3224 Research Article Cyclin E-dependent localization of MCM5 regulates centrosome duplication

Rebecca L. Ferguson and James L. Maller* Howard Hughes Medical Institute and Program in Molecular Biology, University of Colorado School of Medicine, Aurora, CO 80045, USA *Author for correspondence (e-mail: [email protected])

Accepted 24 June 2008 Journal of Cell Science 121, 3224-3232 Published by The Company of Biologists 2008 doi:10.1242/jcs.034702

Summary Centrosomes are the primary microtubule-organizing centers dependent but Cdk2-independent manner. The domain in in animal cells and are required for bipolar spindle assembly MCM5 that is responsible for interaction with cyclin E is distinct during mitosis. Amplification of centrosome number is from any previously described for MCM5 function and is highly commonly observed in human cancer cells and might contribute conserved in MCM5 from yeast to mammals. to genomic instability. Cyclin E–Cdk2 has been implicated in Expression of MCM5 or its cyclin E-interacting domain, but regulating centrosome duplication both in Xenopus embryos and not MCM2, significantly inhibits over-duplication of extracts and in mammalian cells. Localization of cyclin E on centrosomes in CHO cells arrested in S-phase. These results centrosomes is mediated by a 20-amino acid domain termed indicate that proteins involved in DNA replication might also the centrosomal localization sequence (CLS). In this paper, regulate centrosome duplication. cyclin E is shown to directly interact with and colocalize on centrosomes with the DNA replication factor MCM5 in a CLS- Key words: Centrosome, Cyclin E, Cdk2, MCM5

Introduction (Geisen and Moroy, 2002; Geng et al., 2007; Matsumoto and Maller, During mitosis, equal segregation of chromosomal DNA into two 2004; Mazumder et al., 2007). Furthermore, localization of cyclin E daughter cells is achieved by the formation of a bipolar spindle in on centrosomes is also independent of Cdk2 binding (Matsumoto which each is organized by a centrosome. At the end of mitosis, and Maller, 2004). This laboratory previously identified a 20-amino each daughter cell inherits a single centrosome, duplication of which acid domain in rat cyclin E (aa 231-250) as a modular centrosomal is initiated near the subsequent G1-S transition of the cell cycle localization signal (CLS) necessary for both the centrosomal (Hinchcliffe and Sluder, 2002; Kochanski and Borisy, 1990; localization of cyclin E and cyclin E-mediated acceleration of S-phase Kuriyama and Borisy, 1981). Control of centrosome number is entry. Mutation of four highly conserved residues within the CLS to

Journal of Cell Science essential for accurate segregation, and centrosome alanine (aa S234, W235, N237 and Q241) produces a quadruple cyclin amplification is commonly associated with aneuploidy in many E mutant (SWNQ-A) that no longer localizes to centrosomes or human tumors, suggesting a role in the pathogenesis of cancer accelerates the G1-S transition. Additionally, expression of the GFP- (Duensing, 2005; Fukasawa, 2005; Wang et al., 2004). These tagged CLS domain of cyclin E was sufficient to target GFP to observations have focused recent attention on the mechanism of centrosomes and prevented endogenous cyclin E and cyclin A from centrosome duplication. Several independent groups have provided localizing to centrosomes (Matsumoto and Maller, 2004). The cyclin evidence that, during G1, cyclin-dependent kinase 2 (Cdk2) is an E CLS binding partners on the centrosome are currently unknown. important regulator of the initiation and progression of both To identify potential interactions mediated by the CLS of centrosome duplication and DNA replication (Coverley et al., 2002; cyclin E, we conducted a bacterial two-hybrid screen of a HeLa cDNA Hinchcliffe et al., 1999; Lacey et al., 1999; Matsumoto et al., 1999; library using the CLS domain as interaction bait. With this approach, Woo and Poon, 2003). In Xenopus egg extracts, up to three rounds we discovered that minichromosome maintenance 5 (MCM5), a of centrosome duplication occur if the cell cycle is arrested in S- protein well documented as an essential DNA prereplication complex phase by aphidicolin (Hinchcliffe et al., 1999), and this amplification factor, interacted specifically with the wild-type CLS but not the is dependent on active cyclin E–Cdk2 (Hinchcliffe et al., 1999; SWNQ-A CLS mutant. Lacey et al., 1999). Similarly, certain mammalian cell lines undergo We show here that a direct interaction between cyclin E and multiple rounds of centrosome duplication during prolonged S-phase MCM5 is dependent on the CLS of cyclin E but independent of arrest, and expression of the Cdk2 inhibitor p21 blocks duplication the binding or activity of Cdk2. The minimal interacting region (Balczon et al., 1995; Matsumoto et al., 1999). A crucial role for within MCM5 comprises a 37-amino acid domain (533-569) that cyclin E–Cdk2 during centrosome replication in mammalian cells has no reported role or function in DNA replication and shares no has been supported by the identification of specific substrates that significant homology with other MCM family members. are directly involved in centrosome duplication, including Additionally, centrosomal localization of MCM5 is dependent on nucleophosmin B (Okuda et al., 2000; Tokuyama et al., 2001) and the CLS of cyclin E, and expression of MCM5 but not MCM2 CP110 (Chen et al., 2002). inhibits centrosome over-duplication in CHO cells arrested in S- Recent studies have begun to uncover Cdk2-independent functions phase. These studies support an emerging trend in which proteins of cyclin E, including roles in re-entry into the cell cycle from previously thought to function only in DNA replication also regulate quiescence, DNA endoreduplication, oncogene-mediated the centrosome duplication cycle (Prasanth et al., 2004b; Stuermer transformation, apoptosis and acceleration of the G1-S transition et al., 2007; Tachibana et al., 2005). MCM5 and centrosome duplication 3225

