Cyclin A/cdk2 has a role in progression through S phase, and a large pool is also activated in G2 phase. Here we report that this G2 phase pool regulates the timing of progression into mitosis. Knock down of cyclin A by siRNA or addition of a specific cdk2 small molecule inhibitor delayed entry into mitosis by delaying cells in G2 phase. The G2 phase-delayed cells contained elevated levels of inactive cyclin B/cdk1. However, increased microtubule nucleation at the centrosomes was observed, and the centrosomes stained for markers of cyclin B/cdk1 activity. Both microtubule nucleation at the centrosomes and the phosphoprotein markers were lost with short-term treatment of the cdk1/2 inhibitor roscovitine but not the Mek1/2 inhibitor U0126. Cyclin A/cdk2 localized at the centrosomes in late G2 phase after separation of the centrosomes but before the start of prophase. Thus G2 phase cyclin A/cdk2 controls the timing of entry into mitosis by controlling the subsequent activation of cyclin B/cdk1, but also has an unexpected role in coordinating the activation of cyclin B/cdk1 at the centrosome and in the nucleus.
Cell cycle progression is regulated by the sequential activation and inactivation of cyclin/cdk complexes (Sherr, 2000). Binding of the cyclin regulatory subunit is essential for activation and determines the substrate specificity of these complexes. In eukaryotes, cyclin E/cdk2 drives initiation of DNA replication, and cyclin B/cdk1 is required for both entry into and progression through mitosis. Cyclin A/cdk2 is unusual, as it has biphasic activity (Gu et al., 1992; Goldstone et al., 2001). The complex is initially activated at the onset of S phase, while a much more robust activation occurs in early G2 phase. This precedes the activation of the cyclin B/cdk1 complex at G2/M, placing it in an ideal position to regulate G2 phase progression. The biphasic activity of cyclin A/cdk2 is regulated not only by the levels of cyclin A protein (Pines and Hunter, 1990), but also by the removal of the inhibitory tyrosine-15 phosphorylation on cdk2 by the dual specific phosphatase cdc25A in S phase (Blomberg and Hoffmann, 1999) and cdc25B in G2 phase (Goldstone et al., 2001).
To date, studies of cyclin A have centred mainly on its S-phase role, where it has been implicated in DNA replication. The G2 phase function of cyclin A/cdk2 is controversial, and the majority of studies have focused on the cdk2 subunit of the complex. In fact, the role of cyclin A/cdk2 in regulation of the cell cycle has been questioned recently due to the lack of phenotype of cdk2 knockout mice (Berthet et al., 2003; Ortega et al., 2003) and cdk2 knockdown with siRNA or antisense oligonucleotides in cancer cells (Tetsu and McCormick, 2003). However, other studies have suggested a function for cyclin A/cdk2 in G2 phase progression. Microinjection of cyclin A antibodies into tissue culture cells delayed entry into mitosis (Clarke et al., 1992), and induction of a dominant-negative cdk2 resulted in G2 phase delay (Hu et al., 2001). Furthermore, cells from a Drosophila mutant lacking cyclin A arrest in G2 after the maternal cyclin A has been exhausted (Knoblich and Lehner, 1993), indicating the importance of the cyclin A/cdk complex. Microinjection of active cyclin A/cdk2 into early G2 phase HeLa cells promotes premature entry into mitosis, and it has been suggested that the complex is a rate-limiting component required for entry into and progression through mitosis until late prophase (Furuno et al., 1999).
The involvement of cyclin A in the G2/M phase has been implied in a number of studies but its precise role and potential substrates remain to be elucidated. Cyclin A/cdk2 appears to be involved in the activation (Furuno et al., 1999; Mitra and Enders, 2004), and possibly the stabilization of cyclin B, the latter by indirectly inhibiting the anaphase-promoting complex/cyclosome (APC/C) (Lukas et al., 1999). Cyclin A is itself destroyed by the APC/C at prometaphase (den Elzen and Pines, 2001) inferring that its activity is required until that time. In vitro studies using Xenopus extracts have demonstrated that cyclin A/cdk is capable of increasing microtubule nucleation at the centrosomes (Buendia et al., 1992). Thus it is likely that cyclin A in association with its cdk partner has roles in not only promoting entry into mitosis but also in establishing mitosis, possibly by influencing the mitotic machinery.
