Regulation of G2/M events by Cdc25A through phosphorylation-dependent modulation of its stability
2002; Springer Nature; Volume: 21; Issue: 21 Linguagem: Inglês
10.1093/emboj/cdf567
ISSN1460-2075
Autores Tópico(s)Cancer-related Molecular Pathways
ResumoArticle1 November 2002free access Regulation of G2/M events by Cdc25A through phosphorylation-dependent modulation of its stability Niels Mailand Niels Mailand Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Alexandre V. Podtelejnikov Alexandre V. Podtelejnikov Protein Interaction Laboratory, Odense University, Campusvej 55, DK-5230 Odense M, Denmark Search for more papers by this author Anja Groth Anja Groth Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Matthias Mann Matthias Mann Protein Interaction Laboratory, Odense University, Campusvej 55, DK-5230 Odense M, Denmark Search for more papers by this author Jiri Bartek Corresponding Author Jiri Bartek Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Jiri Lukas Jiri Lukas Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Niels Mailand Niels Mailand Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Alexandre V. Podtelejnikov Alexandre V. Podtelejnikov Protein Interaction Laboratory, Odense University, Campusvej 55, DK-5230 Odense M, Denmark Search for more papers by this author Anja Groth Anja Groth Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Matthias Mann Matthias Mann Protein Interaction Laboratory, Odense University, Campusvej 55, DK-5230 Odense M, Denmark Search for more papers by this author Jiri Bartek Corresponding Author Jiri Bartek Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Jiri Lukas Jiri Lukas Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Author Information Niels Mailand1, Alexandre V. Podtelejnikov2, Anja Groth1, Matthias Mann2, Jiri Bartek 1 and Jiri Lukas1 1Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark 2Protein Interaction Laboratory, Odense University, Campusvej 55, DK-5230 Odense M, Denmark *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:5911-5920https://doi.org/10.1093/emboj/cdf567 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info DNA replication in higher eukaryotes requires activation of a Cdk2 kinase by Cdc25A, a labile phosphatase subject to further destabilization upon genotoxic stress. We describe a distinct, markedly stable form of Cdc25A, which plays a previously unrecognized role in mitosis. Mitotic stabilization of Cdc25A reflects its phosphorylation on Ser17 and Ser115 by cyclin B–Cdk1, modifications required to uncouple Cdc25A from its ubiquitin–proteasome-mediated turnover. Cdc25A binds and activates cyclin B–Cdk1, accelerates cell division when overexpressed, and its downregulation by RNA interference (RNAi) delays mitotic entry. DNA damage-induced G2 arrest, in contrast, is accompanied by proteasome-dependent destruction of Cdc25A, and ectopic Cdc25A abrogates the G2 checkpoint. Thus, phosphorylation-mediated switches among three differentially stable forms ensure distinct thresholds, and thereby distinct roles for Cdc25A in multiple cell cycle transitions and checkpoints. Introduction The molecular mechanisms underlying cell multiplication and maintenance of genomic integrity require precise timing, velocity and spatial distribution of phosphorylation events carried out by cyclin-dependent kinases (CDKs) (Nigg, 1995; Nurse et al., 1998; Zhou and Elledge, 2000). Owing to their central role in such fundamental biological processes, CDKs are subject to complex regulation in response to signals from the extracellular environment or cell cycle checkpoints (Morgan, 1995). One important mode of CDK control reflects phosphorylation of the conserved N-terminal ATP-binding region (Lew and Cornbluth, 1996). Phosphorylations of Tyr15 by the Wee1 kinase, and Thr14 by Myt1, hinder phosphate transfer to CDK-bound substrates and ATP binding to CDKs, respectively (Atherton-Fessler et al., 1993; Booher et al., 1997). Conversely, activation of CDKs requires dephosphorylation of Thr14 and Tyr15 by Cdc25 phosphatases (Nilsson and Hoffmann, 2000). The balance between the inhibiting and activating effects on CDKs, mediated