GSK3-SCFFBXW7 Targets Junb for Degradation in G2 to Preserve Chromatid Cohesion Before Anaphase

ORIGINAL ARTICLE GSK3-SCFFBXW7 targets JunB for degradation in G2 to preserve chromatid cohesion before anaphase

BPe´ rez-Benavente1, JL Garcı´a2, MS Rodrı´guez3, A Pineda-Lucena4, M Piechaczyk5, J Font de Mora6 and R Farra`s1

JunB, an activator protein-1 (AP-1) transcription factor component, acts either as a tumor suppressor or as an oncogene depending on the cell context. In particular, JunB is strongly upregulated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) where it enhances cell proliferation. Although its overexpression is linked to lymphomagenesis, the mechanisms whereby JunB promotes neoplastic growth are still largely obscure. Here, we show that JunB undergoes coordinated phosphorylation-dependent ubiquitylation during the G2 phase of the cell cycle. We characterized a critical consensus phospho- degron that controls JunB turnover and identiﬁed GSK3 and SCFFBXW7 as, respectively, the kinase and the E3 ubiquitin ligase responsible for its degradation in G2. Pharmacological or genetic inactivation of the GSK3-FBXW7-JunB axis induced accumulation of JunB in G2/M and entailed transcriptional repression of the DNA helicase DDX11, leading to premature sister chromatid separation. This abnormal phenotype due to dysregulation of the GSK3b/JunB/DDX11 pathway is phenocopied in ALK-positive ALCL. Thus, our results reveal a novel mechanism by which mitosis progression and chromatid cohesion are regulated through GSK3/SCFFBXW7-mediated proteolysis of JunB, and suggest that JunB proteolysis in G2 is an essential step in maintaining genetic ﬁdelity during mitosis.

Oncogene (2013) 32, 2189–2199; doi:10.1038/onc.2012.235; published online 18 June 2012 Keywords: JunB; G2 phase; ubiquitin-proteasome; NPM-ALK; GSK3; DDX11

INTRODUCTION JunB is a member of the Jun family, which also comprises c-Jun Anaplastic large cell lymphoma (ALCL) is an aggressive type of and JunD, and is one of the components of the Activator Protein-1 non-Hodgkin lymphoma of the T-cell/null lineage frequently (AP-1) transcription complex. AP-1 is a collection of dimers formed associated with chromosomal translocations involving the ana- by members of the Jun-, Fos-, ATF- and Maf multigene families plastic lymphoma kinase (ALK) locus on chromosome 2.1 The most that bind to speciﬁc DNA regulatory elements called AP-1/12-O- common translocation is t(2;5)(p23;q35), which produces a fusion tetradecanoylphorbol-13-acetate-responsive elements (TREs) 11–13 between the kinase coding portion of ALK to the oligomerization and cAMP-responsive elements (CREs). It can exert opposite domain coding a portion of the nucleophosmin gene (NPM1) actions depending on the cell and physiopathological 14–21 resulting in the expression of the NPM-ALK fusion oncogenic context. For example, JunB can show cell-cycle inhibitory kinase.2 NPM-ALK activates oncogenic signaling via phospholipase and tumor suppressor activities by transcriptional induction of the C-g, phosphotidylinositol 3-kinase (PI3K)/serine threonine kinase cyclin kinase-dependent inhibitor p16INK4a and transcriptional 22,23 AKT, RAS/MAPK and Janus kinase/signal transducer and activator repression of Cyclin D1. On the other hand, JunB can exert of transcription pathways resulting in enhanced cell proliferation positive actions on the cell cycle by activating Cyclin A2 (CCNA2) 24,25 and inhibition of apoptosis.3 Chromosomal abnormalities transcription. Moreover, overexpression of JunB was shown to 10,20 occurring in association with ALK translocation in ALCLs have contribute to lymphomagenesis. In this context, activation of been also described. In fact, ALCLs are characterized by a complex the CD30 promoter by abnormally overexpressed JunB is essential karyotype, different numerical and structural chromosomal for the pathogenesis of ALCL, Hodgkin lymphoma and 26–29 abnormalities and chromosomal imbalances.4–9 The JunB lymphomatoid papulosis. transcription factor has been shown to contribute to the Therefore, proper regulation of JunB protein levels and activity pathogenesis of ALK-positive ALCLs10 where its abnormally high are crucial for the preservation of normal cellular functions. In accumulation is explained by at least two mechanisms: particular, JunB is subjected to regulated destruction via the 30–32 (i) increased JUNB transcription dependent on Erk1/2 kinase ubiquitin-proteasome pathway in various situations. For activation by NPM-ALK and (ii) increased JUNB translation example, activation of the Jun amino-terminal kinase accelerates mediated by mTOR activation by AKT, which is induced by JunB degradation after T-cell stimulation via phosphorylation- 32 NPM-ALK via activation of PI3K. However, how JunB promotes dependent activation of the E3 ligase AIP4, which recognizes a 30 ALCL lymphomagenesis is still unclear. PPVY motif within JunB sequence. JunB expression is tightly