We have used siRNA to knock down cyclin A2, the major cyclin A isoform in somatic cells, and a small molecule inhibitor specific for cdk2, to examine the role of the G2 phase cyclin A/cdk2 complex in cell cycle progression. We demonstrate that knock down of cyclin A and inhibition of cdk2 have similar effects in delaying progression into mitosis by delaying activation of cyclin B/cdk1. We also report that knock down of cyclin A or cdk2 inhibition resulted in uncoupling of the coordination of microtubule nucleation at the spindle poles from nuclear mitotic events. Microtubule nucleation was observed more than 2 h prior to evidence of nuclear mitotic events, whereas this was normally less than 30 min prior to chromosome condensation. Mitosis-specific phosphorylation and markers of cyclin B/cdk1 activation were detected on centrosomes in the cyclin A knockdown cells. Finally we demonstrate that cyclin A/cdk2 associates with the centrosomes soon after separation of the centrosome pairs in late G2 phase. Thus cyclin A/cdk2 localizes to the centrosome in late G2 phase and coordinates nuclear and centrosomal mitotic events.
Depletion of cyclin A/cdk2 delays entry into mitosis
We developed three independent cyclin A-specific siRNAs, which knock down cyclin A protein levels and cyclin A/cdk2 kinase activity by >80%, but had no effect on cyclin B/cdk1 activity (Figures 1a and b). Knockdown was observed as early as 6 h post transfection and maintained for over 48 h. Analysis by bromodeoxyuridine (BrdU) pulse labelling demonstrated that cyclin A knockdown had little effect on S-phase progression (Supplementary Figure 1). All three cyclin A siRNAs delayed cells in G2/M phase (Figure 1c), with the degree of accumulation correlating with the level of cyclin A knockdown achieved by each siRNA (Figure 1a). SiRNA A1 significantly increased the proportion of G2/M phase HeLa cells to 40% (P>0.001) by 24 h after transfection compared to nonsense (26%) and lipofection control-treated cells (22%) (Figure 1d). Cyclin A siRNA knockdown produced similar results in U20S cells (Supplementary Figure 2) and HEK293T cells (data not shown). Analysis of cyclin B1/cdk1 demonstrated an increase in cyclin B1 and tyrosine-15-phosphorylated cdk1 in the cyclin A knockdown cells, similar to that found in G2-arrested etoposide-treated cells, indicative of a G2 phase delay (Figure 1e). Examination of both cyclin A and cyclin B by immunofluorescence revealed cells with cytoplasmic cyclin B but lacking nuclear cyclin A (Supplementary Figure 3), which was rarely observed in control cells.
Time-lapse microscopy of cyclin A siRNA-transfected HeLa cells synchronized in early S phase by thymidine block showed cells delayed entry into mitosis by 3–4 h compared to the control siRNA-transfected cells, and this delay was observed with all three cyclin A siRNAs (Figure 2a). The contribution of cdk2 to the G2 phase delay was examined using a cdk2-specific small molecule inhibitor Ro09-3033, a highly selective inhibitor of cdk2 (IC50 20 nM), which binds irreversibly to the ATP-binding site of cdk2 (Stead et al., 2002). Addition of Ro09-3033 (cdk2i) to HeLa cells in early G2 phase irreversibly inactivated cyclin A/cdk2 and delayed progression into mitosis (Figure 2b). In contrast to the inactivation of cyclin A/cdk2, cyclin B1/cdk1 was recovered by in vitro activation with recombinant cdc25B, demonstrating the selectivity of the Ro09-3033. In addition, the inhibition of cyclin A/cdk2 blocks the subsequent cdc25-dependent dephosphorylation of the tyrosine-15-phosphorylated cdk1. Time-lapse microscopy revealed that cdk2i addition to G2 phase cells produced dose-dependent delay in progression into mitosis (Figure 2c). The doses of cdk2i used did not affect cyclin B/cdk1 activity as HeLa cells treated with cdk2i had similar levels of MPM2 staining as controls, whereas treatment with the cdk1/2 inhibitor roscovitine completely ablated MPM2 staining (Figure 2d). Therefore knock down of cyclin A and inhibition of cdk2 caused a G2 phase delay with accumulation of the inactive tyrosine-15-phosphorylated cdk1/cyclin B1 complex in the cytoplasm, as observed in normal G2 phase and G2 phase checkpoint-arrested cells.
