Current Biology 16, 2239–2244, November 21, 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2006.09.034

ReportCK2 Controls the Recruitmentof Wnt Regulatorsto Target Genes In Vivo

Song Wang1 and Katherine A. Jones1,*1Regulatory Biology LaboratoryThe Salk Institute10010 N. Torrey Pines RoadLa Jolla, California 92037

Summary

Nuclear b-catenin is a transcriptional coactivator ofLEF-1/TCF DNA-binding proteins in the Wnt/Wg signal-

ing pathway [1]. Casein Kinase 2 (CK2), a positiveregulator of Wnt signaling [2–4], is present in b-catenin

complexes [5, 6] and activated in Wnt-signaling cells[7]. We show here that CK2 enhances b-catenin:

LEF-1 transactivation in vivo and in vitro and that b-catenin and CK2 cycle on and off the DNA in an alter-

nating manner with the TLE1 corepressor at Wnt targetgenes. Interestingly, CK2 phosphorylates hLEF-1

directly and stimulates binding and transactivationof b-catenin:LEF-1 complexes on chromatin templates

in vitro. In vitro, CK2 phosphorylation of hLEF-1strongly enhances its affinity for b-catenin and re-

duces its affinity for TLE1. MALDI-TOF mass spectrom-etry (MS) identified two CK2 phosphorylation sites

(S42, S61) within the amino terminus of hLEF-1, and

mutation of these sites reduced binding to b-cateninin vitro and transactivation in vivo. Remarkably, treat-

ment of cells with TBB, a pharmaceutical inhibitor ofCK2, blocked the recruitment and cycling of b-catenin

and TLE1 at Wnt target genes in vivo. Taken together,these data indicate that CK2 is required for the assem-

bly and cycling of Wnt-enhancer complexes in vivo.

Results and Discussion

Wnt Coregulators Cycle at Target GenesCK2 associates tightly with nuclear b-catenin and theWnt transducer, Dishevelled, in Wnt-signaling cells [5, 6]and is required for the expression of Wnt target genes[2–7]. For determining whether CK2 is recruited withb-catenin to Wnt-induced genes, chromatin immuno-precipitation (ChIP) experiments were used to analyzethe endogenous c-Myc and CycD1 genes in synchro-nized myoblast C2C12 cells treated either with lithium,a GSK3b inhibitor that stabilizes b-catenin, or withWnt3A-conditioned medium (WCM). Native hLEF-1bound constitutively to the c-Myc (Figure 1A) and CycD1(Figure 1B) genes in C2C12 cells. By contrast, b-cateninappeared at each gene 15–30 min after induction bylithium and oscillated on and off the DNA at repeating75 min intervals. CK2 was also recruited to these Wnt en-hancers and cycled together with b-catenin and othercoactivators, including TIP60 and the FACT/SSRP1elongation factor. No binding of LEF-1 or b-catenin was

*Correspondence: jones@salk.edu

observed at distal (5 kB) upstream sites (data not shown).Negative-acting Wnt regulators, including the TLE1corepressor, Mi-2, a subunit of the Mi-2/NURD complex[8], HDAC1, and GSK3b, were also present at the DNA,but cycled in an alternating manner with b-catenin.Thus GSK3b, which has recently been proposed to re-press nuclear-receptor genes [9], may also function inthe nucleus as a transcriptional repressor of Wnt genes.

The observation that b-catenin and CK2 alternate withTLE1 for binding to the CycD1 gene was confirmed byreal-time PCR ChIP (Figure 1C) and indicates that LEF-1cannot bind simultaneously to b-catenin and TLE1 in vivo,consistent with previous biochemical studies [10]. Asequential ChIP (reChIP) analysis (Figure 1D) revealedthat CK2 is present in chromatin complexes isolatedwith b-catenin antibodies and confirms that these pro-teins can bind simultaneously to Wnt-responsive genes.Importantly, endogenous b-catenin and CK2 cycledwith similar kinetics in cells treated with Wnt3A-condi-tioned medium (WCM) (Figure 1E), concomitant withinduction of CycD1 mRNA in these cells (Figure S1 in theSupplemental Data available online). Importantly,siRNA-mediated depletion of endogenous CK2 signifi-cantly impaired b-catenin transactivation of the TOP-FLASH reporter gene in 293 cells, as well as endogenousc-Myc transcription in SW480 cells where Wnt signalingis constitutively active (Figure 1F).

