Mitotic Wnt Signaling Promotes Protein Stabilization and Regulates Cell Size

Please cite this article in press as: Acebron et al., Mitotic Wnt Signaling Promotes Protein Stabilization and Regulates Cell Size, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.04.014

Molecular Cell

Article Mitotic Wnt Signaling Promotes Protein Stabilization and Regulates Cell Size Sergio P. Acebron,1,* Emil Karaulanov,2 Birgit S. Berger,1 Ya-Lin Huang,1 and Christof Niehrs1,2,* 1DKFZ-ZMBH

Alliance, Division of Molecular Embryology, DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany of Molecular Biology, Ackermannweg 4, D-55128 Mainz, Germany *Correspondence: [email protected] (S.P.A.), [email protected] (C.N.) http://dx.doi.org/10.1016/j.molcel.2014.04.014 2Institute

SUMMARY

Canonical Wnt signaling is thought to regulate cell behavior mainly by inducing b-catenin-dependent transcription of target genes. In proliferating cells Wnt signaling peaks in the G2/M phase of the cell cycle, but the significance of this ‘‘mitotic Wnt signaling’’ is unclear. Here we introduce Wnt-dependent stabilization of proteins (Wnt/STOP), which is independent of b-catenin and peaks during mitosis. We show that Wnt/STOP plays a critical role in protecting proteins, including c-MYC, from GSK3dependent polyubiquitination and degradation. Wnt/ STOP signaling increases cellular protein levels and cell size. Wnt/STOP, rather than b-catenin signaling, is the dominant mode of Wnt signaling in several cancer cell lines, where it is required for cell growth. We propose that Wnt/STOP signaling slows down protein degradation as cells prepare to divide.

INTRODUCTION Canonical Wnt signaling plays a pivotal role in development by regulating stem cell differentiation, embryonic axes, patterning, and organogenesis (De Robertis, 2010; Clevers and Nusse, 2012; Hikasa and Sokol, 2013; Holland et al., 2013). In adult organisms, canonical Wnt signaling modulates tissue homeostasis in many organs, and its misregulation is implicated in numerous diseases, including cancer (Clevers and Nusse, 2012; Anastas and Moon, 2013). Notably, in cancer and stem cells canonical Wnt signaling can promote cell proliferation (Reya and Clevers, 2005). There is a complex interaction between canonical Wnt signaling and the cell cycle. On one hand, mitogenic Wnt signaling promotes cell proliferation, and this is thought to occur by transcriptional induction of the G1 regulators CCND1 and MYC (Reya and Clevers, 2005). On the other hand, components of the Wnt signaling cascade function directly in spindle formation during mitosis (Niehrs and Acebron, 2012). At the core of canonical Wnt signaling is the transcriptional regulator b-catenin, which in unstimulated cells is phosphorylated by glycogen synthase kinase 3 (GSK3), then polyubiquitinated by b-transducin repeat containing protein 1 (b-TrCP), and thereby targeted for proteasomal degradation (Angers and

Moon, 2009; MacDonald et al., 2009). A key Wnt signaling transducer at the plasma membrane is the Wnt coreceptor low-density lipoprotein receptor-related 5 or 6 (LRP5/6). Wnt induces clustering of its receptors Frizzled and LRP6 in signalosomes, and phosphorylation of the LRP6 intracellular domain (ICD) by GSK3 and casein kinase 1g (CK1g) (Davidson et al., 2005; Zeng et al., 2005; Bilic et al., 2007; Schwarz-Romond et al., 2007). ICD phosphorylation triggers the inhibition of GSK3 by various mechanisms (Piao et al., 2008; Metcalfe et al., 2010; Taelman et al., 2010; Li et al., 2012; Kim et al., 2013; Vinyoles et al., 2014). Thus, Wnt pathway activation ultimately prevents b-catenin phosphorylation by GSK3 and stabilizes the protein, allowing it to enter the nucleus and modulate target gene transcription (Aberle et al., 1997; Liu et al., 2002). We previously showed that LRP6 phosphorylation is under cell cycle control, peaking in G2/M (Davidson et al., 2009). This is because the cyclin-dependent kinase CDK14 and its associated G2/M Cyclin Y phosphorylate ICD and thereby prime LRP6 for Wnt-dependent phosphorylation by CK1g. Hence, the competence of LRP6 to respond to Wnt is under cell cycle control and peaks at G2/M. This explains why cytoplasmic b-catenin and AXIN2 levels also oscillate with the cell cycle, peaking at G2/M (Olmeda et al., 2003; Davidson et al., 2009; Hadjihannas et al., 2012). It is curious that Wnt signaling should peak during transcriptionally silent mitosis, when its major effects are thought to require b-catenin-dependent gene transcription (MacDonald et al., 2009; Clevers and Nusse, 2012; Niehrs, 2012). Interestingly, there is mounting evidence that some of the physiological consequences of canonical Wnt signaling are b-catenin independent (see Table S1 online). Bioinformatic analyses suggested that GSK3 motifs are abundant in the proteome (Xu et al., 2009; Taelman et al., 2010). Once phosphorylated, GSK3 substrates can be recognized by E3 ubiquitin ligases, including F box/WD repeat-containing protein 7 (FBW7), b-TrCP and Neural precursor cell expressed, developmentally downregulated 4-like (NEDD4L), which target these substrates for proteasomal degradation (Hart et al., 1999; Welcker et al., 2004; Fuentealba et al., 2007; Xu et al., 2009; Arago´n et al., 2011). Recent data suggest that GSK3 indeed mediates not only degradation of b-catenin, but potentially of hundreds of proteins in a Wnt-dependent manner (Kim et al., 2009; Taelman et al., 2010). Consistent with this model, 70% of GSK3 activity is inhibited upon Wnt treatment by sequestration of GSK3 in multivesicular bodies (Taelman et al., 2010). This raises important questions: What is the relationship between mitotic Wnt Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc. 1

