Polo-like kinases (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.
Transitions between the different phases of the cell cycle, and successful growth and division, require the coordinated action of numerous protein kinases. Among these are the polo-like kinases (Plks), which play pivotal roles during mitosis and the centrosome cycle (Archambault and Glover, 2009). Mammalian cells express four polo-like kinase family members, Plk1–4. The founding member of the Plk family, polo, was first described in fruit flies as a mitotic regulator (Llamazares et al., 1991). Plk1 is expressed in G2 and M phases and localizes to the centrosomes, kinetochores and central spindle during mitosis, and is required for a normal metaphase spindle (Lane and Nigg, 1996; Llamazares et al., 1991; Tokuyama et al., 2001) and cytokinesis (Litvak et al., 2004; Neef et al., 2003). In addition, Plk1 is implicated in centrosome maturation (Barr et al., 2004). Plk3 appears to be expressed at constant levels throughout the cell cycle, and plays a role in stress response pathways, including those activated by DNA damage and spindle disruption (Bahassi el et al., 2002; Donohue et al., 1995; Xie et al., 2001). Plk4 activity is implicated in centriole duplication (Bettencourt-Dias et al., 2005; Habedanck et al., 2005). Following activation of Plk4 and its recruitment to the centrosome by its interacting protein Cep152 (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010), a sequential assembly of several crucial proteins including Sas6, Cep135, CPAP, γ-tubulin and CP110 is induced (Kleylein-Sohn et al., 2007). It is suggested that Plk4 stability is placed under direct control of its own activity and this could define an important mechanism for limiting normal centriole duplication to once per cell cycle (Cunha-Ferreira et al., 2009; Guderian et al., 2010; Holland et al., 2010; Rogers et al., 2009; Sillibourne et al., 2010). However, cells need to reach a permissive cell cycle window (G1–S transition and early S phase) before they can respond to Plk4 activity (Kleylein-Sohn et al., 2007).
Analysis of the growth and development of Plk2−/− mice indicated that Plk2 is involved in embryonic development and cell cycle progression at the G1–S transition (Ma et al., 2003). In addition, Plk2 is localized to the centrosome. Plk2 kinase is first activated at the G1–S phase transition and its activity is required for centriole duplication (Cizmecioglu et al., 2008; Warnke et al., 2004). Silencing of Plk2 by RNAi leads to apoptosis in the presence of Taxol (Burns et al., 2003), an effect that might be explained by a failure to duplicate centrosomes. Recently, CPAP, a protein that controls centriole length has been identified as a Plk2 substrate in this process (Chang et al., 2010); however insight into how Plk2 substrates are involved in the control of centriole duplication remains scarce.
Centrosomes are the microtubule-organizing centers (MTOCs) of animal cells. Centrosome duplication must occur in coordination with other cell cycle events, including DNA synthesis. Indeed, duplication of the centrioles begins near the G1–S boundary and is completed in G2 (Doxsey et al., 2005). Centriole duplication is regulated by different protein kinases, but their interplay is incompletely understood (Strnad and Gonczy, 2008). Cdk2–cyclin-E kinase activity is required for initiation of centriole duplication (Hinchcliffe et al., 1999; Matsumoto et al., 1999; Meraldi et al., 1999), whereas the continuation of this process during S phase seems to depend on the Cdk2–cyclin-A complex. A centrosomal localization domain within cyclin E is essential for promoting S-phase entry in a Cdk2-independent manner (Matsumoto and Maller, 2004). Two centrosomal substrates of Cdk2–cyclin E have been identified, nucleophosmin (NPM; also known as B23) and CP110 (Chen et al., 2002; Okuda et al., 2000). Npm1−/− mouse embryonic fibroblasts (MEFs) exhibit aberrant centrosome numbers as a consequence of unrestrained centrosome duplication (Grisendi et al., 2005).
Deregulation of cyclin E abundance can trigger premature S-phase entry, genomic instability and cancer (Hwang and Clurman, 2005; Spruck et al., 2002). Cyclin E is targeted for ubiquitin-mediated degradation by Fbxw7 (human Cdc4) an F-box protein, which is the specificity component of the Skp–Cullin–F-Box (SCF)–Fbxw7 multi-subunit E3 ubiquitin ligase. Inactivation of Fbxw7 leads to cyclin E accumulation (Koepp et al., 2001; Strohmaier et al., 2001) but cyclin E protein levels are also controlled by the cullin-3 pathway (Singer et al., 1999). Fbxw7 recognizes a short, phosphothreonine-containing motif known as the Fbxw7 phosphodegron (CPD) present in each of its substrates, including cyclin E (Nash et al., 2001). Cyclin E contains two CPDs that are phosphorylated by glycogen synthase kinase 3β and autophosphorylated by Cdk2 (Koepp et al., 2001; Strohmaier et al., 2001; Welcker et al., 2003). To date, little is known about the regulation of Fbxw7 itself.
In the present study we investigated the function of Plk2 in centriole duplication. We show that Plk2 phosphorylates the F-box protein Fbxw7 at three conserved serine residues, leading to its destabilization. This in turn results in both increased cyclin E levels and capability to duplicate centrioles.