Cyclin A/cdk2 coordinates centrosomal and nuclear cyclin B/cdk1 activation
Mitotic entry commences at prophase with activation of a small pool of centrosomal cyclin B1/cdk1, which increases microtubule nucleation at the centrosome (De Souza et al., 2000; Jackman et al., 2003). This precedes complete activation of cyclin B1/cdk1, which promotes the dramatic nuclear and cell morphology in mitosis. All of the measures used to assess the G2-phase arrest imposed by cyclin A/cdk2 depletion are markers of the activation status of the major pool of cyclin B/cdk1. To assess whether the centrosomal mitotic events are also affected by knockdown or inhibition of G2 phase cyclin A/cdk2, HeLa cells expressing cherry-tagged α-tubulin were treated with either cyclin A siRNA or low-dose cdk2i and observed by time-lapse microscopy. In control cells, two microtubule foci were observed in 50% of cells prior to disassembly of the interphase microtubule structures and formation of the mitotic spindle. The cells retained their normal flattened appearance, and did not assume the rounded mitotic morphology until spindle formation had occurred. The microtubule foci persisted for an average of 20–30 min before forming a spindle (Figures 3a and c). In cyclin A knockdown cells, again 50% of cells that entered mitosis displayed two microtubule foci, but their appearance was delayed compared to controls to a similar degree as mitotic entry (compare Figures 2c–3d). However, once the microtubule foci appeared they persisted for an average of 70 min before forming a spindle (Figures 3b and c). These stabilized microtubule foci were also observed with 0.3 μM cdk2i treatment and persisted for a similar length of time (data not shown), indicating that the effect was due to the specific inhibition of G2 phase cyclin A/cdk2 activity.
In normal prophase, microtubule foci are associated with markers of early mitotic events such as chromosome condensation, increased histone H3 phosphoserine 10 and nuclear MPM2 immunostaining. To examine whether this was also the case in the cyclin A-depleted cells, immunofluorescent staining for mitotic markers was performed. Staining cyclin A knockdown cells for microtubules and DNA revealed the microtubule foci where centrosome containing structures (γ-tubulin containing cytoplasmic structures; data not shown), but no chromosome condensation or staining with the mitotic marker, histone H3 phosphoserine 10 (pH3) was detected in these cells (Figure 4a, cell indicated with arrowhead). These cells were termed preprophase to differentiate them from normal prophase where microtubule foci are also observed associated with chromosome condensation and strong staining for pH3. The mitotic phosphoprotein antibody MPM2 also strongly stained the nucleus of prophase cells, but little increase over interphase staining was detected in preprophase cells (Figure 4b). Interestingly, the microtubule foci stained with MPM2 in both normal prophase and preprophase cells (Figure 4b, arrowheads). Quantitation of all cells with these microtubule foci revealed that 60% of cyclin A knockdown cells displayed the preprophase morphology. This was a significant increase compared to only 10% of controls cells, where the majority of prophase cells had condensed chromosomes and elevated pH3 and nuclear MPM2 staining (Figure 4c). Similar levels of preprophase were detected using A3 siRNA, and only 30% of cells with microtubule foci had the preprophase phenotype in A2 siRNA-treated cells. This reflected the efficiency of cyclin A knockdown achieved with these siRNA (Figure 1a).