1Cytomics Laboratory, Centro de Investigacio´ nPrıńcipe Felipe, Valencia, Spain; 2Unidad de Investigacioń, Instituto Estudios Ciencias de la Salud de Castilla y Leon-(IECSCYL)- Hospital Universitario de Salamanca, Centro de Investigacioń del Cańcer Universidad de Salamanca-CSIC, Salamanca, Spain; 3Proteomics Unit, CIC bioGUNE, Bizkaia, Spain; 4Structural Biochemistry Laboratory, Centro de InvestigaciońPrıńcipe Felipe, Valencia, Spain; 5Institut de Geńe´tique Molećulaire de Montpellier UMR 5535 CNRS, Universite´ Montpellier 2, Universite´ Montpellier 1, Montpellier Cedex 2, France and 6Cellular and Molecular Biology Laboratory, Centro de InvestigaciońPrıńcipe Felipe, Valencia, Spain. Correspondence: Dr R Farra`s, Cytomics Laboratory, Centro de InvestigaciońPrıńcipe Felipe, c/EP Autopista del Saler 16, 46012 Valencia, Spain. E-mail: [email protected] Received 1 January 2012; revised 30 March 2012; accepted 2 May 2012; published online 18 June 2012 Regulation of JunB turnover in G2 by GSK3b and FBXW7 BPe´rez-Benavente et al 2190 regulated during the cell cycle. Its levels are high in S phase and Supplementary Figure S2). Altogether, our data point to a role for decrease by mid-to-late G2 due to accelerated proteasome- FBXW7 in JunB degradation. dependent degradation.22,25 However, the molecular mechanisms involved in JunB turnover in G2 are unknown as well as the genes Phosphorylation of JunB at GSK3 sites is required for FBXW7- which might be affected by JunB accumulation in G2/M. dependent ubiquitylation Here, we identified SCFFBXW7 as the E3 ubiquitin ligase targeting Phosphorylation at position þ 4 of the CPD serves as a priming JunB for degradation in mid/late G2 and excluded any role for signal for GSK3 phosphorylation that allows the efficient recogni- b 33 AIP4 in this process. We also describe GSK3 as the kinase tion of the phosphorylated substrate by FBXW7. In addition to responsible for the initiation of JunB ubiquitylation via phosphor- the JunB T255, the Prosite Motif Scan Program identified JunB ylation of a phospho-degron that is conserved among critical cell- S251 as potential phosphorylation sites for GSK3 (Figure 1c). To cycle regulators. Impaired JunB degradation in late G2 resulted in investigate whether GSK3b was involved in the phosphorylation of transcriptional repression of the DNA helicase DDX11 and sister JunB at S251 and/or T255, we generated specific anti-JunB chromatid cohesion defects, underlining that physiological phospho-antibodies. Ectopic expression in HeLa cells of a degradation of JunB in late G2 is critical for proper mitosis. constitutively active form of AKT (AKT-E40K),39 which inhibits Finally, we report that, in ALK-positive ALCLs, inactivation of GSK3b activity via its phosphorylation at S9, reduced b GSK3 mediated by the ALK/PI3K/AKT signaling pathway con- phosphorylation of JunB at T255, but not at S259 (Figure 2a). tributes to JunB protein stabilization and DDX11 repression in Similarly, pharmacological inhibition of GSK3b with the GSK3b G2/M, resulting in chromatid cohesion defects. Hence, our results inhibitor VIII reduced phosphorylation of ectopic JunB at T255 and describe the molecular events that coordinate JunB degradation also at S251 (Figure 2b). Finally, ectopic expression of JunB mutants during G2/M transition and elucidate a new mechanism whereby in which S251, T255 and S259 were mutated into alanine indicated JunB may contribute to tumorigenesis. that phosphorylation at S251 was not affected by mutation of T255 and vice-versa, whereas phosphorylation of both S251 and T255 was suppressed by mutation of S259 (Figures 2c and d). Thus, JunB RESULTS phosphorylation at S251 and T255 by GSK3b is primed by AIP4 is not involved in targeting JunB for degradation in mid/late G2 phosphorylation at S259 by a yet to-be-identified kinase. We first investigated the mechanisms involved in JunB turnover We also observed that JunB levels were increased in the during G2 phase in cycling cells that exhibit normal regulation of presence of the GSK3b inhibitor VIII (Figure 2b; see the JunB-HA JunB. In particular, we asked whether AIP4 was involved in JunB lane), suggesting that GSK3b phosphorylation of JunB CPD might degradation in this specific phase of the cell cycle. Although AIP4 be instrumental for its degradation by the ubiquitin-proteasome is stably expressed in synchronized HeLa cells from mid-S to system. To test this hypothesis, we first compared the stability of mitosis (Figure 1a), its siRNA-mediated depletion did not lead to non-phosphorylatable JunB mutants where residues S251, T255 JunB accumulation in G2 and prometaphase (M) HeLa cells and S259 were replaced by alanines (see Supplementary Figure (Figure 1b). In line with this, JunB protein with a mutation in the S3a for a detailed description of the different JunB mutants). AIP4 recognition motif (Y179F-JunB) followed similar pattern of Cycloheximide chase experiments indicated that the double and expression of wild-type JunB during cell-cycle progression. Both triple mutants T255A/S259A-JunB and S251A/T255A/S259A-JunB Y179F-JunB and wild-type JunB protein levels rose during S-phase strongly stabilized JunB (Supplementary Figures S3b and c). progression and rapidly decreased at late G2 before Cyclin A2 Moreover, S251A/T255A/S259A-JunB did not interact any longer degradation (Supplementary Figure S1). This further excluded a with FBXW7a in co-immunoprecipitation assays (Figure 2e) and major role for AIP4 in the degradation of JunB in G2. FBXW7a stimulated ubiquitylation of wild-type JunB, but not of S251A/T255A/S259A-JunB (Figure 2f). Taken together, our data FBXW7 promotes JunB degradation in mid/late G2 indicate that FBXW7a promotes ubiquitin-dependent degradation of JunB in a GSK3b-dependent manner. In our search for the E3 ligase involved in JunB degradation in G2, we noted that the JunB sequence contains a potential FBXW7 phospho-degron motif, known as Cdc4 phospho-degron (CPD) GSK3b regulates JunB turnover in G2 that included T255 and S259 (Figure 1c). The F-box and WD40 To assess whether JunB degradation in mid/late G2 was repeat domain-containing 7 (FBXW7/hCdc4) protein is the dependent on GSK3b activity, we analyzed JunB decay and substrate recognition component of the so-called SCF ubiquitin GSK3b activity as determined by its S9 phosphorylation state in ligase SCFFBXW7 complex. FBXW7 is recruited and mediates synchronized U2OS cells. Following the release of G1/S block, JunB ubiquitylation and degradation of proteins after they are protein levels increased as cells progressed through S phase and phosphorylated on the CPD. It is a tumor suppressor that targets dropped 12–14 h after, concomitant with an increase of the Cyclin E, c-Myc and c-Jun for degradation during the G1-to-S mitotic index. Importantly, degradation of JunB coincided with the transition33–36 and recently it has been identified to target Mcl1 window of maximum GSK3b activity, as indicated by the decrease for degradation during mitotic arrest.37,38 Physical interaction of in GSK3b phosphorylation at S9 (Figure 3a). Phosphorylation of JunB and FBXW7a isoform was demonstrated in U2OS cells co- JunB at S259, but not at S251 and T255 (not shown), was clearly transfected with HA-tagged JunB and Flag-tagged FBXW7a. detected 4 h after release from the thymidine block, concomitant Conversely, the negative trans-dominant FBXW7a(R465A) mutant with JunB expression levels. This was consistent with the idea that that cannot bind to its substrate33 did not interact with JunB phosphorylation at S259 can prime JunB phosphorylation at S251 (Figure 1d). Moreover, other E3 ligases involved in the degradation and T255 by GSK3b that is rapidly followed by degradation. of cell-cycle regulators, namely SKP2, CDC20 and CDH1, could not Indeed, inhibition of GSK3b by the GSK3 inhibitor VIII in immunoprecipitate JunB (Figure 1e), confirming the specificity of synchronized G2 cells led to JunB stabilization without affecting this interaction. In addition, JunB turnover was slower in U2OS JunB phosphorylation at S259 (Figure 3b). cells that ectopically express FBXW7a(R465A) than in cells Similar data were obtained upon pharmacological inhibition of transfected with FBXW7a (Figure 1f). Finally, we used FBXW7 À / À GSK3b in G2-enriched cells (Figure 3c). Moreover, Cyclin A2 levels cells (DLD1FBXW7 À / À ) to demonstrate the role of FBXW7 in JunB increased after inhibition of JunB degradation, corroborating degradation during mid/late G2. Absence of FBXW7 resulted in previous reports showing that CCNA2 gene is a transcriptional slightly increased levels of JunB in G2-enriched cells and in a target of JunB.24,25 Additionally, RNAi depletion of GSK3b, but not dramatic accumulation of JunB in prometaphase cells (Figure 1g; of GSK3a, resulted in decreased phosphorylation of JunB at T255