CK2 Phosphoryation of LEF-1 Enhances

Binding to Beta-CateninTo test whether CK2 affects transcription directly, weused an in vitro system in which a LEF-1 promotertemplate (pBRE) was assembled into nucleosomes byincubation with a crude Drosophila embryo S190 chro-matin-assembly extract and purified core histones, inthe presence or absence of b-catenin and LEF-1 [11].Interestingly, CK2 enhanced b-catenin:LEF-1 transcrip-tion in this system (Figure 2A, top panel, compare lanes9 and 10), and this effect was blocked by the pharma-ceutical CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole(TBB; lane 11). As noted previously [11], b-cateninstrongly enhances binding of LEF-1 to chromatin tem-plates (Figure 2A, bottom panel, compare lanes 6 and9), and CK2 enhanced this effect (Figure 2A, bottompanel, compare lanes 9 and 10; see also Figure 3). Bycontrast, CK2 had no effect on Notch-enhancer-depen-dent transcription in vitro (data not shown).

In vitro kinase assays indicated that CK2 phosphory-lates full-length His-LEF-1 and His-LEF-1DAD, a minimalform of LEF-1 that supports b-catenin activation, but didnot phosphorylate DNß-catenin (Figure S2A). HumanTCF4 is phosphorylated by CK2 at N-terminal residuesS58, S59, and S60, which lowers its affinity for plakoglo-bin (g-catenin) [12]. Although only one of these residues(corresponding to S67) is conserved in human LEF-1,protein analysis of CK2-phosphorylated His-LEF-1DADby MALDI-TOF mass spectrometry revealed mono-,di-, and tri-phosphorylation of an N-terminal peptide

Current Biology2240

Figure 1. Cyclic Recruitment of Wnt Coregulators at Wnt/b-Catenin-Induced Genes In Vivo

(A and B) ChIP analysis of the c-Myc (A) and CycD1 (B) genes in synchronized C2C12 cells treated with LiCl for the times indicated.

(C) Quantitative analysis of the binding of b-catenin, CK2, and TLE1 to the CycD1 gene by real-time PCR.

(D) A sequential ChIP (re-ChIP) analysis of binding of b-catenin and CK2 to the CycD1 gene is shown.

(E) Cycling of Wnt coactivators at the CycD1 gene in C2C12 cells exposed to Wnt3A-conditioned medium for the different times indicated.

(F) Analysis of b-catenin transactivation of the TOPFLASH reporter gene in 293 cells (left panel) or of endogenous CycD1 transcription in SW480

CRC cells. Where indicated, cells were depleted of endogenous CK2 by treatment with si-CK2aRNA or with a nonspecific control siRNA (si-con-

RNA). The error bars are based on the standard deviations of triplicate experiments.

(aa 37–74, Figure 2B), which includes S67 as well as twoseries (S42 and S61) that lie within an acidic environmenttypical of CK2 sites. In vitro kinase assays with mutantHis-LEF-1DAD proteins containing either single (S42A,S61A) or double (S42A:S61A) substitutions indicatethat both S42 and S61 are key CK2 phosphorylationsites in vitro (Figure 2C).

The observation that CK2 phosphorylates at least tworesidues (S42 and S61) within the LEF-1–b-catenin

interaction domain led us to examine whether it influ-ences binding to b-catenin. First, purified GST-b-cateninwas coupled to glutathione-S-agarose beads and incu-bated with purified untreated or CK2-phosphorylatedwild-type or mutant S42A:S61A His-LEF-1DAD proteinsin GST pull-down experiments. As shown in Figure 3A,CK2 strongly enhanced the binding of b-catenin towild-type (compare lanes 5 and 6), but not mutantS42A:S61A (compare lanes 3 and 4), His-LEF-1DAD

CK2 Controls Assembly of Wnt Complexes2241

proteins. CK2 also facilitated binding of full-lengthHis-LEF-1 to GST-b-catenin (Figure 3B, compare lanes3 and 4). A triple-point mutation affecting S42, S61, andS67 was as defective as the S42A:S61A mutant in itsability to respond to CK2 (Figure S2C), whereas a sin-gle-point mutation affecting S42, a double mutationaffecting S61 and S67, and other mutations retainedthe ability to be activated by CK2 in vitro (Figures S2Band S2C). Thus CK2 phosphorylates LEF-1 at Ser-42and Ser-61 to increase its affinity for b-catenin and chro-matin templates in vitro.

