CELL CYCLE 2016, VOL. 15, NO. 20, 2742–2752 http://dx.doi.org/10.1080/15384101.2016.1220455
REPORT
Phosphorylation of CDC25A on SER283 in late S/G2 by CDK/cyclin complexes accelerates mitotic entry Laurent Mazzolinia,b, Ana€ıs Brobana, Carine Fromentc, Odile Burlet-Schiltzc, Arnaud Bessona,b, Stephane Manentia,b, and Christine Doziera,b a Centre de Recherche en Cancerologie de Toulouse, INSERM UMR1037, CNRS ERL5294, Universite Toulouse III Paul Sabatier, Toulouse, France; bEquipe labellisee LIGUE contre le Cancer, CNRS ERL5294, Toulouse, France; cInstitut de Pharmacologie et de Biologie Structurale, Universite Toulouse III Paul Sabatier Toulouse, CNRS UMR5089, Toulouse, France
ABSTRACT
ARTICLE HISTORY
The Cdc25A phosphatase is an essential activator of CDK-cyclin complexes at all steps of the eukaryotic cell cycle. The activity of Cdc25A is itself regulated in part by positive and negative feedback regulatory loops performed by its CDK-cyclin substrates that occur in G1 as well as during the G1/S and G2/M transitions. However, the regulation of Cdc25A during G2 phase progression before mitotic entry has not been intensively characterized. Here, we identify by mass spectrometry analysis a new phosphorylation event of Cdc25A on Serine283. Phospho-specific antibodies revealed that the phosphorylation of this residue appears in late S/G2 phase of an unperturbed cell cycle and is performed by CDK-cyclin complexes. Overexpression studies of wild-type and non-phosphorylatable mutant forms of Cdc25A indicated that Ser283 phosphorylation increases the G2/M-promoting activity of the phosphatase without impacting its stability or subcellular localization. Our results therefore identify a new positive regulatory loop between Cdc25A and its CDK-cyclin substrates which contributes to accelerate entry into mitosis through the regulation of Cdc25A activity in G2.
Received 11 March 2016 Revised 29 July 2016 Accepted 31 July 2016
Introduction The sequential activation and inactivation of cyclin-dependent kinases (CDKs) play a critical role during cell cycle progression.1 A crucial step in the activation of CDKcyclin complexes consists in the removal of inhibitory phosphorylations on the CDK by dual-specificity phosphatases of the Cdc25 family. In mammals, 3 Cdc25 isoforms have been identified: Cdc25A, Cdc25B and Cdc25C.2,3 Mouse knockout models have revealed that a certain degree of functional redundancy exists between these isoforms. Indeed, double knockout Cdc25B¡/¡- Cdc25C¡/¡ mice develop normally and cells from these mice display normal cell cycle profiles.4 Cdc25A therefore appears to fulfill the most important functions of the other Cdc25 isoforms. On the contrary, Cdc25A knockout is lethal at a very early stage during embryogenesis5 indicating that Cdc25A plays essential non redundant functions during cell division. Previous studies revealed that the regulation of Cdc25A activity in dividing cells involves different interconnected positive and negative feedback loops with its CDK-cyclin substrates and this reciprocal regulation contributes to control cell cycle transitions.6 At the end of G1, Cdc25A activates CDK2-Cyclin A/E complexes to drive entry into S phase.7 Moreover, CDK2-Cyclin E complexes directly
KEYWORDS
activating phosphorylation; Cdc25A; CDK-cyclin; cell cycle; G2/M transition
phosphorylate and activate Cdc25A in a positive feedback loop which further accelerates the G1/S transition.8 Cdc25A also contributes, together with Cdc25B, to the activation of CDK1-cyclin B at the G2/M transition,9,10 both phosphatases performing at least partially non-overlapping functions during this step.11 During the G2/M transition, phosphorylation of Cdc25A on Ser17, Ser115 and Ser320 by CDK1cyclin B complexes leads to a robust stabilization of the phosphatase12,13 again generating a positive activation loop amplifying mitosis promoting activity. Previous studies have shown that during G2, Cdc25A is activated earlier than Cdc25B14 and may be primarily responsible for the activation of CDK-cyclin pools until a point near the G2/M transition where Cdc25B synergizes with Cdc25A to complete CDK1-cyclin B activation, leading to mitotic entry. So far, the mechanisms that regulate Cdc25A function in G2 are still largely unclear. Inhibition and knockdown studies performed on CDK2 have indicated that CDK2 activity increases Cdc25A turnover in interphase cells15,16 and may contribute to avoid uncontrolled Cdc25A activation in S and G2 phases. Here we report the characterization of a phosphorylation event occurring on serine 283 of Cdc25A and mediated by CDK-cyclin complexes during the late S/G2 phase of an unperturbed cell cycle. We show that this event contributes to increase the intracellular activity of this phosphatase and to accelerate entry into mitosis.
Centre de Recherche en Cancérologie de Toulouse, CONTACT Christine Dozier [email protected]; Laurent Mazzolini [email protected] INSERM UMR1037, CNRS ERL5294, UPSToulouse III, 2, avenue Hubert Curien, Oncopole entrée C, 31037 Toulouse Cedex 1, France. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy. Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis
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Results Cdc25A is phosphorylated on serine 283 during G2 phase of the cell cycle To identify new phosphorylation sites that may contribute to the functional regulation of Cdc25A, a plasmid encoding human Cdc25A was transiently transfected in exponentially growing HEK293 cells. Mass spectrometry analyses of immunoprecipitated Cdc25A allowed the unambiguous identification of a Ser283 monophosphorylated peptide (Fig. 1A). Phosphorylation of Cdc25A on ser283 had been previously detected by mass spectrometry in U2OS cells conditionally overexpressing the phosphatase13 and more recently on recombinant Cdc25A phosphorylated in vitro by Cdk1/cyclin B complexes immunopurified from Hela cell mitotic extracts.17 However, the role of this phosphorylation is still unknown. Ser283 is conserved in mammals and birds Cdc25A orthologues (Fig. 1B). This residue is located within a previously described nuclear localization signal (NLS, consensus motif: KRX10-12KRRK) present in the 3 Cdc25 isoforms and important for nuclear relocalization of Cdc25A.18 A phosphorylation-specific antibody was raised against a phosphopeptide derived from the Ser283 phosphorylation site. As shown in Figure 1C this antibody specifically reacts with wild-type Cdc25A transiently transfected in HEK293 cells, but not with a non-phosphorylatable mutant (serine 283 mutated to alanine; S283A). This antibody also allows the specific detection of endogenous Cdc25A phosphorylated on Ser283 in whole protein extracts of asynchronous H1299 human lung carcinoma cells which were mostly used in our studies (Fig. 1D). To determine at which step Ser283 phosphorylation occurs during the cell cycle we used the H1299 cell line which can be efficiently synchronized at different phases of the cell cycle. First, H1299 ells were arrested in G0/G1 by serum starvation and lysates were collected at various time points after release from the block and probed with the anti-phospho-Ser283 antibody. In these conditions phosphorylation of Ser283 could be detected in total extracts 20 h after release when cells reached G2 phase as determined by flow cytometry analysis of DNA content (Fig. 2A) and as soon as 18h post release in purified Cdc25A fractions, obtained by immunoprecipitation, when cells were still mainly in S phase (Fig. 2A, B). To further define the Ser283 phosphorylation pattern during the cell cycle, H1299 cells were synchronized at the onset of S phase by double thymidine block. Immunoblot analysis of cells after release confirmed that Ser283 phosphorylation could be detected in cells which were accumulating in late S/G2 (4h post-release time point) (Fig. 2C). This was confirmed by additional synchronization experiments performed with H1299 cells expressing HA tagged-Cdc25A in which western blot analysis of immunopurified Cdc25A allowed the detection of phosphorylated serine 283 as soon as 4h after release from thymidine block when cells were highly predominantly in S phase (Fig. S1). Moreover quantifications of serine283 phosphorylation in these experiments revealed that this phosphorylation was not enhanced upon entry into mitosis when cell positive for phospho-Ser10 histone H3 began to
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massively accumulate. Altogether these studies led us to conclude that serine 283 phosphorylation must take place in late S and during the G2 phase of the cell cycle and is maximal before mitotic onset. Cdc25A phosphorylation on serine 283 is mediated by CDK-cyclin complexes Ser283 and its adjacent proline form a candidate motif for proline-directed kinases such as CDKs and mitogen-activated protein kinases.19 Treatment of H1299 and HEK293 cells with the CDK1/2 inhibitor roscovitine20 strongly decreased phosphorylation of endogenous Cdc25A on Ser283 (Fig. 3A). Treatment with roscovitine and with the more specific CDK1 inhibitor RO330621 also strongly reduced Ser283 phosphorylation of Cdc25A transiently overexpressed in HEK293 cells (Fig. 3B). Moreover, a strong increase of Ser283 phosphorylation was observed when plasmids encoding either CDK1 and cyclin A2/ B1 or CDK2 and cyclin A2 were cotransfected with the Cdc25A expression vector in H1299 cells (Fig. 4A). Finally we found that both CDK1-cyclin B1 and CDK2-cyclin A2 were able to efficiently phosphorylate Cdc25A in vitro on Ser283 and this reaction was fully inhibited in the presence of roscovitine or RO3306 (Fig. 4B). Altogether these results argue for a direct involvement of the 2 main CDK-cyclin complexes acting during G2 phase and at the G2/M transition in the phosphorylation of Cdc25A on Ser283. Phosphorylation on serine 283 does not modify Cdc25A intracellular localization Since Ser283 is localized in the NLS of Cdc25A we investigated whether its phosphorylation could affect its intracellular localization. For this, expression plasmids coding for the wild-type and S283A mutant forms of Cdc25A fused either with mRFP (Cdc25A wild type) or with GFP (Cdc25A wild type or S283A mutant) were cotransfected in U2OS cells. In this cell type Cdc25A is predominantly nuclear but can also have pancellular localization in a small fraction of the cells. As expected, the cotransfected control constructs (i.e. RFP-Cdc25A and GFP Cdc25A) showed identical localization, whether Cdc25A was localized in the nucleus or distributed in the whole cell, confirming that the presence of the RFP or GFP did not alter the localization of the fusion protein (Fig. S2). When Cdc25A wild type and Cdc25A-S283A were cotransfected, no differences could be observed between wild-type CDC25A and its nonphosphorylatable S283A mutant form. The distribution of transiently expressed wild-type and S283A mutant forms of cdc25A was also studied by immunofluorescence in Hela cells in which Cdc25A is highly predominantly nuclear. High content analysis of the transfected cells revealed only a mild increase of nuclear accumulation of the mutant Cdc25A relative to its wild-type counterpart (i.e., 92,1 % vs 87,0%, Fig. S3). Finally, no differences in intracellular localization could be evidenced in H1299 cells inducibly expressing HA-tagged wild type or mutant Cdc25A (see below) and synchronized in interphase or during mitosis (data not shown). Altogether these studies indicated that phosphorylation of Ser283 has no major impact on Cdc25A nucleocytoplasmic shuttling.