The detection of increased microtubule nucleation and MPM2 staining at the centrosomes in cyclin A-depleted cells suggested the presence of activated cyclin B/cdk1. Cyclin B/cdk1 activity at the centrosomes was assessed using a phosphospecific antibody detecting MEK1 phosphorylated on Thr286 (pMEK1 T286), a mitosis-specific phosphorylation catalysed by cyclin B/cdk1 (Rossomando et al., 1994). The antibody strongly stained all mitotic cells, as well as the nucleus and spindle poles of prophase cells (Figure 4d). In cyclin A siRNA-treated cells, no nuclear staining with pMEK1 T286 was detected in preprophase cells although it was clearly present once chromosome condensation was observed. pMEK1 T286 staining was also detected at the microtubule foci in cyclin A knockdown preprophase cells as well as control prophase cells (Figure 4d, arrowheads). Pre-treatment with the cdk1/2 inhibitor roscovitine for 2 h prior to fixing resulted in the complete loss of microtubule foci, and completely ablated nuclear and spindle pole MPM2 and pMEK1 T286 staining, the very few mitotic cells being the only exceptions. Pre-treatment with MEK inhibitor U0126 at a concentration that ablated ERK1 activation had no effect on the presence of preprophase, prophase or mitotic cells, or MPM2 and pMEK1 T286 staining (data not shown). This demonstrated that the MPM2 and pMEK1 T286 staining at the microtubule foci in the preprophase cyclin A knockdown cells was dependent on cyclin B/cdk1 activity.
Cyclin A/cdk2 localizes to the centrosome in late G2 phase
The unexpected centrosomal effects of blocking cyclin A/cdk2 activity in G2 phase would predict that cyclin A/cdk2 localized to centrosomes. Cyclin A has been detected at centrosomes although its binding partner was not identified (Bailly et al., 1992; Pagano et al., 1992; den Elzen and Pines, 2001). Mild permeabilization of cells prior to fixing to remove cytoplasmic cyclin A revealed that the cyclin A co-localized with the γ-tubulin stained centrosomes (Figure 5a). Cyclin A centrosomal localization was only detected after separation of centrosome pair, but prior to increased microtubule nucleation. Cdk2 was also localized to the centrosome at the same stage, and cyclin A knockdown depleted cdk2 from the centrosome, demonstrating that it was the cyclin A/cdk2 complex that localized to the centrosome (Figure 5b). Cyclin A and cdk2 were also detected in centrosomal preparations of G2 phase cells, whereas PCNA, another prominent nuclear protein was not detected, indicating the specificity of this association (Supplementary Figure 4). The association was transient and occurred prior to cyclin B1/cdk1 association with the centrosomes, indicating the association occurred in G2 phase.
A number of laboratories have provided evidence that G2 phase cyclin A/cdk2 has a critical role in the timing of entry into mitosis (Furuno et al., 1999; Goldstone et al., 2001; Hu et al., 2001; Mitra and Enders, 2004; Fung et al., 2007; Gong et al., 2007), which is confirmed by our siRNA and cdk2-specific inhibitor studies. In agreement with these reports, the present study demonstrates that inhibition of G2 phase cyclin A/cdk2 delays the cdc25-dependent activation (Mitra and Enders, 2004; Fung et al., 2007), and nuclear translocation (Gong et al., 2007) of cyclin B/cdk1. The accumulated evidence now presents a convincing case that the regulation of the G2 phase pool of cyclin A/cdk2 is critical for cyclin B/cdk1 activation and G2/M progression.
Our study also reveals that cyclin A/cdk2 coordinates the activation of the centrosomal and major soluble pool of cyclin B/cdk1. Activation of cyclin B/cdk1 is coordinated such that it is first detected at the centrosome during prophase, preceding activation of the remaining cytoplasmic and translocated nuclear pools, by no more than 20–30 min (De Souza et al., 2000; Kramer et al., 2004). Inhibition of G2 phase cyclin A/cdk2 by either depletion of cyclin A or inhibition of cdk2 resulted in the loss of this coordination of the activation of the centrosomal and soluble pools of cyclin B/cdk1. In cyclin A-depleted cells, cyclin B/cdk1 activation occurred on average 70 min prior to nuclear events, and in some cases as much as 3 h earlier, but the centrosomal cyclin B/cdk1 activation was still significantly delayed compared with controls. Despite premature activation of the centrosomal pool, there is little evidence of activation of the remaining cytoplasmic cyclin B/cdk1. This was indicated by the lack of change in the general microtubule structures or increase in cytoplasmic phosphorylated Mek1 Thr286, indicators of cyclin B/cdk1 activity.