Results Given the observation in the two-hybrid assay that interaction To biochemically verify a potential interaction of cyclin E with between cyclin E and MCM5 is potentially dependent on the CLS MCM5, endogenous cyclin E immunoprecipitates from lysates of of cyclin E, we next tested the necessity of a wild-type CLS for asynchronous HeLa S3 cells were analyzed for co-precipitation of MCM5 binding to cyclin E in mammalian cells. To this end, multiple MCM5 by immunoblotting (Fig. 1A). MCM5 was clearly associated tetracycline-inducible stable CHO cell lines were generated that with cyclin E, and there was no significant background binding of expressed either full-length 6Myc-tagged wild-type rat cyclin E, MCM5 to an IgG control antibody, demonstrating a specific full-length 6Myc-tagged rat cyclin E containing the four (SWNQ- interaction. To exclude the possibility that co-precipitation was A) CLS point mutations, or full-length 6Myc-tagged rat cyclin E mediated by a bridging protein, the interaction was also evaluated containing a single point mutation (S180D) that blocks nearly all in an in vitro GST pull-down assay (Fig. 1B). GST or GST–cyclin Cdk2 binding and activity (Matsumoto and Maller, 2004) (Fig. 1C, E purified from baculovirus-infected Sf9 insect cells was incubated lower panel). Expression of the various Myc-tagged cyclin E with MCM5 protein that was produced and radiolabeled with constructs was induced for 18 hours by addition of doxycycline, [35S]methionine in an in vitro transcription/translation reaction. The and Myc immunoprecipitates were analyzed for the presence of resultant pull-down was separated by SDS PAGE and analyzed by MCM5 by immunoblot (Fig. 1C, top panel). Binding to MCM5 autoradiography. Whereas GST alone had little background binding was essentially equivalent with wild-type cyclin E and the S180D to MCM5, GST–cyclin E exhibited a strong specific interaction mutant of cyclin E, whereas binding to MCM5 was greatly reduced with MCM5 (Fig. 1B). Taken together, these data indicate that cyclin with cyclin E containing the SWNQ-A mutant CLS. The finding E is capable of directly interacting with MCM5 in vivo. that an in vivo interaction between cyclin E and MCM5 does not require binding to Cdk2 but does require a wild-type CLS verifies the binding observed in the two-hybrid screen. A number of domains in MCM5 have been identified that function in DNA replication or regulation of transcription (Barry et al., 2007; Forsburg, 2004; Jenkinson and Chong, 2006; Kearsey and Labib, 1998). It was therefore important to determine what region within MCM5 mediates an interaction with cyclin E. Sequencing during the initial two-hybrid screen resulted in isolation of eight different partial cDNA constructs encoding various regions of MCM5 (data not shown). Alignment of all eight sequences revealed a minimal 72-amino acid region (aa 496-569) within MCM5 that was shared among all constructs. We tested both a C-terminal HA-tagged construct of MCM5 (aa 496-734) as well as an HA-tagged construct encoding only the 74 shared amino acids (aa 496-569) in an in vitro GST pull-down assay (Fig. 2A). Both of the truncated MCM5 proteins were able to specifically bind cyclin E, with little or no

Journal of Cell Science background binding to GST alone. To further elucidate the region necessary for an interaction with cyclin E, aa 496-569 of MCM5 were subdivided into two equal GFP-tagged segments and examined for their ability to interact with GST–cyclin E (Fig. 2B). Whereas neither GFP alone nor GFP-MCM5 aa 496-532 showed any significant ability to interact with cyclin E, aa 533-569 of MCM5 specifically bound to cyclin E, albeit at a lower level than that observed for longer constructs. However, it is evident that aa 533- 569 of MCM5 are sufficient for an interaction with cyclin E, and this region in MCM5 is largely conserved from yeast to human (Fig. 3). Given the specificity of this interaction for a wild-type CLS in Fig. 1. MCM5 specifically interacts with the wild-type CLS of cyclin E. cyclin E, we next investigated the potential for centrosomal (A) Endogenous cyclin E co-immunoprecipitates MCM5. Lysates from localization of endogenous MCM5. Both CHO-K1 and HeLa S3 asynchronous HeLa S3 cells were subjected to immunoprecipitation, as cells were fixed and immunostained with antibodies to MCM5 and described in the Materials and Methods, with anti-cyclin E antibody or control γ IgG, as indicated, and immunoprecipitates were western blotted with anti- -tubulin, a marker for centrosomes (Fig. 4A). In an asynchronous MCM5 and -cyclin E antibodies. (B) Cyclin E directly interacts with full- population, endogenous MCM5 colocalized with γ-tubulin in a length MCM5. MCM5 was radiolabeled with [35S]methionine in an in vitro majority of the cells. Additionally, ectopically expressed HA-tagged transcription-translation system as described in the Materials and Methods. full-length MCM5 also localized to centrosomes (Fig. 4B). In order The radiolabeled protein was incubated with glutathione-agarose beads bound to GST or GST–cyclin E. Beads were washed, eluted with SDS-PAGE sample to determine whether an interaction with cyclin E is necessary for buffer and electrophoresed on 10% gels. Left: Coomassie-stained gel of 25% MCM5 to be centrosomally localized, the same MCM5 constructs of GST and GST–cyclin E. Right: an autoradiograph of MCM5 is shown. used in GST pulldown assays (Fig. 2) were transiently transfected (C) Mutation of the cyclin E CLS disrupts MCM5–cyclin E interaction. Flp-In into CHO-K1 cells and examined by indirect immunofluorescence T-Rex CHO cells were induced to express the indicated Myc-tagged cyclin E for localization. Both the full C-terminal fragment (aa 496-734) as constructs as described in the Materials and Methods. Lysates were prepared and anti-Myc immunoprecipitates were electrophoresed on 10% SDS gels, well as the 72-amino-acid ‘shared region’ (aa 496-569) of MCM5 followed by western blotting for MCM5 (top panel), Myc (middle panel) and colocalized with γ-tubulin (Fig. 4B), but GFP alone or GFP-MCM5 Cdk2 (lower panel). aa 496-532 – the fragment that did not bind cyclin E (Fig. 2B) – 3226 Journal of Cell Science 121 (19)

