. None declared. REFERENCES 1. Cordera,F. and Jordan,V.C. (2006) Steroid receptors and their role in the biology and control of breast cancer growth. Semin. Oncol., 33, 631–641. 2. Ali,S. and Coombes,R.C. (2002) Endocrine-responsive breast cancer and strategies for combating resistance. Nat. Rev. Cancer, 2, 101–112. 3. Lannigan,D.A. (2003) Estrogen receptor phosphorylation. Steroids, 68, 1–9. 4. Alarid,E.T., Bakopoulos,N. and Solodin,N. (1999) Proteasome- mediated proteolysis of estrogen receptor: a novel component in autologous down-regulation. Mol. Endocrinol., 13, 1522–1534. 5. Lonard,D.M., Nawaz,Z., Smith,C.L. and O’Malley,B.W. (2000) The 26S proteasome is required for estrogen receptor-alpha and coactivator turnover and for eﬃcient estrogen receptor-alpha transactivation. Mol. Cell, 5, 939–948. 6. Nawaz,Z., Lonard,D.M., Dennis,A.P., Smith,C.L. and O’Malley,B.W. (1999) Proteasome-dependent degradation of the human estrogen receptor. Proc. Natl Acad. Sci. USA, 96, 1858–1862. 7. Preisler-Mashek,M.T., Solodin,N., Stark,B.L., Tyriver,M.K. and Alarid,E.T. (2002) Ligand-speciﬁc regulation of proteasome- mediated proteolysis of estrogen receptor-alpha. Am. J. Physiol. Endocrinol. Metab., 282, E891–898. 8. Grisouard,J., Medunjanin,S., Hermani,A., Shukla,A. and Mayer,D. (2007) Glycogen synthase kinase-3 protects estrogen receptor alpha from proteasomal degradation and is required for full transcrip- tional activity of the receptor. Mol. Endocrinol., 21, 2427–2439. 9. Valley,C.C., Metivier,R., Solodin,N.M., Fowler,A.M., Mashek,M.T., Hill,L. and Alarid,E.T. (2005) Diﬀerential regulation of estrogen-inducible proteolysis and transcription by the estrogen receptor alpha N terminus. Mol. Cell Biol., 25, 5417–5428. 10. Yi,P., Feng,Q., Amazit,L., Lonard,D.M., Tsai,S.Y., Tsai,M.J. and O’Malley,B.W. (2008) Atypical protein kinase C regulates dual pathways for degradation of the oncogenic coactivator SRC-3/ AIB1. Mol. Cell, 29, 465–476. 11. Anzick,S.L., Kononen,J., Walker,R.L., Azorsa,D.O., Tanner,M.M., Guan,X.Y., Sauter,G., Kallioniemi,O.P., Trent,J.M. and Meltzer,P.S. (1997) AIB1, a steroid receptor coactivator ampliﬁed in breast and ovarian cancer. Science, 277, 965–968. 12. Li,Q., Wrange,O. and Eriksson,P. (1997) The role of chromatin in transcriptional regulation. Int. J. Biochem. Cell Biol., 29, 731–742. 13. Suen,C.S., Berrodin,T.J., Mastroeni,R., Cheskis,B.J., Lyttle,C.R. and Frail,D.E. (1998) A transcriptional coactivator, steroid receptor coactivator-3, selectively augments steroid receptor transcriptional activity. J. Biol. Chem., 273, 27645–27653. 14. Torchia,J., Rose,D.W., Inostroza,J., Kamei,Y., Westin,S., Glass,C.K. and Rosenfeld,M.G. (1997) The transcriptional co-acti- vator p/CIP binds CBP and mediates nuclear-receptor function. Nature, 387, 677–684. 15. Shao,W., Keeton,E.K., McDonnell,D.P. and Brown,M. (2004) Coactivator AIB1 links estrogen receptor transcriptional activity and stability. Proc. Natl Acad. Sci. USA, 101, 11599–11604. 16. Gianni,M., Parrella,E., Raska,I. Jr., Gaillard,E., Nigro,E.A., Gaudon,C., Garattini,E. and Rochette-Egly,C. (2006) P38MAPK- dependent phosphorylation and degradation of SRC-3/AIB1 and RARalpha-mediated transcription. EMBO J., 25, 739–751. 17. Wu,R.C., Feng,Q., Lonard,D.M. and O’Malley,B.W. (2007) SRC-3 coactivator functional lifetime is regulated by a phospho-dependent ubiquitin time clock. Cell, 129, 1125–1140. 18. Gross,S.D. and Anderson,R.A. (1998) Casein kinase I: spatial organization and positioning of a multifunctional protein kinase family. Cell Signal., 10, 699–711. 19. Knippschild,U., Gocht,A., Wolﬀ,S., Huber,N., Lohler,J. and Stoter,M. (2005) The casein kinase 1 family: participation in mul- tiple cellular processes in eukaryotes. Cell Signal., 17, 675–689. 20. Fuja,T.J., Lin,F., Osann,K.E. and Bryant,P.J. (2004) Somatic mutations and altered expression of the candidate tumor suppres- sors CSNK1 epsilon, DLG1, and EDD/hHYD in mammary ductal carcinoma. Cancer Res., 64, 942–951. 21. von Blume,J., Knippschild,U., Dequiedt,F., Giamas,G., Beck,A., Auer,A., Van Lint,J., Adler,G. and Seuﬀerlein,T. (2007) Phosphorylation at Ser244 by CK1 determines nuclear localization and substrate targeting of PKD2. EMBO J., 26, 4619–4633. 22. Brockschmidt,C., Hirner,H., Huber,N., Eismann,T., Hillenbrand,A., Giamas,G., Radunsky,B., Ammerpohl,O., Bohm,B., Henne-Bruns,D. et al. (2008) Anti-apoptotic and growth- stimulatory functions of CK1 delta and epsilon in ductal