To determine how Plk2 affects centriole duplication and S-phase entry we set out to identify substrates of Plk2. Probable candidates were proteins that are present in G1 or those regulating the G1–S phase transition. We therefore investigated whether Plk2 might have an effect on protein levels of known G1 regulators. Ablation of Plk2 function led to a specific downregulation of cyclin E protein in both S-phase-synchronized and exponentially growing human U2OS cells and was accompanied by a reduced cyclin-E-associated kinase activity (Fig. 1a). Plk2 short interfering RNA (siRNA) had only a minor effect on the G1–S phase transition in U2OS cells (supplementary material Fig. S1a–c) and did not impair the expression of Plk1, Cdk2, p27 or Cdc25A, whereas cyclin A protein levels were slightly decreased (supplementary material Fig. S1d). In addition, upon Plk2 downregulation, the timing of the events at the G1–S phase transition, as determined by cyclin E accumulation or Plk1 degradation, remained unaltered (supplementary material Fig. S1e). We observed centrosomal staining of cyclin E in early G1 phase. In addition to the overall reduction of cyclin E in the cell, its localization to the centrosome was also markedly affected in response to Plk2 RNA interference (RNAi; supplementary material Fig. S2a). To further confirm these results, a plasmid expressing kinase-inactive Plk2 (GFP–Plk2 kd) was transfected into HeLa cells and the effect on cyclin E protein levels in comparison to transfection of an empty vector (GFP) was analyzed. Similar to Plk2 siRNA treatment, competing out the endogenous Plk2 kinase upon transfection of Plk2 kd led to reduced levels of cyclin E protein, without significantly altering the cell cycle distribution of the transfected population of cells (supplementary material Fig. S2b). By contrast, upon expression of wild-type Plk2 (Plk2 wt), cyclin E levels were slightly increased (Fig. 1b).
To analyze whether the decrease in the amount of cyclin E protein in response to Plk2 RNAi is due to an accelerated rate of cyclin E degradation by the proteasome, we used U2OS cells treated with Plk2 siRNA or control siRNA that were synchronized at G1–S and treated with the proteasome inhibitor MG132. Plk2 RNAi followed by treatment of cells with MG132 lead to a stabilization of cyclin E protein (Fig. 1c). Next, we tested whether the stability of cyclin E is altered by Plk2 RNAi by treating cells with the protein synthesis inhibitor cycloheximide. In response to Plk2 siRNA transfection cyclin E decayed faster than in control siRNA-treated cells (Fig. 1d). To further evaluate the role of Plk2 in regulation of cyclin E stability we explored ubiquitylation and proteasome-dependent degradation of cyclin E upon expression of Plk2 kd. Ubiquitylated proteins from cells overexpressing His-tagged ubiquitin were purified on nickel columns and analyzed by western blotting to detect cyclin E. Interfering with Plk2 activity by expression of the Plk2 kd mutant promoted ubiquitylation of cyclin E (supplementary material Fig. S2c). Taken together, these results indicate that Plk2 kinase activity is required for stabilization of cyclin E protein.
Because cyclin E is an activator of Cdk2 we investigated whether expression of Plk2 kd might interfere with Cdk2 function in centriole reduplication. Cdk2 was coexpressed with Plk2 kd in the presence of aphidicolin in U2OS cells. Upon expression of Cdk2 wt alone the number of cells with additional centrioles (more than four) increased by ~15% in comparison with cells that were untransfected but aphidicolin treated. Indeed co-transfection of Cdk2 wt and Plk2 kd led to a decrease in the number of cells with more than four centrioles by about 40% (Fig. 1e). Taken together, these results suggest that Cdk2 cannot regulate centriole duplication when Plk2 kinase activity is impaired. Thus, Cdk2 and Plk2 cooperate either in the same or in parallel pathways in the regulation of centriole reduplication.
Because Plk2 neither interacted with nor phosphorylated Cdk2–cyclin E (data not shown) we focused on regulators of cyclin E protein stability. The abundance of cyclin E is controlled primarily at the level of gene transcription and ubiquitin-dependent proteolysis. Cyclin E can be ubiquitylated by the SCF Fbxw7 ubiquitin ligase (Strohmaier et al., 2001; Koepp et al., 2001). In addition, cyclin E turnover is catalyzed by at least two kinases, GSK3 and Cdk2 (Welcker et al., 2003). We found that Plk2 neither phosphorylated nor interacted with GSK3 (data not shown). Our data also indicate that interfering with Plk2 function does not lead to degradation of cyclin E when the GSK3 and Cdk2 phosphorylation site, T395, which is located within the phosphodegron that binds Fbxw7, is mutated to alanine. siRNA-induced knockdown of Plk2 did not promote cyclin E phosphorylation on T395 (supplementary material Fig. S3a,b) suggesting that Plk2 might target Fbxw7. Treatment of U2OS cells or non-transformed human telomerase reverse transcriptase (hTERT) RPE1 cells with Plk2 siRNA led to an accumulation of Fbxw7 protein (Fig. 2a,b) but did not affect Fbxw7 mRNA levels (supplementary material Fig. S3c). Transfection of U2OS cells with siRNA oligonucleotides targeting Plk4 did not alter cyclin E or Fbxw7 levels (Fig. 2c), suggesting that the effects on cyclin E and Fbxw7 are specific for Plk2. Similarly, expression of Plk2 kd lead to an accumulation of endogenous Fbxw7 protein, whereas upon expression of Plk2 wt Fbxw7 levels declined, suggesting that Plk2 might regulate its stability (Fig. 2d). Addition of the proteasome inhibitor MG132 rescued the decline in Fbxw7 protein levels, which could indicate that the process is proteasome dependent (Fig. 2d). We also analyzed whether the levels of two other Fbxw7 substrates, Myc and Jun (reviewed by Welcker and Clurman, 2008), were affected. Although the levels of Myc were clearly lower upon Plk2 RNAi treatment, the effect on Jun levels was less pronounced (supplementary material Fig. S3d). Fbxw7 recognizes pre-phosphorylated substrates, so the level of priming phosphorylation on a particular substrate might determine its susceptibility to the changes in Fbxw7.