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Figure 1. Mass spectrometric identification of Cdc25A phosphorylation at serine 283. (A) The HCD MS/MS spectrum of the monophosphorylated peptide, 279-SQEEpSPPGSTKR-290 (doubly charged precursor ion, MH2+, at m/z 691.80157) displays series of y- and b-ions. Intense simply charged y7 (at m/z 742.4204) together with simply charged b2 (at m/z 216.0978) indicate that serine 283 is phosphorylated but not serine 279, serine 287 or threonine 288. (B) Multiple sequence alignment of the NLS region of various Cdc25A orthologues. Arrow: position of ser283 (human sequence). (C) HEK293 cells were transfected with a bicistronic plasmid expression vector encoding GFP (control for transfection efficiency), and either wild-type (WT) Cdc25A or the S283A mutant. Twenty-four h post-transfection, total protein extracts were immunoblotted with the indicated antibodies. p-S283: phospho-ser283 antibody. (D) Total H1299 extracts were subjected to protein gel blot analysis. Treatments with the protein synthesis inhibitor cycloheximide (Chx, 50 mg/ml for 1 h) or with a siRNA directed against Cdc25A, which both lead to a strong reduction of Cdc25A protein level were included as negative controls for specificity of the antibody reaction. Arrow: position of Cdc25A. Asterisk: nonspecific cross reacting polypeptide. Respective molecular weights of the proteins are indicated on the right of the immunoblots.
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Figure 2. Cdc25A is phosphorylated on serine 283 in late S and G2 phase of the cell cycle. H1299 cells were either synchronized in G0/G1 (A-B) or G1/S (C) by serum starvation and thymidine block, respectively. Following release, cells were collected and their nuclear DNA content analyzed by flow cytometry (A,C). In parallel, total protein extracts (A,C) or Cdc25A immunoprecipitates (B) obtained from the same fractions used in (A) were immunoblotted with the indicated antibodies. Cyclin A, cyclin E and phospho-ser10 histone H3 (pS10 HH3) were included as cell cycle and mitosis entry markers. Starv.: serum-deprived cells. Thy: cells blocked in G1/S. Arrow: position of Cdc25A. Asterisk: nonspecific cross reacting protein. Respective molecular weights of the proteins are indicated on the right of the immunoblots.
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Figure 3. Cdc25A serine 283 phosphorylation is reduced upon treatment of cells with cyclin-dependent kinases inhibitors. (A) Exponentially growing H1299 (upper panel) or HEK293 cells (lower panel) were treated for 1 h with either 50 mg/ml cycloheximide (Chx) or 50 mM roscovitine (Ros.). n.t.: untreated control cells. The corresponding cell extracts were analyzed by western blot with the indicated antibodies. Arrow: position of Cdc25A. Asterisks: nonspecific cross-reacting polypeptides. (B) HEK293 cells were transfected with a bicistronic plasmid vector encoding wild-type Cdc25A together with GFP, used as a control for transfection efficiency. Twenty-four h after transfection the cells were either left untreated (n.t.) or treated for 2 h with different CDK inhibitors: Ros. : roscovitine (50 mM), RO: RO3306 (10 mM), PD: PD0332991 (CDK 4/6 inhibitor,1 mM, used as a control). The corresponding protein samples were immunoblotted with the indicated antibodies.
Phosphorylation on serine 283 does not impact the stability of Cdc25A
Mutation of serine 283 reduces the G2/M promoting activity of Cdc25A
Previous studies have shown that phosphorylation of Cdc25A by CDK-cyclin complexes can modify its turnover during interphase and mitosis.6 Moreover Ser283 is in close proximity to 2 previously described CHK1/2 phosphorylation sites (i.e. Ser279 and Ser293) which together with additional phosphorylations are involved in proteasomaldependent degradation of Cdc25A in the S and G2 phase of the cell cycle.22 We therefore investigated the impact of Ser283 phosphorylation on Cdc25A stability. For this, we used tetracycline-inducible H1299 cells conditionally expressing HA-tagged wild type or S283A-mutated Cdc25A proteins. The tetracycline inducible cell populations consist in pools of several hundred of puromycin-resistant clones. No background expression of the HA-tagged proteins was detected in absence of the inducer doxycycline (Fig. S4). Although only a fraction of the cells were found to express the HA-tagged Cdc25A upon induction with doxycycline (14 % for the wild-type construct and 20% for the S283A mutant as determined by immunofluorescence analysis, data not shown) the use of pools formed by a large number of independent clones allows to minimize variations in cell responses that may originate from clonal heterogeneity. Stability studies on the HA-tagged Cdc25A proteins expressed in H1299 inducible cells performed either in exponentially growing cells (data not shown) or in cells synchronized in G2 (Fig. S5) indicated that the turnover rate of the Cdc25A S283A mutant was not significantly different from that of the wild type protein. Moreover, no difference in stability between the wild type and S283A mutant form of Cdc25A could be evidenced in transiently transfected HEK293 cells either exponentially growing (data not shown) or arrested in mitosis after treatment with nocodazole (Fig. S6). We therefore concluded that the phosphorylation of Ser283 does not significantly modify interphasic or mitotic Cdc25A stability during a normal cell cycle.
Since phosphorylation of Ser283 was found to take place in late S/G2 (Fig. 2 and Fig. S1), we asked whether it could affect Cdc25A activity in promoting entry into mitosis. For this, H1299 inducible cell pools were synchronized in G1/S by thymidine block and then released from the block in presence of doxycycline. Cell cycle profiles were determined by flow cytometry analysis of DNA content (Fig. S7). Cells entering mitosis were identified by immunofluorescence using anti-phosphoSer10 histone H3 antibodies (Fig. 5A). Phosphorylation of histone H3 on Ser10 takes place before nuclear envelope breakdown occurs and therefore allows detection of the very early step of prophase.23 The subpopulations of cells overexpressing wild type or S283A-mutated HA-Cdc25A after induction with doxycycline were identified by immunofluorescence using an anti-HA antibody (Fig. 5A, arrows). To determine whether overexpression of wild type or mutant HA-Cdc25A could affect entry into mitosis, we compared, in each pool and at a fixed time point after release from thymidine block, the percentage of phospho-Ser10 histone H3-positive cells in the cell subpopulation overexpressing HA-Cdc25A (HA-positive cells) with the one in the remaining non inducible cell subpopulation (HA-negative). In order to focus our analyses on the early steps of mitosis only phosphoSer10 histone H3 positive cells corresponding to cells in early to late prophase were counted. In three independent experiments, a strong increase (4 to 20-fold, depending on the synchronization experiment) in the percentage of phospho-Ser10 histone H3-positive cells was observed in cells overexpressing HA-Cdc25A relative to those only expressing endogenous Cdc25A (see Fig. 5B for a representative experiment). These results indicate that overexpression of Cdc25A accelerates entry into mitosis in H1299 cells, as previously described in other model cell lines. This effect was observed in both cell pools, however the relative enrichment in cells entering mitosis was significantly lower in cells expressing the S283A mutant when compared to the pool expressing wild
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This indicated that the reduction in the percent of cells entering mitosis could not be attributed to lower induction levels of the mutant. The low differences in mitosis promoting activity between the wild type and S283A mutant in our experiments may in part originate from the fact that both proteins are expressed in significant excess (5 to 18-fold ; data not shown) with respect to endogenous Cdc25A in the induced cells. Additional microscopy analyses suggested that the overexpression of Cdc25A or the phosphorylation mutant does not modify mitotic progression as cells in all phases of mitosis could be observed (data not shown). Moreover, flow cytometry analysis of exponentially growing cells showed that the duration of mitosis was identical in exponentially growing cells overexpressing the wild type or the S283A mutant protein (Fig. S8). Altogether, these results led us to conclude that the phosphorylation of Ser283 does not play an essential role during progression through mitosis but rather contributes to increase the G2/M promoting activity of Cdc25A in interphase.
Discussion
Figure 4. CDK-cyclin complexes are involved in the phosphorylation of serine 283 of Cdc25A. (A) H1299 cells were transfected with the Cdc25A bicistronic plasmid used in Fig. 3 B either alone (-) or in combination with different plasmids encoding CDK1, CDK2, cyclin A2 (CycA) and cyclin B1 (CycB). Twenty-four h after transfection total protein extracts were analyzed by protein gel blot. The anti-HA antibody allows the detection of the transfected CDKs which are N-terminally tagged with the HA epitope. (B) Bacterially expressed recombinant wild type Cdc25A was incubated for 1 h with either recombinant CDK1-Cyclin B1 complexes in the presence or absence of 10 mM RO3306 or with recombinant CDK2-Cyclin A2 complexes in the presence or absence of 50 mM roscovitine. The corresponding protein samples were immunoblotted with the indicated antibodies. Cdk: anti-CDK1 (left panel) or anti-CDK2 (right panel) antibodies. Cyclin: anti-Cyclin B1 (left panel) or anti-Cyclin A2 (right panel) antibodies.
type Cdc25A (mean reduction: 20%, Fig. 5C). This results was confirmed by additional flow cytometry analyses which allowed to simultaneously analyze HA- and phospho-Ser10 histone H3-labelings as well as cell cycle profiles in the synchronized cell pools (Fig. 5D) and therefore to accurately follow the appearance of mitotic (phospho-HH3 positive) cells at different time points after release for the thymidine block. These studies revealed that, whereas the kinetics of entry into mitosis appeared highly reproducible between the 2 cell pools in the non-induced cell population (Fig. 5E), again a small delay in the entry into mitosis (which becomes highly statistically significant from 7h post-release) was observed in the cell subpopulation overexpressing the Ser283A mutant when compared to the cells overexpressing wild type Cdc25A (Fig. 5F). Quantitative analyses of Cdc25A expression showed that the induction levels of wild type and mutant Cdc25A were very similar (Table S1).
Positive regulatory loops between the Cdc25A phosphatase and its CDK-cyclin substrates have previously been observed both at the G1/S and G2/M transitions. During G1/S transition phosphorylation of Cdc25A by CDK2-cyclin E increases its enzymatic activity8 whereas during entry into mitosis phosphorylation by the CDK1-cyclin B complexes strongly stabilizes the protein.13 Many studies on the regulation of Cdc25A focused on the G2/M transition but molecular mechanisms possibly regulating Cdc25A activity during progression from S phase until mitosis i.e., G2 phase remain largely unknown. Previous studies showed that inhibition of CDK2 activity lead to an increase in Cdc25A protein levels suggesting that CDK2-containing complexes are also involved in the destabilization of Cdc25A in interphase cells although most probably indirectly through ATR-CHK1 signaling.15,16 This may contribute to counteract Cdc25A enzymatic activation occurring at the G1/S transition in order to prevent excessive accumulation of active Cdc25A in both S and G2, which could lead to premature mitotic entry during an unperturbed cell cycle and/or ineffective cell cycle arrest following DNA replicative stress. This last point is strongly suggested by the fact that the stabilization of Cdc25A in CDK2-deficient cells contributes to G2/M checkpoint defects following DNA damage.15 The CHK1 kinase which maintains a basal level of activity during and unperturbed cell cycle was also reported to perform an additional regulation of Cdc25A stability22,24 These data indicate that the expression and activity of Cdc25A6 must be tightly controlled in the interphase cell in order to avoid premature cell cycle transitions. Herein, we characterized a novel positive feedback loop that involves the phosphorylation of serine 283 of Cdc25A performed by CDK-cyclin complexes in late S/G2 and found that this phosphorylation event increases the activity of Cdc25A in promoting mitotic entry. Although the mechanism leading to a raise in Cdc25A activity remains unknown we showed that it does not involve a stabilization of the phosphatase or a modification of its subcellular localization. This phosphorylation
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Figure 5. Phosphorylation on serine 283 contributes to the activation of Cdc25A in G2. (A) H1299 inducible cells expressing either wild type or mutant HA-tagged Cdc25A were synchronized at the G1/S transition and released in the presence of doxycycline. At 7.5 h post-release, cells were fixed on coverslips and stained with anti-phosphoser10 H3 antibody (Phospho-HH3 ; green) and anti-HA antibody (red). DNA was stained with DAPI (blue). Scale bar: 20 mm. Arrows indicate HA-positive cells. Representative images are shown. (B) At 7.5 h post-release the percentages of mitotic cells corresponding to cells ranging from very early prophase (complete phospho-ser10 H3-positive labeling of the nucleus but no apparent chromatin condensation in DAPI staining41) to late prophase (strong phospho-ser10 H3-labeling and full condensation of chromatin but no reorganization of chromosomes indicative of entry into prometaphase) were determined in each subpopulation. Values obtained with the HA-negative subpopulations were then set to 1 for each pool. A representative experiment is shown. At least 3,000 cells were analyzed in each pool. (C) For each synchronization experiment, the enrichment factor in early mitotic cells associated with the overexpression of wild type Cdc25A (as determined in B) was set to 100 and the enrichment factor obtained with the S283A mutant recalculated accordingly. Graphs represent mean values of 3 independent experiments. Bar represents s.e.m.; p D 0.0225 according to Student’s unpaired t test. (D-F): H1299 inducible cells were synchronized and induced with doxycycline as described above and collected at different time points after release for the thymidine block for flow cytometry analysis. (D) Multiparametric flow cytometry analysis. Cell cycle progression, expression of HA-Cdc25A and mitotic status were simultaneously analyzed by labeling of DNA with Hoechst 33342 (left panel) and labeling with antibodies directed against HA and anti-phospho-ser10 H3 histone, respectively (right panel). A representative experiment is shown (cell pool expressing wild type HA-Cdc25A; 7h post-release). (E) Kinetics of entry into mitosis of the cells in the induced pools following release from the block. Cells were analyzed by flow cytometry as described in (D) A representative synchronization experiment is shown. (F) For each synchronization experiment and at each time point after release, the enrichment factor in mitotic (phospho-HH3 positive) cells in the cell subpopulation overexpressing wild type HA-Cdc25A (phospho-HH3 positive) relative to the non-induced cell subpopulation (phospho-HH3 negative) was set to 100 and the enrichment factor obtained under the same conditions with the S283A mutant recalculated accordingly. Graphs represent mean values of 4 independent experiments. Bar represents s.e.m.; p < 0 .01 according to Student’s unpaired t test.