by Wee1/Myt1 and Cdc25, respectively, determines key cellular decisions such as cell cycle arrest in response to DNA damage (Zhou and Elledge, 2000; Kastan, 2001). In mammals, the Cdc25 family includes three homologs, Cdc25A, B and C (Sadhu et al., 1990; Galaktionov and Beach, 1991; Nagata et al., 1991). Cdc25B and C are regarded as mitotic regulators (Nilsson and Hoffmann, 2000) and serve as targets of checkpoint pathways that delay entry into mitosis in response to DNA damage or stalled replication (Peng et al., 1997; Kumagai et al., 1998; Bulavin et al., 2001). Cdc25B probably represents a ‘starter’ phosphatase that triggers the initial activation of mitosis-promoting CDKs including cyclin B–Cdk1, which in turn activate Cdc25C and create a positive amplification loop required for irreversible commitment to mitosis (Nilsson and Hoffmann, 2000). Cdc25A appears to regulate earlier cell cycle transitions. Both c-Myc and E2F transcription factors stimulate Cdc25A expression in mid to late G1 (Galaktionov et al., 1996; Vigo et al., 1999) and, through activation of cyclin E(A)–Cdk2 complexes (Hoffmann et al., 1994; Blomberg and Hoffmann, 1999), Cdc25A induces S-phase entry. Microinjection of Cdc25A antibodies or transfection of Cdc25A antisense oligonucleotides arrests cells in G1 (Hoffmann et al., 1994; Jinno et al., 1994; Vigo et al., 1999; Cangi et al., 2000). Conversely, overexpressed Cdc25A shortens G1 and prematurely activates Cdk2 (Blomberg and Hoffmann, 1999; Sexl et al., 1999). The role of Cdc25A in initiation of DNA replication is also consistent with the ubiquitin–proteasome-mediated destruction of Cdc25A in G1- and intra-S-phase checkpoint responses to DNA damage and replicational stress (Mailand et al., 2000; Molinari et al., 2000; Falck et al., 2001). This requires Chk1- or Chk2-mediated phosphorylation of Cdc25A, and phosphorylation of Ser123 is a prerequisite for such accelerated ubiquitylation and degradation (Mailand et al., 2000; Falck et al., 2001). Consequently, the absence of Cdc25A in damaged cells precludes dephosphorylation of Thr14 and Tyr15 of Cdk2, and locks this essential S-phase-promoting kinase in its inactive form. In Xenopus laevis, an analogous checkpoint mechanism inhibits Cdk2 and disallows loading of Cdc45, an essential component of the pre-replicative complexes, onto replication origins, thereby preventing initiation of DNA replication (Costanzo et al., 2000). Despite recent progress, our understanding of overlapping and unique roles for the Cdc25 phosphatases at various cell cycle transitions is incomplete. One crucial question has emerged from a finding that homozygous disruption of Cdc25C in mice yielded healthy animals whose cells suffered no mitotic defect, had no alteration of Cdk1 phosphorylation and responded normally to DNA damage (Chen et al., 2001). What then performs the function of what has been widely regarded as a ‘master regulator’ of mitosis? Cdc25B is a plausible candidate, although the fact that this phosphatase is destabilized by cyclin A–Cdk2 (Baldin et al., 1997), highly active in G2, indicates that Cdc25B may not be the complete answer. Unexpectedly, during our studies of protein turnover of human Cdc25 phosphatases, we found that Cdc25A stability undergoes dramatic changes at the G2/M transition. Thus, Cdc25A became abruptly stabilized upon entry into mitosis and contributed to the cellular phosphatase pool required to dephosphorylate Cdk1 fully. In addition, DNA damage-induced inhibition of mitotic entry was accompanied by destruction of Cdc25A, an event required for the productive G2/M arrest. Our results point to novel functions and regulatory modes of Cdc25A, and highlight a concept of phosphorylation-mediated switches among multiple, differentially stable states of a cell cycle-regulatory protein as a flexible way to re-set its activity thresholds under diverse biological conditions. Results Cdc25A is stabilized in mitosis To study mechanisms that determine the abundance of Cdc25A, we measured the stability of the endogenous protein in synchronized U-2-OS cells. Short inhibition of protein synthesis by cycloheximide showed