Please cite this article in press as: Acebron et al., Mitotic Wnt Signaling Promotes Protein Stabilization and Regulates Cell Size, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.04.014

Molecular Cell Wnt/STOP Signaling Regulates Cell Size

(legend on next page)

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Molecular Cell Wnt/STOP Signaling Regulates Cell Size

signaling and GSK3-mediated protein degradation? Is mitotic Wnt signaling b-catenin independent and transcription independent? What role does mitotic Wnt signaling play during the cell cycle? Here we show that protein stabilization of a GSK3-biosensor is oscillating with the cell cycle in a Wnt-dependent manner, peaking at G2/M. Likewise, endogenous Wnt/GSK3 targets are maximally stabilized at G2/M. Using protein microarrays in HeLa cells, we identify over 100 candidate proteins whose polyubiquitination depends on GSK3 and which are stabilized by mitotic Wnt signaling. Unexpectedly, in HeLa cells this Wntdependent stabilization of proteins (Wnt/STOP), rather than transcriptional activation, appears to be the dominant mode of Wnt signaling. Activation of Wnt/STOP increases cellular protein content during G2/M and the size of daughter cells. We propose that Wnt/STOP is an alternative mode of canonical Wnt signaling to regulate cell size by slowing down GSK3-dependent protein degradation as cells prepare to divide. RESULTS Wnt Signaling Stabilizes GSK3 Target Proteins in G2/M To analyze the relationship between mitotic Wnt signaling and GSK3-mediated protein degradation, we first tested if cell cycle oscillation of Wnt signaling may entrain periodic stabilization of GSK3 target proteins. As a model we used an established GSK3-biosensor in which GFP is fused to three GSK3 phosphorylation sites, followed by a MAPK priming site and an E3 ligase recognition motif. Thus, the GSK3-biosensor is degraded via E3 ligases, unless stabilized by Wnt signaling (Taelman et al., 2010). Live-cell imaging of HeLa cells transfected with the GSK3biosensor showed a stable fluorescence in control cells (Figures 1A and 1B). Importantly, in the presence of Wnt3a, GSK3biosensor fluorescence increased in G2, peaked at mitosis, and returned close to control levels by the end of S phase (Figures 1A and 1B; Movie S1). Upon the subsequent mitosis, a second but lower-fluorescence peak was observed (Figure S1A). Depletion of either LRP6 (siLRP5/6) or Cyclin Y (CCNY and CCNYL1, ‘‘siCycY’’) using validated siRNAs (Davidson et al.,

2009) completely blocked GSK3-biosensor stabilization by Wnt3a (Figures 1A and 1B). Since a GSK3-biosensor is stabilized by Wnt signaling, we hypothesized that bona fide GSK3 target proteins such as c-MYC, CDC25A, and cyclin D1 (CCND1) (Diehl et al., 1998; Welcker et al., 2004; Kang et al., 2008), for which Wnt-dependent stabilization is yet unknown, may also be protected from phosphorylation, ubiquitination, and proteasomal degradation (Figure 1C). To test this hypothesis, HeLa cells were cell cycle synchronized and treated with Wnt3a or Dkk1 to activate or inhibit endogenous Wnt signaling, respectively (see Figure 1D for experimental scheme). The cytosolic levels of the GSK3-target proteins were indeed consistently higher in Wnt- than in Dkk1-treated G2/M cells (Figures 1E and 1F). In contrast, Dkk1 or Wnt3a had little effect in G1 cells. The effect of Wnt3a was mediated by the canonical pathway, since siLRP5/6 or siCycY reduced GSK3 target protein levels (Figure S1B). In untreated control cells target protein levels were intermediate between Dkk1- and Wnt3a- treated cells (Figure 1E), since HeLa cells harbor endogenous Wnt signaling (Davidson et al., 2009; Kikuchi et al., 2010). Since we aimed for a full on/off situation, we omitted untreated samples in some of the following experiments and only compared Wnt3a versus Dkk1. Wnt-dependent increase of protein levels was mediated by protein stabilization rather than by increased transcription or translation since (1) GSK3 target proteins were also stabilized when protein translation was blocked with cycloheximide (Figure 1G); (2) phosphorylation of the GSK3 sites in c-MYC (T58/ S62) and CCND1 (T286) was reduced by mitotic Wnt signaling (Figure 1H); (3) Dkk1 induced K48-linked ubiquitination of the GSK3 targets, and mostly so in G2/M cells, consistent with Wnt signaling protecting target proteins from proteasomal degradation (Figures 1I and S1C); and (4) gene expression of CCND1 and MYC, which are Wnt-transcriptional targets in other cell types (He et al., 1998; Tetsu and McCormick, 1999), were not induced in HeLa cells (Figure 1J). We corroborated these results in MDA-MB-231 breast cancer cells, which also showed Wntdependent increase of c-MYC protein levels specifically in

