LETTERS

Casein kinase 1a governs antigen-receptor-inducedNF-kB activation and human lymphoma cell survivalNicolas Bidere1,4*, Vu N. Ngo2*, Jeansun Lee1, Cailin Collins2, Lixin Zheng1, Fengyi Wan1, R. Eric Davis2, Georg Lenz2,D. Eric Anderson3, Damien Arnoult4, Aime Vazquez4, Keiko Sakai1{, Jun Zhang1, Zhaojing Meng5,Timothy D. Veenstra5, Louis M. Staudt2* & Michael J. Lenardo1*

The transcription factor NF-kB is required for lymphocyte activationand proliferation as well as the survival of certain lymphoma types1,2.Antigen receptor stimulation assembles an NF-kB activatingplatform containing the scaffold protein CARMA1 (also calledCARD11), the adaptor BCL10 and the paracaspase MALT1 (theCBM complex), linked to the inhibitor of NF-kB kinase complex3–12,but signal transduction is not fully understood1. We conductedparallel screens involving a mass spectrometry analysis ofCARMA1 binding partners and an RNA interference screen forgrowth inhibition of the CBM-dependent ‘activated B-cell-like’(ABC) subtype of diffuse large B-cell lymphoma (DLBCL)12. Herewe report that both screens identified casein kinase 1a (CK1a) as abifunctional regulator of NF-kB. CK1a dynamically associates withthe CBM complex on T-cell-receptor (TCR) engagement to partici-pate in cytokine production and lymphocyte proliferation. However,CK1a kinase activity has a contrasting role by subsequently promot-ing the phosphorylation and inactivation of CARMA1. CK1a hasthus a dual ‘gating’ function which first promotes and then termi-nates receptor-induced NF-kB. ABC DLBCL cells required CK1a forconstitutive NF-kB activity, indicating that CK1a functions as aconditionally essential malignancy gene—a member of a new classof potential cancer therapeutic targets.

For a better understanding of signal regulation by the CBM com-plex, we performed a mass spectrometry proteomic screen afterCARMA1 immunoprecipitation. Sixteen peptides covering 54% ofthe length of CK1a were isolated from an excised band (Fig. 1a andSupplementary Fig. 1). CK1a belongs to the CK1 family of serine/threonine protein kinases, which regulates developmental andhomeostatic processes including the Wnt/b-catenin pathway andcircadian rhythm13. Co-immunoprecipitations in HEK293T cellsshowed that haemagglutinin (HA)-tagged CK1a interacted withV5–CARMA1 (Fig. 1b). CARMA1, BCL10 and MALT1 associatedwith CK1a after TCR stimulation in Jurkat T lymphocytes, and inBJAB B cells stimulated with phorbol 12-myristate 13-acetate (PMA)and ionomycin (Fig. 1c–e). Notably, phosphorylated forms of BCL10and ubiquitinated species of MALT1, modifications due to signal-ling14, associated with CK1a. Moreover, MALT1 and BCL10 prec-ipitations revealed TCR-induced recruitment of CK1a concomitantlywith inhibitor of NF-kB kinase (IKK)-b and phosphorylated IKK-b(p-IKK-b; Fig. 1d and data not shown). Also, cytosolic CK1a reor-ganized into punctate structures that co-localized with CD3 clusterson TCR activation (Fig. 1f), suggesting an association with CBMcomponents within membrane microdomains2,15. In contrast to

TCR agonists, tumour necrosis factor-a (TNF-a), which does notuse the CBM, induced no interaction between CK1a and CBMsubstituents (Supplementary Fig. 2). Finally, antibody depletion ofCK1a from cell lysates removed nearly the entire active CBM complex(Supplementary Fig. 3). Hence, CK1a is a new component selectivelyentering the active CBM complex after antigen receptor stimulation.

CK1a harbours a short and unique carboxy-terminal portionattached to the conserved kinase domain13 (Fig. 1a). Removing thisregion (D283–337) abolished CK1a binding to CARMA1 (Fig. 1g). Inaddition, the human (residues 283–337) and mouse (283–325) CK1aC-terminal domains were sufficient, with Y292 and D293 as the keyresidues, for CARMA1 association (Fig. 1h and SupplementaryFig. 4a, b). We also found that both the coiled-coil (CC) and linkerregions (CCL) of CARMA1 together were critical for CK1a binding.(Fig. 1i and Supplementary Fig. 4c).

The CBM complex is an obligate gateway from lymphocyte anti-gen receptors to NF-kB activation1,3,8–11. To define the functionalimportance of CK1a, we first decreased its endogenous levels byRNA interference in primary human T cells. CK1a (also calledCSNK1A1) silencing reduced TCR-induced interleukin-2 (IL-2)production as did silencing of messenger RNA for NF-kB p65(RELA) and BCL10 (Fig. 2a). This was accompanied by diminishedIL-2 receptor (CD25) upregulation, and reduced proliferation(Fig. 2b). In Jurkat cells, CK1a knockdown with small interferingRNAs (siRNAs) decreased the TCR induction of an NF-kB luciferasereporter as efficiently as a BCL10 siRNA (Fig. 2c). Again, TNF-a-mediated NF-kB was unaffected, underscoring the selective involve-ment of CK1a in the TCR–NF-kB pathway. Accordingly, CK1a-silenced primary T cells and Jurkat cells also displayed diminishedp65 NF-kB nuclear translocation after TCR, but not TNF-a, stimu-lation (Supplementary Fig. 5a–c). By contrast, NF-kB activationproceeded normally when levels of CK1d or CK1e were reduced(Supplementary Fig. 6). Thus, among the CK1s, CK1a provides anessential, non-redundant function in regulating TCR-inducedNF-kB activation.

