Research Article 981 Plk2 regulates centriole duplication through phosphorylation-mediated degradation of Fbxw7 (human Cdc4)

Onur Cizmecioglu1, Annekatrin Krause1, Ramona Bahtz1, Lena Ehret1, Nisar Malek2 and Ingrid Hoffmann1,* 1Cell cycle Control and Carcinogenesis (F045), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany 2University Hospital Tu¨bingen, Department of Internal Medicine 1, Otfried-Mu¨ller-Str. 10, 72076 Tu¨bingen, Germany *Author for correspondence ([email protected])

Accepted 26 September 2011 Journal of Cell Science 125, 981–992 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.095075

Summary Polo-like (Plks) perform crucial functions during mitosis, cytokinesis and centriole duplication. Plk2 is activated in early G1 phase and is involved in the reproduction of centrosomes. However, the mechanisms underlying Plk2-induced centriole duplication are incompletely understood. Here, we show that Plk2 directly targets the F-box protein F-box/WD repeat-containing protein 7 (Fbxw7), which is a regulator of the ubiquitin-mediated degradation of cyclin E. Plk2 phosphorylates Fbxw7 on serine 176 and the two proteins form a complex in vitro and in vivo. Phosphorylation of Fbxw7 by Plk2 induces destabilization of the F-box protein resulting in accumulation of cyclin E and increased potential for centriole reproduction. In addition, loss of Fbxw7 in human cells leads to uncontrolled centriole duplication, highlighting the importance of Fbxw7 regulation by Plk2. These findings define a previously unknown Plk2-dependent pathway involved at the onset of S phase and in centrosome duplication.

Key words: Plk2, Fbxw7, human Cdc4, Cyclin E, Cell cycle, Centrosome

Introduction permissive cell cycle window (G1–S transition and early S phase) Transitions between the different phases of the cell cycle, and before they can respond to Plk4 activity (Kleylein-Sohn et al., successful growth and division, require the coordinated action of 2007). 2/2 Journal of Cell Science numerous protein kinases. Among these are the polo-like kinases Analysis of the growth and development of Plk2 mice (Plks), which play pivotal roles during mitosis and the centrosome indicated that Plk2 is involved in embryonic development and cycle (Archambault and Glover, 2009). Mammalian cells express cell cycle progression at the G1–S transition (Ma et al., 2003). In four polo-like family members, –4. The founding addition, Plk2 is localized to the centrosome. Plk2 kinase is first member of the Plk family, polo, was first described in fruit flies as activated at the G1–S phase transition and its activity is required a mitotic regulator (Llamazares et al., 1991). Plk1 is expressed in for centriole duplication (Cizmecioglu et al., 2008; Warnke et al., G2 and M phases and localizes to the centrosomes, kinetochores 2004). Silencing of Plk2 by RNAi leads to apoptosis in the and central spindle during mitosis, and is required for a normal presence of Taxol (Burns et al., 2003), an effect that might be metaphase spindle (Lane and Nigg, 1996; Llamazares et al., 1991; explained by a failure to duplicate centrosomes. Recently, CPAP, Tokuyama et al., 2001) and cytokinesis (Litvak et al., 2004; Neef a protein that controls centriole length has been identified as a et al., 2003). In addition, Plk1 is implicated in centrosome Plk2 substrate in this process (Chang et al., 2010); however maturation (Barr et al., 2004). Plk3 appears to be expressed at insight into how Plk2 substrates are involved in the control of constant levels throughout the cell cycle, and plays a role in stress centriole duplication remains scarce. response pathways, including those activated by DNA damage and Centrosomes are the microtubule-organizing centers (MTOCs) spindle disruption (Bahassi el et al., 2002; Donohue et al., 1995; of animal cells. Centrosome duplication must occur in Xie et al., 2001). Plk4 activity is implicated in centriole duplication coordination with other cell cycle events, including DNA (Bettencourt-Dias et al., 2005; Habedanck et al., 2005). Following synthesis. Indeed, duplication of the centrioles begins near the activation of Plk4 and its recruitment to the centrosome by its G1–S boundary and is completed in G2 (Doxsey et al., 2005). interacting protein Cep152 (Cizmecioglu et al., 2010; Dzhindzhev Centriole duplication is regulated by different protein kinases, but et al., 2010; Hatch et al., 2010), a sequential assembly of several their interplay is incompletely understood (Strnad and Gonczy, crucial proteins including Sas6, Cep135, CPAP, c-tubulin and 2008). Cdk2–cyclin-E kinase activity is required for initiation of CP110 is induced (Kleylein-Sohn et al., 2007). It is suggested that centriole duplication (Hinchcliffe et al., 1999; Matsumoto et al., Plk4 stability is placed under direct control of its own activity and 1999; Meraldi et al., 1999), whereas the continuation of this this could define an important mechanism for limiting normal process during S phase seems to depend on the Cdk2–cyclin-A centriole duplication to once per cell cycle (Cunha-Ferreira et al., complex. A centrosomal localization domain within cyclin E is 2009; Guderian et al., 2010; Holland et al., 2010; Rogers et al., essential for promoting S-phase entry in a Cdk2-independent 2009; Sillibourne et al., 2010). However, cells need to reach a manner (Matsumoto and Maller, 2004). Two centrosomal 982 Journal of Cell Science 125 (4)