Figure 1. Wnt Signaling Stabilizes GSK3 Target Proteins in G2/M (A) Live-cell imaging of HeLa cells transiently transfected with a GSK3-dependent GFP-degron (GSK3-biosensor). HeLa cells were continuously treated with control or Wnt3a-conditioned medium, starting 3 hr before recording, and filmed for 50 hr. Media were supplemented with 100 ng/ml bFGF to activate MAPK. GSK3-biosensor fluorescence intensity is represented by false color gradient. (B) Quantification of relative GSK3-biosensor fluorescence intensity during HeLa cell cycle as shown in Figure 1A (SD, n = 13). The indicated time refers to minutes before and after mitosis (M). (C) Schematic example of GSK3-mediated protein degradation. GSK3 phosphorylates c-MYC. The resulting phosphodegron is recognized by the E3 ligase FBW7, which targets c-MYC to K48-linked ubiquitination and proteasomal degradation. Note that other GSK3 targets are degraded similarly, while the priming kinases and the E3 ligases may vary. This study establishes that c-MYC and other GSK3 targets are stabilized in a Wnt-dependent manner. (D) Scheme of the cell-synchronization experiments. Cells arrested in G1/S by a double thymidine block were released by media change. Cells were treated during G2/M or during G1 for 3 hr with Wnt3a or Dkk1 (to inhibit endogenous Wnt signaling) and then analyzed for protein or RNA levels of GSK3 targets (E–H and J), or for 2 hr and then analyzed for their polyubiquitination status (I). (E, F, and H) Western blots of the indicated proteins in synchronized HeLa cells after 3 hr Wnt3a or Dkk1 treatment at different cell cycle phases. Note that the phosphorylation sites shown in (H) are GSK3-dependent degrons. (F) Protein levels from (E) were normalized to the loading control (ERK1/2). The green line indicates no change between Wnt3a versus Dkk1 treatment (SD; n = 4). (G) Western blots of the indicated proteins from synchronized HeLa cells after 1 hr Control, or Wnt3a or Dkk1 treatment at G2/M in the presence of 40 mg/ml cycloheximide (CHX). (I) Western blots of immunoprecipitated c-MYC from synchronized HeLa cells treated for 2 hr with Wnt3a or Dkk1 in the presence of the proteasome inhibitor MG-132 and immunoblotted (IB) with an anti-K48-Ub antibody. (J) Wnt-dependent expression of the indicated genes in G2/M- and G1-synchronized HeLa cells treated as in Figure 1E (SD; n = 3).

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Molecular Cell Wnt/STOP Signaling Regulates Cell Size

Figure 2. Identification of Wnt/STOP Target Proteins (A) Scheme of the microarray analysis of proteins polyubiquitinated by HeLa cell extracts in a GSK3-dependent manner. (B) Volcano plot depicting the polyubiquitination changes of 831 in vitro-polyubiquitinated array proteins following GSK3 inhibition by SB-216763 (SB). GSK3 inhibition significantly reduced polyubiquitination of 119 proteins (yellow-red) and increased polyubiquitination of 4 proteins (blue). The color-selected hits remain significant upon multiple testing correction at FDR levels below 28% (n = 3). (C) Expression changes of the indicated genes determined by RNA expression microarrays of G1- and G2/M-sorted HeLa cells upon Wnt3a or control treatment as indicated in Figure 3B. Green line, no fold change. (legend continued on next page)

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Molecular Cell Wnt/STOP Signaling Regulates Cell Size