To achieve a better knockdown, single Jurkat clones stably expres-sing a small hairpin RNA (shRNA) against CK1a were generated. Asexpected, NF-kB activation was markedly reduced on stimulation, asIkBa phosphorylation and degradation were inhibited (Fig. 2d–f).Other early TCR signalling events, such as ERK1/2 phosphorylation,overall tyrosine phosphorylation, calcium mobilization and NF-ATactivation occurred normally (Fig. 2e and Supplementary Fig. 5d–f).Of note, NF-kB activity was restored when CK1a-silenced cells were

1Molecular Development Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases. 2Metabolism Branch, Center for Cancer Research, National CancerInstitute, 3Proteomics and Mass Spectrometry Facility, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892,USA. 4U542, INSERM, Universite Paris-Sud, Hopital Paul Brousse, Villejuif 94800, France. 5Laboratory of Proteomics and Analytical Technologies (LPAT), National Cancer Institute,Frederick, Maryland 21702, USA. {Present address: Division of Viral Immunology, Center for AIDS Research, Kumamoto University, 2-2-1 Honjhou, Kumamoto-shi, Kumamoto-ken860-0811, Japan.*These authors contributed equally to this work.

Vol 458 | 5 March 2009 | doi:10.1038/nature07613

92 Macmillan Publishers Limited. All rights reserved©2009

a b

IP V5

HA–CK1α +–

Input

– +++– –V5–CARMA1

anti-V5

anti-HA

anti-HA

d c

10 20 5 0 Anti-CD3/28(min)

Anti-CD3/28(min)

BCL10

CK1α

CARMA1

MALT1Ub

IP CK1α e

CD3 CK1α Merge

4 °C

37 °C

f g

10 20 5 0

IKK-β

BCL10

CK1α

IP MALT1

MALT1MASSSGSKAEFIVGGKYKLVRKIGSGSFGDIYLAINITNGEEVAVKLESQKARHPQLLYESKLYKILQGGVGIPHIRWYGQEKDYNVLVMDLLGPSLEDLFNFCSRRFTMKTVLMLADQMISRIEYVHTKNFIHRDIKPDNFLMGIGRHCNKLFLIDFGLAKKYRDNRTRQHIPYREDKNLTGTARYASINAHLGIEQSRRDDMESLGYVLMYFNRTSLPWQGLKAATKKQKYEKISEKKMSTPVEVLCKGFPAEFAMYLNYCRGLRFEEAPDYMYLRQLFRILFRTLNHQYDYTFDWTMLKQKAAQQAASSSGQGQQAQTPTGKQTDKTKSNMKGF

CK1α1 12 337 282

h

V5–CARMA1HA–CK1α

+ + + – – – FL ∆C –

anti-V5

anti-HA IP V5

Lysates

anti-V5

anti-HA

CARMA1 CC CARD PDZ GUK Linker 1 104 443 752 1147 660

1–660

1–443

104–1147

443–1147

104–660

443–660

104–443

CK1αinteraction

+

+

–+

–+

–

–

i

WT

anti-HA

anti-Myc

M/V–CARMA1 + + –

IP GFP

WT

Y29

2A

+

HA–CK1α

anti-HA Lysates

D29

3A

10 15 5 0 P/I (min)

BCL10

CK1α

IP CK1α

CARMA1

MALT1

BJAB

Ub

Kinase domain

Figure 1 | Identification of CK1a as a CARMA1-binding partner.a, Schematic and sequence of CK1a. Peptides identified by a massspectrometry analysis of CARMA1-containing complexes are highlighted ingrey. b, Interaction between HA–CK1a and V5–CARMA1 in HEK293T cellsby immunoprecipitation (IP) and immunoblot (IB).c–e, Immunoprecipitation/immunoblot, as indicated, in Jurkat Tlymphocytes stimulated with 1mg ml21 anti-CD3 and anti-CD28 (c, d), andin BJAB B cells stimulated with 20 ng ml21 PMA and 300 ng ml21 ionomycin(e). Filled and open symbols indicate non-phosphorylated andphosphorylated forms, respectively. Ub, ubiquitin. f, Confocal images of

CD3 and CK1a after CD3 crosslinking in Jurkat T lymphocytes. Nucleicounterstaining is shown in blue. g, Immunoprecipitation/immunoblot ofV5–CARMA1 binding to HA-tagged full-length (FL) CK1a or CK1a lackingresidues 283–337 (DC) in HEK293T cells. h, Myc/Venus (M/V)-taggedCARMA1 association with HA–CK1a mutants in HEK293T cells byimmunoprecipitation/immunoblot. i, Mapping of the minimal CK1a-binding domain of CARMA1 by immunoprecipitation/immunoblot inHEK293T cells expressing HA–CK1a and M/V–CARMA1 truncationmutants.

b

NS

BC

L10

CK

1α.2

CK

1α.3

CK1α

BCL10

β-actin

siRNA

c

Control

0 5 10 20

CK1αsh18 Control CK1αsh18

p-IκBα

IκBα

ERK

p-ERK

β-actin

CK1α

p-IκBα

IκBα

IKK-β

p-IKK

CK1αIKK-β

IKK-β

CK1α

0 5 10 20 P/I

CK1αsiRNA

0 100 101 102 103 104 100 101 102 103 104

101 102 103 104

100 101 102 103 104 100 101 102 103 104

20 40 60 80

100

0 20 40 60 80

100

12.7

0

20

40

60 69.6

10.1 40.8

Unstimulated Unstimulated Anti-CD3/28

CD25

NSsiRNA

0

20

40

60

CFSE

0 50

100 150 200 250

0 50

100 150 200 250

Anti-CD3/28

0 50

100 150 200 250

0 50

100 150 200 250

g f

0

1,500

3,000

4,500

6,000

7,500

9,000

0 100 200 400

IL-2

(pg

ml–1

)