We examined whether Plk2 kinase activity during G1 phase correlated with a change in Fbxw7 protein levels. Plk2 kinase activity gradually increased in HeLa cells in early G1 phase after release from a prometaphase block. At the same time point, Fbxw7 protein levels started to decrease, demonstrating that an increase of Plk2 kinase activity directly correlates with a decrease in Fbxw7 protein levels (Fig. 2e). Taken together, these results suggest that Plk2 regulates cyclin E abundance by interfering with Fbxw7 stability.
We next investigated whether complexes between endogenous Plk2 and Fbxw7 could be detected in vivo. As seen in Fig. 3a, endogenous Fbxw7 was present in Plk2 immunoprecipitates. In support of this notion, interactions between ectopically expressed FLAG-tagged Plk2 kd and Myc-tagged Fbxwα the isoform that was used throughout this study, could also be detected in reciprocal co-immunoprecipitations (Fig. 3b). These results demonstrate that Plk2 and Fbxw7 associate in vivo. To determine whether the two proteins directly interact we performed pulldown assays using Fbxw7 that was in vitro translated in the presence of [35S]methionine and then precipitated with Zz-tagged Plk2 bound to IgG–Sepharose. An efficient recovery of Fbxw7 was observed, suggesting that the proteins probably directly bind to each other (Fig. 3c). Owing to the absence of suitable antibodies we were unable to detect the endogenous Fbxw7 protein in immunofluorescence assays. To show an association of Fbxw7 with the centrosome we made use of centrosome-enriched fractions prepared by sucrose gradient centrifugation. We observed that Fbxw7 was associated with centrosomes and found in the same fractions as Plk2 and the centrosomal proteins γ-tubulin and centrin 2 (Fig. 3d). Together these results suggest that Fbxw7 and Plk2 are localized together at the centrosome.
To explore the possibility that Fbxw7 is phosphorylated directly by Plk2 we carried out in vitro kinase assays. Active Plk2 but not its kinase inactive form phosphorylated Myc–Fbxw7 (Fig. 4a). Phosphorylated Myc–Fbxw7 was then subjected to mass spectrometry to identify Plk2-specific phosphorylation sites. Three sites were identified, namely serine (S)25, S176 and S349 (Fig. 4b). To verify that these sites are major sites phosphorylated by Plk2 the same three serine residues were mutated to alanine and this mutant was used in an in vitro kinase assay. The triple mutant Fbxw7AAA exhibited a markedly reduced phosphorylation signal in comparison with the wild-type protein (Fig. 4c). To strengthen these findings we examined the phylogenetic conservation of Fbxw7 phosphorylation sites. This analysis revealed that all three sites are conserved in Fbxw7 orthologs from human to Xenopus laevis (Fig. 4d). S176 and S349 are also present in zebrafish (Danio rerio). Furthermore, we found that mutant Fbxw7AAA is functional in Fbxw7–SCF complex formation (supplementary material Fig. S4a). To show that Fbxw7 was phosphorylated in vivo by Plk2, we generated a polyclonal phosphorylation-specific antibody against phosphorylated S176 (S176-P) and S349 (S349-P) as these are the more conserved residues through evolution. However, the anti-S349-P antibody did not work in the in vitro and in vivo experiments. The anti-S176-P antibody recognized Fbxw7 wt but not the Fbxw7 S176A mutant upon incubation with Plk2 and ATP in vitro (Fig. 5a). Phosphorylated Fbxw7 was detected by anti-S176-P and the signal was increased when Plk2 was expressed along with Fbxw7 wt but not with the S176A mutant (Fig. 5b). Moreover, we found that endogenous Fbxw7 had increased levels of S176 phosphorylation upon elevated expression of Plk2 (Fig. 5c). Reciprocally, S-phase enrichment of cells through aphidicolin treatment lead to an increase in S176 phosphorylation, whereas siRNA-mediated downregulation of either Plk2 or Fbxw7 substantially reduced the signal (Fig. 5d). Thus, Plk2 phosphorylates Fbxw7 in vivo.