mitosis without increased mRNA levels (Figures S1D and S1E). Taken together, we conclude that Wnt signaling stabilizes GSK3 target proteins predominantly in G2/M. Identification of Wnt/STOP-Target Proteins We used commercial protein microarrays to identify proteins that are polyubiquitinated in a GSK3-dependent manner (Merbl and Kirschner, 2009; Merbl et al., 2013). To this end, protein microarrays were exposed to extracts of HeLa cells with or without GSK3 inhibitor (SB-216763; SB), and the polyubiquitinated products were detected by polyubiquitin-specific antibodies (see Figure 2A for experimental overview). Of the more than 9,000 interrogated proteins, 831 were polyubiquitinated in vitro, a proportion matching previous estimates (Merbl and Kirschner, 2009). GSK3 inhibition significantly reduced polyubiquitination of 119 proteins (14%), while only four proteins showed increased polyubiquitination (Figures 2B and S2A– S2C; Table S2). While most candidate GSK3 target proteins contain one or more putative GSK3 phosphorylation sites (data not shown), there was no strong enrichment of any particular gene ontology class among these proteins. Interestingly, there was a 4-fold depletion of predicted short-lived proteins (Song et al., 2011) in the GSK3-target group compared to a matched control group of non-GSK3-targets (Figure S2D). It appears plausible that the dynamic range of protein degradation control via GSK3 would suffer if its targets were constitutively shortlived. Based on commercial antibody availability, we selected several candidate proteins for further validation, including CENPT, HDAC7, HINT2, MARK3, and RPSA. RNA levels of the corresponding candidate genes were unchanged by Wnt3a (Figure 2C; and see below). In contrast, not only were the protein levels of all these candidates Dkk1 sensitive, but specifically so in G2/M (Figure 2D). Consistent with this protein increase being due to protection from proteasomal degradation, the K48-ubiquitinated fraction of the candidates decreased Wnt dependently (Figure 2E). Based on these results, we expect that many of the other identified array candidates are also bona fide (nontranscriptional) Wnt targets. Degradation of known GSK3 target proteins is mediated by E3 ligases including FBW7, b-TrCP, and NEDD4L, which recognize distinct phosphodegrons (Hart et al., 1999; Welcker et al., 2004; Xu et al., 2009; Arago´n et al., 2011). Only for the canonical FBW7/ GSK3 degron T/SPxxS we found a 2-fold increase in the GSK3 target—over the control protein group (Figure 2F). In contrast, there was no enrichment for the FBW7 degron T/SPxxE, which is not associated with GSK3-dependent degradation (Welcker and Clurman, 2008), nor for the b-TrCP and NEDD4L degrons, nor for degrons of the GSK3-unrelated E3 ligase APC/C. Among

the proteins containing putative FBW7/GSK3 degrons is HDAC7, and overexpression of FBW7 indeed could override the Wnt3adependent stabilization of HDAC7 in G2/M (Figure 2G, lane 5 versus lane 7). In contrast, FBW7 had no effect on b-catenin, which depends on b-TrCP. Collectively, these data suggest that FBW7 may play a prominent role in the Wnt-dependent stabilization of proteins in G2/M. Wnt Signaling Induces Only a Few Genes in HeLa Cells The fraction of proteins whose polyubiquitination was under GSK3 control in the protein microarrays (14%) is close to previous GSK3 target estimates (20% of the proteome) based on bioinformatic analyses (Xu et al., 2009; Taelman et al., 2010). To relate the Wnt effects in HeLa cells on protein stabilization to its well-established transcriptional response, we also performed gene expression microarray profiling. Unsynchronized HeLa cells were treated with Wnt3a for 3 hr, a time point when 10-fold induction of the immediate response gene AXIN2 was at plateau (Figure 3A). We FACS sorted G1 and G2/M cells for mRNA expression profiling, but unexpectedly, other than AXIN2, we only found three more genes (PDK4, OLR1, and SKIDA1) to be induced by Wnt3a more than 2-fold (Figure 3B). While other cell lines show significant transcriptional responses (van de Wetering et al., 2002; Jackson et al., 2005), at least in HeLa cells protein stabilization rather than transcriptional activation appears to be the dominant mode of Wnt3a signaling. These results support the notion that a significant fraction of the proteome is stabilized by Wnt signaling (Xu et al., 2009; Taelman et al., 2010) and corroborates that this occurs preferentially in G2/M. Moreover, the data suggest that in certain cell types protein stabilization rather than transcriptional activation can be the dominant mode of Wnt signaling. We refer to this Wntdependent stabilization of proteins pathway as Wnt/STOP. Wnt/STOP Signaling Stabilizes Proteins in G2/M The steady-state level of proteins is determined by the ratio between protein biosynthesis and protein degradation. Wnt signaling increases protein steady state levels (1) by b-catenindependent transcriptional activation, (2) by increased protein translation via TOR pathway activation (Inoki et al., 2006), and (3) by Wnt/STOP signaling, as proposed here (Figure 4A). We analyzed the contribution of Wnt/STOP to the regulation of total cellular protein levels by blocking b-catenin signaling with siRNA and TOR signaling by Rapamycin and cycloheximide treatment, respectively. To analyze protein levels in single HeLa cells, we cytometrically monitored incorporation of N-hydroxysuccinimide ester (NHS)-fluorescein, which binds to lysine-side chains. Wnt signaling increased total protein content during G2/M, but

(D) Western blots of the indicated proteins at the indicated cell cycle phases in synchronized HeLa cells after 3 hr of Wnt3a or Dkk1 treatment. (E) Western blots of the indicated immunoprecipitated proteins from synchronized G2/M HeLa cells treated for 2 hr with Wnt3a or Dkk1 in the presence of the proteasome inhibitor MG-132 and developed with an anti-K48-Ub antibody (IB). (F) Occurence of known E3 ligase degrons in the GSK3 target and matched non-target (control) group from Figure 2B (see Experimental Procedures). FBW7 and b-TrCP canonical degrons are indicated. NEDD4L degron, SPxLSN. APC/C degron (KEN), xKENx. APC/C degron (D box), RxxLxxxxN/D/E. (G) Western blots of immunoprecipitated b-catenin and HDAC7 from synchronized G2/M HeLa cells treated for 2 hr with Wnt3a or Dkk1 in the presence of the proteasome inhibitor MG-132 and developed with an anti-K48-Ub antibody (IB). Cells were transfected 24 hr before the treatments with FBW7 or empty vector as indicated.