Figure 2. CK2 Enhances Transcription and Chromatin-Binding

Activity of LEF-1:b-Catenin In Vitro

(A) Analysis of the transcriptional activity (primer extension, upper

panel) and DNA binding (DNase I footprint, lower panel) of LEF-1

and b-catenin to chromatin-assembled pBRE templates in vitro.

The a-globin gene (a-glo) was a nonchromatin control.

(B) MALDI-TOF mass-spectrometry identification of a CK2-phos-

phorylated LEF-1 peptide.

(C) Analysis of the ability of wild-type LEF-1DAD, or various mutant

LEF-1DAD proteins, to be phosphorylated by CK2 in vitro.

Interestingly, b-catenin and the Gro/TLE1 corepressorcompete for partially overlapping interaction sites withinthe central and high-mobility group (HMG) domains ofLEF-1 [10]. Because this region contains multiple poten-tial CK2 phosphorylation sites, we tested whether CK2might affect binding of LEF-1 to Gro/TLE1. FLAG-taggedDrosophila Groucho protein, purified from DrosophilaS2 cells [13], was coupled to M2 FLAG-agarose beadsand used in pull-down assays with the recombinantfull-length His-LEF-1 protein. In these experiments,His-LEF-1 bound avidly to either unphosphorylated orCK2-phosphorylated FLAG-Gro beads (Figure 3C, com-pare lanes 3 and 4 with lanes 5 and 7). By contrast, CK2phosphorylation of LEF-1 strongly blocked binding toboth unphosphorylated and CK2-phosphorylatedFLAG-Gro proteins (compare lane 5 with 6 and lane 7with 8, respectively). Both wild-type and S42A:S61ALEF-1DAD proteins bound only weakly to Groucho;however, this interaction was nevertheless inhibited byCK2 (Figure S3), indicating that CK2 phosphorylatesthe TLE1 binding region of LEF-1DAD. Importantly,CK2 failed to stimulate transcription (Figure 3D, toppanel, compare lanes 3 and 4) or chromatin binding(Figure 3D, bottom panel, compare lanes 3 and 4) ofthe mutant S42A:S61A His-LEF-1DAD protein in vitro.

LEF-1 N-Terminal Residues S42 and S61 Facilitate

Transactivation In VivoFor determining whether LEF-1 activity requires S42 andS61, Raji B cells were cotransfected with the TOPFLASHreporter, an activated b-catenin (S33Y), and FLAG-tagged wild-type or S42A:S61A mutant LEF-1 con-structs, and TOPFLASH reporter-gene activity was mon-itored relative to a TK-Renilla luciferase control plasmid.As shown in Figure 3E, b-catenin activity was signifi-cantly higher when coexpressed with wild-type LEF-1than with the S42A:S61A mutant LEF-1 protein. Immuno-blot analysis confirmed that the wild-type and S42A:S61A mutant LEF-1 proteins were equivalently ex-pressed in the cells, indicating that the predominantN-terminal CK2 phosphorylation sites in LEF-1 arerequired for transactivation in vivo and in vitro.