that in late G1, S and G2, Cdc25A was degraded with a half-life of ∼20 min (Figure 1A). In contrast, Cdc25A became remarkably stabilized in purified mitotic cells synchronized by the microtubule-depolymerizing drug nocodazole (Figure 1A), as judged from the extended half-life of well over 2 h (Figure 1B). Cdc25C, on the other hand, remained stable under all conditions (Figure 1B). Pulse–chase measurement of the radioactively labeled proteins confirmed a rapid turnover of Cdc25A in asynchronously growing U-2-OS cells and its marked stabilization in cells arrested in mitosis (Figure 1C). Since Cdc25A has been regarded as a G1/S regulator, its accumulation and stabilization in mitosis were surprising and inspired us to elucidate this phenomenon. Figure 1.Mitotic stabilization of Cdc25A. (A) Cdc25A is labile in interphase and stabilized in mitosis. U-2-OS cells were released from a double thymidine block for 4, 9 or 18 h to obtain cells in S, G2 or G1 phase, respectively, or arrested in prometaphase (M) by treatment with nocodazole. After addition of cycloheximide (CHX), cells were harvested at the indicated times and analyzed for Cdc25A levels by western blotting. (B) Differential stability of Cdc25A in asynchronous (AS) versus mitotic (M, purified by shake-off after nocodazole treatment) cells, compared with Cdc25C control, in an extended CHX experiment analogous to that in (A). (C) Pulse–chase measurement of the Cdc25A protein turnover in exponentially growing (AS) and nocodazole-arrested M U-2-OS cells labeled with [35S]methionine. (D) Abundance of Cdc25A, Cdc25C and a control hPBGD mRNA, determined by RT–PCR of poly(A)+ RNA from asynchronous or mitotic cells. (E) Interphase, but not mitotic, Cdc25A accumulates after proteasome inhibition. Asynchronous or mitotic cells were treated with LLnL (25 μM) for 6 h, lysed and analyzed for Cdc25A by western blotting. (F) Cdc25A is hyperphosphorylated in mitosis independently of nocodazole treatment. Mitotic cells were obtained by shake-off after nocodazole (+), or upon release for 14 h from a double thymidine block (−). Cdc25A was analyzed by western blotting. (G) M-phase-specific phosphorylation regulates Cdc25C, but not Cdc25A, activity. Cdc25 proteins were immunoprecipitated from asynchronous or mitotic cells, left untreated or dephosphorylated with λ phosphatase (PPase) and assayed for Cdc25 phosphatase activity. (H) Cdc25A is destabilized after release from metaphase arrest. Cells were released from a nocodazole block by replating into a drug-free medium. At the indicated times, cell lysates were processed for western blotting or cyclin B–Cdk1 activity measurement. Download figure Download PowerPoint First, we excluded that the mitotic accumulation of Cdc25A reflected increased transcription. In fact, a semi-quantitative RT–PCR analysis showed that the Cdc25A (but not Cdc25C) mRNA was downregulated in nocodazole-arrested cells (Figure 1D), consistent with Cdc25A gene expression being positively regulated by E2F (Vigo et al., 1999), and with E2F silencing upon S-phase exit (Krek et al., 1994). Secondly, while treatment of asynchronous U-2-OS cells with a proteasome inhibitor stabilized the endogenous Cdc25A protein, it did not have any effect on Cdc25A in nocodazole-arrested cells (Figure 1E), suggesting that the mitotic form of Cdc25A is already fully stable. Finally, we found that the Cdc25A protein was also highly elevated in cells naturally progressing through mitosis (Figure 1F), eliminating the possibility that the observed stabilization could be a consequence of any side effects elicited by nocodazole. We conclude that the accumulation of human Cdc25A in mitosis reflects its protein stabilization. Next, we determined whether the stabilized form of Cdc25A retains its activity. Indeed, in vitro phosphatase assays showed that Cdc25A immunoprecipitated from mitotic cells was highly active, as was the known regulator of mitosis, Cdc25C (Figure 1G). Like Cdc25C, the mitotic Cdc25A was modified, as indicated by its retarded electrophoretic migration (Figure 1B and E–H). Treatment of immunopurified Cdc25A and C with λ phosphatase reverted