substrates of Cdk2–cyclin E have been identified, nucleophosmin To analyze whether the decrease in the amount of cyclin E (NPM; also known as B23) and CP110 (Chen et al., 2002; Okuda protein in response to Plk2 RNAi is due to an accelerated rate of et al., 2000). Npm12/2 mouse embryonic fibroblasts (MEFs) cyclin E degradation by the proteasome, we used U2OS cells exhibit aberrant centrosome numbers as a consequence of treated with Plk2 siRNA or control siRNA that were synchronized unrestrained centrosome duplication (Grisendi et al., 2005). at G1–S and treated with the proteasome inhibitor MG132. Plk2 Deregulation of cyclin E abundance can trigger premature S- RNAi followed by treatment of cells with MG132 lead to a phase entry, genomic instability and cancer (Hwang and stabilization of cyclin E protein (Fig. 1c). Next, we tested whether Clurman, 2005; Spruck et al., 2002). Cyclin E is targeted for the stability of cyclin E is altered by Plk2 RNAi by treating cells ubiquitin-mediated degradation by Fbxw7 (human Cdc4) an F- with the protein synthesis inhibitor cycloheximide. In response to box protein, which is the specificity component of the Skp– Plk2 siRNA transfection cyclin E decayed faster than in control Cullin–F-Box (SCF)–Fbxw7 multi-subunit E3 ubiquitin . siRNA-treated cells (Fig. 1d). To further evaluate the role of Plk2 Inactivation of Fbxw7 leads to cyclin E accumulation (Koepp in regulation of cyclin E stability we explored ubiquitylation and et al., 2001; Strohmaier et al., 2001) but cyclin E protein levels proteasome-dependent degradation of cyclin E upon expression of are also controlled by the cullin-3 pathway (Singer et al., 1999). Plk2 kd. Ubiquitylated proteins from cells overexpressing His- Fbxw7 recognizes a short, phosphothreonine-containing motif tagged ubiquitin were purified on nickel columns and analyzed by known as the Fbxw7 phosphodegron (CPD) present in each of its western blotting to detect cyclin E. Interfering with Plk2 activity substrates, including cyclin E (Nash et al., 2001). Cyclin E by expression of the Plk2 kd mutant promoted ubiquitylation of contains two CPDs that are phosphorylated by glycogen synthase cyclin E (supplementary material Fig. S2c). Taken together, these kinase 3b and autophosphorylated by Cdk2 (Koepp et al., 2001; results indicate that Plk2 kinase activity is required for stabilization Strohmaier et al., 2001; Welcker et al., 2003). To date, little is of cyclin E protein. known about the regulation of Fbxw7 itself. Because cyclin E is an activator of Cdk2 we investigated In the present study we investigated the function of Plk2 in whether expression of Plk2 kd might interfere with Cdk2 function centriole duplication. We show that Plk2 phosphorylates the F- in centriole reduplication. Cdk2 was coexpressed with Plk2 kd in box protein Fbxw7 at three conserved serine residues, leading to the presence of aphidicolin in U2OS cells. Upon expression of its destabilization. This in turn results in both increased cyclin E Cdk2 wt alone the number of cells with additional centrioles (more than four) increased by ,15% in comparison with cells levels and capability to duplicate centrioles. that were untransfected but aphidicolin treated. Indeed co- transfection of Cdk2 wt and Plk2 kd led to a decrease in the Results number of cells with more than four centrioles by about 40% To determine how Plk2 affects centriole duplication and S-phase (Fig. 1e). Taken together, these results suggest that Cdk2 cannot entry we set out to identify substrates of Plk2. Probable regulate centriole duplication when Plk2 kinase activity is candidates were proteins that are present in G1 or those impaired. Thus, Cdk2 and Plk2 cooperate either in the same or regulating the G1–S phase transition. We therefore investigated in parallel pathways in the regulation of centriole reduplication. whether Plk2 might have an effect on protein levels of known Because Plk2 neither interacted with nor phosphorylated G1 regulators. Ablation of Plk2 function led to a specific Journal of Cell Science Cdk2–cyclin E (data not shown) we focused on regulators of downregulation of cyclin E protein in both S-phase-synchronized cyclin E protein stability. The abundance of cyclin E is controlled and exponentially growing human U2OS cells and was primarily at the level of transcription and ubiquitin- accompanied by a reduced cyclin-E-associated kinase activity dependent proteolysis. Cyclin E can be ubiquitylated by the (Fig. 1a). Plk2 short interfering RNA (siRNA) had only a minor SCF Fbxw7 ubiquitin ligase (Strohmaier et al., 2001; Koepp et al., effect on the G1–S phase transition in U2OS cells (supplementary 2001). In addition, cyclin E turnover is catalyzed by at least two material Fig. S1a–c) and did not impair the expression of Plk1, kinases, GSK3 and Cdk2 (Welcker et al., 2003). We found that Cdk2, p27 or Cdc25A, whereas cyclin A protein levels were Plk2 neither phosphorylated nor interacted with GSK3 (data not slightly decreased (supplementary material Fig. S1d). In addition, shown). Our data also indicate that interfering with Plk2 function upon Plk2 downregulation, the timing of the events at the G1–S does not lead to degradation of cyclin E when the GSK3 and phase transition, as determined by cyclin E accumulation or Plk1 Cdk2 phosphorylation site, T395, which is located within the degradation, remained unaltered (supplementary material Fig. phosphodegron that binds Fbxw7, is mutated to alanine. siRNA- S1e). We observed centrosomal staining of cyclin E in early G1 induced knockdown of Plk2 did not promote cyclin E phase. In addition to the overall reduction of cyclin E in the cell, phosphorylation on T395 (supplementary material Fig. S3a,b) its localization to the centrosome was also markedly affected in suggesting that Plk2 might target Fbxw7. Treatment of U2OS response to Plk2 RNA interference (RNAi; supplementary cells or non-transformed human telomerase reverse transcriptase material Fig. S2a). To further confirm these results, a plasmid (hTERT) RPE1 cells with Plk2 siRNA led to an accumulation of expressing kinase-inactive Plk2 (GFP–Plk2 kd) was transfected Fbxw7 protein (Fig. 2a,b) but did not affect Fbxw7 mRNA levels into HeLa cells and the effect on cyclin E protein levels in (supplementary material Fig. S3c). Transfection of U2OS cells comparison to transfection of an empty vector (GFP) was with siRNA oligonucleotides targeting Plk4 did not alter cyclin E analyzed. Similar to Plk2 siRNA treatment, competing out the or Fbxw7 levels (Fig. 2c), suggesting that the effects on cyclin E endogenous Plk2 kinase upon transfection of Plk2 kd led to and Fbxw7 are specific for Plk2. Similarly, expression of Plk2 kd reduced levels of cyclin E protein, without significantly altering lead to an accumulation of endogenous Fbxw7 protein, whereas the cell cycle distribution of the transfected population of cells upon expression of Plk2 wt Fbxw7 levels declined, suggesting (supplementary material Fig. S2b). By contrast, upon expression that Plk2 might regulate its stability (Fig. 2d). Addition of the of wild-type Plk2 (Plk2 wt), cyclin E levels were slightly proteasome inhibitor MG132 rescued the decline in Fbxw7 increased (Fig. 1b). protein levels, which could indicate that the process is Plk2 regulates Fbxw7 stability 983 Journal of Cell Science

Fig. 1. Plk2 downregulation affects cyclin E stability and cyclin E-associated kinase activity. (a) Western blots of extracts from U2OS cells that were transfected with either control (GL-2) or Plk2 siRNAs for 60 hours and immunoblotted with anti-Plk2 CDL21 or anti-cyclin E antibodies in exponential or aphidicolin-treated cells. Cyclin-E-associated kinase activity of cells transfected with control or Plk2 siRNAs was determined on histone H1 in an in vitro kinase assay. (b) HeLa cells were transfected with GFP, GFP–Plk2 wt and GFP–Plk2 kd for 36 hours and cell extracts were assessed for endogenous cyclin E. Anti-GFP immunoblotting demonstrated the expression of transfected constructs. *Crossreacting bands observed in GFP immunoblots. (c) U2OS cells were transfected with control or Plk2 siRNA and 20 hours post-transfection were treated with aphidicolin for 40 hours. To analyze cyclin E stability cells were incubated with MG132 for 6 hours. Cyclin E levels before and after MG132 treatment were determined by anti-cyclin E immunoblotting. (d) Cycloheximide was added to inhibit new protein synthesis, and cyclin E levels at the indicated time points were analyzed by western blotting. The numbers above lanes denote relative band intensities. (e) U2OS cells were co-transfected with Cdk2 wt and GFP–Plk2 kd. At 12 hours post-transfection, cells were treated with aphidicolin for 60 hours and then fixed for immunostaining. Overexpression of Cdk2 was detected with anti-Cdk2 (blue) antibody, GFP–Plk2 kd was fluorescently detected (green) and GT335 was used to stain the centrioles (red). Arrows indicate the position of nascently formed centrioles. Scale bar: 10 mm. Right: quantification of the centriole reduplication experiments. Error bars denote the standard deviation of three independent experiments (n5200). ***P50.005 (Student’s t-test) for centriole numbers in control versus Cdk2-wt-transfected cells.