CK2 is Required for Recruitment and Cycling

of Wnt Coregulators at Target GenesThese data lead us to consider whether CK2 is requiredfor targeting of b-catenin to Wnt target genes in vivo.Moreover, CK2 phosphorylation of TLE1 is required forits repressive activity and association with chromatin[14, 15]. Consequently, we reasoned that CK2 mightregulate the binding of both b-catenin and TLE1 to Wnttarget genes. To examine this possibility, we carriedout ChIP analyses of the CycD1 gene in cells treatedwith the CK2 inhibitor, TBB, or control dimethyl sulfox-ide (DMSO), for 1 hr prior to lithium induction of b-cate-nin. As shown in Figure 4A, recruitment of b-catenin tothe CycD1 gene was dramatically impaired in cellstreated with TBB. Similarly, the level of TLE1 at theCycD1 promoter before and after lithium induction wasgreatly reduced in the presence of TBB (Figure 4B). Bycontrast, TBB had no effect on binding of LEF-1 (Fig-ure 4C). Induction of CycD1 transcription by lithiumwas strongly blocked by TBB (Figure 4D), which didnot alter b-catenin or TLE1 levels in the nucleus

Current Biology2242

Figure 3. CK2 Enhances Binding of LEF-1 to b-Catenin and Blocks Binding to Gro/TLE1 In Vitro

(A) Untreated or CK2-phosphorylated mutant (S42A:S61A; lanes 3 and 4) or wild-type (lanes 5–8) LEF-1DAD proteins were analyzed for binding to

GST-b-catenin in vitro.

(B) Binding of untreated or CK2-phosphorylated full-length His-LEF-1 to GST-b-catenin in vitro.

(C) Binding of untreated or CK2-phosphorylated full-length His-LEF-1 to unphosphorylated or CK2-phosphorylated FLAG-Gro beads in vitro.

(D) The mutant (S42A:S61A) LEF-1DAD protein is not activated by CK2 in vitro.

(E) The TOPFLASH reporter gene was transfected into Raji cells together with vectors expressing wild-type or mutant (S42A:S61A) FLAG-LEF-1,

and b-catenin, as indicated. Insert shows protein expression by immunoblot. The error bars are based on the standard deviations of triplicate

experiments.

(Figure 4E). A more extensive ChIP analysis revealedthat TBB also blocks the cycling of b-catenin at theCycD1 gene (Figure 4F), indicating that CK2 is requiredfor assembly and turnover of Wnt-enhancer complexesin vivo.

The finding that CK2 phosphorylates LEF-1 and en-hances binding to b-catenin, while blocking binding toTLE1, indicated that it might regulate the assembly of ac-tive Wnt enhancers. In this respect, CK2 would functionoppositely to CK1, which promotes binding of b-cateninto the APC tumor suppressor, disfavors binding to LEF-1/TCF proteins [16–18], and thereby inhibiting Wnt sig-naling. Thus CK1 and CK2 may control, in oppositeways, the switching of b-catenin and Gro/TLE1 on theLEF-1/TCF platform. LEF-1 residues S42, S61, and S67are highly conserved, and TCF proteins contain multipleserine and acidic residues in the vicinity of this region,suggesting that activation by CK2 may be conservedamong LEF/TCF family members. Interestingly, X-raycrystallography studies indicate that the interaction of

b-catenin with E-cadherin, which closely resembles itsinteraction with TCF4, is similarly enhanced by CK2phosphorylation [19]. Remarkably, this region of LEF-1is frequently mutated in human sebaceous-gland tumors[20], which express a mutant LEF-1(E45K:S61P) proteinthat is unable to interact with b-catenin. Our findingssuggest that the reduced affinity of the mutant LEF-1(E45K:S61P) protein for b-catenin may arise, in part,from its inability to be phosphorylated by CK2.

It is notable that b-catenin and associated coactiva-tors, including CK2 and FACT, are recruited in a cyclicmanner to Wnt-responsive genes. In yeast, CK2 associ-ates with Polymerase-associated Factor 1 (Paf1) andFACT complexes [21]. The observation that b-catenin as-sociates with the Paf1 subunit, Parafibromin [22], andwith Trithorax-related MLL1/2 complexes, to promotehistone H3K4 trimethylation in vivo [23] suggests thatCK2 might also stimulate transcription elongation at Wntgenes. Moreover, the ability of the wild-type APC tumorsuppressor, but not cancer-associated APC mutant

CK2 Controls Assembly of Wnt Complexes2243

Figure 4. CK2 Is Required for Recruitment of b-Catenin and TLE1 to the CycD1 Gene In Vivo

(A–C)Synchronized C2C12 cells were stimulated with LiCl for the various amounts of time and analyzed by real-time PCR ChIP for (A) b-catenin,

(B) TLE1, and (C) LEF-1, with or without TBB.