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could directly affect the phosphatase activity of Cdc25A or its interaction with the CDK-cyclin substrates. No activating phosphorylation of Cdc25A by CDKs has previously been described during G2 phase. Moreover our biochemical studies show that this phosphorylation may be directly performed by CDK1/2 -cyclin complexes themselves. This result suggests that CDKs active during G2 have an overall positive impact on Cdc25A activity despite some members such as CDK2 also contribute to Cdc25A destabilization. The simultaneous increase of Cdc25A activity and of its turnover rate may constitute a mean to increase the responsiveness of this phosphatase to the regulatory pathways controlling G2/M entry in an unperturbed cell cycle as well as in response to various cell stresses such as DNA damage. This coupling between protein activation and protein instability has been frequently observed for proteins whose function need to be quickly and efficiently regulated such as receptor kinases or cell signaling intermediates.25 The Cdc25A and Cdc25B phosphatases both independently contribute to the assembly and activation of CDK1-cyclin B complexes in G29,10 and Cdc25A overexpression accelerates entry into mitosis9,13,14 and can prevent cell cycle arrest following checkpoint activation thus leading to premature mitotic entry.26 Conversely, Cdc25A targeting by RNAi delays G2/M transition.11,13 Studies of Lindqvist et al.11 have shown that Cdc25A and Cdc25B may cooperate to induce mitotic entry by activating specific nuclear and centrosomal CDK-cyclin pools during this step. Interestingly, data of Timofeev et al.14 have revealed that Cdc25A is activated earlier than Cdc25B in G2. Moreover, these authors showed that Cdc25A is specifically involved in the activation of CDK2-Cyclin A complexes. These complexes are assembled and activated during S phase but show biphasic activity with a second major boost of activation occurring in early G2.27 CDK2-cyclin A complexes contribute to mitotic both directly28 and through the activation of CDK1-cyclinB complexes29-34 and their activity appears rate-limiting for G2/M transition. Based on our results, we propose that an initial phosphorylation of Cdc25A on serine 283 by CDK2-Cyclin A complexes could be involved in the establishment of a positive activatory loop between Cdc25A and CDK-cyclin complexes acting during the G2 phase. Control of Cdc25A activity appears essential to ensure the correct timing of entry into mitosis and phosphatases and kinase activities distinct from CDKs have been found to regulate mitotic entry through the regulation of Cdc25A. Indeed, Vazquez-Novelle et al.10 previously showed that the Cdc14A phosphatase may also control the timing of G2/M transition by inhibiting the catalytic activity of Cdc25A. Since Cdc14A was also shown to inhibit CDK1-cyclinB1 activity at the G2/M transition, it was tempting to speculate that inhibition of Cdc25A activity by Cdc14A overexpression involved dephosphorylation of Ser283. However, the mode of action of Cdc14A on Cdc25A appears to be an indirect effect exerted by a still unknown protein.10 In agreement of this, Tumurbaatar et al. showed that phospho-Ser283 was not dephosphorylated by Cdc14A in vitro.17 The ribosomal S6 kinase has also been recently reported to phosphorylate Cdc25A on serine 293 and 295 and increase its M phase-inducing activity but the mechanism of action of these phosphorylations on Cdc25A function was not investigated in this work.35
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Our studies further emphasize the essential role of Cdc25A in the regulation of G2/M transition. The numerous regulatory loops established between Cdc25A and its CDK substrates at all the phases of the cell cycle most probably represent a first essential layer in the regulation of progression through the cell division cycle.
Materials and methods Cell culture, generation of inducible cell pools and cell transfections Human non-small-cell lung carcinoma H1299 cells (ATCC validated) and human embryonic kidney HEK293 cells (obtained from DSMZ) were grown respectively in RPMI 1640 and DMEM medium, supplemented with 10% calf serum and 1X penicillin-streptomycin (Gibco, LifeTechnologies, Carlsbad, CA, USA). Tetracycline inducible H1299 cell pools were generated by a one-step transformation process with the “all-in-one” TET-Cdc25A plasmid vectors. Plasmids were transfected with the Amaxa nucleofection system (Lonza, Koeln, Germany). Puromycin-resistant pools were selected in culture medium supplemented with 1 mg/ml puromycin (InVivogen, San Diego, CA, USA). H1299 cells were transfected with siRNAs using INTERFERin reagent (Polyplus, Illkirch, France). Transfections of plasmid vectors were performed with Jet PEI (Polyplus). NanoLC–MS/MS analysis and database searches After SDS-PAGE separation of the immunoprecipitated FLAGtagged Cdc25A, the Coomassie blue-stained corresponding band was in-gel digested with trypsin and analyzed by online nanoLC using an Ultimate 3000 System (Dionex, Amsterdam, The Netherlands) coupled to an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) for data-dependent HCD fragmentation experiments. Antibodies Anti-Cdc25A (mouse F6, dil.1/500), anti-CDK1 (mouse P34, dil.1/1,000), anti-CDK2 (rabbit M2, dil.1/1,000), anti-cyclin B1 (mouse GNS1, dil.1/1,000) and anti-Cyclin A2 (rabbit C-19, dil.1/1,000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-actin (mouse C4 , dil.1/5,000) was from Chemicon (Temecula, CA, USA). Anti-GFP (rabbit D5.1, dil.1/500) and anti-HA (rabbit C29FA, dil.1/2,000) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-phospho-ser283 Cdc25A rabbit antibody was raised against peptide RSQEES(PO3H2) PPGSTKC (Eurogentec SA, Belgium). Plasmids The bicistronic Cdc25A expression plasmids were constructed by inserting the coding sequence of human Cdc25A in the pIRES2-EGFP vector (Clontech, Mountain View, CA). The S283A mutation was introduced by site-directed mutagenesis using inverted PCR. Vectors encoding human HA-tagged CDK1 and CDK2 were a gift from Sander van den Heuvel36
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(Addgene plasmids # 1888 and 1884). Expression vectors for human cyclin A2 and Cyclin B1 were respective gifts from Robert Weinberg37 (Addgene plasmid # 8959) and Jonathon Pines38 (Addgene plasmid # 26060). The TET-Cdc25A vectors were constructed by first inserting the coding sequence of human Cdc25A downstream of the Tet promoter in the pTREtight plasmid (Clontech). This TET cassette was then introduced in the pZDonor-AAVS1 plasmid (Sigma-Aldrich, St Louis, MO, USA) together with the tricistronic expression cassette of vector pWHE64439 which allows the constitutive expression of Tet activator, Tet repressor and puromycin resistance genes. siRNAs and inhibitors Cdc25A siRNA (Hs_CDC25A_9) was obtained from Qiagen (Hilden, Germany). Control siRNA (ON-TARGET plus Nontargeting Control Pool) was from Dharmacon (Chicago, IL, USA). Roscovitine, RO3306 and PD0332991 were obtained from Selleck Chemicals (Houston, TX, USA). Cell synchronizations and flow cytometry analyses H1299 cells were synchronized in G0/G1 by serum deprivation for 48 h in RPMI medium containing 0.1% BSA and then released in 10% serum-supplemented medium. For synchronization in G1/S, 5 mM thymidine was added to the medium 7 h after release from the G0/G1 block and the cells incubated for an additional 16 h time period. Cells were released for the block by extensive washing with PBS and addition of fresh RPMI medium. Flow cytometry analyses of cell cycle distribution were performed on propidium-iodide-stained cells. Flow cytometry analysis of cell cycle profiles, HA-labeling patterns and phospho-Ser10 histone H3 status of the synchronized cells were performed by simultaneous labeling of ethanol fixed cells with anti HA antibodies, anti phospho-Ser10 histone H3 antibodies and Hoechst 33342 dye. Flow cytometry measurements were performed using MACSQuant VYB and MQ10 flow cytometers (Miltenyi Biotech, Bergisch Gladbach, Germany) Immunoprecipitations, total protein extraction, protein gel blotting and quantifications Cell lysates for immunoprecipitations were prepared in cold lysis buffer (20 mM, Tris pH 7.4, 100 mM NaCl, 5 mM EGTA, 0.5% NP-40) supplemented with phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich) and CompleteTM protease inhibitor cocktail (Roche Diagnostics, Basel, Stwitzerland). Prior to immunoprecipitation extracts were clarified by centrifugation at 16,000 g for 10 min at 4 C). For mass spectrometry analyses, FLAG-tagged Cdc25A was purified from transfected HEK293 cells with anti-FLAG (mouse M2, Sigma-Aldrich) antibodies and G protein-coupled sepharose (EZviewTM Red Protein G, Sigma Aldrich). HA-tagged Cdc25A was purified with an antiHA agarose affinity gel (EZ view Red, Sigma-Aldrich). Total protein extractions and western blotting were done as previously described.40 Image acquisition of immunoblots and quantifications were performed with the PxiTM imager and GeneTools software from Syngene (Cambridge, UK).
Fixed cells staining and image acquisition Cells were fixed in 4% paraformaldehyde in PBS and processed for immunofluorescence following conventional protocols. Images were taken with a Plan-Apochromat 40x/1.4 Oil DIC objective by using an Axio observer.Z1 (Carl Zeiss, Iena, Germany) microscope. In vitro kinase reactions Recombinant human Cdc25A (200 ng, Jena Bioscience, Jena, Germany) and Cdk1-cyclin B1 or Cdk2-cyclin A2 proteins (20ng, Sigma-Aldrich) were incubated for 1 h at 30 C in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0,1mM EDTA, 0,01% Brij35, 2 mM DTT and 200 mM ATP).
Abbreviations CDK Cdc ATR CHK1
cyclin-dependent kinases cell-dependent cycle ataxia telangiectasia and rad3-related checkpoint kinase 1
Disclosure of potential conflicts of interests No potential conflicts of interest were disclosed.