proteasome dependent (Fig. 2d). We also analyzed whether the activity gradually increased in HeLa cells in early G1 phase after levels of two other Fbxw7 substrates, Myc and Jun (reviewed by release from a prometaphase block. At the same time point, Welcker and Clurman, 2008), were affected. Although the levels Fbxw7 protein levels started to decrease, demonstrating that an of Myc were clearly lower upon Plk2 RNAi treatment, the effect increase of Plk2 kinase activity directly correlates with a decrease on Jun levels was less pronounced (supplementary material Fig. in Fbxw7 protein levels (Fig. 2e). Taken together, these results S3d). Fbxw7 recognizes pre-phosphorylated substrates, so the suggest that Plk2 regulates cyclin E abundance by interfering level of priming phosphorylation on a particular substrate might with Fbxw7 stability. determine its susceptibility to the changes in Fbxw7. We next investigated whether complexes between endogenous We examined whether Plk2 kinase activity during G1 phase Plk2 and Fbxw7 could be detected in vivo. As seen in Fig. 3a, correlated with a change in Fbxw7 protein levels. Plk2 kinase endogenous Fbxw7 was present in Plk2 immunoprecipitates. 984 Journal of Cell Science 125 (4) Journal of Cell Science

Fig. 2. Plk2 regulates Fbxw7 protein levels. (a) U2OS cells were transfected with either control or Plk2-specific siRNAs. Cell lysates were prepared 72 hours after transfections and samples were probed with anti-Fbxw7, anti-Plk2 and anti-cyclin E antibodies. (b) hTERT RPE1 cells were transfected with control or Plk2 siRNAs. Cell lysates were prepared 72 hours after transfections; samples were probed with the indicated primary antibodies. The numbers above lanes in the Fbxw7 blot denote relative band intensities. (c) U2OS cells were transfected with control, Plk2 and Plk4 siRNAs for 60 hours and probed with anti-Plk2, anti-Plk4, anti-Fbxw7 and anti-cyclin E antibodies. (d) FLAG-empty, FLAG–Plk2 kd or FLAG–Plk2 wt was expressed in HEK293T cells. Cell lysates were prepared 24 hours post-transfection and immunoblotted with the indicated antibodies. MG132 was added to the reactions 3 hours before harvesting, where indicated. (e) HeLa cells were blocked with nocodazole, released and harvested for the indicated time points. Cell extracts were immunoblotted with the indicated antibodies. For in vitro kinase assays endogenous Plk2 was immunoprecipitated and kinase activity was determined with a-casein as substrate in the presence of [c-32P]ATP. *Crossreacting band in Fbxw7 immunoblots.

In support of this notion, interactions between ectopically with the centrosome we made use of centrosome-enriched fractions expressed FLAG-tagged Plk2 kd and Myc-tagged Fbxwa the prepared by sucrose gradient centrifugation. We observed that isoform that was used throughout this study, could also be detected Fbxw7 was associated with centrosomes and found in the same in reciprocal co-immunoprecipitations (Fig. 3b). These results fractions as Plk2 and the centrosomal proteins c-tubulin and centrin demonstrate that Plk2 and Fbxw7 associate in vivo. To determine 2 (Fig. 3d). Together these results suggest that Fbxw7 and Plk2 are whether the two proteins directly interact we performed pulldown localized together at the centrosome. assays using Fbxw7 that was in vitro translated in the presence of To explore the possibility that Fbxw7 is phosphorylated [35S]methionine and then precipitated with Zz-tagged Plk2 bound directly by Plk2 we carried out in vitro kinase assays. Active to IgG–Sepharose. An efficient recovery of Fbxw7 was observed, Plk2 but not its kinase inactive form phosphorylated Myc–Fbxw7 suggesting that the proteins probably directly bind to each other (Fig. 4a). Phosphorylated Myc–Fbxw7 was then subjected to (Fig. 3c). Owing to the absence of suitable antibodies we mass spectrometry to identify Plk2-specific phosphorylation were unable to detect the endogenous Fbxw7 protein in sites. Three sites were identified, namely serine (S)25, S176 immunofluorescence assays. To show an association of Fbxw7 and S349 (Fig. 4b). To verify that these sites are major sites Plk2 regulates Fbxw7 stability 985

Fig. 3. Biochemical evidence for Plk2–Fbxw7 interaction and association to centrosomes. (a) Endogenous Plk2 was immunoprecipitated from U2OS S-phase lysates and the association with Fbxw7 was detected with anti-Fbxw7 antibodies. Random rabbit IgGs were used as an IP control. (b) Myc–Fbxw7 and FLAG– Plk2 kinase dead (kd) constructs were transfected into HEK293T cells. Reciprocal IP western blots were performed with anti-Myc and anti-FLAG antibodies to determine the association of the expressed proteins. (c) In-vitro-translated (IVT) Myc–Fbxw7 was used in a binding assay with bacterially expressed and purified Journal of Cell Science wild-type (wt) and kinase dead versions of Zz-Plk2. IVT Myc–Fbxw7 was loaded to indicate the appropriate size. (d) Fbxw7 protein was detected in centrosome fractions purified using a discontinuous sucrose gradient. Immunoblots were performed on whole cell lysates (WCL) and sucrose fractions (centrosome preparations) from HeLa cells with the indicated antibodies. Akt and lamin B1 immunoblots were performed to detect possible cytoplasmic and nuclear contamination.

phosphorylated by Plk2 the same three serine residues were (Fig. 5b). Moreover, we found that endogenous Fbxw7 had mutated to alanine and this mutant was used in an in vitro kinase increased levels of S176 phosphorylation upon elevated assay. The triple mutant Fbxw7AAA exhibited a markedly reduced expression of Plk2 (Fig. 5c). Reciprocally, S-phase enrichment phosphorylation signal in comparison with the wild-type protein of cells through aphidicolin treatment lead to an increase in S176 (Fig. 4c). To strengthen these findings we examined the phosphorylation, whereas siRNA-mediated downregulation of phylogenetic conservation of Fbxw7 phosphorylation sites. This either Plk2 or Fbxw7 substantially reduced the signal (Fig. 5d). analysis revealed that all three sites are conserved in Fbxw7 Thus, Plk2 phosphorylates Fbxw7 in vivo. orthologs from human to Xenopus laevis (Fig. 4d). S176 and To investigate the effect of Plk2-dependent phosphorylation on S349 are also present in zebrafish (Danio rerio). Furthermore, we the stability of Fbxw7, the stability of non-phosphorylatable found that mutant Fbxw7AAA is functional in Fbxw7–SCF alanine (Fbxw7AAA) and phosphomimetic aspartic acid complex formation (supplementary material Fig. S4a). To show (Fbxw7DDD) mutant proteins was examined in comparison to that Fbxw7 was phosphorylated in vivo by Plk2, we generated wild-type Fbxw7 in cells treated with cycloheximide. Although a polyclonal phosphorylation-specific antibody against Fbxw7 wt started to decline after 1.5 hours the Fbxw7AAA mutant phosphorylated S176 (S176-P) and S349 (S349-P) as these are was more stable with a 1.5-hour longer half-life. By contrast, the more conserved residues through evolution. However, the Fbxw7DDD was much more labile than the wild-type protein anti-S349-P antibody did not work in the in vitro and in vivo and started declining even after 0.5 hours suggesting that experiments. The anti-S176-P antibody recognized Fbxw7 wt but phosphorylation by Plk2 on the three serine residues markedly not the Fbxw7 S176A mutant upon incubation with Plk2 and reduced the stability of Fbxw7 (Fig. 6a). Moreover, when Plk2 ATP in vitro (Fig. 5a). Phosphorylated Fbxw7 was detected by wt was expressed in HEK293T cells together with either Fbxw7 anti-S176-P and the signal was increased when Plk2 was or Fbxw7AAA only the Fbxw7 wt protein levels declined with expressed along with Fbxw7 wt but not with the S176A mutant increasing amounts of Plk2 (Fig. 6b). In an attempt to find out 986 Journal of Cell Science 125 (4)

