Regulation of G(2)/M events by Cdc25A through phosphorylation-dependent modulation of its stability - PubMed (original) (raw)
Regulation of G(2)/M events by Cdc25A through phosphorylation-dependent modulation of its stability
Niels Mailand et al. EMBO J. 2002.
Abstract
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 G(2) arrest, in contrast, is accompanied by proteasome-dependent destruction of Cdc25A, and ectopic Cdc25A abrogates the G(2) 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.
Figures
Fig. 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.
Fig. 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.
Fig. 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).
Fig. 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.
Fig. 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.
Fig. 6. Cdc25A degradation in response to DNA damage in G2. (A) Etoposide-induced G2/M arrest causes loss of Cdc25A protein and associated activity. Asynchronous U-2-OS cells were treated with etoposide for 12 h to arrest cells in G2, and levels and activities of Cdc25A, B and C were determined. (B) Overexpression of Cdc25A but not Cdc25C compromises the G2/M checkpoint. Etoposide was added to asynchronous Cdc25A- and Cdc25C-inducible, or parental U-2-OS/TA cells. After 4 h, nocodazole was added to the medium and cells concomitantly were induced to express the transgenes. After an additional 16 h, cells were analyzed by flow cytometry for phospho-histone H3. Following Cdc25A induction, 72% of the phospho-H3-positive cells also showed signs of premature chromosome condensation. (C) The mitotic form of Cdc25A is not destabilized by DNA damage. Asynchronous or mitotic U-2-OS cells were γ-irradiated (10 Gy), and Cdc25A analyzed by western blotting 1 h later.
Fig. 7. Models of Cdc25A regulation and function in the mammalian cell cycle. (A) A dual positive feedback loop leading to maximal and irreversible activation of the M-phase-promoting cyclin B–Cdk1 kinase. See Discussion for details. (B) Phosphorylation-dependent switches among three differentially stable forms of Cdc25A, designated labile, ultra-labile and stable, determine the thresholds and roles of Cdc25A in unperturbed or DNA-damaged interphase and mitotic cells, respectively. See Discussion for details.
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