To investigate the effect of Plk2-dependent phosphorylation on the stability of Fbxw7, the stability of non-phosphorylatable alanine (Fbxw7AAA) and phosphomimetic aspartic acid (Fbxw7DDD) mutant proteins was examined in comparison to wild-type Fbxw7 in cells treated with cycloheximide. Although Fbxw7 wt started to decline after 1.5 hours the Fbxw7AAA mutant was more stable with a 1.5-hour longer half-life. By contrast, Fbxw7DDD was much more labile than the wild-type protein and started declining even after 0.5 hours suggesting that phosphorylation by Plk2 on the three serine residues markedly reduced the stability of Fbxw7 (Fig. 6a). Moreover, when Plk2 wt was expressed in HEK293T cells together with either Fbxw7 or Fbxw7AAA only the Fbxw7 wt protein levels declined with increasing amounts of Plk2 (Fig. 6b). In an attempt to find out which of the three sites is crucial for Fbxw7 stability, cycloheximide chase profiles of individual serine to alanine mutants of Fbxw7 were compared. All three mutants appeared to be more resistant to degradation than the wild-type protein. S176A and S349A mutants were more stable than the S25A mutant, which displayed a modest stability (supplementary material Fig. S4b). This suggests that the residues S176 and S349 might be more potent in controlling Fbxw7 stability than S25. Fbxw7 is known to form intermolecular homodimers (Welcker and Clurman, 2007; Zhang and Koepp, 2006). We aimed to find out whether phosphorylation of a dimerization partner could modulate the stability and thus the abundance of the endogenous Fbxw7 protein in trans. Equal amounts of FLAG–Fbxw7 wt were co-transfected with normalized amounts of either GFP–Fbxw7 wt, GFP–Fbxw7AAA or GFP–Fbxw7DDD to achieve comparable expressions in HEK293T cells. Interestingly, FLAG–Fbxw7 wt was markedly stabilized in the presence of GFP–Fbxw7AAA whereas GFP–Fbxw7DDD coexpression substantially destabilized the Fbxw7 wt version. This decrease in Fbxw7 stability could be reversed by MG132 treatment, indicating that it indeed is regulated by proteasome-mediated degradation (Fig. 6c). These findings suggest that phosphorylation of one interaction partner lead to destabilization of Fbxw7 homodimer, thus regulating Fbxw7 protein levels. Taken together, these data indicate that S25, S176 and S349 are the crucial phosphorylation sites within Fbxw7 that are required for Plk2-mediated degradation of Fbxw7.
Although Fbxw7AAA promotes ubiquitylation and degradation of cyclin E similar to Fbxw7 wt (supplementary material Fig. S4c,d), we found that the Fbxw7AAA mutant but not Fbxw7 wt can induce cyclin E ubiquitylation in the presence of Plk2 upon a short treatment of cells with MG132. This short incubation with MG132 allowed us to visualize the extent of cyclin E ubiquitylation before the treatment with the inhibitor leads to saturation of proteins upon stabilization. Cyclin E ubiquitylation was markedly enhanced at 2 hours of MG132 treatment when Plk2 wt and the Fbxw7AAA mutant were coexpressed, whereas the effect of Fbxw7 wt on cyclin E ubiquitylation was minimal in response to Plk2 expression (Fig. 7a). This can be explained by the reduced stability of Fbxw7 wt protein in comparison to the non-phosphorylatable mutant, although alternative explanations such as the mutant Fbxw7 increasing SCF–cyclin E complex formation could not be ruled out. Because Fbxw7 promotes cyclin E degradation we expected that its excessive expression would inhibit centriole reduplication. In fact, centriole reduplication was impaired in a Fbxw7-dependent manner in aphidicolin-arrested U2OS cells expressing GFP–centrin1. This effect could be restored when Plk2 was co-transfected with Fbxw7 wt but not with the Fbxw7AAA mutant (Fig. 7b), suggesting that phosphorylation of Fbxw7 by Plk2 enables centriole reduplication. Interestingly enhanced Plk2 expression could not rescue the deficiency in centriole reduplication upon siRNA-mediated downregulation of cyclin E (Fig. 7b). This indicates the importance of cyclin E in centriole reduplication because Plk2 cannot mediate centriole duplication in the absence of cyclin E.
Finally, to test whether the loss of Fbxw7 promotes centriole duplication, we made use of karyotypically stable HCT116 cells in which both alleles of Fbxw7 were disrupted by homologous recombination. HCT116 FBXW7−/− cells were reported to exhibit chromosomal instability (Rajagopalan et al., 2004). The presence of extra centrosomes might contribute to the manifestation of this phenotype. Therefore, we analyzed centriole numbers in cycling HCT116 FBXW7+/+ and FBXW7−/− cells by staining CP110, a protein that localizes to the distal part of centrioles (Chen et al., 2002). Interestingly, we found that genetic ablation of Fbxw7 resulted in a threefold increase in the percentage of cells harboring extra copies of centrioles. Prolonged incubation with aphidicolin dramatically enhanced the percentage of extra centrioles in HCT116 FBXW7−/− cells, whereas only a moderate effect was observed in wild-type HCT116 cells. Moreover, siRNA-mediated downregulation of either Cdk2 or cyclin E inhibited formation of extra centrioles both in wild-type and FBXW7−/− cells, indicating that the observed reduplication phenotype is dependent on cyclin E and associated Cdk2 kinase activity. Plk2 knockdown only partially inhibited centriole reduplication in HCT116 FBXW7−/− cells, whereas the level of inhibition in wild-type HCT116 cells was comparable to that seen with Cdk2 RNAi (Fig. 7c,d). This is presumably because Plk2 cannot regulate cyclin E levels in the absence of Fbxw7. Taken together, these results suggest that Plk2-mediated phosphorylation of Fbxw7 promotes centriole duplication by preventing ubiquitin-dependent degradation of cyclin E.