cyclin A (Matsumoto and Maller, 2004). Therefore, we examined the localization of MCM5 during expression of the wild-type CLS while also forcing cyclin E to return to centrosomes via a different targeting motif. Subcloning of the pericentrin-AKAP450 centrosomal targeting (PACT) domain (Gillingham and Munro, 2000) upstream and in-frame of each 6Myc-tagged cyclin E construct created fusion constructs that should be centrosomally localized regardless of the CLS. This 91-amino-acid domain (aa 3699-3790) has been reported to be sufficient to target GFP to centrosomes (Gillingham and Munro, 2000). Fusion of this domain to the N-terminus of cyclin E was sufficient for strong centrosomal localization of all cyclin E constructs, including cyclin E containing an SWNQ-A mutant CLS (Fig. 5A). Presumably, if cyclin E is re- targeted to centrosomes despite expression of the wild-type CLS, it could re-establish the centrosomal localization of endogenous MCM5. CHO cells were co-transfected with wild-type GFP-CLS along with the indicated 6Myc-tagged PACT-fused cyclin E constructs, fixed and analyzed for the co-expression of both plasmids within cells as well as for the localization of the PACT–cyclin E proteins. In general, approximately half of the Myc-positive cells (i.e. expressing Myc-tagged PACT-fused cyclin E) were also GFP positive (expressing GFP-CLS), whereas nearly 99% of the GFP- positive cells were also Myc-positive. This allowed analysis of the localization of MCM5 in GFP-positive cells with approximately Fig. 2. The cyclin E-interaction domain within MCM5. (A) HA-tagged MCM5 99% confidence that these cells were also co-expressing the desired cyclin E-interaction fragments. The indicated HA-tagged MCM5 fragments PACT-fused cyclin E protein. Importantly, cells that were GFP were radiolabeled with [35S]methionine in an in vitro transcription-translation positive also displayed a strong centrosomal localization of the system and subjected to pulldown analysis as in Fig. 1B with GST or PACT–cyclin E proteins, indicating that expression of the CLS does GST–cyclin E. Right: an autoradiograph is shown. Left: a Coomassie-stained not interfere with localization that is mediated by the PACT domain gel of 25% of GST and GST cyclin E. (B) GFP-tagged MCM5 cyclin E- interaction fragments. The indicated GFP-tagged fragments were radiolabeled (data not shown). and analyzed by GST pulldown analysis as in A. Right: autoradiograph of Analysis of endogenous MCM5 localization within GFP-positive pulldown. Left: Coomassie-stained gel of 25% of GST and GST proteins. cells showed that co-expression of the PACT-fused wild-type cyclin E or the S180D non-Cdk2-binding mutant of cyclin E restored the

Journal of Cell Science centrosomal localization of MCM5 to near normal levels. However, did not localize to centrosomes. Significantly, GFP-MCM5 aa 533- co-expression of PACT-fused cyclin E containing the SWNQ-A CLS 569 – the fragment that did retain the ability to bind cyclin E – was point mutations that prevent binding to MCM5 (Fig. 1C) did not centrosomally localized (Fig. 4C). rescue MCM5 localization even though the CLS-mutant cyclin E Although these data suggest that an interaction with cyclin E is protein had been re-localized to the centrosome (Fig. 5A,B). Taken necessary for MCM5 to localize to centrosomes, we also examined together, these data strongly indicate that an interaction with cyclin the localization of endogenous MCM5 in cells expressing either E, mediated by the CLS, directly recruits MCM5 to centrosomes the GFP-tagged wild-type CLS or SWNQ-A mutant CLS of cyclin and that MCM5 is co-displaced from centrosomes with cyclin E E. It has previously been shown that endogenous cyclin E is when the CLS domain is expressed. displaced from centrosomes presumably by competition with the We next wanted to explore whether MCM5 has a functional role expressed intact CLS (Matsumoto and Maller, 2004), and therefore in centrosome duplication. In certain cell lines, S-phase inhibition expression of the CLS might also displace MCM5 from uncouples the DNA replication cycle from the centrosome centrosomes. CHO-K1 cells were fixed 20 hours after transient duplication cycle and allows multiple rounds of centrosome transfection of either the wild-type or SWNQ-A-mutant CLS duplication to occur, even in the absence of DNA replication. construct and immunostained for endogenous MCM5. GFP-positive Furthermore, apparent centrosome amplification in this system has cells were identified by fluorescence of the expressed GFP tag rather been shown by electron microscopy to be a result of template-driven than by antibody detection. Importantly, expression of GFP alone (control) or the SWNQ-A mutant CLS had no significant effect on MCM5 localization, whereas the wild-type CLS greatly disrupted centrosomally localized endogenous MCM5 (Fig. 5B). Although these results support the hypothesis that the centrosomal localization of MCM5 is Fig. 3. Evolutionary conservation of the MCM5 domain that interacts with cyclin E. The region dependent on the CLS of cyclin E, it could not be from human MCM5 that is sufficient for binding cyclin E was aligned with MCM5 proteins from excluded that expression of the CLS displaced the indicated species. Red residues are identical, blue residues are highly similar and green MCM5 through removal of a different protein, e.g. residues are moderately similar. MCM5 and centrosome duplication 3227