Fig. 4. Plk2 phosphorylates Fbxw7 in vitro on three conserved serine residues. (a) Myc–Fbxw7 expressed in HEK293T cells, was immunoprecipitated with anti-Myc antibodies and subjected to an in vitro kinase assay with Plk2 in the presence of [c-32P]ATP. Plk2 kd and Myc–Fbxw7 served as negative controls. (b) Scheme representing Fbxw7a with the Plk2 phosphorylation sites identified by mass-spectrometry analysis. DD, dimerization domain. (c) In vitro kinase assays were performed with Fbxw7AAA in comparison to the wild type. Myc–Fbxw7 constructs were transiently expressed in HEK293T and immunoprecipitated with anti-Myc antibodies. (d) Amino acid sequences from a range of species aligned around the phosphorylation sites. Conserved residues are depicted in green and variations are depicted in red. Phosphorylation sites are indicated in black. Although S25 is specific for the a-isoform of Fbxw7, both S176 and S349 are also present in the Fbxw7 b and c isoforms.

which of the three sites is crucial for Fbxw7 stability, can induce cyclin E ubiquitylation in the presence of Plk2 upon a cycloheximide chase profiles of individual serine to alanine short treatment of cells with MG132. This short incubation with mutants of Fbxw7 were compared. All three mutants appeared to MG132 allowed us to visualize the extent of cyclin E

Journal of Cell Science be more resistant to degradation than the wild-type protein. ubiquitylation before the treatment with the inhibitor leads to S176A and S349A mutants were more stable than the S25A saturation of proteins upon stabilization. Cyclin E ubiquitylation mutant, which displayed a modest stability (supplementary was markedly enhanced at 2 hours of MG132 treatment when material Fig. S4b). This suggests that the residues S176 and Plk2 wt and the Fbxw7AAA mutant were coexpressed, whereas S349 might be more potent in controlling Fbxw7 stability than the effect of Fbxw7 wt on cyclin E ubiquitylation was minimal in S25. Fbxw7 is known to form intermolecular homodimers response to Plk2 expression (Fig. 7a). This can be explained by (Welcker and Clurman, 2007; Zhang and Koepp, 2006). We the reduced stability of Fbxw7 wt protein in comparison to the aimed to find out whether phosphorylation of a dimerization non-phosphorylatable mutant, although alternative explanations partner could modulate the stability and thus the abundance of the such as the mutant Fbxw7 increasing SCF–cyclin E complex endogenous Fbxw7 protein in trans. Equal amounts of FLAG– formation could not be ruled out. Because Fbxw7 promotes Fbxw7 wt were co-transfected with normalized amounts of either cyclin E degradation we expected that its excessive expression GFP–Fbxw7 wt, GFP–Fbxw7AAA or GFP–Fbxw7DDD to achieve would inhibit centriole reduplication. In fact, centriole comparable expressions in HEK293T cells. Interestingly, FLAG– reduplication was impaired in a Fbxw7-dependent manner in Fbxw7 wt was markedly stabilized in the presence of GFP– aphidicolin-arrested U2OS cells expressing GFP–centrin1. This Fbxw7AAA whereas GFP–Fbxw7DDD coexpression substantially effect could be restored when Plk2 was co-transfected with destabilized the Fbxw7 wt version. This decrease in Fbxw7 Fbxw7 wt but not with the Fbxw7AAA mutant (Fig. 7b), stability could be reversed by MG132 treatment, indicating that it suggesting that phosphorylation of Fbxw7 by Plk2 enables indeed is regulated by proteasome-mediated degradation centriole reduplication. Interestingly enhanced Plk2 expression (Fig. 6c). These findings suggest that phosphorylation of one could not rescue the deficiency in centriole reduplication upon interaction partner lead to destabilization of Fbxw7 homodimer, siRNA-mediated downregulation of cyclin E (Fig. 7b). This thus regulating Fbxw7 protein levels. Taken together, these data indicates the importance of cyclin E in centriole reduplication indicate that S25, S176 and S349 are the crucial phosphorylation because Plk2 cannot mediate centriole duplication in the absence sites within Fbxw7 that are required for Plk2-mediated of cyclin E. degradation of Fbxw7. Finally, to test whether the loss of Fbxw7 promotes centriole Although Fbxw7AAA promotes ubiquitylation and degradation duplication, we made use of karyotypically stable HCT116 cells of cyclin E similar to Fbxw7 wt (supplementary material Fig. in which both alleles of Fbxw7 were disrupted by homologous S4c,d), we found that the Fbxw7AAA mutant but not Fbxw7 wt recombination. HCT116 FBXW72/2 cells were reported to Plk2 regulates Fbxw7 stability 987

Fig. 5. Plk2 phosphorylates Fbxw7 on S176 in vivo. (a) HEK293T cells were transfected with either FLAG–Fbxw7 wt or FLAG–Fbxw7 S176A constructs. Anti-FLAG immunoprecipitates were then subjected to an in vitro kinase assay with bacterially expressed and purified GST–Plk2 wt as indicated. Boiled samples were analyzed with anti-FLAG, anti-S176-P (pS176) and anti-GST antibodies in a western blot. (b) HEK293T cells were transfected with HA-empty or HA–Plk2 Journal of Cell Science wt together with either FLAG-empty, FLAG–Fbxw7 wt or FLAG–Fbxw7 S176A for 60 hours. Cells were then treated with MG132 for 4 hours. Relative expression levels of transfected plasmids are shown in the bottom panel. Anti-FLAG immunoprecipitates were probed with either anti-FLAG or anti-pS176 antibodies on the top. (c) HEK293T cells were transfected with FLAG-empty or FLAG–Plk2-wt constructs. The relative expression level of FLAG–Plk2-wt transfection is shown on the right. Endogenous Fbxw7 was immunoprecipitated with 15 mg of transfected clear cell lysate and the immunoprecipitates were probed with anti-Fbxw7 or anti-pS176 antibodies (left). Band intensity quantifications are included at the top of the bottom panel. (d) HEK293T cells were transfected with control, Plk2- or Fbxw7-specific siRNAs for 48 hours. 5 mg/ml aphidicolin was added 24 hours before harvesting some of the cells (as indicated). Fbxw7 immunoprecipitations were performed as in c. Transfected cells were treated with 5 mg/ml MG132, 4 hours before harvesting. Band intensity quantifications are included at the top of the bottom panel.

exhibit chromosomal instability (Rajagopalan et al., 2004). The FBXW72/2 cells, whereas the level of inhibition in wild-type presence of extra centrosomes might contribute to the HCT116 cells was comparable to that seen with Cdk2 RNAi manifestation of this phenotype. Therefore, we analyzed (Fig. 7c,d). This is presumably because Plk2 cannot regulate centriole numbers in cycling HCT116 FBXW7+/+ and cyclin E levels in the absence of Fbxw7. Taken together, these FBXW72/2 cells by staining CP110, a protein that localizes to results suggest that Plk2-mediated phosphorylation of Fbxw7 the distal part of centrioles (Chen et al., 2002). Interestingly, we promotes centriole duplication by preventing ubiquitin-dependent found that genetic ablation of Fbxw7 resulted in a threefold degradation of cyclin E. increase in the percentage of cells harboring extra copies of centrioles. Prolonged incubation with aphidicolin dramatically Discussion enhanced the percentage of extra centrioles in HCT116 The F-box protein SCF Fbxw7 is involved in the degradation of FBXW72/2 cells, whereas only a moderate effect was observed proteins with key roles in and cell growth, including in wild-type HCT116 cells. Moreover, siRNA-mediated cyclin E and Myc (Koepp et al., 2001; Strohmaier et al., 2001; downregulation of either Cdk2 or cyclin E inhibited formation Welcker et al., 2004). Fbxw7 inactivation by homologous of extra centrioles both in wild-type and FBXW72/2 cells, recombination in human HCT116 cells caused genetic indicating that the observed reduplication phenotype is dependent instability associated with cyclin E activation (Rajagopalan on cyclin E and associated Cdk2 kinase activity. Plk2 knockdown et al., 2004). Here, we show an interesting and previously only partially inhibited centriole reduplication in HCT116 unknown regulatory function for Plk2: it modulates Fbxw7 988 Journal of Cell Science 125 (4)