Acknowledgments We are very grateful to Drs Christian Berens and Christina Danke for providing the pWHE644 tricistronic transregulator expression plasmid. We thank Sander van den Heuvel, Jonathon Pines and Robert Weinberg for the CDK and Cyclin expression plasmids. We are grateful to Manon Farce and Laetitia Ligat for assistance with flow cytometry and microscopy, respectively. We thank Charlotte Cavanihac and Julie Poulain for technical assistance.
Funding This work was supported by the Institut National de la Sante et de la Recherche Medicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), the University Paul Sabatier Toulouse III, the Ligue Nationale Contre le Cancer, the Fondation ARC pour la Recherche sur le Cancer and the Laboratoire d’Excellence Toulouse Cancer LABEX TOUCAN. It was also supported in part by the Region Midi-Pyrenees, Toulouse Metropole, and European funds FEDER (Fonds Europeens de Developpement Regional) for mass spectrometry.
References [1] Malumbres M, Barbacid M. Mammalian cyclin-dependent kinases. Trends Biochem Sci 2005; 30:630-41; PMID:16236519; http://dx.doi. org/10.1016/j.tibs.2005.09.005 [2] Boutros R, Lobjois V, Ducommun B. CDC25 phosphatases in cancer cells: key players? Good targets? Nat Rev Cancer 2007; 7:495-507; PMID:17568790; http://dx.doi.org/10.1038/nrc2169 [3] Kiyokawa H, Ray D. In vivo roles of CDC25 phosphatases: biological insight into the anti-cancer therapeutic targets. Anticancer Agents Med Chem 2008; 8:832-6; PMID:19075565; http://dx.doi.org/ 10.2174/187152008786847693 [4] Ferguson AM, White LS, Donovan PJ, Piwnica-Worms H. Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and Cdc25C protein phosphatases. Mol Cell Biol
CELL CYCLE
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
2005; 25:2853-60; PMID:15767688; http://dx.doi.org/10.1128/ MCB.25.7.2853-2860.2005 Ray D, Terao Y, Nimbalkar D, Hirai H, Osmundson EC, Zou X, Franks R, Christov K, Kiyokawa H. Hemizygous disruption of Cdc25A inhibits cellular transformation and mammary tumorigenesis in mice. Cancer Res 2007; 67:6605-11; PMID:17638870; http://dx. doi.org/10.1158/0008-5472.CAN-06-4815 Frazer C, Young PG. Phosphorylation Mediated Regulation of Cdc25 Activity, Localization and Stability. In: Cai H, ed. Protein Phosphorylation in Human Health: InTech, 2012; 395-436 Blomberg I, Hoffmann I. Ectopic expression of Cdc25A accelerates the G(1)/S transition and leads to premature activation of cyclin Eand cyclin A-dependent kinases. Mol Cell Biol 1999; 19:6183-94; PMID:10454565; http://dx.doi.org/10.1128/MCB.19.9.6183 Hoffmann I, Draetta G, Karsenti E. Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition. EMBO J 1994; 13:430210; PMID:7523110 Timofeev O, Cizmecioglu O, Settele F, Kempf T, Hoffmann I. Cdc25 phosphatases are required for timely assembly of CDK1-cyclin B at the G2/M transition. J Biol Chem 2010; 285:16978-90; PMID:20360007; http://dx.doi.org/10.1074/jbc.M109.096552 Vazquez-Novelle MD, Mailand N, Ovejero S, Bueno A, Sacristan MP. Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B. J Biol Chem 2010; 285:40544-53; PMID:20956543; http://dx.doi.org/10.1074/jbc.M110.133009 Lindqvist A, K€alstr€ om H, Lundgren A, Barsoum E, Rosenthal CK. Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome. J Cell Biol 2005; 171:35-45; PMID:16216921; http://dx.doi.org/ 10.1083/jcb.200503066 Esteban V, Vazquez-Novelle MD, Calvo E, Bueno A, Sacristan MP. Human Cdc14A reverses CDK1 phosphorylation of Cdc25A on serines 115 and 320. Cell Cycle 2006; 5:2894-8; PMID:17172867; http:// dx.doi.org/10.4161/cc.5.24.3566 Mailand N, Podtelejnikov AV, Groth A, Mann M, Bartek J, Lukas J. Regulation of G(2)/M events by Cdc25A through phosphorylationdependent modulation of its stability. EMBO J 2002; 21:5911-20; PMID:12411508; http://dx.doi.org/10.1093/emboj/cdf567 Timofeev O, Cizmecioglu O, Hu E, Orlik T, Hoffmann I. Human Cdc25A phosphatase has a non-redundant function in G2 phase by activating Cyclin A-dependent kinases. FEBS Lett 2009; 583:841-7; PMID:19192479; http://dx.doi.org/10.1016/j.febslet.2009.01.044 Chung JH, Bunz F. Cdk2 is required for p53-independent G2/M checkpoint control. PLoS Genet 2010; 6:e1000863; PMID:20195506; http://dx.doi.org/10.1371/journal.pgen.1000863 Ducruet AP, Lazo JS. Regulation of Cdc25A half-life in interphase by cyclin-dependent kinase 2 activity. J Biol Chem 2003; 278:31838-42; PMID:12801928; http://dx.doi.org/10.1074/jbc.M303604200 Tumurbaatar I, Cizmecioglu O, Hoffmann I, Grummt I, Voit R. Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity. PloS One 2011; 6:e14711; PMID:21379580; http://dx.doi.org/10.1371/journal. pone.0014711 K€alstr€om H, Lindqvist A, Pospisil V, Lundgren A, Rosenthal CK. Cdc25A localisation and shuttling: characterisation of sequences mediating nuclear export and import. Exp Cell Res 2005; 303:89-100; PMID:15572030; http://dx.doi.org/10.1016/j.yexcr.2004.09.012 Ubersax JA, Ferrell JE, Jr. Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 2007; 8:530-41; PMID:17585314; http://dx.doi.org/10.1038/nrm2203 Meijer L, Borgne A, Mulner O, Chong JP, Blow JJ, Inagaki N, Inagaki M, Delcros JG, Moulinoux JP. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem 1997; 243:527-36; PMID:9030781; http://dx.doi.org/10.1111/j.1432-1033.1997.t01-200527.x Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H, Heimbrook DC, Chen L. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
2751
Acad Sci U S A 2006; 103:10660-5; PMID:16818887; http://dx. doi.org/10.1073/pnas.0600447103 Sørensen CS, Syljuasen RG, Falck J, Schroeder T, Ronnstrand L, Khanna KK, Zhou BB, Bartek J, Lukas J. Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell 2003; 3:247-58; PMID:12676583; http://dx.doi.org/10.1016/S1535-6108 (03)00048-5 Sawicka A, Seiser C. Histone H3 phosphorylation - a versatile chromatin modification for different occasions. Biochimie 2012; 94:2193201; PMID:22564826; http://dx.doi.org/10.1016/j.biochi.2012.04.018 Goloudina A, Yamaguchi H, Chervyakova DB, Appella E, Fornace AJ, Jr., Bulavin DV. Regulation of human Cdc25A stability by Serine 75 phosphorylation is not sufficient to activate a S phase checkpoint. Cell Cycle 2003; 2:473-8; PMID:12963847; http://dx.doi.org/10.4161/ cc.2.5.482 Vazquez F, Ramaswamy S, Nakamura N, Sellers WR. Phosphorylation of the PTEN tail regulates protein stability and function. Mol Cell Biol 2000; 20:5010-8; PMID:10866658; http://dx.doi.org/ 10.1128/MCB.20.14.5010-5018.2000 Molinari M, Mercurio C, Dominguez J, Goubin F, Draetta GF. Human Cdc25 A inactivation in response to S phase inhibition and its role in preventing premature mitosis. EMBO Rep 2000; 1:71-9; PMID:11256629; http://dx.doi.org/10.1093/embo-reports/ kvd018 Goldstone S, Pavey S, Forrest A, Sinnamon J, Gabrielli B. Cdc25dependent activation of cyclin A/cdk2 is blocked in G2 phase arrested cells independently of ATM/ATR. Oncogene 2001; 20:92132; PMID:314027; http://dx.doi.org/10.1038/sj.onc.1204177 Furuno N, den Elzen N, Pines J. Human cyclin A is required for mitosis until mid prophase. J Cell Biol 1999; 147:295-306; PMID:10525536; http://dx.doi.org/10.1083/jcb.147.2.295 De Boer L, Oakes V, Beamish H, Giles N, Stevens F, SomodevillaTorres M, Desouza C, Gabrielli B. Cyclin A/cdk2 coordinates centrosomal and nuclear mitotic events. Oncogene 2008; 27:4261-8; PMID:18372919; http://dx.doi.org/10.1038/onc.2008.74 Fung TK, Ma HT, Poon RY. Specialized roles of the two mitotic cyclins in somatic cells: cyclin A as an activator of M phase-promoting factor. Mol Biol Cell 2007; 18:1861-73; PMID:17344473; http:// dx.doi.org/10.1091/mbc.E06-12-1092 Gong D, Pomerening JR, Myers JW, Gustavsson C, Jones JT, Hahn AT, Meyer T, Ferrell JE, Jr. Cyclin A2 regulates nuclear-envelope breakdown and the nuclear accumulation of cyclin B1. Curr Biol 2007; 17:85-91; PMID:17208191; http://dx.doi.org/10.1016/j. cub.2006.11.066 Li C, Andrake M, Dunbrack R, Enders GH. A bifunctional regulatory element in human somatic Wee1 mediates cyclin A/Cdk2 binding and Crm1-dependent nuclear export. Mol Cell Biol 2010; 30:116-30; PMID:19858290; http://dx.doi.org/10.1128/MCB.01876-08 Mitra J, Enders GH. Cyclin A/Cdk2 complexes regulate activation of Cdk1 and Cdc25 phosphatases in human cells. Oncogene 2004; 23:3361-7; PMID:14767478; http://dx.doi.org/10.1038/sj.onc.1207446 Oakes V, Wang W, Harrington B, Lee WJ, Beamish H, Chia KM, Pinder A, Goto H, Inagaki M, Pavey S, et al. Cyclin A/Cdk2 regulates Cdh1 and claspin during late S/G2 phase of the cell cycle. Cell Cycle 2014; 13:3302-11; PMID:25485510; http://dx.doi.org/10.4161/ 15384101.2014.949111 Wu CF, Liu S, Lee YC, Wang R, Sun S, Yin F, Bornmann WG, Yu-Lee LY, Gallick GE, Zhang W, et al. RSK promotes G2/M transition through activating phosphorylation of Cdc25A and Cdc25B. Oncogene 2013; PMID:23708659 van den Heuvel S, Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science 1993; 262:2050-4; PMID:8266103; http://dx.doi.org/10.1126/science.8266103 Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI, Weinberg RA. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell 1992; 70:993-1006; PMID:1388095; http://dx. doi.org/10.1016/0092-8674(92)90249-C Hagting A, Jackman M, Simpson K, Pines J. Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent
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nuclear import signal. Curr Biol 1999; 9:680-9; PMID:10395539; http://dx.doi.org/10.1016/S0960-9822(99)80308-X [39] Danke C, Grunz X, Wittmann J, Schmidt A, Agha-Mohammadi S, Kutsch O, Jack HM, Hillen W, Berens C. Adjusting transgene expression levels in lymphocytes with a set of inducible promoters. J Gene Med 2010; 12:501-15; PMID:20527043; http://dx.doi.org/10.1002/ jgm.1461 [40] Bertoli S, Boutzen H, David L, Larrue C, Vergez F, Fernandez-Vidal A, Yuan L, Hospital MA, Tamburini J, Demur C, et al. CDC25A
governs proliferation and differentiation of FLT3-ITD acute myeloid leukemia. Oncotarget 2015; 6:38061-78; PMID:26515730 [41] Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 1997; 106:348-60; PMID:9362543; http://dx.doi.org/ 10.1007/s004120050256
REPORT
Phosphorylation of CDC25A on SER283 in late S/G2 by CDK/cyclin complexes accelerates mitotic entry Laurent Mazzolinia,b, Ana€ıs Brobana, Carine Fromentc, Odile Burlet-Schiltzc, Arnaud Bessona,b, Stephane Manentia,b, and Christine Doziera,b a Centre de Recherche en Cancerologie de Toulouse, INSERM UMR1037, CNRS ERL5294, Universite Toulouse III Paul Sabatier, Toulouse, France; bEquipe labellisee LIGUE contre le Cancer, CNRS ERL5294, Toulouse, France; cInstitut de Pharmacologie et de Biologie Structurale, Universite Toulouse III Paul Sabatier Toulouse, CNRS UMR5089, Toulouse, France
ABSTRACT
ARTICLE HISTORY
The Cdc25A phosphatase is an essential activator of CDK-cyclin complexes at all steps of the eukaryotic cell cycle. The activity of Cdc25A is itself regulated in part by positive and negative feedback regulatory loops performed by its CDK-cyclin substrates that occur in G1 as well as during the G1/S and G2/M transitions. However, the regulation of Cdc25A during G2 phase progression before mitotic entry has not been intensively characterized. Here, we identify by mass spectrometry analysis a new phosphorylation event of Cdc25A on Serine283. Phospho-specific antibodies revealed that the phosphorylation of this residue appears in late S/G2 phase of an unperturbed cell cycle and is performed by CDK-cyclin complexes. Overexpression studies of wild-type and non-phosphorylatable mutant forms of Cdc25A indicated that Ser283 phosphorylation increases the G2/M-promoting activity of the phosphatase without impacting its stability or subcellular localization. Our results therefore identify a new positive regulatory loop between Cdc25A and its CDK-cyclin substrates which contributes to accelerate entry into mitosis through the regulation of Cdc25A activity in G2.