Oncogene (2013) 2189 – 2199 & 2013 Macmillan Publishers Limited Regulation of JunB turnover in G2 by GSK3b and FBXW7 BPe´rez-Benavente et al 2191 Release from AIP4 Control G1/S blockade siRNA siRNA G1/S G1 0 24681012hr G2MM G2 AIP4 AIP4 JunB JunB Cyclin A2 Cyclin B1 Cyclin B1 GAPDH GAPDH

JunB 249 ARSRDATPPVSPI WT-JunB-HA - ++ - + + c-Jun 233 PEMPGETPPLSPI c-Myc 56 LPSGLLTPPQSGK Flag-FBXW7 + + - + + - CyclinE 374 KFELLPTPPLSPS Flag-FBXW7 R465A -+--+- 52 PC LIP PDK DD CyclinE S T E IB: anti-FLAG FBXW7α S/TPXXS/T/E 0 +4 IB: anti-HA JunB FBXW7 phosphodegron Total Extract IP: anti-HA

A FBXW7 FBXW7 R465A R 465 0 264 0 264hr JunB JunB-HAFBXW7FBXW7SKP2 CDH1CDC20 GFP IP: α-FLAG JunB IB: α-JunB DLD-1 DLD-1FBXW7 -/- AS G2 M AS G2 M FBW7α (110 kD) SKP2 (46 kD) JunB Total Extract * CDH1 (55 kD) IB: α-FLAG CDC20 (60 kD) Cyclin B1

JunB GAPDH Figure 1. JunB contains an FBXW7 phospho-degron and specifically interacts with FBXW7a.(a) Expression of AIP4 and JunB proteins in synchronized HeLa cells. Cells were synchronized at the G1/S boundary by a double thymidine/aphidicolin block. They were then washed and allowed to progress through the cell cycle for the indicated times. Protein extracts were analyzed by immunoblotting with antibodies against AIP4, JunB, Cyclin A2, Cyclin B1 and GAPDH. (b) Depletion of AIP4 does not affect JunB turnover in G2 and prometaphase HeLa cells. HeLa cells were transfected twice with control or AIP4-specific siRNAs and treated with nocodazole for 18 h. Prometaphase (M) round cells were collected by shake-off. The remaining attached cells (mainly in G2 phase) were also harvested. Protein extracts were analyzed by immunoblotting with antibodies against the indicated proteins. (c) JunB contains an FBXW7 consensus phospho-degron. The CPD contains a central phospho- threonine or serine and a negative charge at the þ 4 position. Sequence alignment of the c-Jun, c-Myc and Cyclin E FBXW7 phospho-degrons with the JunB sequence reveals a putative phospho-degron motif in JunB. (d) JunB associates with FBXW7a. U2OS cells were co-transfected with HA-tagged JunB and Flag-tagged FBXW7a or the mutant FBXW7a(R465A) and treated with the proteasome inhibitor MG132 for 6 h before harvesting. Proteins were immunoprecipitated with anti-HA resin and immunocomplexes were analyzed by immunoblotting using anti-HA and anti-Flag antibodies. (e) Specific association of JunB with FBXW7a. HEK293T cells were co-transfected with HA-tagged JunB and constructs encoding the Flag-tagged Fbox proteins FBXW7a, FBXW7a(R465A) and SKP2, as well as CDH1 and CDC20. Cells were treated for 6 h with the proteasome inhibitor MG132 before harvesting. Proteins were immunoprecipitated with anti-Flag resin and immunocomplexes were probed by western blotting with anti-JunB and anti-Flag antibodies. Asterisk indicates non-specific immunoreactive bands. (f) Expression of a dominant-negative FBXW7 mutant increases JunB half-life. Immunoblot analysis of U2OS cells co-transfected with HA-tagged JunB and Flag- tagged FBXW7a or FBXW7a(R465A), together with a plasmid encoding GFP. Cell extracts were prepared at the indicated time points after the cells were treated with cycloheximide. (g) Depletion of FBXW7 leads to accumulation of JunB in prometaphase arrested cells. DLD1 and DLD1 FBXW7 À / À cells were treated with nocodazole for 18 h and prometaphase round cells (M) were collected by shake-off. The remaining attached cells (G2) were also harvested. Nocodazole-free cultures were also grown for analysis of asynchronous cells (AS). Protein extracts were analyzed by immunoblotting with antibodies against the indicated proteins.

(Figure 3d), conﬁrming GSK3b as the kinase responsible for JunB of mitotic aberrations (misaligned chromosomes, chromatin phosphorylation at T255 in G2. We supported that JunB bridges, micronuclei and altered cytokinesis).25 Consistently, over- degradation in late G2 depends on phosphorylation of its CPD expression of the degradation-resistant S251A/T255A/S259A-JunB by following the expression of a Tet-inducible S251A/T255A/ mutant in late G2 led to comparable CCNA2 upregulation and S259A-JunB mutant in synchronized UTA6 cells (Figure 3e). mitotic defects in UTA6 cells (not shown). Moreover, due to the Differently from endogenous JunB, S251A/T255A/S259A-JunB role of FBXW7 in JunB degradation in mid/late G2, both Cyclin A2 protein was stabilized in late G2 (Figure 3e) and accumulated in protein and mRNA levels were upregulated in DLD1FBXW7 À / À cells prometaphase cells (Figure 3f). All together, our data indicate that (Figures 4a and b). To assess whether the mitotic aberrations JunB degradation in mid/late G2 depends on phosphorylation of observed upon JunB accumulation in late G2 could be due to the the FBXW7-interacting domain of JunB, with a crucial role for deregulation of other genes, we compared the mRNA abundances GSK3b in phosphorylation of at least T255. of 84 genes involved in cell-cycle regulation by RT–PCR Array in UTA6 cells that express either S251A/T255A/S259A-JunB or Impaired GSK3-FBXW7-JunB axis results in downregulation of the endogenous JunB. In agreement with previous data,22 CCND1 DNA helicase DDX11 mRNA was downregulated by a threefold factor in UTA6 cells We previously reported that overexpression of JunB in late expressing S251A/T255A/S259A-JunB mutant (Supplementary G2 upregulates CCNA2, lengthens mitosis and causes a number Table 1). However, DDX11, a gene coding for a DNA helicase

& 2013 Macmillan Publishers Limited Oncogene (2013) 2189 – 2199 Regulation of JunB turnover in G2 by GSK3b and FBXW7 BPe´rez-Benavente et al 2192 WT-JunB + +- WT-JunB + ++ - S251A/T255A/S259A-JunB -+- AKT(E40K) - +- S251A/T255A/S259A-JunB --- + GSK3 Inhibitor --+ + JunB-pT255 MG132 -++ + JunB-pS259 JunB-pT255