Fig. 6. Plk2-dependent phosphorylation of Fbxw7 negatively regulates its stability. (a) HEK293T cells were transfected with Myc–Fbxw7 wt, non- phosphorylatable Fbxw7 (Fbxw7AAA) and phospho-mimetic Fbxw7 (Fbxw7DDD) constructs for 24 hours and then treated with cycloheximide for the indicated time periods. Lysates were analyzed for Fbxw7 signal with anti-Myc antibodies (left). Relative intensities of Myc–Fbxw7 bands at the indicated time points are graphically depicted (right). The experiment was repeated three times with similar results. (b) Myc–Fbxw7 wt and the AAA mutant were coexpressed with increasing amounts of wild-type (wt) Plk2 in HEK293T cells. The total amount of transfected plasmid DNA was kept constant by adding empty pCMV-Myc vector. Cell lysates were prepared 24 hours post-transfection. MG132 was added to the reactions for 4 hours before harvesting, where indicated. (c) Equal amounts of FLAG–Fbxw7 wt was coexpressed with either GFP alone, GFP–Fbxw7 wt, GFP–Fbxw7AAA or GFP–Fbxw7DDD in HEK293T cells. GFP–Fbxw7 expression levels were normalized by adjusting the Journal of Cell Science amount of plasmid DNA needed to obtain comparable expression levels. The total amount of transfected plasmid DNA was kept constant by adding empty pCMV-FLAG vector. Cell lysates were prepared 36 hours post-transfection. MG132 was added to the reactions for 6 hours before harvesting, where indicated. Immunoblots were performed with the indicated antibodies. *Crossreacting bands in GFP immunoblots. Band intensity quantifications are included above respective panels.

protein levels through direct phosphorylation of three serine substrates are caused by altered levels of the E3 ligase rather than residues. Plk2-mediated phosphorylation of Fbxw7 leads to its its activity. Our data further suggest that a phosphorylated form proteasome-mediated degradation and to subsequent stabilization of Fbxw7 could influence the stability of unmodified Fbxw7 of cyclin E. Analysis of the CDC4 gene transcripts has revealed through homodimerization (Fig. 6c), an effect that might that three splice variant isoforms are expressed, designated a, b potentiate the consequences of Plk2-mediated phosphorylation and c (Spruck et al., 2002). Although S25 is specific for the a- of Fbxw7. isoform, both S176 and S349 are present in the Fbxw7 b and c A large body of evidence suggests that highly conserved polo- isoforms. Phosphorylation of Fbxw7 on serine and glutamine box domains present in the C-terminal region of polo kinases residues was reported in response to DNA damage but not in the play a pivotal role in the function of these . The PBD unperturbed cells (Matsuoka et al., 2007). It would be intriguing serves as an essential molecular mediator that brings the kinase to find out if this DNA-damage-induced phosphorylation plays a domain of Plks into proximity with its substrates, mainly through role in Fbxw7 regulation. Interestingly, the protein levels of the phosphorylation-dependent interactions with its target proteins Fbxw7a isoform decrease in response to oxidative stress (Olson (Elia et al., 2003). Recently, Cdk5 has been identified as a et al., 2008). Of note, Fbxw7a was shown to be phosphorylated priming kinase for Plk2-mediated phosphorylation of SPAR, a by serum and glucocorticoid inducible kinase (SGK1) on postsynaptic RapGAP in homeostatic synaptic plasticity (Seeburg S227, which promotes Notch1-IC degradation through et al., 2008). Of equal importance will be the identification of enhanced ubiquitylation (Mo et al., 2011). Plk2-mediated priming kinases that generate PBD binding sites in Fbxw7. phosphorylation of Fbxw7, however, does not seem to alter Cyclin E is localized to the centrosome and can promote S- intrinsic Fbxw7 activity, because comparable amounts of phase entry (Matsumoto and Maller, 2004). It has been suggested Fbxw7AAA and Fbxw7 wt trigger cyclin E ubiquitylation and that a 20-amino-acid centrosome localization sequence within degradation to similar extents (supplementary material Fig. human cyclin E is essential for promotion of S-phase entry S4c,d). Therefore Plk2-mediated effects manifested on Fbxw7 independently of Cdk2. We detected Fbxw7 cofractionating with Plk2 regulates Fbxw7 stability 989

Journal of Cell Science Fig. 7. Fbxw7 phosphorylation by Plk2 regulates cyclin E ubiquitylation and triggers centriole duplication. (a) The extent of cyclin E ubiquitylation was AAA determined after 1 and 2 hours of MG132 treatment, in the presence of either Myc–Fbxw7 wt or Myc–Fbxw7 coexpressed with FLAG–Plk2 wt and His6- ubiquitin. The 1 hour treatment with MG132 refers to a time point when the drug is not fully effective and poly-ubiquitylation of cyclin E is not efficiently

detectable. Samples were normalized according to His6-ubiquitin expression. The anti-cyclin E immunoblot depicts the level of cyclin E ubiquitylation, whereas the anti-His immunoblot served as loading control. (b) GFP–centrin1 U2OS cells were transfected with Myc-empty, Myc–Fbxw7 wt, Myc–Fbxw7AAA and cyclin E siRNAs together with either FLAG-empty or FLAG–Plk2 wt constructs. Cells were treated with aphidicolin 12 hours after transfection for 60 hours and were then fixed with methanol–acetone. Centrioles in transfected cells were quantified by counting GFP–centrin1-positive dots in individual cells (n.100, three independent experiments; error bars denote standard deviation). Level of cyclin E knockdown is shown in the right panel. (c) HCT116 FBXW7+/+ and FBXW72/2 cells were transfected with control, Cdk2, Plk2 and cyclin E siRNAs. Cells were either left untreated or incubated with aphidicolin 24 hours after siRNA transfections for 60 hours. The number of centrioles in methanol-fixed cells on coverslips were analyzed with the aid of CP110 immunostaining (n.100, in three independent experiments; error bars denote standard deviation). The corresponding immunoblot analysis of aphidicolin-treated samples was performed with the indicated antibodies (right). (d) Either untreated or aphidicolin-treated HCT116 FBXW7+/+ and FBXW772/2 cells on coverslips were stained with anti-CP110 antibodies. Representative images of unduplicated, duplicated and reduplicated centrioles are shown (enlarged in the inset). Scale bar: 10 mm. DNA is shown in blue and CP110 is in green in the merged images.