their migration back to the pattern seen in interphase cells (Figure 1G), indicating that their M-phase shifts were due to phosphorylation. Despite these similarities, at least three pieces of evidence suggest that the regulation of Cdc25A and C in mitosis differs profoundly. First, while the activity of Cdc25C was strictly associated with its shifted, phosphorylated form, Cdc25A was already active during the interphase (Figure 1G; data not shown). Secondly, dephosphorylation by the λ phosphatase completely inhibited the mitotic increase in Cdc25C activity, while it had little effect on the activity of the mitotic Cdc25A (Figure 1G). Thirdly, physiological dephosphoryl ation upon exit from mitosis correlated with gradual and complete destruction of the Cdc25A protein, but did not alter the abundance of Cdc25C (Figure 1H). Together, these data suggest that in addition to activating Cdc25C, the mitosis-specific phosphorylation helps to build up the total activity of Cdc25A through its stabilization. Mitotic Cdc25A is stabilized via cyclin B–Cdk1-mediated phosphorylation of Ser17 and Ser115 The mobility shift and the appearance of stable Cdc25A coincided with the peak of activity of cyclin B–Cdk1, the main mitosis-promoting kinase (Figure 1G). Two additional results supported the link between cyclin B–Cdk1 and phosphorylation-dependent stabilization of Cdc25A. First, immunopurified cyclin B–Cdk1 phosphorylated glutathione S-transferase (GST)–Cdc25A, resulting in a mobility shift similar to that observed on endogenous Cdc25A in mitosis (Figure 2A). This was completely reversed by roscovitine (Meijer et al., 1997), an inhibitor of Cdk2 and Cdk1 (Figure 2A). Secondly, if the stability of the mitotic Cdc25A was dependent on CDK-mediated phosphorylation, inhibition of Cdk1 should destabilize Cdc25A. Indeed, treatment of nocodazole-arrested cells with roscovitine reduced the mitosis-specific mobility shift of Cdc25A and led to a rapid disappearance of the endogenous Cdc25A protein with kinetics closely following those of dephosphorylation of known targets of cyclin B–Cdk1 such as Cdc27 (Figure 2B; data not shown). The major target of roscovitine under these conditions must have been cyclin B–Cdk1 because the other plausible candidates, cyclin A–Cdk2 or cyclin A–Cdk1, are absent in nocodazole-arrested cells due to cyclin A degradation (den Elzen and Pines, 2001; Geley et al., 2001). Figure 2.Cyclin B–Cdk1-targeted phosphorylation sites of Cdc25A. (A) Cyclin B–Cdk1 phosphorylates Cdc25A in vitro. Cyclin B–Cdk1 immunopre cipitated (IP) from asynchronous or nocodazole-arrested cells was assayed using full-length GST–Cdc25A as substrate. Ros, roscovitine; WB, western blot; Cdk1pTyr, inactive, Tyr15-phosphorylated Cdk1. (B) Inhibition of cyclin B–Cdk1 destabilizes Cdc25A in mitosis. Mitotic cells prepared by nocodazole treatment and shake-off were treated with roscovitine for the indicated times. Cdc25A and Cdc27 protein levels and SDS gel migration were analyzed by western blotting. (C) Mass spectrometric identification of Ser17 as an in vivo phosphorylation site on Cdc25A. Nanoelectrospray tandem mass spectrum of a tryptic peptide derived from the in-gel digestion of CDC25A from mitotic cells. Collision fragmentation of triply charged precursor ion at m/z 597.588 led to overlapping series of singly Y″ and doubly Y″2+ charged ions. The partial sequences were determined and sequence tags assigned to both Y″ and Y″2+ series allowing identification of CDC25A peptide and localization of a phospho-group on Ser17. The peptide sequence and localization of phospho-serine residue were confirmed by the existence of partial b and Y″-98 ion series, corresponding to the loss of phosphoric acid from fragment ions containing Ser17. (D) Ser17 and Ser115 of Cdc25A are conserved across species. Download figure Download PowerPoint To identify the residues of Cdc25A targeted by cyclin B–Cdk1 in vivo, we subjected the Cdc25A protein, isolated from either asynchronous or nocodazole-arrested cells, to mass spectrometry. To obtain enough material for this analysis, we employed our U-2-OS cell line conditionally expressing ectopic