JunB-HA * JunB-pS251 AKT JunB-pS259 GSK3-pS9 JunB-HA GSK3α GSK3β GSK3 GFP GFP

GSK3β Unknown kinase phosphorylation priming sites phosphorylation site

WT-JunBJunB JunB(S251A) (T255A)JunB (S259A) P P P JunB WT-JunB ARSRDATPPVSPI 251 255 259 JunB-pS251 * P P JunB-pT255 JunB(S251A) ARARDATPPVSPI P P JunB-pS259 JunB(T255A) ARSRDAAPPVSPI GFP P P JunB(S259A) ARSRDATPPVAPI

WT-JunB ++ -- WT-JunB --+++ - S A/T A-JunB - --+ 251 255 S A/T A/S A-JunB + +-+ - - S A/T A/S A-JunB -- - + 251 255 259 251 255 259 His -Ubiquitin - + + - ++ Flag-FBXW7 - + ++ 6 Flag-FBXW7 -+---+ IB: α-Flag IP: -HA α IB: -HA JunB(Ub)n

α Total IB: -Flag Extract Total Extract IB: α-HA IB: -JunB Figure 2. Phosphorylation at S251, T255 and S259 is required for JunB degradation. (a) AKT inhibits JunB phosphorylation at T255. HeLa cells were transfected with JunB alone or together with a mutant AKT(E40K) which is constitutively active. Six hours before harvesting, cells were treated with 30 mM MG132. AKT(E40K) expression led to increased phosphorylation of its direct target, GSK3, as determined with an antibody against phosphorylated GSK3b at pS9. The effect of AKT(E40K) expression on JunB phosphorylation was determined by immunoblotting with each of the phospho-specific antibodies. The JunB expression plasmid in which S251, T255 and S259 are replaced by alanines (S251A/T255A/ S259A-JunB) was transfected and used as control for JunB phospho-specific antibodies. (b) Pharmacological inhibition of GSK3b reduces JunB phosphorylation at S251 and T255. HeLa cells were transfected with JunB or the triple S251A/T255A/S259A-JunB mutant. Cells were then incubated with dimethyl sulfoxide alone or the GSK3b inhibitor VIII. Where indicated, cells were incubated with MG132 6 h before harvesting. The effect of GSK3b inhibition on JunB phosphorylation was determined by immunoblotting with JunB phospho-specific antibodies. (c) Phosphorylation at S259 primes JunB for phosphorylation at S251 and T255. To determine the GSK3 priming phosphorylation site in JunB, U2OS cells were transfected with JunB or JunB single mutants, in which the S251, T255 or S259 residue was replaced by alanine. Phosphorylation of S251, T255 and S259 at JunB was assessed by immunoblotting, as described in (b). (d) Schematic of JunB phosphorylation. Phosphorylation at S259 primes JunB for GSK3-dependent phosphorylation at S251 and T255. Asterisks in (b) and (c) indicate non-specific immunoreactive bands. (e) Phospho-serine 251, phospho-threonine 255 and phospho-serine 259 are necessary for interaction of JunB with FBXW7 in vivo. Immunoblot (IB) analysis of immunoprecipitates (IP) from U2OS cells transfected with Flag-tagged FBXW7a and the indicated HA-tagged JunB proteins. (f) Ubiquitylation of JunB mediated by FBXW7 is dependent on phosphorylation at S251, T255 and S259. U2OS cells were transiently co-transfected with the indicated plasmids and treated with the proteasome inhibitor MG132 for 6 h before cell lysates were prepared. Ubiquitin conjugates were affinity purified from protein extracts by talon affinity chromatography and probed for JunB by immunoblotting.

involved in the regulation of the G2/M transition and required DLD1 cells demonstrated the in-vivo binding of endogenous JunB for sister chromatid cohesion,40–42 showed the strongest to a putative CRE binding site at position À 399 of DDX11 downregulation. Consistently, DDX11 mRNA (Figure 5a) and (Figure 5d). Furthermore, chromatin immunoprecipitation assay in protein expression (Figures 5b and c) were downregulated in DLD1FBXW7 À / À cells and S251A/T255A/S259A-JunB-UTA6 cells synchronized S251A/T255A/S259A-JunB-UTA6 and DLD1FBXW7 À / À showed enhanced binding to the same region, as well as to a cells at G2 and prometaphase. Similar, but weaker DDX11 region located downstream of the transcription initiation site downregulation was also observed in asynchronous cells. Addi- (Figure 5d). Taken together, these data support a role for the tional conﬁrmation of these results was obtained by transfecting FBXW7-JunB axis in the regulation of the DDX11 via direct wild-type JunB in HeLa cells (Supplementary Figure S4). Finally, transcriptional repression by JunB. siRNA-mediated depletion of JUNB in DLD1FBXW7 À / À cells resulted in upregulation of DDX11 at G2 and prometaphase, conﬁrming that downregulation of DDX11 is dependent on the accumulation Accumulation of JunB in mitosis results in premature sister of JunB (Supplementary Figure S5). chromatid separation in metaphase Analysis of the DDX11 genomic sequence revealed several Since DDX11 is involved in sister chromatid cohesion, we putative AP-1/CRE and TRE DNA binding sites. Chromatin performed metaphase spread analysis to visualize sister chromatid immunoprecipitation assays using control UTA6 and parental pairs in S251A/T255A/S259A-JunB-UTA6 cells. Measurement of the

Oncogene (2013) 2189 – 2199 & 2013 Macmillan Publishers Limited Regulation of JunB turnover in G2 by GSK3b and FBXW7 BPe´rez-Benavente et al 2193 Release from G1/S blockade Release from G1/S Release from G1/S in the presence of nocodazole blockade in the presence blockade in the presence G1/S M of nocodazole of nocodazole 0 4 8 1012141618 hr +GSK3 Inhibitor JunB 0410 12 14 16 0 4 10 12 14 16 hr JunB-pS259 JunB Cyclin A2 JunB-pS259

Cyclin B1 Cyclin A2 α β GSK3 / Cyclin B1 β GSK3 -pS9 GAPDH

GAPDH

/ / - - ++GSK3 Inhibitor VIII

++--DMSO siControlsiCyclinsiGSK3 A2 siGSK3 siControlsiGSK3siGSK3siCyclin B1 G2 MG2M JunB JunB JunB-HA JunB-pT255 JunB-pT255 JunB-pS259 JunB-pS259 JunB-pS259 Cyclin A2 Cyclin A2 Cyclin B1 Cyclin B1 GSK3α β GSK3 GSK3 GAPDH GSK3 β-Actin β-Actin