Plk2 in centrosome-enriched fractions from sucrose gradient of S phase (Clurman et al., 1996; Won and Reed, 1996). Plk4 centrifugation. Along the same lines, a number of proteasomal kinase has also been shown to cooperate with Cdk2 in regulating subunits have been identified in a mass-spectrometry-based centriole duplication (Habedanck et al., 2005). In the future, it proteomic analysis of human centrosomes (Andersen et al., would be intriguing to determine whether Cdk2–cyclin E and 2003). Moreover, in mammalian cells Skp1 and Cul1, which are Plk2 also function in the centriole duplication pathway in components of the SCF complex are localized to interphase and cooperation with Plk4. Interestingly, Plk22/2 MEFs were mitotic centrosomes (Freed et al., 1999). Our findings further reported to have a prolonged cell cycle and an impaired indicate that Plk2 kinase cooperates with Cdk2–cyclin E in the G1–S transition (Ma et al., 2003). Therefore, Plk2-mediated regulation of centriole reproduction. Our data suggest that Plk2 phosphorylation of Fbxw7 might globally influence cell cycle kinase activity has a crucial function to keep cyclin E levels high kinetics. through destabilization of Fbxw7 in order to ensure faithful In conclusion, Plk2 could contribute to aneuploidy and duplication of centrioles before cyclin E is degraded at the onset tumorigenesis by decreasing the levels of Fbxw7, leading to a 990 Journal of Cell Science 125 (4)

continuous expression of cyclin E. More intriguingly, Fbxw7 has immunoprecipitations for determining Plk2 kinase activity were performed using 2 mg of HeLa lysate and 2.5 mg anti-Plk2 antibodies. The antibody–antigen been implicated in tumorigenesis, and mutations in Fbxw7 have complexes were incubated for 3 hours at 4˚C and then were collected with protein- been found in a rapidly expanding number of human neoplasms A–Sepharose beads for 1 hour at 4˚C. For the kinase assays 3 mg a-casein (Sigma) (reviewed by Welcker and Clurman, 2008). However, Fbxw7 can was used as exogenous substrate in the presence of [a-32P]ATP, and the reactions also be deregulated in tumors without mutations in Fbxw7. For were incubated for 20 minutes at 30˚C. Fbxw7 immunoprecipitations for S176-P signal detection were conducted using 15 mg of HEK293T lysates with 6 mg rabbit example oncogenic ras prevents Fbxw7-dependent cyclin E anti-Fbxw7 (Santa Cruz, H300) antibodies per IP. Transfected cells were treated ubiquitylation and degradation by altering the physical with 5 mg/ml MG132 for 4 hours before harvesting. Lysis buffer contained interaction between Fbxw7 and cyclin E (Minella et al., 2005). 500 ng/ml calyculin A (Cell Signaling) in addition to the protease inhibitors. siRNA-transfected HEK293T cells were treated with 5 mg/ml aphidicolin 24 hours Thus phosphorylation-dependent regulation of Fbxw7 by Plk2 is before harvesting. Cyclin E immunoprecipitations for determining cyclin E- a new mechanism for Fbxw7 deregulation that is linked to associated kinase activity were performed as previously described (Warnke et al., chromosomal instability and tumorigenesis. 2004) and tested for histone H1 (Roche; 2 mg protein/reaction) kinase activity. The antibody–antigen complexes were collected with protein-G–Sepharose beads for 1 hour at 4˚C, washed twice with the lysis buffer and used for in vitro kinase Materials and Methods assays. Kinase assays were performed as described previously (Warnke et al., Cell culture, transfections and centriole duplication assay 2004). Various Myc or FLAG–Fbxw7 constructs were expressed in HEK293T, and HeLa, HEK293T, U2OS and GFP–centrin1 U2OS cells were grown at 37˚Cina immunoprecipitated using 2 mg anti-Myc (9E10; Sigma) or anti-FLAG (M2; 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM), Sigma) antibodies. For kinase assays of FLAG–Fbxw7, 1 mg of GST–Plk2 wt supplemented with 10% fetal bovine serum, 2 mM L-glutamine (Sigma), and (Calbiochem) was used. penicillin and streptomycin (100 IU/ml and 100 mg/ml, respectively). HCT116 FBXW7+/+ and FBXW72/2 cells were cultured in McCoy’s 5A medium (Sigma) under standard conditions. hTERT-RPE1 cells were cultured in Ham’s 12 medium Preparation of centrosomes by sucrose gradients under standard conditions. HeLa and U2OS cells were arrested in prometaphase by Centrosomes were isolated from HeLa S3 cells by discontinuous sucrose gradient addition of 50 ng/ml nocodazole (Sigma) for 15 hours. Mitotic cells were then ultracentrifugation. Briefly, the cell pellet was washed with TBS and 0.16 TBS/ harvested by shake off, washed five times and reincubated with fresh medium for 8% sucrose. Cells were resuspended in 0.16 TBS/8% sucrose and mixed with the release from the block. HEK293T cells were transfected using calcium 0.5% NP-40 lysis buffer. The suspension was shaken slowly for 30 minutes at 4˚C phosphate and HBS buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 3 mM and spun at 2500 g for 10 minutes. HEPES buffer and DNase were added to the EDTA). HeLa cells were transfected using calcium phosphate and BES buffer supernatant to a final concentrations of 10 mM and 1 mg/ml, respectively. After N (50 mM BES, pH 6.9, 280 mM NaCl, 1.5 mM Na2HPO4 2H2O). U2OS, HCT116 incubation for 30 minutes at 4˚C, the mixture was gently underlaid with 60% and HEK293T cells were transfected with DNA plasmids and small-interfering sucrose solution and spun at 10,000 g for 30 minutes. The obtained centrosomal RNAs using Lipofectamine 2000 (Invitrogen) or Polyfect (Qiagen) according to suspension was loaded onto a discontinuous sucrose gradient (70, 50 and 40% the manufacturer’s instructions. sucrose solutions from the bottom), and spun at 120,000 g for 1 hour. Fractions In order to inhibit proteasome-mediated