Fig. 4. Localization of MCM5 on centrosomes. (A) Endogenous MCM5. Asynchronous CHO-K1 or HeLa S3 cells were methanol- fixed and stained with antibody to γ-tubulin and MCM5, as indicated. Arrowheads mark the centrosome. Inset: enlarged image of the centrosomal area. (B) Transfection of HA-tagged MCM5. CHO-K1 cells were transiently transfected with the indicated MCM5 constructs as described in the Materials and Methods. Cells were methanol- fixed and stained with antibodies to γ-tubulin and HA, as indicated. Arrowheads mark the

Journal of Cell Science centrosome. Inset: enlarged image of the centrosomal area. (C) Transfection of GFP-tagged MCM5 fragments. The indicated constructs were expressed in CHO-K1 cells and stained with antibody to γ-tubulin. GFP was visualized by excitation of the expressed GFP tag. Arrowheads mark the centrosome. Inset: enlarged image of the centrosomal area. Scale bars: 5 μm.

centrosome duplication events rather than from centrosome splitting MCM family, exhibited no significant inhibition of centrosome or de novo generation (Balczon et al., 1995). Using this system, duplication (Fig. 6B). Taken together, these results suggest that we examined centrosome duplication in hydroxyurea (HU)-arrested MCM5 has a specific negative regulatory function in centrosome CHO-K1 cells expressing MCM5. After 48 hours of HU-mediated duplication. Finally, we examined the localization of endogenous S-phase arrest, control CHO cells contained an average of seven γ- cyclin E while expressing MCM5 (Fig. 7). If expression of MCM5 tubulin staining foci per cell, whereas cells transfected with displaced cyclin E from centrosomes, this could be predicted to untagged or HA-tagged full-length wild-type MCM5 contained an result in inhibition of duplication. However, the presence of average of only four γ-tubulin staining foci (Fig. 6A). Strikingly, endogenous cyclin E at centrosomes was evident even in cells highly the same degree of inhibition of centrosome duplication was expressing MCM5 (Fig. 7). observed in HU-arrested cells expressing only the HA-tagged 74 amino acid (496-569) fragment of MCM5 (Fig. 6B), a fragment Discussion that binds cyclin E (Fig. 1A) and localizes to centrosomes (Fig. In many cell types, both centrosome duplication and DNA 2C). Importantly, as an additional control for an MCM5-specific replication are initiated at the G1-S transition. This essential effect, cells that were expressing MCM2, a related member of the coordination of DNA and centrosome duplication is achieved at 3228 Journal of Cell Science 121 (19)