Cdc25A (Mailand et al., 2000). Mass spectrometry analysis identified the peptides 11RLLFACS*PPPASQPVVK27, 12LLFACS*PPPASQPV VK27, 111LLGCS*PALK119 and 278SQEES*PPGSTK288 as phosphorylated in mitotic cells (Figure 2C). Comparisons of CDC25A from synchronized and non-synchronized cells showed that phosphorylation on Ser17 and Ser115 was specific to mitotic cells. The regions surrounding these serines are highly homologous in human, mouse and rat proteins (Figure 2D), suggesting that modification of Ser17/Ser115 by cyclin B–Cdk1 could represent a conserved regulatory mechanism. Mutation of Ser17/Ser115 abolishes Cdc25A phosphorylation in vitro, and restores ubiquitylation and instability of the mitotic Cdc25A in vivo To test whether the identified serines were phosphorylated in mitosis, we purified, from bacteria, short GST-tagged fragments of Cdc25A spanning Ser17 and Ser115, respectively, and their derivatives where the serines were substituted by alanines. While both fusion proteins with the wild-type serines were phosphorylated efficiently by cyclin B–Cdk1, the alanine mutants remained virtually unmodified (Figure 3A). Furthermore, concomitant mutations of Ser17 and Ser115 in the context of the entire regulatory domain of Cdc25A containing most of the CDK consensus sites also impaired (although did not completely abolish) its phosphorylation by cyclin B–Cdk1 in this in vitro kinase assay (Figure 3B). Figure 3.Phosphorylation by cyclin B–Cdk1 uncouples Cdc25A from ubiquitylation and degradation. (A) Cyclin B–Cdk1 phosphorylates Cdc25A on Ser17 and Ser115 in vitro. Cyclin B–Cdk1 was immunoprecipitated from mitotic U-2-OS cells and assayed using the indicated GST-tagged fragments of Cdc25A containing Ser17 or Ser115, or alanine substitutions as substrates, with or without roscovitine (Ros). (B) Reduced phosphorylation of the Ser17/Ser115(2A) mutant in the context of the entire regulatory region of Cdc25A. Experimental conditions were as in (A); incorporation of 32P was reduced to ∼55% in the 2A mutant. (C) U-2-OS T-Rex cells were transfected with plasmids encoding wild-type Cdc25A or the Ser17/Ser115(2A) mutant, treated with nocodazole (M) or left asynchronous (AS), and induced by addition of tetracycline for 3 or 6 h. Where indicated, LLnL was added to the medium at the time of the transgene induction. Cdc25A proteins were analyzed by western blotting. (D) Destabilization of Cdc25A(2A) in mitosis. U-2-OS T-Rex cells were treated as in (C), followed by addition of cycloheximide for 2 h, and Cdc25A was assessed by western blotting. (E) Cdc25A is not ubiquitylated in mitosis. Asynchronous (AS) or mitotic (M) U-2-OS/B3C4 cells transiently transfected with His-ubiquitin were kept uninduced or induced to express ectopic Cdc25A for 12 h as indicated, and processed for detection of Cdc25A-associated ubiquitin (Ub) conjugates. (F) Mutation of the cyclin B–Cdk1-targeted sites in Cdc25A restores its ubiquitylation in mitosis. U-2-OS/Cdc25A(2A) cells were treated and analyzed as in (E). Download figure Download PowerPoint Next, we substituted both Ser17 and Ser115 with alanines in the full-length Cdc25A and generated U-2-OS clones conditionally expressing the wild-type or the double alanine (2A) mutant of Cdc25A in a tetracycline-dependent manner. Upon induction in asynchronous cells, both forms of Cdc25A accumulated with very similar kinetics (Figure 3C). Strikingly, when induced in nocodazole-arrested cells, the phosphorylation-deficient Cdc25A(2A) mutant accumulated more slowly than the wild-type protein (Figure 3C), a difference abolished in the presence of the proteasome inhibitor N-acetyl-Leu-Leu-norleucine (LLnL) (Figure 3C). These results are consistent with the interpretation that the cyclin B–Cdk1-mediated phosphorylation of Ser17/Ser115 is instrumental for stabilization and accumulation of Cdc25A specifically in mitotic cells. This conclusion was also supported by direct protein stability estimation, as wild-type Cdc25A was stable in mitosis, while the Cdc25A(2A) mutant (but not the one where both serines were substituted by acidic, and presumably phospho-mimicking, amino acids) was turned