Anti-CD3/28 (ng ml–1)

NS CK1α RELA BCL10

a

CARMA1

BCL10

CK1αNS

IPBCL10

– + Anti-CD3/28

Lysates

– +

MALT1

siRNA:

30 0 7.5 15

30 0 7.5 15

0

100

200

300

400

500

600

700

Ctrlsh

14sh

18 Ctrlsh

14sh

18Ctr

l

sh14

sh18

Ctr

l

sh14

sh18

mCK1αEmpty vector

0 1 10

PMA (ng ml–1)

e

d

5.8 74.3

1.4 43.2

0

2

4

6

8 CK1α.2CK1α.3BCL10

NS

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100

NF-

κB a

ctiv

ity (R

LU)

NF-

κB a

ctiv

ity (R

LU)

Unstim. TNF-αAnti-CD3/28

Figure 2 | Requirement of CK1a for NF-kBactivation and proliferation in lymphocytes.a, b, Human peripheral blood T lymphocyteswere transfected with siRNA for CK1a, NF-kBp65 (RELA), BCL10, or scrambled nonspecific(NS) siRNA. IL-2 secretion (mean and s.d. oftriplicate measurements) and CD25 induction12 h after stimulation is shown.Carboxyfluorescein succinimidyl ester (CFSE)dilution was assessed after 96 h. The percentageof CD25-positive cells, and of dividing cells, isshown. c, NF-kB luciferase assay (mean and s.d.of triplicate experiments) of Jurkat cellstransfected with CK1a (CK1a.2 and CK1a.3),BCL10 or NS siRNA, and stimulated with1mg ml21 anti-CD3 and anti-CD28, or with25 ng ml21 TNF-a. Left panel: immunoblot forCK1a, BCL10 and b-actin. RLU, relative lightunits. d, NF-kB luciferase assay of Jurkat cellsstably expressing CK1a shRNA (sh14 and sh18)reconstituted with a control vector or with mouseCK1a (mCK1a), stimulated with PMA and100 ng ml21 ionomycin, and analysed as inc. e, f, Immunoblot of control and CK1a shRNA(sh18) expressing Jurkat cells exposed to10 ng ml21 PMA and 100 ng ml21 ionomycin.g, Immunoprecipitation/immunoblot of NS- andCK1a-silenced Jurkat cells stimulated with1mg ml21 anti-CD3 and anti-CD28 for 20 min.Arrowhead indicates CARMA1; black and opensquares indicate IKK-b and phosphorylatedIKK-b, respectively.

NATURE | Vol 458 | 5 March 2009 LETTERS

93 Macmillan Publishers Limited. All rights reserved©2009

rescued with ectopic mouse CK1a (mCK1a), but not with mCK1amutants lacking CARMA1 binding ability, indicating that CK1afunction requires association with CARMA1 (Fig. 2d andSupplementary Fig. 7). Hence, CK1a is selectively required foroptimal physiological activation of NF-kB on TCR stimulation innormal lymphocytes.

Promptly following TCR ligation, protein kinase C (PKC)-hphosphorylates CARMA1 and unleashes its adaptor functions16,17.CARMA1 then promotes IkBa phosphorylation by recruiting signal-ling molecules, including BCL10, MALT1 and IKK17,18. Although IKKwas phosphorylated after stimulation of CK1a-silenced cells, p-IKK-bwas no longer recruited to the CBM complex (Fig. 2f, g). This parallelsprevious observations in CARMA1- and BCL10-deficient cells19.Hence, the recruitment of p-IKK and consequent alterations ofIkBa are defective without CK1a. Nonetheless, CK1a still associatedwith CARMA1 in PKC-h- or BCL10-silenced cells, or after treat-ment with the PKC inhibitor rottlerin, although BCL10/MALT1recruitment to CK1a/CARMA1 and NF-kB activation were dimi-nished (Supplementary Fig. 8). Thus, CK1a associates withCARMA1 independently of PKC-h and BCL10, and does not requirePKC-h-dependent modifications of CARMA1.

To investigate the role of CK1a enzyme activity, we generatedkinase-dead CK1a by introducing point mutations in the ATPasedomain (K46R, D136N, or D136A)20,21. In CK1a-silenced Jurkatcells, mCK1a(D136N) markedly enhanced TCR-induced NF-kBactivation, surpassing the response in cells reconstituted with wild-type CK1a (Fig. 3a). Human K46R, D136N and D136A CK1amutants gave similar results (Supplementary Fig. 9a). Accordingly,IkBa phosphorylation and degradation was augmented inmCK1a(D136N) expressing cells compared with wild-type mCK1a(Fig. 3b). The CK1a(D136N) synergistic effect was abolished inBCL10-silenced cells, or when mCK1a(D136N/D293A) was used(Supplementary Fig. 9d, e), suggesting that enhanced NF-kB activityrequires BCL10 and binding to CARMA1. Thus, CK1a kinase activityhas a negative role in TCR-induced NF-kB activation, in addition tothe positive signalling role of CK1a in the CBM complex.