Hours after Hours after release from G1/S release from G1/S UTA6- -Tc -Tc UTA6- S251A/T255A/S259A- EV JunB 0 4 8 10 12 14 16 18 0 4 8 10 12 14 16 18 AS G2 M AS G2 M α-JunB α-JunB α-JunB-pS259 α-Cyclin A2 α-Cyclin A2 α-Cyclin B1 α-Cyclin B1 α β α-β-Actin - -actin UTA6-EV UTA6- S251A/T255A/S259A-JunB Figure 3. GSK3 regulates JunB degradation in late G2. (a) JunB levels inversely correlates with GSK3 activity during cell-cycle progression. U2OS cells were synchronized at the G1/S boundary by double thymidine block (indicated as time 0). Cells were then washed and allowed to progress through the cell cycle for the indicated times in the presence of nocodazole to arrest cells in mitosis. Prometaphase nocodazole- arrested cells showed higher Cyclin B1 levels, conﬁrming the progression from G1/S to M blockade. Protein extracts were analyzed by immunoblotting with antibodies against the indicated proteins. (b, c) Pharmacological inhibition of GSK3b impairs JunB degradation in mid/ late G2. In (b), U2OS cells were synchronized as in (a) and the GSK3b inhibitor VIII was added to the cells 10 h after release from the thymidine block. Protein extracts at the indicated times were analyzed by immunoblotting with antibodies against the indicated proteins. In (c), U2OS cells were treated with nocodazole for 18 h, and prometaphase round cells were collected by shake-off. The remaining attached cells (mainly in G2 phase) were also harvested. Cells were incubated with dimethyl sulfoxide or GSK3b inhibitor 6 h before collection. Protein extracts were analyzed by immunoblotting with antibodies against the indicated proteins. (d) Depletion of GSK3b inhibits phosphorylation of JunB at S251 and T255. U2OS cells were transfected twice with control or Cyclin A2-, Cyclin B1-, GSK3a-, GSK3b- or both GSK3a- and GSK3b-speciﬁc siRNAs. Nocodazole was added 24 h after the last transfection for 16 h. G2/M-enriched cells were harvested, and cell extracts analyzed by immunoblotting with antibodies against the indicated proteins. (e) S251A/T255A/S259A-JunB is not degraded and accumulates during cell- cycle progression. UTA6 cells stably transfected with the S251A/T255A/S259A-JunB Tet-off bicistronic expression plasmid (UTA6-S251A/T255A/ S259A-JunB), or empty vector (UTA6-EV) as a control, were synchronized at the G1/S boundary by double thymidine block (indicated as time 0). Cells were then washed and allowed to progress through the cell cycle for the indicated times in the presence of nocodazole. Full transcriptional activation of S251A/T255A/S259A-JunB occurred within 4 h in the absence of tetracycline. Protein extracts were analyzed by immunoblotting with antibodies against the indicated proteins. (f) S251A/T255A/S259A-JunB protein accumulates in prometaphase cells. UTA6-EV and UTA6-S251A/T255A/S259A-JunB cells were grown in the absence of tetracycline and in the presence of nocodazole for 16 h. Prometaphase round cells were collected by shake-off (M). The remaining attached cells (enriched in G2) were also harvested. Nocodazole untreated cultures were also grown for analysis of asynchronous cells (AS). Protein extracts were analyzed by immunoblotting with antibodies against the indicated proteins. distance between chromosome 7 centromeres revealed prema- S251A/T255A/S259A-JunB-UTA6 cells rescued the loss of chroma- ture separation in 25% of analyzed metaphases (Figure 6a). Direct tid cohesion phenotype (Figure 6c). Thus, repression of DDX11 microscopic analysis of metaphasic cells showed that S251A/ due to accumulation of JunB in late G2 is critical for the T255A/S259A-JunB-UTA6 chromatids appeared to be loosely manifestation of the mitotic phenotype observed in S251A/ paired (Figure 6b). In addition, ectopic expression of DDX11 in T255A/S259A-JunB-UTA6 cells.

& 2013 Macmillan Publishers Limited Oncogene (2013) 2189 – 2199 Regulation of JunB turnover in G2 by GSK3b and FBXW7 BPe´rez-Benavente et al 2194 DLD-1 DLD-1FBXW7 -/- to a concentration-dependent decrease of JunB and S259- phosphorylated-JunB concomitantly with activation of GSK3b as AS G2 M AS G2 M determined by the decreased level of S9-phoshorylated GSK3b (Figure 7b). Addition of the proteasome inhibitor MG132 in these JunB experiments inhibited JunB degradation, highlighting the crucial role of protein destabilization. Finally, metaphase spread analysis revealed premature centromere separation and completely JunB-pS259 separated sister chromatids in both Karpas-299 cells and SU- DHL1 cells (Figures 7c and d) and RNAi-mediated depletion of JUNB resulted in upregulation of DDX11 and downregulation of JunB-pT255 Cyclin A2 in G2/M Karpas-299 cells (Figure 7e). Taken together, these data indicate that stabilized JunB protein results in abnormal sister chromatid cohesion in ALK-positive ALCLs via Cyclin A2 repression of DDX11 and underline the importance of its physiological disappearance by mid/late G2 for normal mitosis. GSK3α GSK3 GSK3β DISCUSSION GSK3β-pS9 Under physiological conditions, JunB expression is cell cycle- regulated. In particular, its levels are high in S phase and decrease Cyclin B1 abruptly by mid/late G2. Here, we identified GSK3b and SCFFBXW7 GAPDH as, respectively, the kinase and the E3 ubiquitin ligase responsible for efficient JunB degradation in mid/late G2 and demonstrate that its GSK3b-SCFFBXW7-dependent degradation is required to Cyclin A2 ensure sufficiently high levels of DDX11 to maintain sister 4 DLD1 chromatid cohesion before anaphase. We also show that in ALK- 3.5 positive ALCL cells inactivation of GSK3b by PI3K/AKT impairs JunB DLD1FBXW7-/- 3 degradation. Accumulation of JunB in mitosis in these cells leads 2.5 to JunB-dependent transcriptional repression of DDX11 and subsequent abnormal sister chromatid cohesion in metaphase, 2 which is most likely a cause of genome instability in these tumors. 1.5 Fold change 1 Regulation of JunB turnover in G2 by GSK3b-dependent 0.5 phosphorylation and FBXW7 0 AP-1 is an important regulator of the G0–G1 transition. Similarly to AS G2 M c-Jun, JunB levels are very low in quiescent cells. Its expression is Figure 4. Depletion of FBXW7 leads to overexpression of Cyclin A2 rapidly and transiently induced by mitogenic stimuli during the in prometaphase-arrested cells. (a) Depletion of FBXW7 in DLD1 G0/G1 transition before it returns to an intermediate level, both of cells leads to accumulation of JunB and Cyclin A2 proteins in these events being instrumental for progression towards S prometaphase. Moreover, JunB phosphorylation at S259 in prome- phase.44,45 Wei et al.34 have shown that c-Jun is targeted for taphase-arrested cells and at T255 in G2 cells was higher in degradation in late G1 after serum-stimulated cells by FBXW7 in a DLD1FBXW7 À / À cells than in the DLD1 parental cell line. DLD1 and GSK3-dependent phosphorylation manner. However, in contrast FBXW7 À / À DLD1 cells were treated as described in Figure 1g. Protein to JunB, whose abundance decreases in mid/late G2, c-Jun levels extracts were analyzed by immunoblotting with antibodies against do not change during the cell cycle, including in G2 and M. the indicated proteins. (b) CCNA2 mRNA is upregulated in Moreover, c-Jun is phosphorylated on its N-terminal serines by the prometaphase DLD1FBXW7 À / À cells. The graph shows CCNA2 Jun amino-terminal kinase increasing its transactivational expression in asynchronously growing (AS), G2-enriched and 22 prometaphase (M) DLD1 and DLD1FBXW7 À / À cells. Cultures were potential and stability from G2 to M. Therefore, member- treated as described in (a). CCNA2 levels were determined by specific Jun modifications seem to be important for the regulation RT–qPCR, normalized to GUS and relativized to those in DLD1 of their protein abundance during cell-cycle progression. parental cells. Data represent the average of three independent It has previously been reported that the combined mutations of experiments. Bars correspond to standard deviations. the phospho-acceptors S23, T150 and S186 into alanines increased JunB stability in mid/late G2.22,25 However, this triple alanine mutant remained relatively unstable,25 suggesting that other Increased JunB stability and DDX11 repression in ALK-positive determinants in the JunB sequence are required for its ALCL cell lines degradation in G2. Here, we reveal that initiation of JunB JunB protein is strongly overexpressed in ALK-positive ALCL. degradation in G2 depends on the phosphorylation of JunB CPD Recently, constitutive activation of PI3K/AKT pathway in ALK- that, in turn, is primed by phosphorylation of S259 and the positive ALCLs has been reported to entail a substantial decrease subsequent phosphorylation of S251 and T255 by GSK3b in G2. In in GSK3b activity through phosphorylation at S9 by AKT.43 line with this, we report here that GSK3b activity is also transiently Therefore, we asked whether, in ALK-positive ALCLs, JunB stimulated during a narrow window in G2 (Figure 4a), concomitant protein was stabilized and whether CCNA2 and DDX11 were to JunB degradation. Moreover, our results support the idea that deregulated in a JunB-dependent manner. JunB levels were low in JunB phosphorylation at S251 and T255 by GSK3b is essential for nocodazole-synchronized G2/M Jurkat T lymphoma cells, whereas accelerating JunB degradation in mid/late G2. they remained high in G2/M Karpas-299 and SU-DHL1 ALK- Targeted degradation of JunB by the E3 ligase AIP4 upon the positive ALCL cells (Figure 7a). Similarly, at G2/M, DDX11 levels activation of mouse helper T cells was suggested to be involved in increased and Cyclin A2 decreased only in Jurkat cells (Figure 7a). the regulation of T-cell differentiation.30 However, our results PI3K inhibition by LY294002 in Karpas-299 and SU-DHL1 cells led show that depletion of AIP4 does not affect JunB protein levels