The F-box protein SCF Fbxw7 is involved in the degradation of proteins with key roles in cell division and cell growth, including cyclin E and Myc (Koepp et al., 2001; Strohmaier et al., 2001; Welcker et al., 2004). Fbxw7 inactivation by homologous recombination in human HCT116 cells caused genetic instability associated with cyclin E activation (Rajagopalan et al., 2004). Here, we show an interesting and previously unknown regulatory function for Plk2: it modulates Fbxw7 protein levels through direct phosphorylation of three serine residues. Plk2-mediated phosphorylation of Fbxw7 leads to its proteasome-mediated degradation and to subsequent stabilization of cyclin E. Analysis of the CDC4 gene transcripts has revealed that three splice variant isoforms are expressed, designated α, β and γ (Spruck et al., 2002). Although S25 is specific for the α-isoform, both S176 and S349 are present in the Fbxw7 β and γ isoforms. Phosphorylation of Fbxw7 on serine and glutamine residues was reported in response to DNA damage but not in the unperturbed cells (Matsuoka et al., 2007). It would be intriguing to find out if this DNA-damage-induced phosphorylation plays a role in Fbxw7 regulation. Interestingly, the protein levels of the Fbxw7α isoform decrease in response to oxidative stress (Olson et al., 2008). Of note, Fbxw7α was shown to be phosphorylated by serum and glucocorticoid inducible kinase (SGK1) on S227, which promotes Notch1-IC degradation through enhanced ubiquitylation (Mo et al., 2011). Plk2-mediated phosphorylation of Fbxw7, however, does not seem to alter intrinsic Fbxw7 activity, because comparable amounts of Fbxw7AAA and Fbxw7 wt trigger cyclin E ubiquitylation and degradation to similar extents (supplementary material Fig. S4c,d). Therefore Plk2-mediated effects manifested on Fbxw7 substrates are caused by altered levels of the E3 ligase rather than its activity. Our data further suggest that a phosphorylated form of Fbxw7 could influence the stability of unmodified Fbxw7 through homodimerization (Fig. 6c), an effect that might potentiate the consequences of Plk2-mediated phosphorylation of Fbxw7.
A large body of evidence suggests that highly conserved polo-box domains present in the C-terminal region of polo kinases play a pivotal role in the function of these enzymes. The PBD serves as an essential molecular mediator that brings the kinase domain of Plks into proximity with its substrates, mainly through phosphorylation-dependent interactions with its target proteins (Elia et al., 2003). Recently, Cdk5 has been identified as a priming kinase for Plk2-mediated phosphorylation of SPAR, a postsynaptic RapGAP in homeostatic synaptic plasticity (Seeburg et al., 2008). Of equal importance will be the identification of priming kinases that generate PBD binding sites in Fbxw7.
Cyclin E is localized to the centrosome and can promote S-phase entry (Matsumoto and Maller, 2004). It has been suggested that a 20-amino-acid centrosome localization sequence within human cyclin E is essential for promotion of S-phase entry independently of Cdk2. We detected Fbxw7 cofractionating with Plk2 in centrosome-enriched fractions from sucrose gradient centrifugation. Along the same lines, a number of proteasomal subunits have been identified in a mass-spectrometry-based proteomic analysis of human centrosomes (Andersen et al., 2003). Moreover, in mammalian cells Skp1 and Cul1, which are components of the SCF complex are localized to interphase and mitotic centrosomes (Freed et al., 1999). Our findings further indicate that Plk2 kinase cooperates with Cdk2–cyclin E in the regulation of centriole reproduction. Our data suggest that Plk2 kinase activity has a crucial function to keep cyclin E levels high through destabilization of Fbxw7 in order to ensure faithful duplication of centrioles before cyclin E is degraded at the onset of S phase (Clurman et al., 1996; Won and Reed, 1996). Plk4 kinase has also been shown to cooperate with Cdk2 in regulating centriole duplication (Habedanck et al., 2005). In the future, it would be intriguing to determine whether Cdk2–cyclin E and Plk2 also function in the centriole duplication pathway in cooperation with Plk4. Interestingly, Plk2−/− MEFs were reported to have a prolonged cell cycle and an impaired G1–S transition (Ma et al., 2003). Therefore, Plk2-mediated phosphorylation of Fbxw7 might globally influence cell cycle kinetics.