degradation, U2OS enriched in early S were collected from the top, diluted with PIPES buffer (10 mM PIPES), and spun phase with 5 mg/ml aphidicolin for 40 hours, and asynchronous HEK293T cells, at 20,400 g for 15 minutes. were treated with 5 mg/ml MG132 (Sigma) for up to 6 hours. For the centriole reduplication assay, U2OS, GFP–centrin1 U2OS or HCT116 cells were grown on coverslips, transfected with Lipofectamine 2000, and then treated with 1.6 mg/ml In-gel tryptic digestion and liquid chromatography–tandem mass aphidicolin for 12 hours. Cells on coverslips were fixed after 60 hours of spectrometry (LC-MS/MS) analysis aphidicolin treatment. For inhibition of protein synthesis, U2OS or HEK293T cells Either untreated or Plk2 wt pre-treated Myc–Fbxw7 immunoprecipitates were treated with 200 mg/ml cycloheximide (Sigma) for up to 4 hours. were resolved by one-dimensional SDS-PAGE. Proteins present in the gel lane were visualized with Coomassie Blue (G250, Roth) staining. Myc–Fbxw7 Construction of expression vectors bands were cut out with a scalpel. Gel slices were transferred to a 96-well plate and reduced, alkylated and digested with trypsin (Catrein et al., 2005) using a Journal of Cell Science Plk2, Fbxw7 and cyclin E mutations were generated using the QuikChange Site- Directed Mutagenesis Kit (Stratagene). Human FBXW7 cDNA was obtained from Digest pro MS liquid handling system (Intavis AG, Germany). Following Molekulare Genomanalyse, DKFZ (Heidelberg, Germany). The insert was PCR digestion, tryptic peptides were extracted from the gel pieces with 50% amplified with EcoRI- and HindIII-compatible primers and inserted into pCMV- acetonitrile, 0.1% trifluoroacetic acid (TFA), concentrated nearly to dryness in a 3Tag-1C (or 2C) vector (Stratagene) and the sequence verified. pQE80zz vector speedVac vacuum centrifuge and diluted to a total volume of 30 ml with 0.1% was a gift from Dirk Go¨rlich (Go¨ttingen, Germany) (Jakel and Go¨rlich, 1998). Plk2 TFA. 25 ml of the sample was analyzed using a nanoHPLC system (Eksigent, was cloned into the pQE80zz vector upon PCR amplification with NcoI- and Dublin Ca; Axel Semrau) coupled to a ESI LTQ Orbitrap mass spectrometer BamHI-compatible primers. SmaI- and HindIII-compatible primers were used to (Thermo Fisher). The sample was loaded on a C18 trapping column (Inertsil, LC clone FBXW7 into pEGFP C2 vector (Clontech). Packings, Amsterdam, The Netherlands) with a flow rate of 10 ml/minute 0.1% TFA. Peptides were eluted and separated on an analytical column (75 mm6150 mm) packed with Inertsil 3 mm C18 material (LC Packings) with Antibodies for western blotting a flow rate of 200 nl/minute in a gradient of buffer A (0.1% formic acid) and buffer A standard protocol for immunoblotting was used (Hassepass et al., 2003). Primary B (0.1% formic acid, acetonitrile): 0–6 minutes, 3% B; 6–60 minutes, 3–40% B; antibodies used were anti-Cdk2 (Blomberg and Hoffmann, 1999), anti-centrin2 (H40), 60–65 minutes, 60–90% B. The column was connected to a nano-ESI emitter anti-cyclin E (HE12), anti-pT395 cyclin E, anti-cyclin A (H432), anti-Myc (E910), (New Objectives). 1500 V were applied by a liquid junction. One survey scan (res: anti-p27, anti-His (C19), anti-Skp1, anti-cullin1, anti-Fbxw7 (D16 and H300) all from 60,000) was followed by five information-dependent product ion scans in the LTQ. Santa Cruz Biotechnology (Heidelberg, Germany), anti-Fbxw7 (Invitrogen), anti- Only doubly and triply charged ions were selected for fragmentation. FLAG, anti-GFP (Ab290, Abcam), anti-cyclin B (Hoffmann et al., 1993), anti-Plk1 The peptide sequences of the phosphorylated peptides were confirmed by (Zymed), anti-Plk2 (Warnke et al., 2004), anti-Plk4 (Cizmecioglu et al., 2010), anti- manual evaluation of the fragment spectra. lamin B1 (Zwerger et al., 2010), anti-c-Myc (no. 9402, Cell Signaling), anti-c-Jun (no. 9165, Cell Signaling), anti-Akt (BD, no. 610860), anti-a-tubulin (Sigma) and anti-b- actin (clone C4, ICN). Secondary antibodies were horseradish-peroxidase-conjugated Flow cytometry anti-rabbit and anti-mouse IgGs (Jackson), anti-rabbit Trueblot HRP (eBioscience) or For cytofluorometric analysis of DNA content, an aliquot of 26105 cells was anti-goat HRP from Santa Cruz Biotechnology. Enhanced chemiluminescence collected by centrifugation and treated with RNase (50 mM Tris-HCl, pH 7.5, immunoblotting detection reagents were from PerkinElmer Life Sciences. 10 mM MgCl2,10mg of RNase A/ml; Roche) for 30 minutes at 37˚C. The cells Quantification of band intensities was performed with ImageJ (NIH, Bethesda, were stained with propidium iodide (35 mg/ml, Sigma) for 30 minutes on ice. MD). Rabbit anti-pS176 Fbxw7 antibodies were raised against a synthetic peptide with Analysis was carried out by flow cytometry with a FACSSort (Becton Dickinson the following sequence; (NH2-)CRKLDHGS(P)EVRSFS(-COOH). After the second Biosciences) using the CellQuest software for at least 10,000 cells. booster, rabbits were killed and antibodies in the immune serum were tandemly affinity purified against non-phosphorylated and phosphorylated peptides, respectively siRNA experiments (Innovagen, Lund, Sweden). The Plk2-targeting oligonucleotides were based on a 19-mer sequence present in the coding sequence of human Plk2 (PLK2 siRNA1: 59-GGCAAGAU- Protein purification, immunoprecipitations and kinase assays AUAUUGACACA-39; PLK2 siRNA2: 59-GAGCAGCUGAGCACAUCAU-39), Fbxw7 immunoprecipitations (IP) were conducted using 5 mg S-phase-enriched which is absent in the other human polo-like kinases. The oligonucleotides were U2OS lysates, using mouse anti-Fbxw7 (Invitrogen) antibodies. Plk2 synthesized as dTdT capped patches of RNA (Qiagen, Dharmacon and Ambion). Plk2 regulates Fbxw7 stability 991