over rapidly (Figure 3D; data not shown). Finally, if the lack of Ser17/Ser115 phosphorylation indeed caused destruction of Cdc25A in mitosis, it should be reflected by the increased polyubiquitylation of the mutated protein, a prerequisite for targeting of many proteins by the proteasome (Hershko and Ciechanover, 1998). An established in vivo assay (Treier et al., 1994) showed that wild-type Cdc25A was ubiquitylated in asynchronous cells but not in mitosis (Figure 3E). In contrast, Cdc25A(2A) also became polyubiquitylated in mitotic cells (Figure 3F). These data demonstrate that the mitotic phosphorylation of Ser17/Ser115 uncouples Cdc25A from the ubiquitin–proteasome-mediated degradation and leads to accumulation of an active Cdc25A phosphatase. Cdc25A activates cyclin B–Cdk1 and is rate limiting for the G2/M transition Next, we asked what is the purpose of preserving the activity of Cdc25A at and beyond the G2/M transition. It is well known that the sharp increase of cyclin B–Cdk1 activity early in mitosis is essential for proper execution of mitotic events (Nigg, 2001). We reasoned that Cdc25A may help to generate and/or maintain the threshold required for mitosis-promoting CDK activity once cells become committed to cell division. To address this issue, we first immunodepleted each member of the Cdc25 family (Figure 4A) and assayed the ability of the resulting cell extracts to activate cyclin B–Cdk1 in vitro. Depletion of each individual Cdc25 phosphatase reduced the total cellular cyclin B–Cdk1-activating potential to ∼50% (Figure 4B). Combined depletion of Cdc25B and C was more effective, yet a complete inhibition, mimicking the effect of the phosphatase inhibitor, sodium vanadate, required depletion of all three Cdc25s (Figure 4B). Re-addition of purified GST–Cdc25A into the cell extracts depleted for all the Cdc25s substantially restored the lysate's capacity to activate the cyclin B–Cdk1 kinase complex (Figure 4C). These data suggest that Cdc25A, together with Cdc25B and C, jointly generate a cellular phosphatase pool required for full activation of cyclin B–Cdk1. Figure 4.Cdc25A activates cyclin B–Cdk1 and promotes mitotic entry. (A) Western blot-verified depletion of Cdc25A, B and C from U-2-OS cells lysed in a kinase buffer. Individual and/or the indicated combinations of Cdc25 proteins were depleted by specific antibodies or control non-specific immunoglobulin (IgG). (B) Cdc25 isoforms contribute to activation of cyclin B–Cdk1. Cell lysates were prepared and depleted as in (A), supplemented with 10 mM EDTA to inhibit endogenous kinases and incubated for 20 min at 30°C. Cyclin B–Cdk1 was then immunoprecipitated (IP) and its activity measured using histone H1 as a substrate. Sodium vanadate was added into the control reaction to inhibit all Cdc25 phosphatase activity. Numbers indicate the extent (%) of cyclin B–Cdk1 activation relative to the depletion with non-specific immunoglobulin (asterisk: control reaction where the lysate was not incubated at 30°C). (C) U-2-OS cell lysates were depleted with non-specific (−) or Cdc25A/B/C antibodies (+) as in (A) and incubated for 20 min at 30°C in the presence of 10 mM EDTA. Where indicated, 100 ng of purified GST–Cdc25A was added to the reaction. (D) Cyclin B–Cdk1 interacts with all Cdc25 family members. U-2-OS cells were transfected with the indicated plasmids (bottom) and the presence of HA-tagged Cdc25s and Cdk1 was analyzed in anti-cyclin B immunoprecipitates (IgG, non-specific control antibody). (E) U-2-OS/HA-Cdc25A cells were induced to express the transgene, and histone H1 kinase activities were measured in lysates harvested at the indicated times. (F) Elevated Cdc25A induces premature mitotic entry. U-2-OS/B3C4 cells were arrested in S phase by a double thymidine block, released and induced to express ectopic Cdc25A (−Tet) or kept uninduced (+Tet). After 5 h, cells were analyzed by flow cytometry for phospho-histone H3, a marker of productive entry into mitosis. About 60% of the phospho-H3-positive cells also displayed other morphological signs of mitosis such as condensed chromatin. Download figure Download PowerPoint Such a