In conclusion, Plk2 could contribute to a neuploidy and tumorigenesis by decreasing the levels of Fbxw7, leading to acontinuous expression of cyclin E. More intriguingly, Fbxw7 has been implicated in tumorigenesis, and mutations in Fbxw7 have been found in a rapidly expanding number of human neoplasms (reviewed by Welcker and Clurman, 2008). However, Fbxw7 can also be deregulated in tumors without mutations in Fbxw7. For example oncogenic ras prevents Fbxw7-dependent cyclin E ubiquitylation and degradation by altering the physical interaction between Fbxw7 and cyclin E (Minella et al., 2005). Thus phosphorylation-dependent regulation of Fbxw7 by Plk2 is a new mechanism for Fbxw7 deregulation that is linked to chromosomal instability and tumorigenesis.
Materials and Methods
Cell culture, transfections and centriole duplication assay
HeLa, HEK293T, U2OS and GFP–centrin1 U2OS cells were grown at 37°C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum, 2 mM L-glutamine (Sigma), and penicillin and streptomycin (100 IU/ml and 100 μg/ml, respectively). HCT116 FBXW7+/+ and FBXW7−/− cells were cultured in McCoy's 5A medium (Sigma) under standard conditions. hTERT-RPE1 cells were cultured in Ham's 12 medium under standard conditions. HeLa and U2OS cells were arrested in prometaphase by addition of 50 ng/ml nocodazole (Sigma) for 15 hours. Mitotic cells were then harvested by shake off, washed five times and reincubated with fresh medium for the release from the block. HEK293T cells were transfected using calcium phosphate and HBS buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 3 mM EDTA). HeLa cells were transfected using calcium phosphate and BES buffer (50 mM BES, pH 6.9, 280 mM NaCl, 1.5 mM Na2HPO4•2H2O). U2OS, HCT116 and HEK293T cells were transfected with DNA plasmids and small-interfering RNAs using Lipofectamine 2000 (Invitrogen) or Polyfect (Qiagen) according to the manufacturer's instructions.
In order to inhibit proteasome-mediated degradation, U2OS enriched in early S phase with 5 μg/ml aphidicolin for 40 hours, and asynchronous HEK293T cells, were treated with 5 μg/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 μg/ml aphidicolin for 12 hours. Cells on coverslips were fixed after 60 hours of aphidicolin treatment. For inhibition of protein synthesis, U2OS or HEK293T cells were treated with 200 μg/ml cycloheximide (Sigma) for up to 4 hours.
Construction of expression vectors
Plk2, Fbxw7 and cyclin E mutations were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Human FBXW7 cDNA was obtained from Molekulare Genomanalyse, DKFZ (Heidelberg, Germany). The insert was PCR amplified with EcoRI- and HindIII-compatible primers and inserted into pCMV-3Tag-1C (or 2C) vector (Stratagene) and the sequence verified. pQE80zz vector was a gift from Dirk Görlich (Göttingen, Germany) (Jakel and Görlich, 1998). Plk2 was cloned into the pQE80zz vector upon PCR amplification with NcoI- and BamHI-compatible primers. SmaI- and HindIII-compatible primers were used to clone FBXW7 into pEGFP C2 vector (Clontech).
Antibodies for western blotting
A standard protocol for immunoblotting was used (Hassepass et al., 2003). Primary antibodies used were anti-Cdk2 (Blomberg and Hoffmann, 1999), anti-centrin2 (H40), anti-cyclin E (HE12), anti-pT395 cyclin E, anti-cyclin A (H432), anti-Myc (E910), anti-p27, anti-His (C19), anti-Skp1, anti-cullin1, anti-Fbxw7 (D16 and H300) all from Santa Cruz Biotechnology (Heidelberg, Germany), anti-Fbxw7 (Invitrogen), anti-FLAG, anti-GFP (Ab290, Abcam), anti-cyclin B (Hoffmann et al., 1993), anti-Plk1 (Zymed), anti-Plk2 (Warnke et al., 2004), anti-Plk4 (Cizmecioglu et al., 2010), anti-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-α-tubulin (Sigma) and anti-β-actin (clone C4, ICN). Secondary antibodies were horseradish-peroxidase-conjugated anti-rabbit and anti-mouse IgGs (Jackson), anti-rabbit Trueblot HRP (eBioscience) or anti-goat HRP from Santa Cruz Biotechnology. Enhanced chemiluminescence immunoblotting detection reagents were from PerkinElmer Life Sciences. Quantification of band intensities was performed with ImageJ (NIH, Bethesda, MD). Rabbit anti-pS176 Fbxw7 antibodies were raised against a synthetic peptide with the following sequence; (NH2-)CRKLDHGS(P)EVRSFS(-COOH). After the second booster, rabbits were killed and antibodies in the immune serum were tandemly affinity purified against non-phosphorylated and phosphorylated peptides, respectively (Innovagen, Lund, Sweden).