By analogy with IKK-b, which marks BCL10 for degradation byphosphorylation22, we inferred that CK1a might phosphorylate asubstrate to downregulate the CBM complex. CARMA1, which bindsCK1a, was an attractive candidate. We observed that wild-type CK1a,but not CK1a(D136N), retarded the electrophoretic migration ofCARMA1 in a l-phosphatase-dependent manner, suggesting thatCK1a phosphorylates it (Fig. 3c and Supplementary Fig. 10).Because CARMA1 contains 142 potential phospho-acceptors, we usedtruncation mutants to identify CK1a phosphorylation sites. InHEK293T cells, CARMA1-CCL(104–660) existed as two species,and wild-type CK1a, but not CK1a(D136N), shifted essentially allthe CARMA1-CCL to the slow-migrating, l-phosphatase-sensitivespecies (Fig. 3d and Supplementary Fig. 10a, b). CARMA1-CCL(104–616) was the shortest CK1a-sensitive construct, as a mutantlacking residues 606–616 was not modified (Fig. 3e). The substitutionto alanine of serines S603, S607 and S608 showed that only S608A andS603A/608A (2SA) were insensitive to CK1a, suggesting that S608might be the key CK1aphosphorylation site (Fig. 3f). NF-kB enhance-ment was observed in CARMA1-deficient Jurkat cells11 reconstitutedwith CARMA1(2SA) and CARMA1(S608A) (Fig. 3g andSupplementary Fig. 10c, d). This evidence indicates that CK1aspecifically phosphorylates CARMA1 at S608, which impairs its abilityto activate NF-kB.

Using a doxycycline-inducible shRNA retroviral library, we con-ducted a systematic screen for essential survival genes for twomolecularly and clinically distinct lymphoma types, ABC and GCB(germinal centre B-cell-like) DLBCL12,23,24. ABC, but not GCB,DLBCL cells rely on constitutive NF-kB activation for proliferationand survival25. Previously, this screen identified CARMA1, BCL10and MALT1 as critical components for ABC DLBCL cell survival12.Additional screening uncovered two CK1a shRNAs that were

selectively lethal for ABC DLBCL cell lines (Fig. 4a). We cloned theseCK1a shRNA sequences into a GFP-expressing retroviral vector12,and infected cell lines representing ABC DLBCL, GCB DLBCL andmultiple myeloma. CK1a shRNA expression decreased the fraction ofGFP-positive, shRNA-expressing cells over time in all four ABCDLBCL cell lines but not in GCB DLBCL or multiple myeloma celllines, confirming that CK1a knockdown is specifically lethal to ABCDLBCL cells (Fig. 4b).

To evaluate CK1a participation in the NF-kB pathway in ABCDLBCL, the OCI-Ly3 cell line harbouring an IKK activity reporterconsisting of an IkBa–luciferase fusion protein was used12. If IKK isinactivated, there is less phosphorylation and degradation of theIkBa–luciferase fusion protein and luciferase activity rises.Induction of CK1a shRNA expression increased luciferase activity,as did shRNAs targeting IKK-b (also called IKBKB), CARMA1,MALT1 and BCL10, whereas control shRNAs targeting the Spi-Btranscription factor (SPIB) and EGFP had no effect (Fig. 4c).Consistently, CK1a knockdown decreased NF-kB in the nuclei of

0

5

10

15

20

25

30

NF-

κB a

ctiv

ity (R

LU)

NF-

κB a

ctiv

ity (R

LU)

siRNA

mCK1α

NS CK1α

–

NS

WT

CK1α NS CK1α

D136N

0 0.25 0.5 1

Anti-CD3/28(µg ml–1)

a b

WT CK1α CK1α (D136N)

p-IκBαIκBα

Anti-CD3/28 (min) 30 0 7.5 15 30 0 7.5 15

β-actin

p-ERK

+ + +M/V–CARMA1

WT

D13

6N

mCKα

Anti-Myc(CARMA1)

Hsp90

–c d

e f g

104 660 CC Linker

600 610

CARMA1-CCL

ERYSFGPSSIH

WT

WT

D13

6N

WT

D13

6N

M/Cer–CCL 104–616

Anti-Myc

2SA

CK1α WT

D13

6N

S60

3A

S60

7A

WT

D13

6N

WT

D13

6N

S60

8A

mDIA1 Anti-Myc

M/V–CCL

CK1α WT

D13

6N

WT

D13

6N

104–

606

104–

616

mDIA10

20406080

100120140160180

0 1 10PMA (ng ml–1)

EVWT2SAS603AS607AS608A

–

WT HA–CK1α

+ + M/V–CCL

Anti-Myc

Anti-HA

D13

6N +

CK1α

CCL

Figure 3 | CK1a kinase activity contributes to the negative feedbackcontrol of the CBM complex and NF-kB activation. a, NF-kB luciferase assay(mean and s.d. of triplicate experiments) of nonspecific (NS)- and CK1a-silenced Jurkat cells reconstituted with an empty vector (2), wild-type CK1a(WT) or a D136N mutant mouse CK1a (mCK1a). RLU, relative luciferaseunits. b, Immunoblot of Jurkat cells expressing wild-type CK1a ormCK1a(D136N) stimulated with 1 mg ml21 anti-CD3 and anti-CD28.c, Immunoblot of HEK293T cells expressing Myc/Venus (M/V)–CARMA1together with an empty vector (2), wild-type CK1a, or mCK1a(D136N).Filled and open triangles indicate CARMA1 and its shifted form,respectively; Hsp90, loading control. d, Schematic of CARMA1 coiled-coillinker region (CARMA1-CCL(104–660)). Red circles indicate serine/threonine residues. An immunoblot of HEK293T cells overexpressing avector alone (2), wild-type CK1a, and CK1a(D136N) with M/V–CARMA1-CCL(104–660) is shown. Open and filled arrowheads indicatephosphorylated and dephosphorylated CCL, respectively. e, Experiment asin d using M/V-tagged CCL residues 104–616 or 104–606. Diaphanous(mDIA1) was used as a loading control. f, Experiment as in d using Myc/cerulean (M/Cer)-tagged CCL(104–616) with the indicated serine to alaninesubstitution. g, NF-kB luciferase assay (mean and s.d. of triplicateexperiments) of CARMA1-deficient Jurkat JPM50.6 cells reconstituted witheither an empty vector (EV) or the indicated CARMA1 plasmid andstimulated with PMA and 1 mg ml21 anti-CD28.