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Molecular Cell Wnt/STOP Signaling Regulates Cell Size

Figure 3. Wnt/b-Catenin Signaling Regulates Few Transcriptional Targets in HeLa Cells (A) AXIN2 expression upon the indicated treatments. AXIN2 expression was measured by qRTPCR and normalized against GAPDH (SD; n = 3). Note that Wnt-dependent expression plateaus after 3 hr (arrow). (B) Genome-wide gene expression microarrays of G1- and G2/M-sorted HeLa cells following 3 hr of Wnt3a, Dkk1, or control treatment. The scatterplots (log10 scale) compare the mean Wnt3adependent expression against control or Dkk1 treatment. Differentially expressed genes are indicated in blue (5% FDR; n = 4). Red lines indicate 2-fold expression changes.

not during G1, by approximately 15% (Figure 4B; Wnt3a versus Dkk1). Knockdown of LRP6 and Cyclin Y phenocopied the Dkk1 effect and reduced G2/M protein content, while sib-catenin, although effectively blocking TOPFLASH reporter signaling (data not shown), had no significant effect (Figures 4C and S3A). Moreover, overexpression of FBW7 also blocked the Wnt3a effect but did not further reduce protein content upon Dkk1 treatment (Figure 4D), suggesting that much of the Wnt/ STOP effect on protein content is mediated by this E3 ligase. Rapamycin treatment expectedly reduced protein levels (5% in G2/M, data not shown). However, on top of Rapamycin Wnt signaling still increased protein levels, suggesting a TOR signaling-independent effect (Figure S3B). We used incorporation of the methionine analog L-azidohomoalanine (AHA) as a proxy for protein translation (Kobayashi et al., 2009). Wnt3a treatment had no effect on protein translation in G2/M (Figure 4E), while in G1 it expectedly increased protein translation TOR dependently (Inoki et al., 2006) (Figure S3C). Finally, even in cycloheximide-treated cells, Wnt3a increased protein content (Figures S3D and S3E). This further corroborates that Wntinduced protein increase in G2/M is not due to TOR signaling/ protein translation but due to protein stabilization. NHS-Fluorescein labels proteins in all cellular compartments, yet most GSK3 target proteins are cytoplasmic, and hence we probably underestimated the contribution of Wnt/STOP for cytoplasmic proteins. We therefore extracted cytoplasmic proteins of G2/M cells with saponin, which perforates the plasma membrane. Saponin-treated cells had 45% less protein, and importantly, the saponin-resistant, noncytoplasmic protein fraction was unaffected by Wnt/Dkk1 treatment (Figure 4F), confirming that up to 25% of cytoplasmic proteins are predominantly stabilized by mitotic Wnt signaling. Wnt/STOP Signaling Regulates Cell Size When cells divide without growth, they become smaller. Therefore, one reason why Wnt signaling peaks in mitosis may be to stabilize proteins in preparation for division so that daughter cells inherit enough protein. If true, Wnt signaling may impact G1 cell size, which is largely determined by its protein content. Indeed, HeLa cells treated with Wnt3a were on average 14% larger than Dkk1-treated cells (Figure 5A). This is a significant change, 6 Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc.

in the same range as that obtained with TOR pathway inhibition (12%) (Figure 5B), which is considered as the master regulator of cell size, and also similar to other cell size studies (Ohtsubo and Roberts, 1993; Fingar et al., 2002; Rosner et al., 2003). Moreover, under TOR pathway inhibition Wnt3a-treated cells were still 15% larger than Dkk1-treated cells (Figure 5B), indicating TORindependent cell size regulation by Wnt signaling. To confirm that Dkk1 functions through Wnt signaling, we also blocked endogenous Wnt secretion with the porcupine inhibitor IWP-2, which mimicked Dkk1 effects (Figure S4A). The following experiments further indicate that the Wnt effect on cell size involves Wnt/STOP, but not b-catenin-dependent transcription: (1) siLRP5/6 and siCycY both reduced cell size, while sib-catenin had no significant effect (Figure 5C), although it efficiently blocked TOPFLASH reporter activity (Figures S4B and S4C); (2) in siLRP5/6-treated cells the GSK3 inhibitor SB216763 (SB) fully rescued cell size reduction (Figure 5D); (3) overexpression of either GSK3 or the E3 ubiquitin ligases b-TrCP and especially FBW7 (both recognizing GSK3 degrons) reduced G1 cell size (Figures 5E and 5F); (4) to further corroborate b-catenin independency, we saturated b-catenin-dependent transcription via overexpression, which, unlike Wnt3a treatment, did not increase cell size (Figures S4D and S4E); (5), even with b-catenin overexpression, siLRP5/6 and siCycY reduced cell size, which was once again rescued by the GSK3 inhibitor SB (Figure S4F). We conclude that Wnt/STOP promotes cell growth and cell size in HeLa cells. Wnt3a-induced increase in cell size was not only limited to HeLa cells but also occurred in MDA-MB-231, DLD-1, and HEK293T cells, as well as mouse embryonic fibroblasts (MEFs) obtained from E14.5 embryos as a model for untransformed cells. In MDA-MB-231 cells Wnt3a treatment stabilized c-MYC during mitosis (Figures S1D and S1E) and increased the average cell size by 17.6% (Figure 6A). This increase occurred under Rapamycin treatment, and hence was independent of TOR pathway and S6K activation (Figure 6B). In the colon cancerderived cell line DLD-1 harboring mutated APC, Wnt3a increased cell size by 9.1% (Figures 6C and 6D). Similarly, Wnt3a increased cell size of HEK293T cells by 15.5% without activating TOR pathway (Figures 6E and 6F). Cell size increase was blocked by siCycY but not by sib-catenin (Figures S5A