Received 11 March 2016 Revised 29 July 2016 Accepted 31 July 2016
Introduction The sequential activation and inactivation of cyclin-dependent kinases (CDKs) play a critical role during cell cycle progression.1 A crucial step in the activation of CDKcyclin complexes consists in the removal of inhibitory phosphorylations on the CDK by dual-specificity phosphatases of the Cdc25 family. In mammals, 3 Cdc25 isoforms have been identified: Cdc25A, Cdc25B and Cdc25C.2,3 Mouse knockout models have revealed that a certain degree of functional redundancy exists between these isoforms. Indeed, double knockout Cdc25B¡/¡- Cdc25C¡/¡ mice develop normally and cells from these mice display normal cell cycle profiles.4 Cdc25A therefore appears to fulfill the most important functions of the other Cdc25 isoforms. On the contrary, Cdc25A knockout is lethal at a very early stage during embryogenesis5 indicating that Cdc25A plays essential non redundant functions during cell division. Previous studies revealed that the regulation of Cdc25A activity in dividing cells involves different interconnected positive and negative feedback loops with its CDK-cyclin substrates and this reciprocal regulation contributes to control cell cycle transitions.6 At the end of G1, Cdc25A activates CDK2-Cyclin A/E complexes to drive entry into S phase.7 Moreover, CDK2-Cyclin E complexes directly
KEYWORDS
activating phosphorylation; Cdc25A; CDK-cyclin; cell cycle; G2/M transition
phosphorylate and activate Cdc25A in a positive feedback loop which further accelerates the G1/S transition.8 Cdc25A also contributes, together with Cdc25B, to the activation of CDK1-cyclin B at the G2/M transition,9,10 both phosphatases performing at least partially non-overlapping functions during this step.11 During the G2/M transition, phosphorylation of Cdc25A on Ser17, Ser115 and Ser320 by CDK1cyclin B complexes leads to a robust stabilization of the phosphatase12,13 again generating a positive activation loop amplifying mitosis promoting activity. Previous studies have shown that during G2, Cdc25A is activated earlier than Cdc25B14 and may be primarily responsible for the activation of CDK-cyclin pools until a point near the G2/M transition where Cdc25B synergizes with Cdc25A to complete CDK1-cyclin B activation, leading to mitotic entry. So far, the mechanisms that regulate Cdc25A function in G2 are still largely unclear. Inhibition and knockdown studies performed on CDK2 have indicated that CDK2 activity increases Cdc25A turnover in interphase cells15,16 and may contribute to avoid uncontrolled Cdc25A activation in S and G2 phases. Here we report the characterization of a phosphorylation event occurring on serine 283 of Cdc25A and mediated by CDK-cyclin complexes during the late S/G2 phase of an unperturbed cell cycle. We show that this event contributes to increase the intracellular activity of this phosphatase and to accelerate entry into mitosis.
Centre de Recherche en Cancérologie de Toulouse, CONTACT Christine Dozier [email protected]; Laurent Mazzolini [email protected] INSERM UMR1037, CNRS ERL5294, UPSToulouse III, 2, avenue Hubert Curien, Oncopole entrée C, 31037 Toulouse Cedex 1, France. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy. Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis
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Results Cdc25A is phosphorylated on serine 283 during G2 phase of the cell cycle To identify new phosphorylation sites that may contribute to the functional regulation of Cdc25A, a plasmid encoding human Cdc25A was transiently transfected in exponentially growing HEK293 cells. Mass spectrometry analyses of immunoprecipitated Cdc25A allowed the unambiguous identification of a Ser283 monophosphorylated peptide (Fig. 1A). Phosphorylation of Cdc25A on ser283 had been previously detected by mass spectrometry in U2OS cells conditionally overexpressing the phosphatase13 and more recently on recombinant Cdc25A phosphorylated in vitro by Cdk1/cyclin B complexes immunopurified from Hela cell mitotic extracts.17 However, the role of this phosphorylation is still unknown. Ser283 is conserved in mammals and birds Cdc25A orthologues (Fig. 1B). This residue is located within a previously described nuclear localization signal (NLS, consensus motif: KRX10-12KRRK) present in the 3 Cdc25 isoforms and important for nuclear relocalization of Cdc25A.18 A phosphorylation-specific antibody was raised against a phosphopeptide derived from the Ser283 phosphorylation site. As shown in Figure 1C this antibody specifically reacts with wild-type Cdc25A transiently transfected in HEK293 cells, but not with a non-phosphorylatable mutant (serine 283 mutated to alanine; S283A). This antibody also allows the specific detection of endogenous Cdc25A phosphorylated on Ser283 in whole protein extracts of asynchronous H1299 human lung carcinoma cells which were mostly used in our studies (Fig. 1D). To determine at which step Ser283 phosphorylation occurs during the cell cycle we used the H1299 cell line which can be efficiently synchronized at different phases of the cell cycle. First, H1299 ells were arrested in G0/G1 by serum starvation and lysates were collected at various time points after release from the block and probed with the anti-phospho-Ser283 antibody. In these conditions phosphorylation of Ser283 could be detected in total extracts 20 h after release when cells reached G2 phase as determined by flow cytometry analysis of DNA content (Fig. 2A) and as soon as 18h post release in purified Cdc25A fractions, obtained by immunoprecipitation, when cells were still mainly in S phase (Fig. 2A, B). To further define the Ser283 phosphorylation pattern during the cell cycle, H1299 cells were synchronized at the onset of S phase by double thymidine block. Immunoblot analysis of cells after release confirmed that Ser283 phosphorylation could be detected in cells which were accumulating in late S/G2 (4h post-release time point) (Fig. 2C). This was confirmed by additional synchronization experiments performed with H1299 cells expressing HA tagged-Cdc25A in which western blot analysis of immunopurified Cdc25A allowed the detection of phosphorylated serine 283 as soon as 4h after release from thymidine block when cells were highly predominantly in S phase (Fig. S1). Moreover quantifications of serine283 phosphorylation in these experiments revealed that this phosphorylation was not enhanced upon entry into mitosis when cell positive for phospho-Ser10 histone H3 began to
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massively accumulate. Altogether these studies led us to conclude that serine 283 phosphorylation must take place in late S and during the G2 phase of the cell cycle and is maximal before mitotic onset. Cdc25A phosphorylation on serine 283 is mediated by CDK-cyclin complexes Ser283 and its adjacent proline form a candidate motif for proline-directed kinases such as CDKs and mitogen-activated protein kinases.19 Treatment of H1299 and HEK293 cells with the CDK1/2 inhibitor roscovitine20 strongly decreased phosphorylation of endogenous Cdc25A on Ser283 (Fig. 3A). Treatment with roscovitine and with the more specific CDK1 inhibitor RO330621 also strongly reduced Ser283 phosphorylation of Cdc25A transiently overexpressed in HEK293 cells (Fig. 3B). Moreover, a strong increase of Ser283 phosphorylation was observed when plasmids encoding either CDK1 and cyclin A2/ B1 or CDK2 and cyclin A2 were cotransfected with the Cdc25A expression vector in H1299 cells (Fig. 4A). Finally we found that both CDK1-cyclin B1 and CDK2-cyclin A2 were able to efficiently phosphorylate Cdc25A in vitro on Ser283 and this reaction was fully inhibited in the presence of roscovitine or RO3306 (Fig. 4B). Altogether these results argue for a direct involvement of the 2 main CDK-cyclin complexes acting during G2 phase and at the G2/M transition in the phosphorylation of Cdc25A on Ser283. Phosphorylation on serine 283 does not modify Cdc25A intracellular localization Since Ser283 is localized in the NLS of Cdc25A we investigated whether its phosphorylation could affect its intracellular localization. For this, expression plasmids coding for the wild-type and S283A mutant forms of Cdc25A fused either with mRFP (Cdc25A wild type) or with GFP (Cdc25A wild type or S283A mutant) were cotransfected in U2OS cells. In this cell type Cdc25A is predominantly nuclear but can also have pancellular localization in a small fraction of the cells. As expected, the cotransfected control constructs (i.e. RFP-Cdc25A and GFP Cdc25A) showed identical localization, whether Cdc25A was localized in the nucleus or distributed in the whole cell, confirming that the presence of the RFP or GFP did not alter the localization of the fusion protein (Fig. S2). When Cdc25A wild type and Cdc25A-S283A were cotransfected, no differences could be observed between wild-type CDC25A and its nonphosphorylatable S283A mutant form. The distribution of transiently expressed wild-type and S283A mutant forms of cdc25A was also studied by immunofluorescence in Hela cells in which Cdc25A is highly predominantly nuclear. High content analysis of the transfected cells revealed only a mild increase of nuclear accumulation of the mutant Cdc25A relative to its wild-type counterpart (i.e., 92,1 % vs 87,0%, Fig. S3). Finally, no differences in intracellular localization could be evidenced in H1299 cells inducibly expressing HA-tagged wild type or mutant Cdc25A (see below) and synchronized in interphase or during mitosis (data not shown). Altogether these studies indicated that phosphorylation of Ser283 has no major impact on Cdc25A nucleocytoplasmic shuttling.
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Figure 1. Mass spectrometric identification of Cdc25A phosphorylation at serine 283. (A) The HCD MS/MS spectrum of the monophosphorylated peptide, 279-SQEEpSPPGSTKR-290 (doubly charged precursor ion, MH2+, at m/z 691.80157) displays series of y- and b-ions. Intense simply charged y7 (at m/z 742.4204) together with simply charged b2 (at m/z 216.0978) indicate that serine 283 is phosphorylated but not serine 279, serine 287 or threonine 288. (B) Multiple sequence alignment of the NLS region of various Cdc25A orthologues. Arrow: position of ser283 (human sequence). (C) HEK293 cells were transfected with a bicistronic plasmid expression vector encoding GFP (control for transfection efficiency), and either wild-type (WT) Cdc25A or the S283A mutant. Twenty-four h post-transfection, total protein extracts were immunoblotted with the indicated antibodies. p-S283: phospho-ser283 antibody. (D) Total H1299 extracts were subjected to protein gel blot analysis. Treatments with the protein synthesis inhibitor cycloheximide (Chx, 50 mg/ml for 1 h) or with a siRNA directed against Cdc25A, which both lead to a strong reduction of Cdc25A protein level were included as negative controls for specificity of the antibody reaction. Arrow: position of Cdc25A. Asterisk: nonspecific cross reacting polypeptide. Respective molecular weights of the proteins are indicated on the right of the immunoblots.