LETTERS NATURE | Vol 458 | 5 March 2009

94 Macmillan Publishers Limited. All rights reserved©2009

OCI-Ly3 cells and reduced IkBa phosphorylation in HBL1, U2932and OCI-Ly3 cell lines (Supplementary Fig. 11). Thus, CK1a isrequired for the IKK–NF-kB signalling pathway in ABC DLBCL cells.We also detected a strong binding of CK1a to MALT1 and ubiqui-tinated MALT1 in ABC DLBCL but not GCB DLBCL cell lines(Fig. 4d). Some ABC DLBCLs harbour mutant forms of CARMA1that constitutively activate NF-kB and generate cytoplasmic aggre-gates that co-localize with MALT1 and IKK26. Such CARMA1 aggre-gates also contain CK1a, further implicating CK1a in CBM complexactivity in ABC DLBCL (Fig. 4e). We next tested whether the lethalityof an shRNA targeting the CK1a 39-untranslated region could beprevented by wild-type CK1a or CK1a(DC), which does not bindCARMA1. Wild-type CK1a, but not CK1a(DC), rescued two ABCDLBCL cell lines from CK1a shRNA toxicity (Fig. 4f). In contrast,neither wild type nor CK1a(DC) rescued ABC DLBCL cells fromCARMA1 shRNA toxicity. Thus, direct interaction of CK1a withCARMA1 is apparently required for ABC DLBCL cell survival.

Our results have shown CK1a to be an important bifunctionalmodulator of lymphocyte adaptive immune responses. WhereasCK1a associates with CARMA1 and positively conveys TCR-inducedNF-kB, it also phosphorylates CARMA1, thereby dampening signal-ling. This provides a conceptual framework for CK1a as a signalling

gate, using both positive and negative influences to control the flowof information leading to gene induction. The phosphorylation andinactivation of CARMA1 by CK1a is reminiscent of the GSK3b/CK1a cooperation to promote b-catenin destruction27, and ourpreliminary data indicate that CK1a might contribute to CARMA1degradation (Supplementary Fig. 10). We also provide genetic, bio-chemical and functional evidence that CK1a is an essential particip-ant in the aberrant NF-kB activity required for ABC DLBCL subtypesurvival. Similar to IKK-b22, the positive function of CK1a supplantsits negative role, probably because of the constitutive upstreamactivation signals. Of note, CK1a was neither mutated nor amplifiedin ABC DLBCL cell lines (data not shown), indicating that this CK1adependency resembles the ‘non-oncogene addiction’ phenomenonin which the cancer cell phenotype depends on specific cellulargenes28,29. These ‘conditionally essential malignancy’ genes may ormay not be oncogenes or the initiator genes for cancer, but they areessential for the propagation of a specific transformed phenotype andtherefore attractive therapeutic targets28,29. Notably, CK1a is requiredfor the survival of ABC DLBCL cells with either mutant or wild-typeCARMA1, revealing it to be a conditionally essential malignancygene. However, CK1a is a complex target for chemotherapy givenits contrasting roles in signalling.

200 250 300

EGFPSPIB

BCL10MALT1

CARMA1IKK-βCK1α

Day 1Day 2Day 3Day 4

Time ofshRNA

induction

IκB–luciferaseshRNA induced/uninduced (%)

shRNA

50 100 1500

3 7 11 14

160

Days after CK1α shRNA transduction

shRNA-transduced

cells(% of day 3

fraction)

020406080

100120140 OCI-Ly3

OCI-Ly10HBL-1U2932

ABC DLBCL

BJAB

H929L363

GCB DLBCL

Myeloma

CK1α 1

OCI-Ly3OCI-Ly10

OCI-Ly7OCI-Ly19

shRNAbar-codedepletion

log2(shRNA uninduced/

shRNA induced)

shRNA: CK1α 2

1

2

3

4

0

ABC DLBCL

GCB DLBCL

a b

c d

f

shRNAtransduced

cells(% of day2 fraction)

0

20

40

60

80

100

120

2 6 9 12 15 2 6 9 12 150

20

40

60

80

100

120OCI-Ly10HBL-1

Days after shRNAtransduction

0

20

40

60

80

100

120

2 6 9 12 15 2 6 9 12 150

20

40

60

80

100

120

Days after shRNAtransduction

Days after shRNAtransduction

Days after shRNAtransduction

OCI-Ly10HBL-1CK1α shRNA CARMA1 shRNA

NoneWT CK1αCK1α(∆C)

Rescuevector

NoneWT CK1αCK1α(∆C)