(D) TBB blocks b-catenin transactivation of the TOPFLASH reporter gene in 293 cells and induction of CycD1 transcription in lithium-treated cells.

(E) TBB does not affect b-catenin protein levels by immunoblot.

(F) TBB blocks the recruitment and cycling of b-catenin at the CycD1 promoter in lithium-treated C2C12 cells, as determined by quantitative real-

time PCR ChIP.

The error bars are based on the standard deviations of triplicate experiments.

proteins, to bind and inhibit CK2 [24] suggests an addi-tional mechanism by which APC may counteract b-cate-nin transactivation in vivo. It is interesting to speculatethat the repetitive cycling of Wnt coregulator complexesmight contribute to the oscillating pattern of Wnt genetranscription that is observed during somitogenesis,where Wnt and Notch target genes are under the controlof the vertebrate segmentation clock [25]. We note thatCK2, CK1, and GSK3b also control circadian gene tran-scription and that inhibition of CK2 abolishes transcriptcycling from these genes in vivo [26]. Thus further studiesare needed to assess whether cyclic coregulator recruit-ment may regulate cyclic transcription, as well as cross-talk among these diverse signaling pathways.

Supplemental Data

Supplemental Data include Experimental Procedures and three

figures and are available with this article online at: http://www.

current-biology.com/cgi/content/full/16/22/2239/DC1/.

Acknowledgments

We thank W. Fischer (Salk) for mass spectrometry, S. Stifani

(Quebec, Canada) for human TLE1 reagents, A. Courey (UCLA) for

Groucho-expressing cells, and K. Willert (UCSD) for Wnt3A-

expressing L cells. This work was funded by a grant to K.A.J. from

the National Institutes of Health.

Received: May 25, 2006

Revised: September 15, 2006

Accepted: September 19, 2006

Published: November 20, 2006

References

1. Gregorieff, A., and Clevers, H. (2005). Wnt signaling in the

intestinal epithelium: From endoderm to cancer. Genes Dev.

19, 877–890.

2. Song, D.H., Dominguez, I., Mizuno, J., Kaut, M., Mohr, S.C., and

Seldin, D.C. (2003). CK2 phosphorylation of the armadillo repeat

Current Biology2244

region of beta-catenin potentiates Wnt signaling. J. Biol. Chem.

278, 24018–24025.

3. Seldin, D.C., Landesman-Bollag, E., Farago, M., Currier, N., Lou,

D., and Dominguez, I. (2005). CK2 as a positive regulator of Wnt

signalling and tumourigenesis. Mol. Cell. Biochem. 274, 63–67.

4. Dominguez, I., Mizuno, J., Wu, H., Song, D.H., Symes, K., and

Seldin, D.C. (2004). Protein kinase CK2 is required for dorsal

axis formation in Xenopus embryos. Dev. Biol. 274, 110–124.

5. Willert, K., Brink, M., Wodarz, A., Varmus, H., and Nusse, R.

(1997). Casein kinase 2 associates with and phosphorylates

dishevelled. EMBO J. 16, 3089–3096.

6. Song, D.H., Sussman, D.J., and Seldin, D.C. (2000). Endogenous

protein kinase CK2 participates in Wnt signaling in mammary

epithelial cells. J. Biol. Chem. 275, 23790–23797.

7. Gao, Y., and Wang, H.Y. (2006). Casein kinase 2 is activated and

essential for Wnt/beta-catenin signaling. J. Biol. Chem. 281,

18394–18400.

8. DasGupta, R., Kaykas, A., Moon, R.T., and Perrimon, N. (2005).

Functional genomic analysis of the Wnt-wingless signaling

pathway. Science 308, 826–833.

9. Yin, L., Wang, J., Klein, P.S., and Lazar, M.A. (2006). Nuclear re-

ceptor Rev-erbalpha is a critical lithium-sensitive component of

the circadian clock. Science 311, 1002–1005.