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Figure 2. Cdc25A is phosphorylated on serine 283 in late S and G2 phase of the cell cycle. H1299 cells were either synchronized in G0/G1 (A-B) or G1/S (C) by serum starvation and thymidine block, respectively. Following release, cells were collected and their nuclear DNA content analyzed by flow cytometry (A,C). In parallel, total protein extracts (A,C) or Cdc25A immunoprecipitates (B) obtained from the same fractions used in (A) were immunoblotted with the indicated antibodies. Cyclin A, cyclin E and phospho-ser10 histone H3 (pS10 HH3) were included as cell cycle and mitosis entry markers. Starv.: serum-deprived cells. Thy: cells blocked in G1/S. Arrow: position of Cdc25A. Asterisk: nonspecific cross reacting protein. Respective molecular weights of the proteins are indicated on the right of the immunoblots.
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Figure 3. Cdc25A serine 283 phosphorylation is reduced upon treatment of cells with cyclin-dependent kinases inhibitors. (A) Exponentially growing H1299 (upper panel) or HEK293 cells (lower panel) were treated for 1 h with either 50 mg/ml cycloheximide (Chx) or 50 mM roscovitine (Ros.). n.t.: untreated control cells. The corresponding cell extracts were analyzed by western blot with the indicated antibodies. Arrow: position of Cdc25A. Asterisks: nonspecific cross-reacting polypeptides. (B) HEK293 cells were transfected with a bicistronic plasmid vector encoding wild-type Cdc25A together with GFP, used as a control for transfection efficiency. Twenty-four h after transfection the cells were either left untreated (n.t.) or treated for 2 h with different CDK inhibitors: Ros. : roscovitine (50 mM), RO: RO3306 (10 mM), PD: PD0332991 (CDK 4/6 inhibitor,1 mM, used as a control). The corresponding protein samples were immunoblotted with the indicated antibodies.
Phosphorylation on serine 283 does not impact the stability of Cdc25A
Mutation of serine 283 reduces the G2/M promoting activity of Cdc25A
Previous studies have shown that phosphorylation of Cdc25A by CDK-cyclin complexes can modify its turnover during interphase and mitosis.6 Moreover Ser283 is in close proximity to 2 previously described CHK1/2 phosphorylation sites (i.e. Ser279 and Ser293) which together with additional phosphorylations are involved in proteasomaldependent degradation of Cdc25A in the S and G2 phase of the cell cycle.22 We therefore investigated the impact of Ser283 phosphorylation on Cdc25A stability. For this, we used tetracycline-inducible H1299 cells conditionally expressing HA-tagged wild type or S283A-mutated Cdc25A proteins. The tetracycline inducible cell populations consist in pools of several hundred of puromycin-resistant clones. No background expression of the HA-tagged proteins was detected in absence of the inducer doxycycline (Fig. S4). Although only a fraction of the cells were found to express the HA-tagged Cdc25A upon induction with doxycycline (14 % for the wild-type construct and 20% for the S283A mutant as determined by immunofluorescence analysis, data not shown) the use of pools formed by a large number of independent clones allows to minimize variations in cell responses that may originate from clonal heterogeneity. Stability studies on the HA-tagged Cdc25A proteins expressed in H1299 inducible cells performed either in exponentially growing cells (data not shown) or in cells synchronized in G2 (Fig. S5) indicated that the turnover rate of the Cdc25A S283A mutant was not significantly different from that of the wild type protein. Moreover, no difference in stability between the wild type and S283A mutant form of Cdc25A could be evidenced in transiently transfected HEK293 cells either exponentially growing (data not shown) or arrested in mitosis after treatment with nocodazole (Fig. S6). We therefore concluded that the phosphorylation of Ser283 does not significantly modify interphasic or mitotic Cdc25A stability during a normal cell cycle.
Since phosphorylation of Ser283 was found to take place in late S/G2 (Fig. 2 and Fig. S1), we asked whether it could affect Cdc25A activity in promoting entry into mitosis. For this, H1299 inducible cell pools were synchronized in G1/S by thymidine block and then released from the block in presence of doxycycline. Cell cycle profiles were determined by flow cytometry analysis of DNA content (Fig. S7). Cells entering mitosis were identified by immunofluorescence using anti-phosphoSer10 histone H3 antibodies (Fig. 5A). Phosphorylation of histone H3 on Ser10 takes place before nuclear envelope breakdown occurs and therefore allows detection of the very early step of prophase.23 The subpopulations of cells overexpressing wild type or S283A-mutated HA-Cdc25A after induction with doxycycline were identified by immunofluorescence using an anti-HA antibody (Fig. 5A, arrows). To determine whether overexpression of wild type or mutant HA-Cdc25A could affect entry into mitosis, we compared, in each pool and at a fixed time point after release from thymidine block, the percentage of phospho-Ser10 histone H3-positive cells in the cell subpopulation overexpressing HA-Cdc25A (HA-positive cells) with the one in the remaining non inducible cell subpopulation (HA-negative). In order to focus our analyses on the early steps of mitosis only phosphoSer10 histone H3 positive cells corresponding to cells in early to late prophase were counted. In three independent experiments, a strong increase (4 to 20-fold, depending on the synchronization experiment) in the percentage of phospho-Ser10 histone H3-positive cells was observed in cells overexpressing HA-Cdc25A relative to those only expressing endogenous Cdc25A (see Fig. 5B for a representative experiment). These results indicate that overexpression of Cdc25A accelerates entry into mitosis in H1299 cells, as previously described in other model cell lines. This effect was observed in both cell pools, however the relative enrichment in cells entering mitosis was significantly lower in cells expressing the S283A mutant when compared to the pool expressing wild
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This indicated that the reduction in the percent of cells entering mitosis could not be attributed to lower induction levels of the mutant. The low differences in mitosis promoting activity between the wild type and S283A mutant in our experiments may in part originate from the fact that both proteins are expressed in significant excess (5 to 18-fold ; data not shown) with respect to endogenous Cdc25A in the induced cells. Additional microscopy analyses suggested that the overexpression of Cdc25A or the phosphorylation mutant does not modify mitotic progression as cells in all phases of mitosis could be observed (data not shown). Moreover, flow cytometry analysis of exponentially growing cells showed that the duration of mitosis was identical in exponentially growing cells overexpressing the wild type or the S283A mutant protein (Fig. S8). Altogether, these results led us to conclude that the phosphorylation of Ser283 does not play an essential role during progression through mitosis but rather contributes to increase the G2/M promoting activity of Cdc25A in interphase.
Discussion
Figure 4. CDK-cyclin complexes are involved in the phosphorylation of serine 283 of Cdc25A. (A) H1299 cells were transfected with the Cdc25A bicistronic plasmid used in Fig. 3 B either alone (-) or in combination with different plasmids encoding CDK1, CDK2, cyclin A2 (CycA) and cyclin B1 (CycB). Twenty-four h after transfection total protein extracts were analyzed by protein gel blot. The anti-HA antibody allows the detection of the transfected CDKs which are N-terminally tagged with the HA epitope. (B) Bacterially expressed recombinant wild type Cdc25A was incubated for 1 h with either recombinant CDK1-Cyclin B1 complexes in the presence or absence of 10 mM RO3306 or with recombinant CDK2-Cyclin A2 complexes in the presence or absence of 50 mM roscovitine. The corresponding protein samples were immunoblotted with the indicated antibodies. Cdk: anti-CDK1 (left panel) or anti-CDK2 (right panel) antibodies. Cyclin: anti-Cyclin B1 (left panel) or anti-Cyclin A2 (right panel) antibodies.
type Cdc25A (mean reduction: 20%, Fig. 5C). This results was confirmed by additional flow cytometry analyses which allowed to simultaneously analyze HA- and phospho-Ser10 histone H3-labelings as well as cell cycle profiles in the synchronized cell pools (Fig. 5D) and therefore to accurately follow the appearance of mitotic (phospho-HH3 positive) cells at different time points after release for the thymidine block. These studies revealed that, whereas the kinetics of entry into mitosis appeared highly reproducible between the 2 cell pools in the non-induced cell population (Fig. 5E), again a small delay in the entry into mitosis (which becomes highly statistically significant from 7h post-release) was observed in the cell subpopulation overexpressing the Ser283A mutant when compared to the cells overexpressing wild type Cdc25A (Fig. 5F). Quantitative analyses of Cdc25A expression showed that the induction levels of wild type and mutant Cdc25A were very similar (Table S1).
Positive regulatory loops between the Cdc25A phosphatase and its CDK-cyclin substrates have previously been observed both at the G1/S and G2/M transitions. During G1/S transition phosphorylation of Cdc25A by CDK2-cyclin E increases its enzymatic activity8 whereas during entry into mitosis phosphorylation by the CDK1-cyclin B complexes strongly stabilizes the protein.13 Many studies on the regulation of Cdc25A focused on the G2/M transition but molecular mechanisms possibly regulating Cdc25A activity during progression from S phase until mitosis i.e., G2 phase remain largely unknown. Previous studies showed that inhibition of CDK2 activity lead to an increase in Cdc25A protein levels suggesting that CDK2-containing complexes are also involved in the destabilization of Cdc25A in interphase cells although most probably indirectly through ATR-CHK1 signaling.15,16 This may contribute to counteract Cdc25A enzymatic activation occurring at the G1/S transition in order to prevent excessive accumulation of active Cdc25A in both S and G2, which could lead to premature mitotic entry during an unperturbed cell cycle and/or ineffective cell cycle arrest following DNA replicative stress. This last point is strongly suggested by the fact that the stabilization of Cdc25A in CDK2-deficient cells contributes to G2/M checkpoint defects following DNA damage.15 The CHK1 kinase which maintains a basal level of activity during and unperturbed cell cycle was also reported to perform an additional regulation of Cdc25A stability22,24 These data indicate that the expression and activity of Cdc25A6 must be tightly controlled in the interphase cell in order to avoid premature cell cycle transitions. Herein, we characterized a novel positive feedback loop that involves the phosphorylation of serine 283 of Cdc25A performed by CDK-cyclin complexes in late S/G2 and found that this phosphorylation event increases the activity of Cdc25A in promoting mitotic entry. Although the mechanism leading to a raise in Cdc25A activity remains unknown we showed that it does not involve a stabilization of the phosphatase or a modification of its subcellular localization. This phosphorylation
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Figure 5. Phosphorylation on serine 283 contributes to the activation of Cdc25A in G2. (A) H1299 inducible cells expressing either wild type or mutant HA-tagged Cdc25A were synchronized at the G1/S transition and released in the presence of doxycycline. At 7.5 h post-release, cells were fixed on coverslips and stained with anti-phosphoser10 H3 antibody (Phospho-HH3 ; green) and anti-HA antibody (red). DNA was stained with DAPI (blue). Scale bar: 20 mm. Arrows indicate HA-positive cells. Representative images are shown. (B) At 7.5 h post-release the percentages of mitotic cells corresponding to cells ranging from very early prophase (complete phospho-ser10 H3-positive labeling of the nucleus but no apparent chromatin condensation in DAPI staining41) to late prophase (strong phospho-ser10 H3-labeling and full condensation of chromatin but no reorganization of chromosomes indicative of entry into prometaphase) were determined in each subpopulation. Values obtained with the HA-negative subpopulations were then set to 1 for each pool. A representative experiment is shown. At least 3,000 cells were analyzed in each pool. (C) For each synchronization experiment, the enrichment factor in early mitotic cells associated with the overexpression of wild type Cdc25A (as determined in B) was set to 100 and the enrichment factor obtained with the S283A mutant recalculated accordingly. Graphs represent mean values of 3 independent experiments. Bar represents s.e.m.; p D 0.0225 according to Student’s unpaired t test. (D-F): H1299 inducible cells were synchronized and induced with doxycycline as described above and collected at different time points after release for the thymidine block for flow cytometry analysis. (D) Multiparametric flow cytometry analysis. Cell cycle progression, expression of HA-Cdc25A and mitotic status were simultaneously analyzed by labeling of DNA with Hoechst 33342 (left panel) and labeling with antibodies directed against HA and anti-phospho-ser10 H3 histone, respectively (right panel). A representative experiment is shown (cell pool expressing wild type HA-Cdc25A; 7h post-release). (E) Kinetics of entry into mitosis of the cells in the induced pools following release from the block. Cells were analyzed by flow cytometry as described in (D) A representative synchronization experiment is shown. (F) For each synchronization experiment and at each time point after release, the enrichment factor in mitotic (phospho-HH3 positive) cells in the cell subpopulation overexpressing wild type HA-Cdc25A (phospho-HH3 positive) relative to the non-induced cell subpopulation (phospho-HH3 negative) was set to 100 and the enrichment factor obtained under the same conditions with the S283A mutant recalculated accordingly. Graphs represent mean values of 4 independent experiments. Bar represents s.e.m.; p < 0 .01 according to Student’s unpaired t test.