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Molecular Cell Wnt/STOP Signaling Regulates Cell Size

Figure 4. Wnt/STOP Increases Protein Content in G2/M (A) Wnt signaling branches regulating cellular protein levels. (B–D and F) Cytometric analyses of relative protein content of N-hydroxysuccinimide ester (NHS)-fluorescein-labeled HeLa cells synchronized and treated as explained in Figure 1D (SD, n = 3). Where indicated, cells were transfected with siRNA 24 hr before synchronization, and/or treated with 5 nM Rapamycin, or transfected with FBW7, or perforated with saponin. Note that Wnt signaling increases protein content in G2/M but not in G1 (B), and via LRP5/6 and CycY, but not b-catenin (C), and this effect is blocked by FBW7 overexpression (D). This regulation is Rapamycin insensitive and hence TOR independent (see Figure S3B) and affects predominantly cytoplasmic proteins (F). (E) Wnt signaling does not affect protein biosynthesis in G2/M. Methionine-analog L-azidohomoalanine (AHA) incorporation in synchronized mitotic HeLa cells upon Wnt3a or Dkk1 treatment with or without 5 nM Rapamycin. Cells were analyzed by flow cytometry (SD, n = 3).

and S5B), like in HeLa cells. Wnt3a-treated MEFs were 24.5% larger than controls (Figure S5C), which was again TOR independent, occurring even in the presence of Rapamycin (Figures 6G and 6H). The results in MDA-MB-231, DLD-1, HEK293T, and MEF cells strongly point to a Wnt/STOP-dependent regulation of cell size. However, further characterization of these cells is required to unequivocally rule out transcriptional effects, as in HeLa cells. To genetically uncouple Wnt/STOP from Wnt/b-catenin signaling, we analyzed cell size in the non-small-cell lung cancer (NSCLC)-derived cell line HCC15. These cells exhibit endogenous Wnt2b and Wnt9a signaling (Akiri et al., 2009), but they harbor constitutively active b-catenin (CTNNB1 S45A mutation) (Shigemitsu et al., 2001), which makes b-catenin and TOPFLASH reporter assays insensitive to Wnt (Figures 6I and S5D). Thus, in HCC15 cells mitotic Wnt signaling can be uncoupled from Wnt-mediated b-catenin-dependent transcription. When we inhibited endogenous Wnt/STOP signaling with siCycY,

siLRP5/6, or the Wnt secretion inhibitor IWP-2, cells were smaller than controls (Figures 6J, 6K, and S5E), which was TOR pathway independent, occurring even in the presence of Rapamycin (Figure S5F). Thus, inhibition of canonical Wnt signaling can change growth of cancer cells independent of b-catenin and TOR pathway. DISCUSSION We set out to answer why Wnt signaling is high in mitotic cells when transcription is low. The key discoveries of this study are (1) that protein stabilization oscillates with the cell cycle in a Wnt-dependent manner (Wnt/STOP); (2) Wnt/STOP stabilizes the critical cell cycle effector c-MYC; (3) the identification of more than 100 candidate proteins whose polyubiquitination is regulated by GSK3; and (4) that Wnt/STOP promotes cell growth and cell size independent of b-catenin. We propose that the physiological relevance of Wnt/STOP signaling is to regulate Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc. 7

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Molecular Cell Wnt/STOP Signaling Regulates Cell Size

Figure 5. Wnt/STOP Increases Cell Size in HeLa Cells (A) Wnt signaling increases G1 cell size. Forward scatter (FSC-H) profile of G1-gated HeLa cells treated with Wnt3a or Dkk1 for 48 hr. (B–F) Cell size of G1 HeLa cells treated for 48 hr as indicated (SD, n R 3). Where indicated, cells were transfected with the indicated siRNAs. SB, GSK3 inhibitor SB-216763. (B) Bottom, western blots showing that Rapamycin treatment completely abolished TOR-dependent S6K phosphorylation. (F) E3 ligases were transfected 24 hr before the treatment. CDC20, the recognition subunit of APC/C, was used as negative control. Note that Wnt signaling increases G1 cell size independent of TOR (B) and via LRP5/6 and CycY, but not b-catenin (C and D). Cell size effects are SB-216763 and GSK3 sensitive and hence GSK3 dependent (D and E). Wnt-induced cell size effects are also FBW7 sensitive (F).