centrosome duplication, has provided preliminary indications of how centrosome duplication is positively regulated by cyclin E (Chen et al., 2002; Okuda et al., 2000; Tokuyama et al., 2001). The results in this paper suggest that, by recruiting MCM5 to the centrosome, cyclin E–Cdk2 might also provide negative regulation of centrosome replication, thereby preventing over- duplication (Fig. 8). Recent studies have presented compelling evidence for Cdk2-independent functions of cyclin E (Geisen and Moroy, 2002; Geng et al., 2007; Honda et al., 2005; Matsumoto and Maller, 2004; Mazumder et al., 2007). In 2004, Matsumoto and Maller demonstrated that localization of cyclin E to the centrosome requires an intact CLS but not Cdk2 binding (Matsumoto and Maller, 2004). Differences between the knockout phenotypes of mice lacking either cyclin E or Cdk2 have also challenged the traditional view that cyclin E functions only as an activator of Cdk2. Cdk2 knockout mice are viable and do not display anatomical or behavioral abnormalities, except for severe gonadal atrophy (Berthet et al., 2003). Additionally, Cdk2–/– mouse embryo fibroblasts proliferate normally in culture, indicating Fig. 5. Effect of PACT–cyclin E expression. (A) Localization of PACT-domain-fused cyclin E. The indicated 6Myc-tagged PACT–cyclin E fusion proteins were expressed by transient transfection in CHO- that Cdk2 activity is not essential for K1 cells, methanol-fixed, and analyzed by immunofluorescence for γ-tubulin and Myc–cyclin E. mitotic cell division. In these cells, cyclin Arrowheads mark the centrosome. Inset: enlarged image of merged centrosomal area. Scale bars: 5 μm. E expression levels and centrosomes are (B) MCM5 centrosomal localization relies on an interaction with cyclin E. Endogenous MCM5 normal, presumably because cyclin E is centrosomal localization was analyzed as in Fig. 4A in CHO-K1 cells co-expressing either GFP or GFP- Journal of Cell Science CLS along with the indicated Myc-tagged PACT-fused cyclin E fusion constructs. Over 500 cells were bound to Cdk1 (Aleem et al., 2005; Berthet analyzed for each condition in each experiment. Error bars indicate mean ± s.e.m. (n 3). et al., 2003). However, a double knockout of both cyclin E1 and cyclin E2 was embryonic lethal owing to placental defects least in part by the activation in late G1 of Cdk2 coupled to cyclin that were not observed in Cdk2 knockout embryos (Geng et al., E (cyclin E–Cdk2). Numerous studies have demonstrated that cyclin 2003). In culture, fibroblasts isolated from double cyclin E knockout E–Cdk2 plays an essential role in S-phase entry. At the G1-S embryos were found to actively proliferate and increase in cell transition, active cyclin E–Cdk2 phosphorylates Orc-bound pre- number similar to wild-type controls as long as the cells were replication complexes that are assembled on DNA replication continuously cycling. However, cyclin E-deficient cells in G0 after origins, thereby promoting the recruitment of DNA serum starvation failed to re-enter the cell cycle after serum and the initiation of DNA synthesis (Forsburg, 2004; Masuda et al., stimulation, owing to a failure to load MCM proteins onto origins 2003; Strausfeld et al., 1996; Zou and Stillman, 2000). However, of replication (Geng et al., 2003). Further work with these cells continued high levels of Cdk2 activity prevent the loading of pre- showed that cyclin E knockout phenotypes could be at least replication-complex proteins onto DNA and inhibit inappropriate partially rescued after retroviral-mediated expression of cyclin E, re-licensing and re-replication (Dahmann et al., 1995; Forsburg, including a construct impaired for activation of Cdk2 as well as a 2004; Hua et al., 1997; Ishimi and Komamura-Kohno, 2001). construct with a mutant CLS (Geng et al., 2007). This rescue was Although the precise functional roles and target substrates of cyclin associated with restoration of MCM loading and is consistent with E–Cdk2 with regards to DNA replication have been extensively multiple studies in egg extracts, yeast and mammalian cells studied, only within recent years has it become evident that Cdk2 indicating that Cdk2 activity is not needed to load MCM proteins also regulates centrosome duplication (Hinchcliffe et al., 1999; onto origins but is required for initiation of DNA replication (Lei Hinchcliffe and Sluder, 2002; Lacey et al., 1999; Matsumoto et al., and Tye, 2001; Prasanth et al., 2004a). In this connection, our work 1999; Winey, 1999). In both Xenopus embryos and mammalian here demonstrates that cyclin E-mediated recruitment of MCM5 to cells, over-duplication of centrosomes is blocked by Cdk2 inhibitors, centrosomes also does not require Cdk2 binding or activity. and it has been suggested that, under some conditions, cyclin A– We biochemically verified an in vivo interaction between cyclin Cdk2 might also play a crucial role (Meraldi et al., 1999). The E and MCM5 and mapped the region within MCM5 that is identification of centrosomal cyclin E–Cdk2 substrates, such as sufficient for interaction to amino acids 533-569. This portion of nucleophosmin B and CP110, which are essential for initiation of MCM5 is located in the C-terminal region and has not previously MCM5 and centrosome duplication 3229

Fig. 7. Expression of MCM5 does not displace endogenous cyclin E from centrosomes. CHO-K1 cells transiently transfected with full-length HA-tagged MCM5 were methanol-fixed after 24 hours and analyzed by immunofluorescence for the localization of endogenous cyclin E. Arrowheads mark the centrosome. Inset: enlarged image of the centrosomal area. Scale bar: 5 μm.

numbers greatly exceeding the number of replication origins (Bell and Dutta, 2002; Donovan et al., 1997; Lei et al., 1996). Even though only a fraction of the total MCM proteins can be mapped to Fig. 6. Expression of MCM5 but not MCM2 inhibits centrosome duplication. replication sites in S-phase, it has been estimated that anywhere (A) Effect of MCM5 on centrosome number. HU-arrested CHO-K1 cells were from 40 to 100 MCM complexes are loaded onto chromatin for transiently transfected with full-length HA-tagged MCM5. The number of γ- each (Edwards et al., 2002; Forsburg, 2004; tubulin-staining foci (centrosomes) was monitored by immunofluorescence in Young and Tye, 1997). Additionally, this large pool of MCM HA-expressing (white bars) and non-expressing (control) cells (black bars) 24 hours after transfection. (B) Statistical analysis of the effect of MCM5 on proteins is required, because even a partial reduction in MCM level centrosome duplication. Several experiments similar to the one in A were that does not greatly affect DNA replication leads to increased