Oncogene (2013) 2189 – 2199 & 2013 Macmillan Publishers Limited Regulation of JunB turnover in G2 by GSK3b and FBXW7 BPe´rez-Benavente et al 2195 DDX11 DDX11 1.2 1.2 1 1 UTA6-EV 0.8 0.8 DLD1 UTA6-S A/T FBXW7-/- 0.6 251 255 0.6 DLD-1 A/S259A-JunB 0.4 0.4 Fold change 0.2 Fold change 0.2 0 0 AS G2 M AS G2 M

UTA6- UTA6- S A/T A/S A- 251 255 259 FBXW7-/- EV JunB DLD-1 DLD-1 AS G2 M AS G2 M AS G2 M AS G2 M

JunB JunB

DDX11 DDX11

β-Actin β-Actin

-399 +701 +4089 ATG

DDX11-A DDX11-B DDX11-C -361/458 +670/785 +4039/+4175

4 DLD-1 4 UTA6-EV 3.5 DLD-1FBXW7-/- 3.5 UTA6S251A/T255A/S259A-JunB 3 3 2.5 2.5 2 2 1.5 1.5

Fold enrichement 1 1 Fold enrichement 0.5 0.5 0 0 DDX11-A DDX11-B DDX11-C GAPDH DDX11-A DDX11-B DDX11-C GAPDH Figure 5. Impaired JunB degradation in G2 represses DDX11 in mitosis. (a) DDX11 mRNA is downregulated in both S251A/T255A/S259A-JunB- expressing cells (left panel) and in DLD1FBXW7 À / À cells (right panel). Cells were treated as described in Figure 1g to obtain asynchronously growing (AS), G2-enriched and prometaphase (M) cells. DDX11 and GAPDH levels were determined by RT–qPCR and relativized to those of control cells. Data represent the average of three independent experiments. Bars correspond to standard deviations. (b) DDX11 protein levels are repressed in S251A/T255A/S259A-JunB expressing cellss. UTA6- EV and UTA6-S251A/T255A/S259A-JunB cells were obtained as described in (a). Protein extracts were analyzed by immunoblotting with antibodies against JunB, DDX11 and b-actin. (c) DDX11 proteins levels are reduced in DLD1FBXW7 À / À cells. DLD1 and DLD1FBXW7 À / À cells were obtained as described in (a). Protein extracts were analyzed by immunoblotting with antibodies against JunB, DDX11 and b-actin. (d) JunB binds to the DDX11 promoter. The upper panel represents the structure of the DDX11 promoter. Initiation of transcription is indicated by an arrow and initiation of translation is indicated with ATG. Exons are shown as thick boxes and introns are indicated with a hyphenated line. Locations of TRE/CRE sites are shown by ovals and their position relative to the initiation of transcription is indicated above each oval. The PCR-amplified regions and their relative positions are indicated below each TRE/CRE site and are assigned a letter (A, B and C). The lower panel reports the chromatin immunoprecipitation assays with anti- JunB antibody in DLD1 and DLD1FBXW7 À / À cells (left) and in Tet-inducible control and S251A/T255A/S259A-JunB UTA6 cells after 24 h in the absence of Tet (right). Quantitative real-time PCR amplification was performed with the indicated primers (A, B and C pairs) surrounding the CRE and TRE sites in the DDX11 promoter and normalized to the unrelated amplified GAPDH promoter. in G2 or prometaphase cells in HeLa cells, arguing for the prometaphase DLD1FBXW7 À / À JunB strongly accumulates in involvement of another E3 ligase in the degradation of JunB in mitosis leading to overexpression of Cyclin A2 and mid/late G2 in these cell lines. We provide evidence that FBXW7, a downregulation of DDX11. It is thus tempting to speculate that substrate-binding component of an SCF ubiquitin ligase, has a abnormal JunB accumulation may contribute to both chromatid major role in mid/late G2 JunB degradation upon phosphorylation cohesion defects and tumorigenesis in tumors with inactive of JunB by GSK3b. FBXW7. However, as FBXW7 has many substrates, this mechanism FBXW7 is the most frequently mutated gene associated with may not be exclusive of JunB and it may even cooperate with chromosome instability.46 FBXW7 mutations have been described other FBXW7 substrates. Indeed, elevated Cyclin E levels in in many human cancers including breast, endometrial, large DLD1FBXW7 À / À cells have already been shown to contribute to intestine, thyroid, pancreas, hematopoietic and lymphoid tumors, genomic instability.46 as well as in cholangiocarcinoma and T-cell acute lymphoblastic leukemia.33 However, the precise mechanisms by which FBXW7 is associated with oncogenesis are not fully understood because of The GSK3b-JunB axis is a novel regulator of chromatid cohesion in the broad variety of FBXW7 substrates and their role in different ALK-positive ALCL types of cancer.33,38,47 Interestingly, the colorectal cancer cell lines Sister chromatids remain connected until they are separated in HCT116 and DLD1 that lack FBXW7 show genomic instability46 anaphase. Cohesion between sister chromatids allows the and chromatid cohesion defects.48 Here, we show that in accurate separation of sister chromatids into two daughter cells.