Protein purification, immunoprecipitations and kinase assays
Fbxw7 immunoprecipitations (IP) were conducted using 5 mg S-phase-enriched U2OS lysates, using mouse anti-Fbxw7 (Invitrogen) antibodies. Plk2 immunoprecipitations for determining Plk2 kinase activity were performed using 2 mg of HeLa lysate and 2.5 μg anti-Plk2 antibodies. The antibody–antigen complexes were incubated for 3 hours at 4°C and then were collected with protein-A–Sepharose beads for 1 hour at 4°C. For the kinase assays 3 μg α-casein (Sigma) was used as exogenous substrate in the presence of [α-32P]ATP, and the reactions 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 μg rabbit anti-Fbxw7 (Santa Cruz, H300) antibodies per IP. Transfected cells were treated with 5 μg/ml MG132 for 4 hours before harvesting. Lysis buffer contained 500 ng/ml calyculin A (Cell Signaling) in addition to the protease inhibitors. siRNA-transfected HEK293T cells were treated with 5 μg/ml aphidicolin 24 hours before harvesting. Cyclin E immunoprecipitations for determining cyclin E-associated kinase activity were performed as previously described (Warnke et al., 2004) and tested for histone H1 (Roche; 2 μg 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 assays. Kinase assays were performed as described previously (Warnke et al., 2004). Various Myc or FLAG–Fbxw7 constructs were expressed in HEK293T, and immunoprecipitated using 2 μg anti-Myc (9E10; Sigma) or anti-FLAG (M2; Sigma) antibodies. For kinase assays of FLAG–Fbxw7, 1 μg of GST–Plk2 wt (Calbiochem) was used.
Preparation of centrosomes by sucrose gradients
Centrosomes were isolated from HeLa S3 cells by discontinuous sucrose gradient ultracentrifugation. Briefly, the cell pellet was washed with TBS and 0.1× TBS/8% sucrose. Cells were resuspended in 0.1× TBS/8% sucrose and mixed with 0.5% NP-40 lysis buffer. The suspension was shaken slowly for 30 minutes at 4°C and spun at 2500 g for 10 minutes. HEPES buffer and DNase were added to the supernatant to a final concentrations of 10 mM and 1 μg/ml, respectively. After incubation for 30 minutes at 4°C, the mixture was gently underlaid with 60% sucrose solution and spun at 10,000 g for 30 minutes. The obtained centrosomal suspension was loaded onto a discontinuous sucrose gradient (70, 50 and 40% sucrose solutions from the bottom), and spun at 120,000 g for 1 hour. Fractions were collected from the top, diluted with PIPES buffer (10 mM PIPES), and spun at 20,400 g for 15 minutes.
In-gel tryptic digestion and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis
Either untreated or Plk2 wt pre-treated Myc–Fbxw7 immunoprecipitates were resolved by one-dimensional SDS-PAGE. Proteins present in the gel lane were visualized with Coomassie Blue (G250, Roth) staining. Myc–Fbxw7 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 Digest pro MS liquid handling system (Intavis AG, Germany). Following digestion, tryptic peptides were extracted from the gel pieces with 50% acetonitrile, 0.1% trifluoroacetic acid (TFA), concentrated nearly to dryness in a speedVac vacuum centrifuge and diluted to a total volume of 30 μl with 0.1% TFA. 25 μl of the sample was analyzed using a nanoHPLC system (Eksigent, Dublin Ca; Axel Semrau) coupled to a ESI LTQ Orbitrap mass spectrometer (Thermo Fisher). The sample was loaded on a C18 trapping column (Inertsil, LC Packings, Amsterdam, The Netherlands) with a flow rate of 10 μl/minute 0.1% TFA. Peptides were eluted and separated on an analytical column (75 μm×150 mm) packed with Inertsil 3 μm C18 material (LC Packings) with a flow rate of 200 nl/minute in a gradient of buffer A (0.1% formic acid) and buffer B (0.1% formic acid, acetonitrile): 0–6 minutes, 3% B; 6–60 minutes, 3–40% B; 60–65 minutes, 60–90% B. The column was connected to a nano-ESI emitter (New Objectives). 1500 V were applied by a liquid junction. One survey scan (res: 60,000) was followed by five information-dependent product ion scans in the LTQ. Only doubly and triply charged ions were selected for fragmentation.
The peptide sequences of the phosphorylated peptides were confirmed by manual evaluation of the fragment spectra.
For cytofluorometric analysis of DNA content, an aliquot of 2×105 cells was collected by centrifugation and treated with RNase (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 μg of RNase A/ml; Roche) for 30 minutes at 37°C. The cells were stained with propidium iodide (35 μg/ml, Sigma) for 30 minutes on ice. Analysis was carried out by flow cytometry with a FACSSort (Becton Dickinson Biosciences) using the CellQuest software for at least 10,000 cells.
The Plk2-targeting oligonucleotides were based on a 19-mer sequence present in the coding sequence of human Plk2 (PLK2 siRNA1: 5′-GGCAAGAUAUAUUGACACA-3′; PLK2 siRNA2: 5′-GAGCAGCUGAGCACAUCAU-3′), which is absent in the other human polo-like kinases. The oligonucleotides were synthesized as dTdT capped patches of RNA (Qiagen, Dharmacon and Ambion). Firefly luciferase (GL-2) siRNA was used as a control. 25 nM oligonucleotides was used for the RNAi studies where the concentration is not indicated. The Cdk2-targetting oligonucleotides were based on the following sequence: 5′-AAGAUGGACGGAGCUUGUUAU-3′ (Cdk2 siRNA). The sequence of Fbxw7 targeting siRNAs is as follows; 5′-GAGUGGAUCUCUUGAUACA-3′ (Fbxw7 siRNA). The sequence of cyclin E targeting siRNAs is as follows; 5′-GCUUCGGCCUUGUAUCAUU-3′ (CycE siRNA). Plk4-targeting siRNA oligonucleotides were published previously (Habedanck et al., 2005).