conclusion predicts that Cdc25A can activate cyclin B–Cdk1 in vivo and thereby modulate G2/M progression. Indeed, conditionally elevated Cdc25A bound physically to cyclin B and Cdk1 in vivo in a manner comparable with Cdc25B and C (Figure 4D), and increased the endogenous cyclin B–Cdk1 kinase activity (Figure 4E). Consistent with activation of cyclin B–Cdk1, transient induction of wild-type Cdc25A accelerated entry into mitosis from G2, and induced premature mitosis in cells with incompletely replicated DNA (Figure 4F), which indicated (but did not prove) that Cdc25A might collaborate with Cdc25B and C to control G2/M transition. To address this issue directly, we employed RNA interference (RNAi) technology and found that quantitative and specific downregulation of endogenous Cdc25A by short inhibitory RNA (siRNA) duplexes (Figure 5A and B) not only inhibited the cyclin E–Cdk2 kinase activity (Figure 5C) and S-phase entry (Figure 5D), as expected, but also impaired a full-scale activation of the cyclin B–Cdk1 kinase complex (Figure 5E) and delayed the G2/M transition (Figure 5F and G). Remarkably, the G2/M delay in cells lacking endogenous Cdc25A occurred in spite of the fact that the levels and activities of Cdc25B and Cdc25C remained unchanged (Figure 5B; data not shown). Thus, apart from its S-phase-promoting effect, Cdc25A physically and functionally interacts with the main mitosis-promoting cyclin–CDK complex and generates a rate-limiting stimulus for the G2/M transition, and the lack of its activity can delay completion of the cell division cycle. Figure 5.Downregulation of Cdc25A by siRNA impairs both G1/S and G2/M transitions. (A) HeLa cells were transfected with Cdc25A siRNA duplexes, and the level of Cdc25A determined by western blotting at the indicated times after transfection. (B) Cdc25A-specific siRNA does not affect the levels of Cdc25B and C. HeLa cells were treated as in (A) and assayed by western blotting 24 h after transfection for the indicated Cdc25 proteins. (C) Downregulation of cyclin E–Cdk2 activity in cells treated with Cdc25A siRNA. HeLa cells were treated as in (A) and the cyclin E-associated histone H1 kinase activity was measured 24 h after transfection. (D) siRNA-mediated downregulation of endogenous Cdc25A delays G1/S transition. HeLa cells were transfected with Cdc25A siRNA for 24 h, treated with nocodazole for an additional 12 h to prevent re-entry of the transfected cells into G1, and analyzed by flow cytometry. The numbers indicate the G1 fractions in mock- and Cdc25A siRNA-treated cells, respectively, and demonstrate a substantial retention of the latter in G1. (E) Lysates from Cdc25A siRNA-treated cells show attenuated cyclin B–Cdk1 activation. HeLa cells were treated as in (A). After 24 h, the cell lysates were prepared and induced to activate cyclin B–Cdk1 as in Figure 4B for the indicated times. Results of two independent experiments are shown. (F) siRNA-mediated downregulation of endogenous Cdc25A impairs G2/M transition. HeLa cells were transfected as in (E) but exposed only to a short (2 h) pulse of nocodazole to prevent mitotic exit of cells acutely progressing through the G2/M transition. Subsequently, the cells were analyzed by flow cytometry, for either DNA content alone (left panels) or DNA content together with the phospho-histone H3 fluorescence associated specifically with mitotic cells (right panels). Numbers indicate the total percentages of cells in G2/M and pure mitotic (M) compartments, respectively, and show that despite a higher overall G2/M population, the Cdc25A siRNA-treated cells were impaired to proceed beyond this transition into mitosis. (G) Quantification of two independent experiments described in (F) and performed after various times of nocodazole treatment as indicated. The results are presented as a percentage of purely mitotic cells (M) from the total amount of G2/M cells at each time point. The lack of increase of this ratio in the Cdc25A siRNA-treated cells reflects their arrest at the G2/M boundary. Download figure Download PowerPoint Destruction of Cdc25A is re
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