Journal of Cell Science performed with the indicated constructs. The bar indicates mean ± s.e.m. (n 3). recombination events, chromosome loss and checkpoint sensitivity Over 150 cells were counted for each condition in each experiment. (Bell and Dutta, 2002; Liang et al., 1999). The excess level of MCM proteins along with broad binding on chromatin has been termed the ‘MCM paradox’ (Forsburg, 2004; Hyrien et al., 2003; Laskey been attributed to any known function of MCM5. Whereas and Madine, 2003) and further studies have shown that MCM alignment analysis shows no significant conservation within this proteins have multiple functions outside of DNA replication, region to other MCM family members, residues in this region are including transcriptional activation and repression, chromatin highly conserved in MCM5 sequences from different species (Fig. remodeling, meiotic recombination, chromatin cohesion, and 3). Additionally, we found that both endogenous and exogenously checkpoint responses (Agarwal et al., 2007; Forsburg, 2004; expressed MCM5, as well as C-terminal cyclin E-interacting Gauthier et al., 2002; Snyder et al., 2005; Sterner et al., 1998). It fragments, are centrosomally localized. This localization appears is significant that, in addition to the localization of MCM5 on to require an interaction with cyclin E, because only those constructs centrosomes that is reported here, localization of both Orc2 and of MCM5 that interact with cyclin E are also capable of centrosomal geminin has also been reported on centrosomes (Prasanth et al., localization. Moreover, PACT-driven localization of cyclin E is 2004b; Stuermer et al., 2007; Tachibana et al., 2005). These sufficient to direct MCM5 to the centrosome only if the CLS is findings support an emerging trend in which essential DNA- wild type (Fig. 4). These data are in agreement with binding assays replication proteins have other functions that are mediated by distinct indicating that the SWNQ-A CLS mutation greatly impairs MCM5 domains in the protein. binding and should therefore be unable to recruit MCM5 to Because a significant reduction in MCM5 protein level has been centrosomes. Although the CLS is necessary for the binding of reported to cause S-phase arrest (Ryu et al., 2005), we performed MCM5 to cyclin E, it is probably not sufficient, because GFP-CLS MCM5 siRNA knockdown in HeLa S3 cells, a line previously expression displaced MCM5 from centrosomes along with cyclin reported to not reduplicate centrosomes during prolonged S-phase E (Fig. 5B) (Matsumoto and Maller, 2004). Taken together, these arrest (Kasbek et al., 2007; McDermott et al., 2006). At 96 hours data indicate that the most likely mechanism by which MCM5 is after siRNA transfection, a near complete knockdown of MCM5 centrosomally localized is direct recruitment by interaction with protein was observed, with no detectable difference in MCM2 cyclin E that has a wild-type CLS. protein level, and S-phase progression was inhibited (data not The likelihood that MCM proteins have diverse functions is shown). Approximately 30% of the siRNA-treated cells displayed suggested by the finding that they are highly abundant, with their an increase in centrosome number, which was not observed in cells 3230 Journal of Cell Science 121 (19)

(Invitrogen). Stable integration of the tetracycline repressor to create a tetracycline- responsive CHO Flp-In T-Rex cell line was carried out according to the manufacturer’s directions. Briefly, Flp-In CHO cells were transfected with linearized pcDNA6/TR and maintained in medium containing 6.5 g/ml Blasticidin (Invitrogen) for approximately 2 weeks. Colonies were trypsinized, and individual cells isolated and placed into 96 wells. Expanded cells were screened by western blotting for expression of the tetracycline repressor (Abcam, 14075) and colonies displaying the highest expression level of the tetracycline repressor were tested for tetracycline-inducible expression by transient transfection of the pcDNA5/FRT/TO/CAT positive control vector supplied by Invitrogen in the Flp-In T-Rex core kit. Cells were analyzed by western blotting for expression of CAT (Sigma, C9336) with and without doxycycline treatment. The final host CHO Flp-In T-Rex cell line was expanded from the clone that exhibited the lowest level of basal expression without doxycycline and the highest expression of CAT 20 hours after addition of 1 g/ml doxycycline. Establishment of Flp-In T-Rex Myc-tagged cyclin E expression cell lines was performed following the manufacturer’s directions and selection of stable transfectants was carried out by culturing cells in the presence of 600 g/ml Hygromycin B (Invitrogen). All transfections were carried out with Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s directions. At 6 hours after transfection, cells were washed with sterile PBS and given fresh media without antibiotics. Fig. 8. Model for positive and negative regulatory roles of cyclin E–Cdk2 and MCM5 during DNA and centrosome replication. A positive role for cyclin Immunoprecipitation E–Cdk2 is indicated by the arrowhead, e.g. phosphorylation of Cdc45 to For endogenous cyclin E immunoprecipitation (IP), HeLa S3 cells were lysed in NP- 40 lysis buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40) supplemented with initiate origin firing during DNA replication or of nucleophosmin B to initiate 1ϫ complete protease inhibitor (Roche), 1 mM AEBSF (Sigma-Aldrich), 1 mM NaF centrosome duplication. The inhibitory arm denotes the role of cyclin E–Cdk2 and 1 mM Na3VO4. Protein concentration was determined by Bradford assay and 1 in preventing re-replication of DNA or centrosomes in the same cycle (e.g. by mg of lysate was used for each IP. Magnabind protein-G beads (Pierce) were incubated preventing re-loading of MCM proteins onto origins of replication on DNA or with either anti-human cyclin E antibody (Upstate, HE12) or control mouse IgG (Santa recruiting MCM5 to centrosomes). Cruz) for 2 hours at 4°C. Beads were thoroughly washed with NP-40 lysis buffer and blocked with 5% BSA/PBS for 6 hours at 4°C. Beads were then incubated with protein lysate overnight at 4°C, washed twice with NP-40 lysis buffer and twice with high-salt (250 mM NaCl) NP-40 lysis buffer. After elution with 1ϫ Laemmli sample treated with negative-siRNA controls. However, HeLa S3 cells buffer, samples were separated on 10% SDS-PAGE, transferred to PVDF membranes and immunoblotted for the protein of interest. For blotting, the following primary placed under HU arrest alone for 96 hours had nearly the same antibodies were used against cyclin E (Upstate, 06-459), MCM5 (Abcam, 17967) percentage of cells with an abnormal number of centrosomes (data and Cdk2 (Sigma, C5223). As a secondary antibody, HRP-conjugated goat anti-rabbit not shown). Consequently, increased centrosomal numbers in cells (Pierce) was used. after treatment with MCM5 siRNA was probably an indirect result For Myc IP, Flp-In T-Rex stable-expression cell lines were induced for 20 hours using 1 g/ml doxycycline. Cells were lysed using NP-40 lysis buffer supplemented of inhibiting S-phase and cannot be necessarily attributed to loss with 1ϫ complete protease inhibitor, 1 mM AEBSF, 1 mM NaF and 1 mM Na3VO4. of MCM5 function in centrosome duplication. Lysate protein concentration was determined by Bradford assay and equal amounts At present, it is unclear how the recruitment of MCM5 by the of protein were used for each IP (600 g). Preconjugated Myc-antibody agarose beads (Santa Cruz, 9E10) or control preconjugated mouse IgG agarose beads (Santa Cruz) CLS inhibits cyclin E-mediated centrosome over-duplication in HU- were washed in NP-40 lysis buffer and blocked in 5% BSA/PBS for 6 hours at 4°C.