The mechanism by which cyclin A/cdk2 regulates the timing of cyclin B/cdk1 activation is unclear. The previous studies examining the mechanism by which depletion of G2 phase cyclin A/cdk2 regulates progression into mitosis have provided evidence for deregulation of Wee1 activity (Fung et al., 2007) and cytoplasmic retention of cyclin B1 (Gong et al., 2007). The mechanism appears independent of ataxia telangiectasia mutated/ATM and Rad3 related (ATM/ATR) checkpoint signalling (Hu et al., 2001; Fung et al., 2007). The mechanism by which cyclin A/cdk2 coordinates activation of the centrosomal and soluble pools is also unclear. The localization of cyclin A/cdk2 to the centrosome in late G2 phase, prior to evidence of microtubule nucleation, suggests that it may normally have a role in regulating the timing of cyclin B/cdk1 activation at that location. Cyclin B/cdk1 is activated at the centrosome by the cdc25B phosphatase (Lindqvist et al., 2005), and Aurora A has been reported to phosphorylate and regulate cdc25B at the centrosome (Dutertre et al., 2004). This Aurora A-dependent mechanism may ensure activation of the centrosomal cyclin B/cdk1 precedes activation of the major soluble pool. We have found normal spindle formation in cyclin A knockdown cells when they do enter mitosis, indicating that Aurora A activity is normal (Andrews et al., 2003), and therefore unlikely to be a direct target for cyclin A/cdk2. In the absence of cyclin A/cdk2, all cyclin B/cdk1 activation is delayed. At the centrosome, however, Aurora A-dependent regulation of cdc25B may more readily overcome the absence of the cyclin A/cdk2-dependent signal and activate centrosomal cyclin B/cdk1 significantly earlier than the major soluble pool.
The regulation of cyclin B/cdk1 is critical for entry into mitosis, but the evidence presented here demonstrates that G2 phase cyclin A/cdk2 has a critical role in determining the timing of that event. It also has an unexpected role in coordinating nuclear and centrosomal mitotic events. By influencing the timing of cyclin B/cdk1 activation, the mechanisms controlling G2 phase cyclin A/cdk2 activation provides another means of regulating G2/M progression.
Materials and methods
Cell culture and synchronization
The cell lines used were a human cervical cancer cell line (HeLa), a human kidney epithelial cell line (HEK293T), human osteosarcoma cell line U2OS and HeLa cells stably expressing GFP-H2B, generated by transfection with the pBOS-H2GFP vector (BD Biosciences, BD PharMingen, San Jose, CA, USA). Cherry fluorescent protein (cherry) α-tubulin (a gift from R Tsien, University of California, CA, USA) was also stably expressed in HeLa cells. All cells were cultured at 37 °C in a 5% CO2-humidified atmosphere in Dulbecco's modified Eagle's medium (Invitrogen, Mt Waverley, Victoria, Australia), supplemented with 10% Serum Supreme (BioWhittaker Europe, Verviers, Belgium) and 3 mM 4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid. Cells were synchronized with a single thymidine block by adding 2.5 mM thymidine for 16 h. Cells were released by washing the monolayer three times with warm phosphate-buffered saline, and adding fresh drug-free medium. The cdk2 inhibitor Ro09-3033 was kindly provided by Hoffmann-La Roche, Inc. (Nutley, NJ, USA).