Rescuevector

CK1α

MALT1

IP CK1α

ABC DLBCL GCB DLBCL

OC

I-Ly

3

OC

I-Ly

7

OC

I-Ly

19

HB

L1

U29

32

BJA

B

Ub CK1α

HA–CARMA1

Merge

e

Figure 4 | Role of CK1a in ABC DLBCL survival and NF-kB signalling. a, Agenetic screen using 1,854 shRNA vectors targeting 683 genes identified twoCK1a shRNAs that block the survival of ABC but not GCB DLBCL cell lines.Shown are the relative fluorescent signals from bar-code microarrayscomparing shRNA-induced versus uninduced cells 21 days after shRNAinduction. Data are mean 6 s.d. of four independent infections of theshRNA retroviral library. b, Survival analysis by flow cytometry of theindicated lymphoma and multiple myeloma cell lines retrovirally infected toexpress CK1a shRNA and GFP. c, An OCI-Ly3 cell line stably expressing anIkBa–luciferase reporter was retrovirally infected with the indicated

shRNAs. Shown is the percentage of luciferase activity in shRNA-inducedcells compared to uninduced cells. d, Immunoprecipitation/immunoblot ofcell lysates from the indicated cell lines. Ub, ubiquitin. e, Immunofluorescentstaining for CARMA1 mutant 326 (HA epitope), and endogenous CK1a inOCI-Ly19 cells retrovirally transduced to express HA-tagged CARMA1mutant 3. Shown are two adjacent cells with nuclei counterstained (blue).f, Indicated ABC DLBCL cells were transduced with wild-type CK1a orCK1a(DC) (D283–337), and subsequently with vectors co-expressing GFPand either CK1a shRNA or CARMA1 shRNA. The GFP1 cell fraction wasmonitored as in b.

NATURE | Vol 458 | 5 March 2009 LETTERS

95 Macmillan Publishers Limited. All rights reserved©2009

METHODS SUMMARYCell culture and reagents. Jurkat E6.1, BJAB and HEK293T cells were purchased

from ATCC. Peripheral blood T cells were isolated from normal healthy donors

as previously described30. CARMA1-deficient JPM50.6 Jurkat cells were pro-

vided by X. Lin11. Lymphocytes were activated with anti-CD3 and anti-CD28

(BD Biosciences), or with PMA (Sigma) and ionomycin (Sigma), or TNF-a(R&D). siRNAs used (Invitrogen) were BCL10, 59-GCCACGAACAACCUC-

UCCAGAUCAA-39; CK1a.2, 59- CCTAGCCTCGAAGACCTCTTCAATT-39;

CK1a.3, 59-GGCAAGGGCTAAAGGCTGCAACAAA-39; PKCh, 59-AAAUGG-UGAUUUCACUUUCGGCCGG-39; p65, 59-GAGCACCAUCAACUAUGAUG-

AGUUU-39.

CARMA1-binding-partner screen by mass spectrometry. CARMA1 was immu-

noprecipitated from HEK293T cells overexpressing V5-tagged CARMA1.

CARMA1-containing complexes were resolved on SDS–PAGE and stained with

colloidal Coomassie blue (Invitrogen). Bands were excised, in-gel trypsin

digested, and subjected to LC-MS/MS mass spectrometry analysis.

shRNA library screen. A retroviral shRNA library was constructed in the modi-

fied pRSMX plasmid containing a doxycycline-inducible H1 promoter for shRNA

expression and a random 60-mer ‘bar-code’ sequence. The association of each

bar-code with an shRNA sequence in each library plasmid was determined by

sequencing. Screening used engineered cells that express the bacterial tetracycline

repressor (TETR)12. 500–1,000 individual shRNA plasmids were combined togenerate retroviral pools, which were used to infect TETR-expressing lymphoma

and multiple myeloma cell lines. Puromycin-selected, infected cells were induced

with doxycycline for shRNA expression in half of the culture. Three weeks

after shRNA induction, bar-code sequences were amplified from the genomic

DNA of induced and uninduced cells, fluorescently labelled, and hybridized to

bar-code microarrays to identify shRNA vectors that were relatively depleted

from the induced population. The effective CK1a shRNA sequences identified

from the screen are: CK1a 1, 59-GACTCTGCATTAACTCTATAA-39 and CK1a 2,

59-GAGCAAGCTCTATAAGATTCT-39.

Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.

Received 28 July; accepted 5 November 2008.Published online 31 December 2008.

1. Hacker, H. & Karin, M. Regulation and function of IKK and IKK-related kinases. Sci.STKE 2006, re13 (2006).

2. Schulze-Luehrmann, J. & Ghosh, S. Antigen-receptor signaling to nuclear factorkB. Immunity 25, 701–715 (2006).

3. Egawa, T. et al. Requirement for CARMA1 in antigen receptor-induced NF-kBactivation and lymphocyte proliferation. Curr. Biol. 13, 1252–1258 (2003).

4. Gaide, O. et al. CARMA1 is a critical lipid raft-associated regulator of TCR-inducedNF-kB activation. Nature Immunol. 3, 836–843 (2002).

5. Hara, H. et al. The MAGUK family protein CARD11 is essential for lymphocyteactivation. Immunity 18, 763–775 (2003).

6. Jun, J. E. & Goodnow, C. C. Scaffolding of antigen receptors for immunogenicversus tolerogenic signaling. Nature Immunol. 4, 1057–1064 (2003).

7. Newton, K. & Dixit, V. M. Mice lacking the CARD of CARMA1 exhibit defective Blymphocyte development and impaired proliferation of their B and T lymphocytes.Curr. Biol. 13, 1247–1251 (2003).

8. Ruefli-Brasse, A. A., French, D. M. & Dixit, V. M. Regulation of NF-kB-dependentlymphocyte activation and development by paracaspase. Science 302, 1581–1584(2003).

9. Ruland, J. et al. Bcl10 is a positive regulator of antigen receptor-induced activationof NF-kB and neural tube closure. Cell 104, 33–42 (2001).

10. Ruland, J., Duncan, G. S., Wakeham, A. & Mak, T. W. Differential requirement forMalt1 in T and B cell antigen receptor signaling. Immunity 19, 749–758 (2003).