Journal of Cell Science arrested cells. In vitro kinase assays revealed no phosphorylation Beads were then thoroughly washed with lysis buffer and incubated with lysate of MCM5 by purified cyclin E–Cdk2 (data not shown). In addition, overnight at 4°C. Beads were washed three times with NP-40 lysis buffer and eluted ϫ even in cells highly expressing MCM5, colocalization of with 1 Laemmli sample buffer. Samples were separated on 10% SDS-PAGE, transferred to PVDF membranes, and the protein of interest was detected by endogenous cyclin E with γ-tubulin was not altered (Fig. 7). immunoblot analysis. The following primary antibodies were used against Myc (Santa Therefore, inhibition of centrosome duplication by MCM5 Cruz, 9E10-HRP conjugated), MCM5 (Abcam, 17967) and Cdk2 (Sigma, C5223). expression is not due to displacement of endogenous cyclin E from As a secondary antibody, HRP-conjugated goat anti-rabbit (Pierce) was used. the centrosome. Because inhibition of centrosome duplication can GST pulldown be achieved with a discrete small domain of MCM5 (aa 496-569), GST and GST-tagged human cyclin E were purified from baculovirus-infected Sf9 it probably reflects a mechanism that is independent of MCM5 cells obtained from the University of Colorado Cancer Center Tissue Culture Core function in DNA replication or transcription. It has long been known Facility. Briefly, Sf9 cell pellets were resuspended in Sf9 lysis buffer (50 mM Tris pH 7.4, 300 mM NaCl, 1 mM MgCl2, 0.1% TX-100) supplemented with 1ϫ complete that coupling of DNA replication and centrosome duplication is protease inhibitor mix and 1 mM DTT, and sonicated three times for 30 seconds essential for ensuring genomic stability, but the precise mechanisms each. Lysates were spun twice at 20,000 g for 10 minutes and the supernatant incubated by which one cycle exerts control over the other is not fully with glutathione-Sepharose 4B beads (Amersham Biosciences) for 1 hour at 4°C. understood. Our data potentially provide evidence that, in addition Beads were washed twice with Sf9 lysis buffer supplemented with 450 mM NaCl and twice with Sf9 lysis buffer containing 150 mM NaCl. The beads were resuspended to cyclin E–Cdk2, other proteins associated with the initiation and in 50% ethylene glycol/50% SF9 lysis buffer with 1 mM DTT, aliquoted and stored progression of DNA replication also regulate centrosome at –20°C. For pulldown analysis, equal amounts of GST and GST–cyclin E bound duplication. It is attractive to speculate that the release of MCM5 to glutathione beads were washed three times with TIF buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM MgCl2, 0.1% NP-40 and 10% glycerol) and blocked in 5% from chromatin during S-phase progression could potentially act BSA/PBS for 1 hour at 4°C. Beads were washed and incubated overnight at 4°C as a negative feedback mechanism that prevents inappropriate with MCM5 protein produced in an in vitro transcription-translation (TNT)-coupled centrosome re-duplication (Fig. 8). reaction according to the manufacturer’s protocol (Promega). MCM5 protein was radiolabeled by incorporation of [35S]methionine during the TNT reaction. Beads were washed three times, eluted with 1X Laemmli sample buffer and separated on Materials and Methods a 10% SDS-PAGE. Gels were dried and exposed to Kodak Biomax MR film. Cell culture and transfection CHO-K1 and HeLa S3 cells were grown at 37°C in a 5% CO2 atmosphere in Ham’s Immunofluorescence F12K medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) Cells were seeded onto collagen-coated glass coverslips (Iwaki, Japan) and fixed (HyClone) and penicillin-streptomycin (50 U/ml and 50 g/ml, respectively) with –20°C methanol for 15 minutes after the indicated treatments. After rehydration (Invitrogen). Flp-In CHO cells were purchased from Invitrogen and maintained in in PBS for 30 minutes, cells were blocked in 5% BSA/PBS overnight at 4°C and Ham’s F12 media supplemented with 10% FBS, antibiotics and 100 g/ml Zeocin immunostained with the following primary antibodies against MCM5 (Santa Cruz, MCM5 and centrosome duplication 3231