For siRNA-mediated ablation of cyclin A, cells were transfected with siRNAs to the following sequences: A1: 5′-IndexTermaaggatcttcctgtaaatgatgagc-3′; A2: 5′-IndexTermtaaacctaaagtgggttacatg-3′; A3: 5′-IndexTermggaaagtcttaagccttgtctca-3′; and N (nonsense control siRNA): 5′-IndexTermaatgatctacctgttaagagtcctgtctc-3′. All siRNA constructs were made using the Ambion siRNA Construction Kit (Ambion Inc., Austin, TX, USA) as per manufacturer's instructions. Transfections were carried out in six-well plates using Lipofectamine 2000 (Invitrogen) and 50 nM of the appropriate siRNA per well. The medium was replaced with fresh complete medium 4 h later, and cells were harvested at indicated times.
Cells grown on poly-L-lysine-coated cover slips were treated as required and fixed in methanol at −20 °C. Cells were stained with α- and γ-tubulin antibodies (Sigma, St Louis, MO, USA and AbCam, Cambridge, UK), cyclin A and cdk2 (Santa Cruz, Santa Cruz, CA, USA), MPM2 (Dako, Carpinteria, CA, USA), phosphoSer10 H3 and phospho Thr286 MEK1 (Cell Signalling, Ipswich, MA, USA). Antibodies were detected with the appropriate fluorescently labelled secondary antibody and countered stained with 4-6-diamidino-2-phenylindole to detect DNA. Epi-fluorescence microscopy was carried out using a Zeiss Z1 Apotome microscope and a Zeiss Axiocam HRm camera using AxioVision software.
Time-lapse experiments were performed using HeLa, HEK293T, U2OS, GFP-H2B HeLa and cherry α-tubulin HeLa cells. Cells were seeded into six-well plates, transfected, synchronized and images collected at indicated times using a Zeiss Live Cell Observer with a 37 °C incubator and 5% CO2 hood. Images were captured using a × 20 objective on a Zeiss AxioCam MRm camera and AxioVision software. From the image stacks, time of mitosis entry and exit for each cell was recorded, and percent cumulative mitosis of the entire field and length of time in mitosis were determined. Entry into mitosis was marked by the appearance of the rounded mitotic morphology, and in some experiments using GFP-histone H2B-expressing HeLa cells, using chromosome condensation as a marker of mitotic entry. Assessing both these parameters gave identical results.
Cell cycle analysis
For DNA content analysis, floating and attached cells were collected, fixed in ice-cold 70% ethanol and stored at −20 °C. Cells were stained and analysed on a FACSCalibur system (BD Biosciences) using Cell Quest (BD Biosciences) and ModFit (Verity Software, Topsham, ME, USA) data analysis software as previously described (Goldstone et al., 2001). BrdU labelling (Qiu et al., 2000) and MPM2 staining were performed as previously described (Krauer et al., 2004).
Immunoblotting and kinase assays
Cells were lysed in buffer (100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 20 mM Tris, pH 8) supplemented with 5 μg ml−1 aprotinin, 5 μg ml−1 pepstatin, 5 μg ml−1 leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM NaF and 0.1 mM sodium orthovanadate. Samples (20 μg of protein) were resolved on 10% SDS–polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. Proteins were probed with phosphoTyr15 cdk1 (Cell Signalling), cyclin A, cdk2, cdk1 (Santa Cruz), PCNA (Dako) and cyclin B1 (Gabrielli et al., 1996) antibodies and detected by enhanced chemiluminescent detection. Centrosomes were prepared from MOLT4 T-cell leukaemia cells as previously described (De Souza et al., 2000). To evaluate cyclin A- and cyclin B1-specific kinase activity, cyclin A and cyclin B1 complexes were immunoprecipitated and assayed as previously described (Goldstone et al., 2001).
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We thank both the National Health and Medical Research Council of Australia and Queensland Cancer Fund for funding, Dr Nicole den Elzen and Dr Rose Boutros for advice and critical reading of the paper and all other members of the Gabrielli laboratory for critical advice. HB is supported with a Lions Medical Research Fellowship and BG is supported with an NHMRC Senior Research Fellowship.
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De Boer, L., Oakes, V., Beamish, H. et al. Cyclin A/cdk2 coordinates centrosomal and nuclear mitotic events. Oncogene 27, 4261–4268 (2008). https://doi.org/10.1038/onc.2008.74
- cyclin A/cdk2
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