11. Wang, D. et al. A requirement for CARMA1 in TCR-induced NF-kB activation.Nature Immunol. 3, 830–835 (2002).

12. Ngo, V. N. et al. A loss-of-function RNA interference screen for molecular targetsin cancer. Nature 441, 106–110 (2006).

13. Price, M. A. CKI, there’s more than one: casein kinase I family members in Wntand Hedgehog signaling. Genes Dev. 20, 399–410 (2006).

14. Oeckinghaus, A. et al. Malt1 ubiquitination triggers NF-kB signaling upon T-cellactivation. EMBO J. 26, 4634–4645 (2007).

15. Bidere, N., Snow, A. L., Sakai, K., Zheng, L. & Lenardo, M. J. Caspase-8 regulationby direct interaction with TRAF6 in T cell receptor-induced NF-kB activation. Curr.Biol. 16, 1666–1671 (2006).

16. Matsumoto, R. et al. Phosphorylation of CARMA1 plays a critical role in T cellreceptor-mediated NF-kB activation. Immunity 23, 575–585 (2005).

17. Sommer, K. et al. Phosphorylation of the CARMA1 linker controls NF-kBactivation. Immunity 23, 561–574 (2005).

18. Shinohara, H. et al. PKC b regulates BCR-mediated IKK activation by facilitatingthe interaction between TAK1 and CARMA1. J. Exp. Med. 202, 1423–1431 (2005).

19. Shambharkar, P. B. et al. Phosphorylation and ubiquitination of the IkB kinasecomplex by two distinct signaling pathways. EMBO J. 26, 1794–1805 (2007).

20. Davidson, G. et al. Casein kinase 1 c couples Wnt receptor activation tocytoplasmic signal transduction. Nature 438, 867–872 (2005).

21. Peters, J. M., McKay, R. M., McKay, J. P. & Graff, J. M. Casein kinase I transducesWnt signals. Nature 401, 345–350 (1999).

22. Wegener, E. et al. Essential role for IkB kinase b in remodeling Carma1-Bcl10-Malt1 complexes upon T cell activation. Mol. Cell 23, 13–23 (2006).

23. Alizadeh, A. A. et al. Distinct types of diffuse large B-cell lymphoma identified bygene expression profiling. Nature 403, 503–511 (2000).

24. Rosenwald, A. et al. The use of molecular profiling to predict survival afterchemotherapy for diffuse large-B-cell lymphoma. N. Engl. J. Med. 346, 1937–1947(2002).

25. Davis, R. E., Brown, K. D., Siebenlist, U. & Staudt, L. M. Constitutive nuclear factorkB activity is required for survival of activated B cell-like diffuse large B celllymphoma cells. J. Exp. Med. 194, 1861–1874 (2001).

26. Lenz, G. et al. Oncogenic CARD11 mutations in human diffuse large B celllymphoma. Science 319, 1676–1679 (2008).

27. Liu, C. et al. Control of b-catenin phosphorylation/degradation by a dual-kinasemechanism. Cell 108, 837–847 (2002).

28. Shaffer, A. L. et al. IRF4 addiction in multiple myeloma. Nature 454, 226–231(2008).

29. Solimini, N. L., Luo, J. & Elledge, S. J. Non-oncogene addiction and the stressphenotype of cancer cells. Cell 130, 986–988 (2007).

30. Su, H. et al. Requirement for caspase-8 in NF-kB activation by antigen receptor.Science 307, 1465–1468 (2005).

Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements This work was supported by the Intramural Research Programof the NIH, NIAID, NCI, NIDDK and by the Agence Nationale de la Recherche(ANR). We thank M.-T. Auffredou for technical assistance; X. Lin and J. Gavard forreagents; R. Germain, R. Schwartz, U. Siebenlist, P. Schwartzberg, J. Bosco deOliveira, L. Yu, D. Baltimore, P. Sharp, H. Varmus and A. Snow for discussions andcomments; and S. Porcella and the DNA sequencing core facility of the RockyMountain Laboratories, NIAID.

Author Contributions N.B., V.N.N., J.L., C.C., L.Z., F.W., R.E.D., G.L., L.M.S. andM.J.L. carried out experimental design/discussion; N.B., V.N.N., J.L., C.C., L.Z.,F.W., R.E.D., G.L., D.E.A., D.A., A.V., K.S., J.Z., Z.M. and T.D.V. carried outpreparation and performance of experiments; N.B., V.N.N., L.M.S. and M.J.L.carried out manuscript preparation. All authors discussed the results andcommented on the manuscript.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. Correspondence and requests for materials should beaddressed to M.J.L. (lenardo@nih.gov) or L.M.S. (lstaudt@mail.nih.gov).

LETTERS NATURE | Vol 458 | 5 March 2009

96 Macmillan Publishers Limited. All rights reserved©2009

METHODSExpression plasmids, transfections, antibodies and reagents. cDNAs from

Open Biosystems and Origene were further cloned into HA-tagged-pCI-neo,

pcDNA3.1D/V5-His-TOPO (Invitrogen), or pCEFL-Myc/Venus (M/V) express-

ion vectors. CARMA1 mutant expression constructs, and human or mouse CK1akinase-dead constructs (K46R, D136N and D136A), were generated by site-directed

mutagenesis, and were verified by sequencing (Rocky Mountain Laboratories;

Genome express, Cogenics). The truncated CK1a and CARMA1 constructs were

made by polymerase chain reaction. HEK293T cells were transfected with Exgen

(Fermentas). Primary blood T lymphocytes and Jurkat cells were transfected aspreviously described15. Antibodies to CK1a, BCL10, MALT1, IkBa, NF-kB p65 and

mDIA1 were from Santa Cruz. Antibodies tob-actin, to Flag and to HA (Sigma), to

Myc, and to the phosphorylated forms of IkBa, ERK and JNK (Cell Signaling

Technologies), to PARP, IKK-b and to CD25 (BD Pharmingen), to GFP

(Roche), to V5 and to phospho-serine (Invitrogen), to phospho-tyrosine

(Millipore), and to CARMA1 (Alexis) were also used. HRP-conjugated secondary

antibodies were from Southern Biotechnology. Amersham ECL or Pierce

Supersignal West chemiluminescent substrates were used for immunoblot detec-

tion. The calcium-sensitive dye Fluo-3 was from Invitrogen, and CFSE was

purchased from Sigma.