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could directly affect the phosphatase activity of Cdc25A or its interaction with the CDK-cyclin substrates. No activating phosphorylation of Cdc25A by CDKs has previously been described during G2 phase. Moreover our biochemical studies show that this phosphorylation may be directly performed by CDK1/2 -cyclin complexes themselves. This result suggests that CDKs active during G2 have an overall positive impact on Cdc25A activity despite some members such as CDK2 also contribute to Cdc25A destabilization. The simultaneous increase of Cdc25A activity and of its turnover rate may constitute a mean to increase the responsiveness of this phosphatase to the regulatory pathways controlling G2/M entry in an unperturbed cell cycle as well as in response to various cell stresses such as DNA damage. This coupling between protein activation and protein instability has been frequently observed for proteins whose function need to be quickly and efficiently regulated such as receptor kinases or cell signaling intermediates.25 The Cdc25A and Cdc25B phosphatases both independently contribute to the assembly and activation of CDK1-cyclin B complexes in G29,10 and Cdc25A overexpression accelerates entry into mitosis9,13,14 and can prevent cell cycle arrest following checkpoint activation thus leading to premature mitotic entry.26 Conversely, Cdc25A targeting by RNAi delays G2/M transition.11,13 Studies of Lindqvist et al.11 have shown that Cdc25A and Cdc25B may cooperate to induce mitotic entry by activating specific nuclear and centrosomal CDK-cyclin pools during this step. Interestingly, data of Timofeev et al.14 have revealed that Cdc25A is activated earlier than Cdc25B in G2. Moreover, these authors showed that Cdc25A is specifically involved in the activation of CDK2-Cyclin A complexes. These complexes are assembled and activated during S phase but show biphasic activity with a second major boost of activation occurring in early G2.27 CDK2-cyclin A complexes contribute to mitotic both directly28 and through the activation of CDK1-cyclinB complexes29-34 and their activity appears rate-limiting for G2/M transition. Based on our results, we propose that an initial phosphorylation of Cdc25A on serine 283 by CDK2-Cyclin A complexes could be involved in the establishment of a positive activatory loop between Cdc25A and CDK-cyclin complexes acting during the G2 phase. Control of Cdc25A activity appears essential to ensure the correct timing of entry into mitosis and phosphatases and kinase activities distinct from CDKs have been found to regulate mitotic entry through the regulation of Cdc25A. Indeed, Vazquez-Novelle et al.10 previously showed that the Cdc14A phosphatase may also control the timing of G2/M transition by inhibiting the catalytic activity of Cdc25A. Since Cdc14A was also shown to inhibit CDK1-cyclinB1 activity at the G2/M transition, it was tempting to speculate that inhibition of Cdc25A activity by Cdc14A overexpression involved dephosphorylation of Ser283. However, the mode of action of Cdc14A on Cdc25A appears to be an indirect effect exerted by a still unknown protein.10 In agreement of this, Tumurbaatar et al. showed that phospho-Ser283 was not dephosphorylated by Cdc14A in vitro.17 The ribosomal S6 kinase has also been recently reported to phosphorylate Cdc25A on serine 293 and 295 and increase its M phase-inducing activity but the mechanism of action of these phosphorylations on Cdc25A function was not investigated in this work.35
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Our studies further emphasize the essential role of Cdc25A in the regulation of G2/M transition. The numerous regulatory loops established between Cdc25A and its CDK substrates at all the phases of the cell cycle most probably represent a first essential layer in the regulation of progression through the cell division cycle.
Materials and methods Cell culture, generation of inducible cell pools and cell transfections Human non-small-cell lung carcinoma H1299 cells (ATCC validated) and human embryonic kidney HEK293 cells (obtained from DSMZ) were grown respectively in RPMI 1640 and DMEM medium, supplemented with 10% calf serum and 1X penicillin-streptomycin (Gibco, LifeTechnologies, Carlsbad, CA, USA). Tetracycline inducible H1299 cell pools were generated by a one-step transformation process with the “all-in-one” TET-Cdc25A plasmid vectors. Plasmids were transfected with the Amaxa nucleofection system (Lonza, Koeln, Germany). Puromycin-resistant pools were selected in culture medium supplemented with 1 mg/ml puromycin (InVivogen, San Diego, CA, USA). H1299 cells were transfected with siRNAs using INTERFERin reagent (Polyplus, Illkirch, France). Transfections of plasmid vectors were performed with Jet PEI (Polyplus). NanoLC–MS/MS analysis and database searches After SDS-PAGE separation of the immunoprecipitated FLAGtagged Cdc25A, the Coomassie blue-stained corresponding band was in-gel digested with trypsin and analyzed by online nanoLC using an Ultimate 3000 System (Dionex, Amsterdam, The Netherlands) coupled to an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) for data-dependent HCD fragmentation experiments. Antibodies Anti-Cdc25A (mouse F6, dil.1/500), anti-CDK1 (mouse P34, dil.1/1,000), anti-CDK2 (rabbit M2, dil.1/1,000), anti-cyclin B1 (mouse GNS1, dil.1/1,000) and anti-Cyclin A2 (rabbit C-19, dil.1/1,000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-actin (mouse C4 , dil.1/5,000) was from Chemicon (Temecula, CA, USA). Anti-GFP (rabbit D5.1, dil.1/500) and anti-HA (rabbit C29FA, dil.1/2,000) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-phospho-ser283 Cdc25A rabbit antibody was raised against peptide RSQEES(PO3H2) PPGSTKC (Eurogentec SA, Belgium). Plasmids The bicistronic Cdc25A expression plasmids were constructed by inserting the coding sequence of human Cdc25A in the pIRES2-EGFP vector (Clontech, Mountain View, CA). The S283A mutation was introduced by site-directed mutagenesis using inverted PCR. Vectors encoding human HA-tagged CDK1 and CDK2 were a gift from Sander van den Heuvel36
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(Addgene plasmids # 1888 and 1884). Expression vectors for human cyclin A2 and Cyclin B1 were respective gifts from Robert Weinberg37 (Addgene plasmid # 8959) and Jonathon Pines38 (Addgene plasmid # 26060). The TET-Cdc25A vectors were constructed by first inserting the coding sequence of human Cdc25A downstream of the Tet promoter in the pTREtight plasmid (Clontech). This TET cassette was then introduced in the pZDonor-AAVS1 plasmid (Sigma-Aldrich, St Louis, MO, USA) together with the tricistronic expression cassette of vector pWHE64439 which allows the constitutive expression of Tet activator, Tet repressor and puromycin resistance genes. siRNAs and inhibitors Cdc25A siRNA (Hs_CDC25A_9) was obtained from Qiagen (Hilden, Germany). Control siRNA (ON-TARGET plus Nontargeting Control Pool) was from Dharmacon (Chicago, IL, USA). Roscovitine, RO3306 and PD0332991 were obtained from Selleck Chemicals (Houston, TX, USA). Cell synchronizations and flow cytometry analyses H1299 cells were synchronized in G0/G1 by serum deprivation for 48 h in RPMI medium containing 0.1% BSA and then released in 10% serum-supplemented medium. For synchronization in G1/S, 5 mM thymidine was added to the medium 7 h after release from the G0/G1 block and the cells incubated for an additional 16 h time period. Cells were released for the block by extensive washing with PBS and addition of fresh RPMI medium. Flow cytometry analyses of cell cycle distribution were performed on propidium-iodide-stained cells. Flow cytometry analysis of cell cycle profiles, HA-labeling patterns and phospho-Ser10 histone H3 status of the synchronized cells were performed by simultaneous labeling of ethanol fixed cells with anti HA antibodies, anti phospho-Ser10 histone H3 antibodies and Hoechst 33342 dye. Flow cytometry measurements were performed using MACSQuant VYB and MQ10 flow cytometers (Miltenyi Biotech, Bergisch Gladbach, Germany) Immunoprecipitations, total protein extraction, protein gel blotting and quantifications Cell lysates for immunoprecipitations were prepared in cold lysis buffer (20 mM, Tris pH 7.4, 100 mM NaCl, 5 mM EGTA, 0.5% NP-40) supplemented with phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich) and CompleteTM protease inhibitor cocktail (Roche Diagnostics, Basel, Stwitzerland). Prior to immunoprecipitation extracts were clarified by centrifugation at 16,000 g for 10 min at 4 C). For mass spectrometry analyses, FLAG-tagged Cdc25A was purified from transfected HEK293 cells with anti-FLAG (mouse M2, Sigma-Aldrich) antibodies and G protein-coupled sepharose (EZviewTM Red Protein G, Sigma Aldrich). HA-tagged Cdc25A was purified with an antiHA agarose affinity gel (EZ view Red, Sigma-Aldrich). Total protein extractions and western blotting were done as previously described.40 Image acquisition of immunoblots and quantifications were performed with the PxiTM imager and GeneTools software from Syngene (Cambridge, UK).
Fixed cells staining and image acquisition Cells were fixed in 4% paraformaldehyde in PBS and processed for immunofluorescence following conventional protocols. Images were taken with a Plan-Apochromat 40x/1.4 Oil DIC objective by using an Axio observer.Z1 (Carl Zeiss, Iena, Germany) microscope. In vitro kinase reactions Recombinant human Cdc25A (200 ng, Jena Bioscience, Jena, Germany) and Cdk1-cyclin B1 or Cdk2-cyclin A2 proteins (20ng, Sigma-Aldrich) were incubated for 1 h at 30 C in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0,1mM EDTA, 0,01% Brij35, 2 mM DTT and 200 mM ATP).
Abbreviations CDK Cdc ATR CHK1
cyclin-dependent kinases cell-dependent cycle ataxia telangiectasia and rad3-related checkpoint kinase 1
Disclosure of potential conflicts of interests No potential conflicts of interest were disclosed.
Acknowledgments We are very grateful to Drs Christian Berens and Christina Danke for providing the pWHE644 tricistronic transregulator expression plasmid. We thank Sander van den Heuvel, Jonathon Pines and Robert Weinberg for the CDK and Cyclin expression plasmids. We are grateful to Manon Farce and Laetitia Ligat for assistance with flow cytometry and microscopy, respectively. We thank Charlotte Cavanihac and Julie Poulain for technical assistance.