& 2013 Macmillan Publishers Limited Oncogene (2013) 2189 – 2199 Regulation of JunB turnover in G2 by GSK3b and FBXW7 BPe´rez-Benavente et al 2196 Normal metaphase Cohesion Defect in abnormal sister chromatid cohesion, mitotic failure and aneuploidy induction.42 Moreover, DDX11 À / À mice die at embryonic day 10.5 due to perturbed chromosome cohesion, chromosome mis-segregation and aneuploidy.41 Here, we provide evidence that the GSK3b-FBXW7-JunB axis constitutes a novel and essential regulator of sister chromatid cohesion via transcriptional regulation of DDX11. First, accumulation of JunB in mitosis represses DDX11 expression via, most probably, a direct transcriptional effect, as suggested by chromatin immuno- 5 μm precipitation assays. Second, in cells expressing the S251A/ T255A/S259A-JunB mutant DDX11 is repressed and they displayed a phenotype with aberrations in centromeric cohesion Normal metaphase Cohesion Defect and defects in chromatid cohesion. Third, ectopic expression of DDX11 rescues the loss of the chromatid cohesion phenotype in these cells. Finally, downregulation of DDX11 is also found in DLD1FBXW7 À / À and ALK-positive ALCL mitotic cells. Our results corroborate previous reports showing loss of both centromeric and arm cohesion defects after DDX11 depletion.41,42 Interestingly, a patient with biallelic mutations in DDX11 was recently reported.50 The DDX11 defect caused abnormalities in the cohesion of sister chromatids and genetic instability, thus 5 μm 5 μm representing a new example of human cohesinopathy. The fact that the mother and the grandmother of the affected individual, both carriers of the DDX11 splice-site mutation, developed 100 Cohesion defect Hodgkin’s lymphoma and adenocarcinoma of the endo- 80 Normal metaphase metrium50 underlines the potential role of DDX11 in the 60 prevention of aneuploidy and tumorigenesis. JunB is overexpressed and has an oncogenic role in certain 40 lymphoma. Consistently, we have observed aberrant overexpres- 20 sion of JunB protein in mitotic Karpas-299 and SU-DHL1 cells, Metaphases (%) 0 which are derived from ALK-positive ALCLs. In ALK-positive ALCLs, the fusion tyrosine kinase NPM-ALK drives tumorigenesis in part 259 259 through activation of the PI3K/AKT pathway.3 This, in turn, leads to A/S A/S 43 255 255 reduced GSK3b activity. Herein, our data highlight a critical A/T A/T role for the perturbation of the GSK3b-dependent JunB S 251 A-JunB+EV S 251 degradation in ALK-positive ALCL cells. Indeed, GSK3b inhibition A-JunB+DDX11 by PI3K/AKT is involved in higher accumulation of JunB protein in Figure 6. Impaired JunB degradation in G2 leads to chromatid these cells, Cyclin A2 is upregulated and DDX11 downregulated in cohesion defects. (a) Aberrant centromeric cohesion in S251A/ a JunB-dependent manner in mitotic ALK-positive ALCL cells T255A/S259A-JunB expressing cells. UTA6-S251A/T255A/S259A-JunB and, consistently with DDX11 downregulation, abnormal sister cells were cultured for 72 h without tetracycline and metaphase chromatid cohesion in metaphase is observed. In line with our spreads were prepared. Centromeres for chromosome 7 were results, recent cytogenetic analysis revealed that ALCLs often stained with CEP7 (D7Z1)-specific probes. DNA was stained with show numerical chromosomal abnormalities.5 DAPI and chromosomes were viewed by confocal microscopy. The In conclusion, our work strengthens the idea that JunB protein distance between centromeres was measured using the Openlab software and the average distance was calculated as 0.51±0.2 mm. level must be accurately controlled throughout the cell cycle to No separation between centromeres was found in cells with normal ensure strict temporal control of JunB transcriptional activity to metaphase. At least 100 metaphases were analyzed in three avoid mitotic failure (Figure 7f). More specifically, we provide independent assays. (b) Abnormal sister chromatid cohesion in evidence for the existence of a GSK3b-FBXW7-JunB axis operating cells in which JunB expression is stabilized. Panels show represen- in mid/late G2, which is essential for subsequent proper progression tative metaphase spreads in control (UTA6-EV) and S251A/T255A/ through mitosis. We demonstrate that a key target of this axis is S259A-JunB-expressing UTA6 cells. Premature centromere separa- DDX11, the repression of which by abnormally high levels of JunB tion and separated sister chromatids were frequently detected in leads to loss of chromatid cohesion in ALK-positive ALCLs. These UTA6-S251A/T255A/S259A-JunB. In addition, chromosomes were findings provide new molecular insights on JunB-dependent thicker and looked fluffier. (c) Ectopic expression of DDX11 in UTA6- S251A/T255A/S259A-JunB cells rescues the loss of chromatid neoplastic transformation and suggest that inhibition of JunB cohesion. UTA6-S251A/T255A/S259A-JunB cells were transfected activity, in combination with PI3K/AKT and/or NPM-ALK pathway with either empty vector (EV) or with a plasmid encoding DDX11 in blockade may represent a valid therapeutic approach in ALK- the absence of Tet to induce JunB expression. Metaphase spreads positive ALCL. Given the fact that JunB over-expression and GSK3b were prepared 72 h later. The graph shows the percentage of and/or FBXW7 inactivation have been reported in a variety of chromatid pairs with cohesion defects in the transfected S251A/ human tumors,33,51,52 our findings may apply to other tumor types. T255A/S259A-JunB cells. At least 100 metaphases were analyzed in each cell line. MATERIALS AND METHODS Expression vectors Without cohesion, the opposing forces required for bipolar HA-tagged JunB and Tet-repressible vectors were previously described.25 separation of chromatids would not exist and premature JunB mutants were generated from the HA-tagged JunB in pCDNA3 separation of chromatids could occur. This would lead to (Invitrogen, Carlsbad, CA, USA), using the QuikChange MultiSite-Directed 49 inaccurate chromosome segregation and genome instability. Mutagenesis Kit from Stratagene (La Jolla, CA, USA). Myc-tagged GSK3b In this sense, depletion of DDX11 protein in HeLa cells results was from Adgene (Cambridge, MA, USA).