cell size by slowing down GSK3-dependent protein degradation as cells prepare to divide. Wnt/STOP Is an Alternative Mode of Wnt Signaling A main conclusion of our study is that a subset of the proteome, which is under direct or indirect GSK3 control, becomes periodically stabilized during the G2/M phase of the cell cycle in a Wnt-dependent manner (Figure 7). Evidence has been accumulating that there is significant signaling through a Wnt cascade, which bifurcates at the level of GSK3 to stabilize proteins beyond b-catenin (Table S3). Based on bioinformatic analysis of proteins carrying potential GSK3 phosphorylation sites, Taelman et al. suggested that up to 20% of the proteome could be under Wnt/GSK3 control, and showed increased half-life of bulk [35S]-methionine labeled proteins upon Wnt treatment (Taelman et al., 2010). Our results support this notion, since in HeLa cells 15% of total or 25% of cytoplasmic protein is stabilized by Wnt/STOP, and specifically at G2/M. Likewise, our protein microarray experiment revealed 8 Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc.

over 100 candidate proteins whose polyubiquitination is GSK3 dependent. This number is a conservative estimate, because (1) the array interrogated only around 9,000 proteins, and (2) the in vitro ubiquitination of immobilized proteins by a HeLa cell extract is probably less efficient than in intact cells (Merbl and Kirschner, 2009; Merbl et al., 2013), notably since GSK3 requires prephosphorylation (priming) of most substrates (Cohen and Frame, 2001). b-catenin-independent Wnt signaling employing the upstream canonical cascade has been observed from fly to man (Table S1). An interesting perspective is that in certain cell types Wnt/ STOP may be more relevant than Wnt/b-catenin signaling, such as in HeLa cells. Conversely, there are cells where Wnt/ b-catenin signaling is the physiologically dominant pathway, for example in Drosophila embryos, where an armadillo transgene is able to rescue wingless mutants (Chiang et al., 2009), or Xenopus embryonic dorsoventral patterning (White and Heasman, 2008). Thus, the relative physiological importance of Wnt/ STOP is likely cell type dependent.

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Molecular Cell Wnt/STOP Signaling Regulates Cell Size

Figure 6. Wnt/STOP Increases Cell Size in Various Cell Types (A, C, E, and G) Cell-size analysis by forward scatter (FSC-H) profiles of G1-gated cells. Volume variation was monitored as in Figure 5 and is shown as mean ± SD from biological triplicates (A, p = 0.0022; C, p = 0.0092; E, p = 0.0011; G, p = 0.0056). (B, D, F, and H) Western blots showing the effects of Rapamycin and Wnt3a treatments on TOR-dependent S6K phosphorylation. Note that Wnt signaling does not increase p-S6K in HEK293T, indicating that Wnt3a functions independently of TOR pathway. (I) TOPFLASH reporter assays in NSCLC-derived HCC15 cells, which harbor a constitutively active b-catenin mutant (S45P), which cannot be stabilized further by Wnt ligands, unlike in HeLa cells. RLA, relative luciferase activity. (J) Cell size of G1-gated HCC15 cells transfected with the indicated siRNAs and grown with or without Rapamycin. (K) Knockdown control from (J) shown by western blots. Note that both siLRP5/6 and siCycY increased S6K phosphorylation in HCC15 cells instead of reducing it. This occurs also in other NSCLC-derived cell lines (H1299 and H358, data not shown) and could be caused by compensatory upregulation of protein translation upon aberrant GSK3-driven proteolysis.

Wnt/STOP Signaling Regulates Cell Growth The second main conclusion of this study is that a biological role of Wnt/STOP signaling is to increase cellular protein levels and cell size, and hence to promote cell growth. Wnt/STOP controls the stability of 15% of proteins in HeLa cells. This is increased up to 30% in Rapamycin-treated cells, revealing redundancy with mTOR signaling. A similar trend applies for cell size in various cell lines including HeLa, MDA-MB-231, HEK293T, MEF, DLD-1, and HCC15 cells. Hence, our data indicate that canonical Wnt signaling regulates cell growth by three arms,

which trifurcate at the level of GSK3: Wnt/b-catenin signaling induces transcription, Wnt/TOR promotes protein biosynthesis, and Wnt/STOP prevents protein degradation (Figure 4A). We show that Wnt/STOP regulates cell size and growth, which are important parameters for cell cycle progression (Dolznig et al., 2004; Kafri et al., 2013). Cells need to duplicate their mass before division, and Wnt/STOP signaling increases protein levels at the time when cells prepare to divide. It appears reasonable that such cells would be in a growth advantage during the next G1 phase. Beyond a bulk protein effect (Jorgensen and Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc. 9

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Molecular Cell Wnt/STOP Signaling Regulates Cell Size