Funding This work was supported by the Institut National de la Sante et de la Recherche Medicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), the University Paul Sabatier Toulouse III, the Ligue Nationale Contre le Cancer, the Fondation ARC pour la Recherche sur le Cancer and the Laboratoire d’Excellence Toulouse Cancer LABEX TOUCAN. It was also supported in part by the Region Midi-Pyrenees, Toulouse Metropole, and European funds FEDER (Fonds Europeens de Developpement Regional) for mass spectrometry.
References [1] Malumbres M, Barbacid M. Mammalian cyclin-dependent kinases. Trends Biochem Sci 2005; 30:630-41; PMID:16236519; http://dx.doi. org/10.1016/j.tibs.2005.09.005 [2] Boutros R, Lobjois V, Ducommun B. CDC25 phosphatases in cancer cells: key players? Good targets? Nat Rev Cancer 2007; 7:495-507; PMID:17568790; http://dx.doi.org/10.1038/nrc2169 [3] Kiyokawa H, Ray D. In vivo roles of CDC25 phosphatases: biological insight into the anti-cancer therapeutic targets. Anticancer Agents Med Chem 2008; 8:832-6; PMID:19075565; http://dx.doi.org/ 10.2174/187152008786847693 [4] Ferguson AM, White LS, Donovan PJ, Piwnica-Worms H. Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and Cdc25C protein phosphatases. Mol Cell Biol
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[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
2005; 25:2853-60; PMID:15767688; http://dx.doi.org/10.1128/ MCB.25.7.2853-2860.2005 Ray D, Terao Y, Nimbalkar D, Hirai H, Osmundson EC, Zou X, Franks R, Christov K, Kiyokawa H. Hemizygous disruption of Cdc25A inhibits cellular transformation and mammary tumorigenesis in mice. Cancer Res 2007; 67:6605-11; PMID:17638870; http://dx. doi.org/10.1158/0008-5472.CAN-06-4815 Frazer C, Young PG. Phosphorylation Mediated Regulation of Cdc25 Activity, Localization and Stability. In: Cai H, ed. Protein Phosphorylation in Human Health: InTech, 2012; 395-436 Blomberg I, Hoffmann I. Ectopic expression of Cdc25A accelerates the G(1)/S transition and leads to premature activation of cyclin Eand cyclin A-dependent kinases. Mol Cell Biol 1999; 19:6183-94; PMID:10454565; http://dx.doi.org/10.1128/MCB.19.9.6183 Hoffmann I, Draetta G, Karsenti E. Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition. EMBO J 1994; 13:430210; PMID:7523110 Timofeev O, Cizmecioglu O, Settele F, Kempf T, Hoffmann I. Cdc25 phosphatases are required for timely assembly of CDK1-cyclin B at the G2/M transition. J Biol Chem 2010; 285:16978-90; PMID:20360007; http://dx.doi.org/10.1074/jbc.M109.096552 Vazquez-Novelle MD, Mailand N, Ovejero S, Bueno A, Sacristan MP. Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B. J Biol Chem 2010; 285:40544-53; PMID:20956543; http://dx.doi.org/10.1074/jbc.M110.133009 Lindqvist A, K€alstr€ om H, Lundgren A, Barsoum E, Rosenthal CK. Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome. J Cell Biol 2005; 171:35-45; PMID:16216921; http://dx.doi.org/ 10.1083/jcb.200503066 Esteban V, Vazquez-Novelle MD, Calvo E, Bueno A, Sacristan MP. Human Cdc14A reverses CDK1 phosphorylation of Cdc25A on serines 115 and 320. Cell Cycle 2006; 5:2894-8; PMID:17172867; http:// dx.doi.org/10.4161/cc.5.24.3566 Mailand N, Podtelejnikov AV, Groth A, Mann M, Bartek J, Lukas J. Regulation of G(2)/M events by Cdc25A through phosphorylationdependent modulation of its stability. EMBO J 2002; 21:5911-20; PMID:12411508; http://dx.doi.org/10.1093/emboj/cdf567 Timofeev O, Cizmecioglu O, Hu E, Orlik T, Hoffmann I. Human Cdc25A phosphatase has a non-redundant function in G2 phase by activating Cyclin A-dependent kinases. FEBS Lett 2009; 583:841-7; PMID:19192479; http://dx.doi.org/10.1016/j.febslet.2009.01.044 Chung JH, Bunz F. Cdk2 is required for p53-independent G2/M checkpoint control. PLoS Genet 2010; 6:e1000863; PMID:20195506; http://dx.doi.org/10.1371/journal.pgen.1000863 Ducruet AP, Lazo JS. Regulation of Cdc25A half-life in interphase by cyclin-dependent kinase 2 activity. J Biol Chem 2003; 278:31838-42; PMID:12801928; http://dx.doi.org/10.1074/jbc.M303604200 Tumurbaatar I, Cizmecioglu O, Hoffmann I, Grummt I, Voit R. Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity. PloS One 2011; 6:e14711; PMID:21379580; http://dx.doi.org/10.1371/journal. pone.0014711 K€alstr€om H, Lindqvist A, Pospisil V, Lundgren A, Rosenthal CK. Cdc25A localisation and shuttling: characterisation of sequences mediating nuclear export and import. Exp Cell Res 2005; 303:89-100; PMID:15572030; http://dx.doi.org/10.1016/j.yexcr.2004.09.012 Ubersax JA, Ferrell JE, Jr. Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 2007; 8:530-41; PMID:17585314; http://dx.doi.org/10.1038/nrm2203 Meijer L, Borgne A, Mulner O, Chong JP, Blow JJ, Inagaki N, Inagaki M, Delcros JG, Moulinoux JP. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem 1997; 243:527-36; PMID:9030781; http://dx.doi.org/10.1111/j.1432-1033.1997.t01-200527.x Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H, Heimbrook DC, Chen L. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
2751
Acad Sci U S A 2006; 103:10660-5; PMID:16818887; http://dx. doi.org/10.1073/pnas.0600447103 Sørensen CS, Syljuasen RG, Falck J, Schroeder T, Ronnstrand L, Khanna KK, Zhou BB, Bartek J, Lukas J. Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell 2003; 3:247-58; PMID:12676583; http://dx.doi.org/10.1016/S1535-6108 (03)00048-5 Sawicka A, Seiser C. Histone H3 phosphorylation - a versatile chromatin modification for different occasions. Biochimie 2012; 94:2193201; PMID:22564826; http://dx.doi.org/10.1016/j.biochi.2012.04.018 Goloudina A, Yamaguchi H, Chervyakova DB, Appella E, Fornace AJ, Jr., Bulavin DV. Regulation of human Cdc25A stability by Serine 75 phosphorylation is not sufficient to activate a S phase checkpoint. Cell Cycle 2003; 2:473-8; PMID:12963847; http://dx.doi.org/10.4161/ cc.2.5.482 Vazquez F, Ramaswamy S, Nakamura N, Sellers WR. Phosphorylation of the PTEN tail regulates protein stability and function. Mol Cell Biol 2000; 20:5010-8; PMID:10866658; http://dx.doi.org/ 10.1128/MCB.20.14.5010-5018.2000 Molinari M, Mercurio C, Dominguez J, Goubin F, Draetta GF. Human Cdc25 A inactivation in response to S phase inhibition and its role in preventing premature mitosis. EMBO Rep 2000; 1:71-9; PMID:11256629; http://dx.doi.org/10.1093/embo-reports/ kvd018 Goldstone S, Pavey S, Forrest A, Sinnamon J, Gabrielli B. Cdc25dependent activation of cyclin A/cdk2 is blocked in G2 phase arrested cells independently of ATM/ATR. Oncogene 2001; 20:92132; PMID:314027; http://dx.doi.org/10.1038/sj.onc.1204177 Furuno N, den Elzen N, Pines J. Human cyclin A is required for mitosis until mid prophase. J Cell Biol 1999; 147:295-306; PMID:10525536; http://dx.doi.org/10.1083/jcb.147.2.295 De Boer L, Oakes V, Beamish H, Giles N, Stevens F, SomodevillaTorres M, Desouza C, Gabrielli B. Cyclin A/cdk2 coordinates centrosomal and nuclear mitotic events. Oncogene 2008; 27:4261-8; PMID:18372919; http://dx.doi.org/10.1038/onc.2008.74 Fung TK, Ma HT, Poon RY. Specialized roles of the two mitotic cyclins in somatic cells: cyclin A as an activator of M phase-promoting factor. Mol Biol Cell 2007; 18:1861-73; PMID:17344473; http:// dx.doi.org/10.1091/mbc.E06-12-1092 Gong D, Pomerening JR, Myers JW, Gustavsson C, Jones JT, Hahn AT, Meyer T, Ferrell JE, Jr. Cyclin A2 regulates nuclear-envelope breakdown and the nuclear accumulation of cyclin B1. Curr Biol 2007; 17:85-91; PMID:17208191; http://dx.doi.org/10.1016/j. cub.2006.11.066 Li C, Andrake M, Dunbrack R, Enders GH. A bifunctional regulatory element in human somatic Wee1 mediates cyclin A/Cdk2 binding and Crm1-dependent nuclear export. Mol Cell Biol 2010; 30:116-30; PMID:19858290; http://dx.doi.org/10.1128/MCB.01876-08 Mitra J, Enders GH. Cyclin A/Cdk2 complexes regulate activation of Cdk1 and Cdc25 phosphatases in human cells. Oncogene 2004; 23:3361-7; PMID:14767478; http://dx.doi.org/10.1038/sj.onc.1207446 Oakes V, Wang W, Harrington B, Lee WJ, Beamish H, Chia KM, Pinder A, Goto H, Inagaki M, Pavey S, et al. Cyclin A/Cdk2 regulates Cdh1 and claspin during late S/G2 phase of the cell cycle. Cell Cycle 2014; 13:3302-11; PMID:25485510; http://dx.doi.org/10.4161/ 15384101.2014.949111 Wu CF, Liu S, Lee YC, Wang R, Sun S, Yin F, Bornmann WG, Yu-Lee LY, Gallick GE, Zhang W, et al. RSK promotes G2/M transition through activating phosphorylation of Cdc25A and Cdc25B. Oncogene 2013; PMID:23708659 van den Heuvel S, Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science 1993; 262:2050-4; PMID:8266103; http://dx.doi.org/10.1126/science.8266103 Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI, Weinberg RA. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell 1992; 70:993-1006; PMID:1388095; http://dx. doi.org/10.1016/0092-8674(92)90249-C Hagting A, Jackman M, Simpson K, Pines J. Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent
2752
L. MAZZOLINI ET AL.
nuclear import signal. Curr Biol 1999; 9:680-9; PMID:10395539; http://dx.doi.org/10.1016/S0960-9822(99)80308-X [39] Danke C, Grunz X, Wittmann J, Schmidt A, Agha-Mohammadi S, Kutsch O, Jack HM, Hillen W, Berens C. Adjusting transgene expression levels in lymphocytes with a set of inducible promoters. J Gene Med 2010; 12:501-15; PMID:20527043; http://dx.doi.org/10.1002/ jgm.1461 [40] Bertoli S, Boutzen H, David L, Larrue C, Vergez F, Fernandez-Vidal A, Yuan L, Hospital MA, Tamburini J, Demur C, et al. CDC25A
governs proliferation and differentiation of FLT3-ITD acute myeloid leukemia. Oncotarget 2015; 6:38061-78; PMID:26515730 [41] Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 1997; 106:348-60; PMID:9362543; http://dx.doi.org/ 10.1007/s004120050256