In vivo ubiquitylation assay
In vivo ubiquitylation assays were performed by transfecting HEK293T cells with 3 μg pCMV His6-ubiquitin, 3 μg pCMV-FLAG-empty or 5 μg of FLAG-Plk2 kd, together with 2 μg pX-cyclin E (gift from Giulio Superti-Furga, Vienna, Austria). After 20 hours, the cells were treated with 5 μg/ml MG132 (Sigma) for up to 4 hours to inhibit proteasome-mediated proteolysis. For the ubiquitylation assays including Fbxw7 versions, 5 μg of Myc-Fbxw7 wt, 3 μg Myc-Fbxw7AAA and 5 μg pX-cyclin E were co-transfected together with His6-ubiquitin. The cells were then lysed in 1 ml urea buffer [8 M urea, 30 mM imidazole (Sigma), 0.1 M phosphate buffer pH 8.0]; sonicated, and 10 μl of the clear cell lysates were analyzed for immunoblotting to serve as a means to normalize expressed His-ubiquitin levels. Cell lysates that were normalized for the amounts of His-ubiquitin were then incubated with nickel-NTA-coupled agarose beads (Qiagen) for 2 hours on a rotating wheel at room temperature. Beads were then washed 4× with urea buffer and the bound proteins were eluted with 2× SDS sample buffer and a final concentration of 200 mM imidazole, and analyzed for immunoblotting.
Total RNA was isolated from control or PLK2 siRNA-treated U2OS cells using the RNeasy MiniKit (Quiagen). cDNA was generated from 0.5 g total RNA using standard conditions (Superscript reverse transcriptase; Invitrogen), and PCR was performed for Fbxw7 at 95°C for 30 seconds, 61°C for 30 seconds and 72°C for 30 seconds (30 cycles) and for GAPDH at 95°C for 30 seconds, 65°C for 45 seconds and 72°C for 30 seconds (20 cycles). The following primers were used: Fbxw7 forward, 5′-CCCAGCAAGGACAGTTGGAA-3′ and Fbxw7 reverse, 5′-GAACGGGCAGGTCCACAATA-3′; GAPDH forward, 5′-TGGATATTGTTGCCATCAATGACC-3′ and GAPDH reverse, 5′-GATGGCATGGACTGTGGTCATG-3′.
In vitro translation and direct binding assays
Myc–Fbxw7 was in-vitro-translated using the TNT Coupled Reticulocyte Lysate Systems (Promega) in the presence of 35S-labeled methionine according to the manufacturer's instructions (PerkinElmer). In-vitro-translated product (15 μl) was incubated with 5 μg purified Zz-Plk2 or Zz-tag-empty in a final volume of 500 μl lysis buffer for 2 hours at 4°C. The complexes were then pulled down with 5 μl packed IgG–Sepharose beads (GE Healthcare) for 1 hour at 4°C on a rotating wheel. Beads were washed five times with the lysis buffer and processed for loading onto a gel for SDS-PAGE.
Cells were fixed with either ice-cold methanol or methanol–acetone for 10 minutes at −20°C or with 10% formalin–10% methanol for 20 minutes at room temperature. Formalin–methanol-fixed samples were permeabilized with 0.5% NP-40 (Fluka) in PBS for 5 minutes at room temperature. The primary antibodies used were Cdk2 (Blomberg and Hoffmann, 1999), γ-tubulin (T3559, Sigma), BrdU (Roche), glutamylated tubulin (GT-335) (Bobinnec et al., 1998), cyclin E (HE12, Santa Cruz), CP110 (Chang et al., 2010). Primary antibodies were detected with anti-mouse and anti-rabbit Alexa-Fluor-488-conjugated, anti-rabbit Alexa-Fluor-405 and anti-rabbit Alexa-Fluor-594-conjugated secondary antibodies (all from Molecular Probes). DNA was stained with Hoechst 33342. The cells were then analyzed by confocal microscopy (PerkinElmer Spinning Disc Confocal on Nikon TE2000E inverted microscope). The images were processed using Adobe Photoshop (Adobe Systems).
We thank Angel Alonso, Stefan Duensing, Bernard Eddé, Harald Herrmann, Carsten Janke, Philipp Kaldis, Kunsoo Rhee and Bert Vogelstein for reagents. We acknowledge Ulrike Engel and Christian Ackermann from the Nikon Imaging Center at the University of Heidelberg for equipment and assistance in implementation of experiments. Ludger Hengst is thanked for critically reading the manuscript.
This work was supported by a grant from the Deutsche Krebshilfe [grant number 109512 to I.H.].