EXPERIMENTAL PROCEDURES Antibodies Rabbit polyclonal anti-cyclin Y (CycY), anti-LRP6, and anti-Sp1490 antibodies were as described (Davidson et al., 2009). Other antibodies used were antia-tubulin, anti-b-catenin, anti-ERK1/2, anti-RPSA (Sigma); anti-CDC25A, antiS6K, anti-phospho-T389-S6K, anti-phospho-T286-cyclin-D1 (Cell Signaling); anti-phospho-Histone 3-Ser10 (pH3), anti-MARK3, anti-HINT2, anti-CENPT, anti-phospho-T58/S62-MYC (Abcam); anti-c-myc, anti-p27, anti-p130, anticyclin D1, anti-HDAC7 (Santa Cruz Biotechnology); and anti-Lys48-linked ubiquitin (anti-K48-Ub) (Merck Millipore). For western blot, antibodies were diluted in TBST containing 5% BSA and 1 mM EDTA. Anti-K48-Ub antibody binds specifically to lysine-48-linked ubiquitin chains, which are the most abundant ubiquitin chains (80%) (Kaiser et al., 2011) and the most common label for proteasomal degradation. Statistics Data are expressed as mean values ± SD of biological replicates (n R 3). Two-tailed unpaired Student’s t tests were used for most statistical analyses. Differences were considered to be statistically significant for *p < 0.05, **p < 0.01, or ***p < 0.005. No significant difference is indicated by n.s. Figures 2B and 3B were analyzed as described in the Supplemental Information. ACCESSION NUMBERS

Figure 7. Mitotic Wnt/STOP Signaling Wnt-dependent stabilization of proteins (Wnt/STOP) peaks during mitosis and protects proteins from GSK3-dependent polyubiquitination and degradation. Note that Wnt/STOP stabilizes proteins containing GSK3/FBW7 (F) and GSK3/ b-TrCP (T) degrons, but not unrelated proteins (X). As a consequence, mitotic cells harbor more proteins, and daughter cells are bigger.

Tyers, 2004; Kafri et al., 2013), we identified the G1 activators CCND1 and c-MYC as Wnt/STOP targets, which may directly promote growth in daughter cells. Considering that GSK3 is regulated by other signaling pathways, how Wnt specific is STOP signaling? Interestingly, in addition to Wnt, PKB/PI3K signaling peaks at G2/M in epithelial cells (Shtivelman et al., 2002). The PKB/PI3K pathway is triggered by insulin, which is a well-known inhibitor of protein degradation and also inhibits GSK3, although by different mechanisms than Wnt, by phosphorylation of Ser9 (Pap and Cooper, 1998; Faridi et al., 2003). This suggests that multiple pathways may converge on protein stabilization during mitosis. Finally, our results may open options for treatment of Wntdependent cancers. Of the three Wnt signaling arms (b-catenin, TOR, and Wnt/STOP), pharmaceutical efforts to treat Wnt related cancers focus on targeting the b-catenin arm. However, there is evidence that factors upstream of mutated b-catenin or APC are also relevant in tumorigenesis (e.g., SFZD1 and DKK1; Vincan and Barker, 2008; Table S1). Similarly, the Wnt/STOP target c-MYC and the E3 ligase FBW7, whose misexpression blocks Wnt/STOP effects on protein content and cell size (Figures 4D and 5F), are associated with tumorigenesis, including colorectal cancer (He et al., 1998; Welcker and Clurman, 2008). These results and our observation that inhibition of Wnt signaling can change growth in HCC15 and DLD-1 cells despite hyperactive b-catenin or mutated APC, respectively, suggest opportunities for pharmacological cancer intervention. 10 Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc.

The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession numbers GSE50629 and GSE50248. SUPPLEMENTAL INFORMATION Supplemental Information includes five figures, three tables, one movie, and Supplemental Experimental Procedures and can be found with this article at http://dx.doi.org/10.1016/j.molcel.2014.04.014. ACKNOWLEDGMENTS We thank J. Herbst and C. Reinhard for technical help; the DKFZ microarray, flow cytometry, and microscopy core facilities for expert technological support; and E.M. De Robertis, S. Fuchs, N. Daigle, J. Sonntag, U. Koch, and A. Glinka for reagents. We thank A. Teleman and S. Koch for critical reading of the manuscript. This work was supported by the DFG. Received: February 4, 2014 Revised: March 27, 2014 Accepted: April 8, 2014 Published: May 15, 2014 REFERENCES Aberle, H., Bauer, A., Stappert, J., Kispert, A., and Kemler, R. (1997). b-catenin is a target for the ubiquitin-proteasome pathway. EMBO J. 16, 3797–3804. Akiri, G., Cherian, M.M., Vijayakumar, S., Liu, G., Bafico, A., and Aaronson, S.A. (2009). Wnt pathway aberrations including autocrine Wnt activation occur at high frequency in human non-small-cell lung carcinoma. Oncogene 28, 2163–2172. Anastas, J.N., and Moon, R.T. (2013). WNT signalling pathways as therapeutic targets in cancer. Nat. Rev. Cancer 13, 11–26. Angers, S., and Moon, R.T. (2009). Proximal events in Wnt signal transduction. Nat. Rev. Mol. Cell Biol. 10, 468–477. Arago´n, E., Goerner, N., Zaromytidou, A.I., Xi, Q., Escobedo, A., Massague´, J., and Macias, M.J. (2011). A Smad action turnover switch operated by WW domain readers of a phosphoserine code. Genes Dev. 25, 1275–1288. Bilic, J., Huang, Y.L., Davidson, G., Zimmermann, T., Cruciat, C.M., Bienz, M., and Niehrs, C. (2007). Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation. Science 316, 1619–1622.

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Molecular Cell Wnt/STOP Signaling Regulates Cell Size

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