Oncogene (2013) 2189 – 2199 & 2013 Macmillan Publishers Limited Regulation of JunB turnover in G2 by GSK3b and FBXW7 BPe´rez-Benavente et al 2197 Jurkat K-299 SU-DHL1 Karpas 299 SU-DHL1

AS G2/M AS G2/M AS G2/M MG132 ---+ ---+ LYS294002 0201020 010 20 20 nM JunB JunB DDX11 JunB-pS259

Cyclin A2 GSK3 α/β

α β Cyclin B1 GSK3 / -pS21/S9

GAPDH GAPDH

Karpas-299 SU-DHL1 45 Cohesion defect 40 35 30 25 20 15

Metaphases (%) 10 5 5 μm 5 μm 0

SU-DH Karpas-299

Karpas 299 siCt siJunB AS G2/M AS G2/M JunB

DDX11

Cyclin A2

Cyclin B1

GAPDH

Figure 7. Deregulation of the GSK3b-JunB axis and DDX11 downregulation in ALK-positive ALCL cell lines. (a) Expression of JunB and DDX11 in the ALK-positive ALCL cell lines Karpas-299 and SU-DHL1 and in CD30-Jurkat T-cell acute lymphoblastic leukemia cells. Cells were treated with nocodazole (G2/M) for 24 h and harvested. Nocodazole untreated cultures were also grown for asynchronous cells (AS). Protein extracts were analyzed by immunoblotting with antibodies against JunB, DDX11, Cyclin A2, Cyclin B1 and GAPDH. (b) PI3K/AKT activity modulates JunB protein turnover. Karpas-299 and SU-DHL1 cells were treated with increasing concentrations of LY294002 or with both LY294002 and MG132. Immunoblotting showed concentration-dependent decrease in JunB and JunB phosphorylated at S259. A decrease in the level of inactive pGSK3b (phosphorylated at S9) was also observed. (c) Abnormal sister chromatid cohesion in ALK-positive ALCL cells lines. Panels show representative metaphase spreads in Karpas-299 and SU-DHL1 cells. Premature centromere separation and separated sister chromatids were detected in Karpas-299 and SU-DHL1. (d) Graph showing the percentage of chromatid pairs with cohesion defects from cells shown in (c). At least 100 metaphases were analyzed in each cell line in three independent assays. (e) Depletion of JunB results in upregulation of DDX11 and downregulation of Cyclin A2 in G2/M Karpas-299 cells. Karpas-299 cells were transfected twice with control or JunB-speciﬁc siRNAs. Nocodazole was added 24 h after the last transfection for another 24 h to enrich for cells in G2/M. Cells were harvested and cell extracts analyzed by immunoblotting with antibodies against the indicated proteins. (f) Model to explain the negative effect of dysregulation of the GSK3-FBXW7-JunB axis in ALK-positive ALCL. Targeted degradation of JunB in mid/late G2 is dependent on GSK3b phosphorylation and SCFFBXW7 ubiquitylation. Failure to degrade JunB causes deregulation of the cell cycle and defects in chromosome segregation. This phenotype is due to deregulation of speciﬁc molecular species leading to mitosis delay and chromatid cohesion defects. Hence, tight temporal and quantitative control of JunB levels is needed for proper cell-cycle regulation and chromatin stability.

Cell culture, antibodies and reagents Where indicated, 25 mM GSK3b inhibitor VIII (Calbiochem, San Diego, ALCL cell lines Karpas-299 and SU-DHL1 were purchased from the German CA, USA), 30 mM MG132 (Biomol, Farmingdale, NY, USA) or LYS294002 Collection of Microorganisms and Cell Cultures (DSMZ). Stable inducible (Cell Signaling, Danvers, MA, USA) was added to the cell culture medium. UTA6 cell populations were generated following the protocol described Mouse monoclonal antibodies were from Calbiochem (anti-GSK3), Sigma- in Farra`s et al.25 DLD1 and DLD1FBXW7 À / À cells were described in Aldrich (St Louis, MO, USA) (anti-Flag and Cyclin A2) and Novus Biological 46 25 Rajagopalan et al. Cell synchronization was described in Farra`s et al. (Littleton, CO, USA) (anti-DDX11). Rabbit polyclonal antibodies were from

& 2013 Macmillan Publishers Limited Oncogene (2013) 2189 – 2199 Regulation of JunB turnover in G2 by GSK3b and FBXW7 BPe´rez-Benavente et al 2198 Abcam (Cambridge, UK) (JunBpS259 and GSK3bpS9) and Sigma-Aldrich (Cyclin ACKNOWLEDGEMENTS B1). Antibodies against JunB phosphorylated at S251 and T255 were obtained We thank Sandra Gallach and Pablo Mateos for their excellent technical support and after immunization of rabbits with the EPQTVPEAR(p)SRDA and EARSRDA A Ferrando, TM Thomson, G Bossis and O Coux for fruitful discussions and critical (p)TPPVSP peptides coupled to keyhole limpet hemocyanin, respectively. They reading of the manuscript. This research was supported by grants from the Fondo de were produced and affinity chromatography-purified by Eurogentec (Seraing, Investigaciones Sanitarias (PI08/1127) and from Valencia’s Regional Ministry of Health 25 Belgium). Antibodies against HA, JunB and GAPDH are described elsewhere. (AP007/11) to RF, from the Spanish Ministry of Science and Innovation (SAF2009- 08334) to JFM. RF is supported by the Institute of Health Carlos III and by the Regional Cytogenetics analysis and FISH Ministry of Health. MP was supported by the program ‘Equipe Labelliseé’ of the French Ligue against Cancer. We are grateful to B Vogelstein for providing DLD1 and Control and S251A/T255A/S259A-JunB-expressing UTA6 cells were grown in DLD1FBXW7 À / À cells, M Pagano for Flag-tagged SKP2, CDH1 and CDC20 constructs, O the absence of tetracycline for 72 h. Cells were treated with 1 mg/ml colcemid Sangfield for Flag-tagged FBXW7a and FBXW7g constructs, C Bonne-Andrea for Flag- for 2 h before harvesting. Images of G-banded (Wright’s stain) metaphases tagged FBXW7a(R465A), PK Vogt (Scripps Research Institute) for activated AKT and were captured using the CytoVision System (Applied Imaging, Santa Clara, E Noguchi for DDX11. CA, USA). CEP 7 (D7Z1) SpectrumGreen Probe Alpha Satellite DNA 7p11.1- q11.1 was used as centromere-specific probe for chromosome 7. Probes were hybridized using the HYbrite system (Abbot Molecular, Abbott Park, IL, USA) according to manufacturer’s recommendations. Slides were washed REFERENCES and mounted in anti-fade solution with 0.1 mg/ml 40,6-diamidino-2-pheny- lindole-2-HCl (DAPI). The median distance between centromeres was 1 Amin HM, Lai R. Pathobiology of ALK þ anaplastic large-cell lymphoma. Blood calculated using the OpenLab software (Perkin Elmer, Waltham, MA, USA) 2007; 110: 2259–2267. at Â 100 magnification. The frequencies of the different phenotypes were 2 Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL et al. expressed as percentages of the total number of metaphase cells. At least Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s 100 metaphase cells were counted for each experiment. lymphoma. 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