Cell lysates and immunoprecipitations. Stimuli were terminated by addition of

ice-cold PBS, and cell pellets were lysed with TNT buffer (50 mM Tris pH 7.4,

150 mM NaCl, 1% Triton X-100, 1% NP-40, 2 mM EDTA) supplemented with

complete proteases inhibitors (Roche) for 30 min. Samples were cleared by a

centrifugation at 15,000g at 4 uC and pre-cleared with protein G-sepharose

(Roche) for 1 h before immunoprecipitation with antibodies and additional

protein G-sepharose at 4 uC. To evaluate CARMA1 phosphorylation, cells were

lysed with TNT buffer containing 1 mM Na3VO4, 1 mM NaF, and complete prote-ases inhibitors (Roche). Precipitates were washed with TNT buffer without EDTA,

and incubated for 30 min at 30 uC with l-phosphatase (NEB) according to the

manufacturer’s instructions. Cytosolic and nuclear extracts were prepared as

previously described30. For immunodepletion experiments, cell lysates were first

incubated with antibodies against CK1a and protein G-sepharose. Supernatants

were collected and underwent three sequential rounds of protein G-sepharose

treatment previously and were then immunoprecipitated with BCL10 antibodies.

Immunofluorescence, ELISA and luciferase. For immunofluorescence studies,

5 3 105 Jurkat T cells were incubated on ice for 20 min with 10 mg ml21 anti-CD3

(clone OKT3), washed twice with ice-cold PBS and CD3 receptors were cross-

linked with 5mg ml21 donkey anti-mouse for 15 min on ice or at 37 uC. Samples

were then processed as previously described15,30. IL-2 production was deter-

mined 12 h after stimulation in the culture supernatants by an enzyme-linked

immunosorbent assay (ELISA). Briefly, Immulon 4 plates coated with 2 mg ml21

anti-human IL-2 capture antibody (R&D) were washed three times with 0.05%

Tween-20 in PBS (PBS-T) and blocked with 0.5% BSA in PBS, for 2 h. The plates

were then washed three times and incubated with triplicates of hIL-2 standards

and the collected supernatants for 2 h. After three washes, plates were incubated

for 2 h with biotin-anti human IL-2 antibody (R&D). The plates were washed

three times and incubated with streptavidin-HRP (R&D) for 30 min. Wells were

washed again and developed with TMB substrate (Sigma) for 30 min in the dark.

Reactions were stopped by adding 2 N H2SO4 and the optical density at 450 nm

was read through a BioRad plate reader. IL-2 concentrations were calculated

from a regression formula derived from the standard curve. For luciferase

reporter assays, firefly luciferase constructs driven by NF-kB binding sequences

and Renilla luciferase pRL-TK (Int2) plasmid were used (Promega). Lysates

were analysed using the Dual-Luciferase Kit (Promega), with firefly fluorescence

units normalized to Renilla luciferase fluorescence units.

Survival assay. A survival assay was done as previously described12. Briefly,

effective shRNAs identified from the library screen were re-cloned into a variant

of pRSMX plasmid that expresses GFP. Cells were infected with retroviruses

prepared from this plasmid and the fraction of GFP1 cells was measured over

time by flow cytometry. At each time point, the observed GFP1 fractions in

cultures transduced with an shRNA of interest were normalized to the GFP1

fractions in control cultures transduced with luciferase shRNA. The toxicity of an

shRNA is indicated by the reduction over time in the GFP1 fraction compared

with the GFP1 fraction at the earliest time point (day 3) after infection.

IKK reporter assay. IKK reporter cell lines (see ref. 12) were created by sequen-

tially transducing TETR-expressing cells with IkBa–Photinus and Renilla luci-

ferase-expressing retroviruses. IkBa–Photinus luciferase was a fusion protein

used for measuring IKK activity, and Renilla luciferase was for normalizing viable

cell number. IKK reporter cell lines were transduced with shRNA-expressing

retroviruses and selected with puromycin. IKK reporter values were measured

over time with a luminescence microplate reader in uninduced and doxycycline-

induced cultures, using the Dual-Glo luciferase assay system (Promega).

Electrophoretic mobility shift assays. Nuclear protein extracts were obtained

from the OCI-Ly3 cell line, and EMSAs were carried out with an EMSA kit

(Promega) with modifications, as previously described31. IgkB double-stranded

oligonucleotide probes (sense strand shown) used were: 59-AGTTGAGGGG

ACTTTCCCAGG-39. 100-fold unlabelled immunoglobulin kB (WT) was added

for cold competition analyses. Samples were resolved on a 6% DNA retardation

gel (Invitrogen) in 0.253 TBE buffer, and autoradiography was carried out on

dried gels.

31. Wan, F. et al. Ribosomal protein S3: a KH domain subunit in NF-kB complexes thatmediates selective gene regulation. Cell 131, 927–939 (2007).

doi:10.1038/nature07613

Macmillan Publishers Limited. All rights reserved©2009