22780), -tubulin (Sigma, GTU-88), hemagglutinin (HA)-Alexa-Fluor-488 conjugate Coverley, D., Laman, H. and Laskey, R. A. (2002). Distinct roles for cyclins E and A (Molecular Probes, 16B12), c-Myc-FITC conjugate (Santa Cruz, 9E10), -tubulin during DNA replication complex assembly and activation. Nat. Cell Biol. 4, 523-528. (Sigma, AK-15) and cyclin E (Abcam, 2094) as indicated. GFP constructs were Dahmann, C., Diffley, J. F. and Nasmyth, K. A. (1995). S-phase-promoting cyclin- visualized by GFP excitation of the expressed GFP-tagged protein rather than dependent kinases prevent re-replication by inhibiting the transition of replication origins immunostaining. All primary antibody incubations were for 1 hour at room to a pre-replicative state. Curr. Biol. 5, 1257-1269. temperature followed by appropriate secondary antibody staining. Secondary Donovan, S., Harwood, J., Drury, L. S. and Diffley, J. F. (1997). Cdc6p-dependent loading antibodies, either goat anti-rabbit or anti-mouse conjugated to either Alexa Fluor 488 of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc. Natl. Acad. Sci. or Alexa Fluor 594 (Molecular Probes) were incubated with cells for 45 minutes at USA 94, 5611-5616. room temperature. Cells were thoroughly washed, DAPI stained (Invitrogen) and Duensing, S. (2005). A tentative classification of centrosome abnormalities in cancer. Cell Biol. Int. 29, 352-359. mounted with Prolong Gold anti-fade reagent (Invitrogen). Microscopic observation ϫ Edwards, M. C., Tutter, A. V., Cvetic, C., Gilbert, C. H., Prokhorova, T. A. and Walter, was made on a Nikon Eclipse TE 300, PCM 2000 inverted microscope with a 100 J. C. (2002). MCM2-7 complexes bind chromatin in a distributed pattern surrounding oil-immersion objective (NA 1.4). Images were obtained with an air-cooled charge- the origin recognition complex in Xenopus egg extracts. J. Biol. Chem. 277, 33049- ϫ coupled device (CCD) camera (SenSys Photometrics) attached to a 0.76 coupler 33057. (Diagnostic Instruments). For microscopic analysis, Simple PCI (Compix) acquisition Forsburg, S. L. (2004). Eukaryotic MCM proteins: beyond replication initiation. Microbiol. software was used. Mol. Biol. Rev. 68, 109-131. Fukasawa, K. (2005). Centrosome amplification, chromosome instability and cancer Bacterial two-hybrid development. Cancer Lett. 230, 6-19. The CLS of rat cyclin E was cloned into the bait vector (pBT) of the BacterioMatch Gauthier, L., Dziak, R., Kramer, D. J., Leishman, D., Song, X., Ho, J., Radovic, M., II two-hybrid system (Invitrogen) using EcoRI and XhoI digestion sites. Competent Bentley, D. and Yankulov, K. (2002). The role of the carboxyterminal domain of RNA BacterioMatch II validation reporter cells were then co-transformed with the pBT II in regulating origins of DNA replication in Saccharomyces cerevisiae. vector containing the CLS and a commercially prepared HeLa plasmid cDNA library Genetics 162, 1117-1129. (Stratagene). Screening for potential interactions was preformed according to the Geisen, C. and Moroy, T. (2002). The oncogenic activity of cyclin E is not confined to manufacturer’s directions. Briefly, cells were plated onto M9+ His-dropout selective Cdk2 activation alone but relies on several other, distinct functions of the protein. J. Biol. Chem. 277, 39909-39918. screening media containing 5 mM 3-amino-1,2,4-triazole (3-AT) and incubated at Geng, Y., Yu, Q., Sicinska, E., Das, M., Schneider, J. E., Bhattacharya, S., Rideout, 37°C overnight. Verification of an interaction was carried out by patching colonies W. M., Bronson, R. T., Gardner, H. and Sicinski, P. (2003). Cyclin E ablation in the onto M9+ His-dropout dual selective screening media containing 3-AT as well as mouse. Cell 114, 431-443. streptomycin (12.5 g/ml). Target vector was isolated by tetracycline selection, Geng, Y., Lee, Y. M., Welcker, M., Swanger, J., Zagozdzon, A., Winer, J. D., Roberts, sequenced and subjected to BLAST analysis for identification. Clones of interest J. M., Kaldis, P., Clurman, B. E. and Sicinski, P. (2007). Kinase-independent function were then re-screened in the two-hybrid system using a pBT bait vector containing of cyclin E. Mol. Cell 25, 127-139. the SWNQ(A) CLS mutant to exclude any interactions not specific for a wild-type Gillingham, A. K. and Munro, S. (2000). The PACT domain, a conserved centrosomal CLS. targeting motif in the coiled-coil proteins AKAP450 and pericentrin. EMBO Rep. 1, 524-529. Centrosome duplication Hinchcliffe, E. H. and Sluder, G. (2002). Two for two: Cdk2 and its role in centrosome CHO-K1 cells were seeded onto collagen-coated glass coverslips and allowed to attach doubling. Oncogene 21, 6154-6160. overnight. Cells were then placed in media containing 4 mM hydroxyurea for 24 Hinchcliffe, E. H., Li, C., Thompson, E. A., Maller, J. L. and Sluder, G. (1999). hours followed by transient transfection of expression vectors. At 6 hours after Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus transfection, cells were washed and fresh media containing HU was added. At 24 egg extracts. Science 283, 851-854. hours after transfection (for a total of 48 hours of HU treatment), cells were methanol- Honda, R., Lowe, E. D., Dubinina, E., Skamnaki, V., Cook, A., Brown, N. R. and Johnson, L. N. (2005). The structure of cyclin E1/CDK2: implications for CDK2 fixed and processed for immunofluorescent staining. Cells were immunostained for γ activation and CDK2-independent roles. 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