Firefly luciferase (GL-2) siRNA was used as a control. 25 nM oligonucleotides was Funding used for the RNAi studies where the concentration is not indicated. The This work was supported by a grant from the Deutsche Krebshilfe Cdk2-targetting oligonucleotides were based on the following sequence: 59- [grant number 109512 to I.H.]. AAGAUGGACGGAGCUUGUUAU-39 (Cdk2 siRNA). The sequence of Fbxw7 targeting siRNAs is as follows; 59-GAGUGGAUCUCUUGAUACA-39 (Fbxw7 siRNA). The sequence of cyclin E targeting siRNAs is as follows; Supplementary material available online at 59-GCUUCGGCCUUGUAUCAUU-39 (CycE siRNA). Plk4-targeting siRNA http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.095075/-/DC1 oligonucleotides were published previously (Habedanck et al., 2005). References In vivo ubiquitylation assay Andersen, J. S., Wilkinson, C. J., Mayor, T., Mortensen, P., Nigg, E. A. and Mann, M. In vivo ubiquitylation assays were performed by transfecting HEK293T cells with (2003). Proteomic characterization of the human centrosome by protein correlation 3 mgpCMVHis6-ubiquitin, 3 mg pCMV-FLAG-empty or 5 mg of FLAG-Plk2 kd, profiling. Nature 426, 570-574. together with 2 mg pX-cyclin E (gift from Giulio Superti-Furga, Vienna, Austria). Archambault, V. and Glover, D. M. (2009). Polo-like kinases: conservation and After 20 hours, the cells were treated with 5 mg/ml MG132 (Sigma) for up to divergence in their functions and regulation. Nat. Rev. Mol. Cell Biol. 10, 265-275. 4 hours to inhibit proteasome-mediated proteolysis. For the ubiquitylation assays Bahassi el, M., Conn, C. W., Myer, D. L., Hennigan, R. F., McGowan, C. H., AAA including Fbxw7 versions, 5 mgofMyc-Fbxw7wt,3mgMyc-Fbxw7 and Sanchez, Y. and Stambrook, P. J. (2002). Mammalian Polo-like kinase 3 (Plk3) is a 5 mg pX-cyclin E were co-transfected together with His6-ubiquitin. The cells multifunctional protein involved in stress response pathways. Oncogene 21, 6633- were then lysed in 1 ml urea buffer [8 M urea, 30 mM imidazole (Sigma), 0.1 M 6640. phosphate buffer pH 8.0]; sonicated, and 10 ml of the clear cell lysates were Barr, F. A., Sillje, H. H. and Nigg, E. A. (2004). Polo-like kinases and the orchestration analyzed for immunoblotting to serve as a means to normalize expressed His- of cell division. Nat. Rev. Mol. Cell Biol. 5, 429-440. ubiquitin levels. Cell lysates that were normalized for the amounts of His- Bettencourt-Dias, M., Rodrigues-Martins, A., Carpenter, L., Riparbelli, M., ubiquitin were then incubated with nickel-NTA-coupled agarose beads (Qiagen) Lehmann, L., Gatt, M. K., Carmo, N., Balloux, F., Callaini, G. and Glover, for 2 hours on a rotating wheel at room temperature. Beads were then washed 46 D. M. (2005). SAK/PLK4 is required for centriole duplication and flagella with urea buffer and the bound proteins were eluted with 26 SDS sample buffer development. Curr. Biol. 15, 2199-2207. and a final concentration of 200 mM imidazole, and analyzed for Blomberg, I. and Hoffmann, I. (1999). Ectopic expression of Cdc25A accelerates the immunoblotting. G(1)/S transition and leads to premature activation of cyclin E- and cyclin A- dependent kinases. Mol. Cell. Biol. 19, 6183-6194. Bobinnec, Y., Moudjou, M., Fouquet, J. P., Desbruyeres, E., Edde, B. and Bornens, M. RT-PCR (1998). Glutamylation of centriole and cytoplasmic tubulin in proliferating non-neuronal Total RNA was isolated from control or PLK2 siRNA-treated U2OS cells using the cells. Cell Motil. Cytoskeleton 39, 223-232. RNeasy MiniKit (Quiagen). cDNA was generated from 0.5 g total RNA using Burns, T. F., Fei, P., Scata, K. A., Dicker, D. T. and El-Deiry, W. S. (2003). Silencing standard conditions (Superscript reverse transcriptase; Invitrogen), and PCR was of the novel p53 target gene Snk/Plk2 leads to mitotic catastrophe in paclitaxel (taxol)-exposed cells. Mol. Cell. Biol. 23, 5556-5571. performed for Fbxw7 at 95 C for 30 seconds, 61 C for 30 seconds and 72 C for ˚ ˚ ˚ Catrein, I., Herrmann, R., Bosserhoff, A. and Ruppert, T. (2005). Experimental proof 30 seconds (30 cycles) and for GAPDH at 95˚C for 30 seconds, 65˚C for for a signal peptidase I like activity in Mycoplasma pneumoniae, but absence of a 45 seconds and 72˚C for 30 seconds (20 cycles). The following primers were used: gene encoding a conserved bacterial type I SPase. FEBS J. 272, 2892-2900. Fbxw7 forward, 59-CCCAGCAAGGACAGTTGGAA-39 and Fbxw7 reverse, 59- Chang, J., Cizmecioglu, O., Hoffmann, I. and Rhee, K. (2010). PLK2 phosphorylation GAACGGGCAGGTCCACAATA-39; GAPDH forward, 59-TGGATATTGTT- is critical for CPAP function in procentriole formation during the centrosome cycle. GCCATCAATGACC-39 and GAPDH reverse, 59-GATGGCATGGACTGTGG- EMBO J. 29, 2395-2406. TCATG-39. Chen, Z., Indjeian, V. B., McManus, M., Wang,L. and Dynlacht, B. D. (2002). CP110, a cell cycle-dependent CDK substrate, regulates centrosome duplication in human cells. Dev. Cell 3, 339-350. In vitro translation and direct binding assays Cizmecioglu, O., Warnke, S., Arnold, M., Duensing, S. and Hoffmann, I. (2008). Myc–Fbxw7 was in-vitro-translated using the TNT Coupled Reticulocyte Lysate Plk2 regulated centriole duplication is dependent on its localization to the centrioles 35 Systems (Promega) in the presence of S-labeled methionine according to the and a functional polo-box domain. Cell Cycle 7, 3548-3555. Journal of Cell Science manufacturer’s instructions (PerkinElmer). In-vitro-translated product (15 ml) was Cizmecioglu, O., Arnold, M., Bahtz, R., Settele, F., Ehret, L., Haselmann-Weiss, U., incubated with 5 mg purified Zz-Plk2 or Zz-tag-empty in a final volume of 500 ml Antony, C. and Hoffmann, I. (2010). Cep152 acts as a scaffold for recruitment of lysis buffer for 2 hours at 4˚C. The complexes were then pulled down with 5 ml Plk4 and CPAP to the centrosome. J. Cell Biol. 191, 731-739. packed IgG–Sepharose beads (GE Healthcare) for 1 hour at 4˚C on a rotating Clurman, B. E., Sheaff, R. J., Thress, K., Groudine, M. and Roberts, J. M. (1996). wheel. Beads were washed five times with the lysis buffer and processed for Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by cdk2 loading onto a gel for SDS-PAGE. binding and cyclin phosphorylation. Dev. 10, 1979-1990. Cunha-Ferreira, I., Rodrigues-Martins, A., Bento, I., Riparbelli, M., Zhang, W., Laue, E., Callaini, G., Glover, D. M. and Bettencourt-Dias, M. (2009). The SCF/ Immunofluorescence microscopy Slimb ubiquitin ligase limits centrosome amplification through degradation of SAK/ Cells were fixed with either ice-cold methanol or methanol–acetone for PLK4. Curr. Biol. 19, 43-49. 10 minutes at 220˚C or with 10% formalin–10% methanol for 20 minutes at Donohue, P. J., Alberts, G. F., Guo, Y. and Winkles, J. A. (1995). Identification by targeted differential display of an immediate early gene encoding a putative serine/ room temperature. Formalin–methanol-fixed samples were permeabilized with threonine kinase. J. Biol. Chem. 270, 10351-10357. 0.5% NP-40 (Fluka) in PBS for 5 minutes at room temperature. The primary Doxsey, S., McCollum, D. and Theurkauf, W. (2005). Centrosomes in cellular antibodies used were Cdk2 (Blomberg and Hoffmann, 1999), c-tubulin (T3559, regulation. Annu. Rev. Cell Dev. Biol. 21, 411-434. Sigma), BrdU (Roche), glutamylated tubulin (GT-335) (Bobinnec et al., 1998), Dzhindzhev, N. S., Yu, Q. D., Weiskopf, K., Tzolovsky, G., Cunha-Ferreira, I., cyclin E (HE12, Santa Cruz), CP110 (Chang et al., 2010). Primary antibodies Riparbelli, M., Rodrigues-Martins, A., Bettencourt-Dias, M., Callaini, G. and were detected with anti-mouse and anti-rabbit Alexa-Fluor-488-conjugated, anti- Glover, D. M. (2010). 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We thank Angel Alonso, Stefan Duensing, Bernard Edde´, Harald Grisendi, S., Bernardi, R., Rossi, M., Cheng, K., Khandker, L., Manova, K. and Herrmann, Carsten Janke, Philipp Kaldis, Kunsoo Rhee and Bert Pandolfi, P. P. (2005). Role of nucleophosmin in embryonic development and Vogelstein for reagents. We acknowledge Ulrike Engel and Christian tumorigenesis. Nature 437, 147-153. Ackermann from the Nikon Imaging Center at the University of Guderian, G., Westendorf, J., Uldschmid, A. and Nigg, E. A. (2010). Plk4 trans- autophosphorylation regulates centriole number by controlling betaTrCP-mediated Heidelberg for equipment and assistance in implementation of degradation. J. Cell Sci. 123, 2163-2169. experiments. Ludger Hengst is thanked for critically reading the Habedanck, R., Stierhof, Y. D., Wilkinson, C. J. and Nigg, E. A. (2005). The Polo manuscript. kinase Plk4 functions in centriole duplication. Nat. Cell Biol. 7, 1140-1146. 992 Journal of Cell Science 125 (4)

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