10. Daniels, D.L., and Weis, W.I. (2005). Beta-catenin directly dis-

places Groucho/TLE repressors from Tcf/Lef in Wnt-mediated

transcription activation. Nat. Struct. Mol. Biol. 12, 364–371.

11. Tutter, A.V., Fryer, C.J., and Jones, K.A. (2001). Chromatin-spe-

cific regulation of LEF-1-beta-catenin transcription activation

and inhibition in vitro. Genes Dev. 15, 3342–3354.

12. Miravet, S., Piedra, J., Miro, F., Itarte, E., Garcia de Herreros, A.,

and Dunach, M. (2002). The transcriptional factor Tcf-4 contains

different binding sites for beta-catenin and plakoglobin. J. Biol.

Chem. 277, 1884–1891.

13. Song, H., Hasson, P., Paroush, Z., and Courey, A.J. (2004). Grou-

cho oligomerization is required for repression in vivo. Mol. Cell.

Biol. 24, 4341–4350.

14. Nuthall, H.N., Husain, J., McLarren, K.W., and Stifani, S. (2002).

Role for Hes1-induced phosphorylation in Groucho-mediated

transcriptional repression. Mol. Cell. Biol. 22, 389–399.

15. Nuthall, H.N., Joachim, K., and Stifani, S. (2004). Phosphoryla-

tion of serine 239 of Groucho/TLE1 by protein kinase CK2 is

important for inhibition of neuronal differentiation. Mol. Cell.

Biol. 24, 8395–8407.

16. Ha, N.C., Tonozuka, T., Stamos, J.L., Choi, H.J., and Weis, W.I.

(2004). Mechanism of phosphorylation-dependent binding of

APC to beta-catenin and its role in beta-catenin degradation.

Mol. Cell 15, 511–521.

17. Xing, Y., Clements, W.K., Kimelman, D., and Xu, W. (2003). Crys-

tal structure of a beta-catenin/axin complex suggests a mecha-

nism for the beta-catenin destruction complex. Genes Dev. 17,

2753–2764.

18. Xing, Y., Clements, W.K., Le Trong, I., Hinds, T.R., Stenkamp, R.,

Kimelman, D., and Xu, W. (2004). Crystal structure of a beta-

catenin/APC complex reveals a critical role for APC phosphory-

lation in APC function. Mol. Cell 15, 523–533.

19. Huber, A.H., and Weis, W.I. (2001). The structure of the beta-

catenin/E-cadherin complex and the molecular basis of diverse

ligand recognition by beta-catenin. Cell 105, 391–402.

20. Takeda, H., Lyle, S., Lazar, A.J., Zouboulis, C.C., Smyth, I., and

Watt, F.M. (2006). Human sebaceous tumors harbor inactivating

mutations in LEF1. Nat. Med. 12, 395–397.

21. Krogan, N.J., Kim, M., Ahn, S.H., Zhong, G., Kobor, M.S., Cag-

ney, G., Emili, A., Shilatifard, A., Buratowski, S., and Greenblatt,

J.F. (2002). RNA polymerase II elongation factors of Saccharo-

myces cerevisiae: A targeted proteomics approach. Mol. Cell.

Biol. 22, 6979–6992.

22. Mosimann, C., Hausmann, G., and Basler, K. (2006). Parafibro-

min/Hyrax activates Wnt/Wg target gene transcription by direct

association with beta-catenin/Armadillo. Cell 125, 327–341.

23. Sierra, J., Yoshida, T., Joazeiro, C.A., and Jones, K.A. (2006). The

APC tumor suppressor counteracts beta-catenin activation and

H3K4 methylation at Wnt target genes. Genes Dev. 20, 586–600.

24. Homma, M.K., Li, D., Krebs, E.G., Yuasa, Y., and Homma, Y.

(2002). Association and regulation of casein kinase 2 activity

by adenomatous polyposis coli protein. Proc. Natl. Acad. Sci.

USA 99, 5959–5964.

25. Aulehla, A., and Herrmann, B.G. (2004). Segmentation in verte-

brates: clock and gradient finally joined. Genes Dev. 18, 2060–

2067.

26. Blau, J. (2003). A new role for an old kinase: CK2 and the circa-

dian clock. Nat. Neurosci. 6, 208–210.