- Open Access
A human cancer-predisposing polymorphism in Cdc25A is embryonic lethal in the mouse and promotes ASK-1 mediated apoptosis
© Bahassi et al; licensee BioMed Central Ltd. 2011
- Received: 1 October 2010
- Accepted: 10 February 2011
- Published: 10 February 2011
Failure to regulate the levels of Cdc25A phosphatase during the cell cycle or during a checkpoint response causes bypass of DNA damage and replication checkpoints resulting in genomic instability and cancer. During G1 and S and in cellular response to DNA damage, Cdc25A is targeted for degradation through the Skp1-cullin-β-TrCP (SCFβ-TrCP) complex. This complex binds to the Cdc25A DSG motif which contains serine residues at positions 82 and 88. Phosphorylation of one or both residues is necessary for the binding and degradation to occur.
We now show that mutation of serine 88 to phenylalanine, which is a cancer-predisposing polymorphic variant in humans, leads to early embryonic lethality in mice. The mutant protein retains its phosphatase activity both in vitro and in cultured cells. It fails to interact with the apoptosis signal-regulating kinase 1 (ASK1), however, and therefore does not suppress ASK1-mediated apoptosis.
These data suggest that the DSG motif, in addition to its function in Cdc25A-mediated degradation, plays a role in cell survival during early embyogenesis through suppression of ASK1-mediated apoptosis.
- Circular Dichroism
- Phosphatase Activity
- Early Embryonic Lethality
- Replication Checkpoint
- S88F Mutation
When DNA is damaged or when blocks to DNA replication occur, cells activate an extensive network of signaling pathways that promote cell cycle arrest and DNA repair . Cell cycle arrest is mediated by checkpoints in which the activities of cyclin-dependent kinases (Cdks) are inhibited. Cell cycle arrest presumably provides time for damage to be repaired and contributes to the preservation of genomic stability and reduction of risk for diseases such as cancer. There are two well established mechanisms by which Cdks are inhibited in response to DNA damage in mammals. The first involves activation of the Cdk inhibitor p21Cip through activation of p53 . The second is through inactivation of the Cdc25 family of phosphatases. The most fully understood mechanism regulating the level of a phosphatase in this family is the proteasome-mediated degradation of Cdc25A [3–5].
Cdc25A phosphatase is an essential activator of cell cycle progression and its expression is tightly regulated at many levels, including transcriptional activation, reversible phosphorylation, protein-protein interaction and ubiquitin-mediated degradation [6–9]. Ubiquitin-dependent degradation of Cdc25A is a major mechanism for damage-induced S-phase checkpoint arrest. Two ubiquitin ligases, the Skp1-cullin-β-TrCP (SCFβ-TrCP) complex and the anaphase-promoting complex (APCCdh1), participate in Cdc25A turnover. The APC/C proteasome complex helps regulate Cdc25A at the exit of mitosis while SCFβ-TrCP regulates the abundance of Cdc25A in S phase and G2 [8, 10–13]. When DNA is damaged or when cells respond to stalled replication forks, ATM and ATR protein kinases are activated leading to subsequent activation of Chk1 and Chk2 and to hyperphosphorylation of Cdc25A. This cascade of events stimulates SCF-mediated ubiquitinylation of Cdc25A and its proteolysis [3, 5], contributing to cell cycle arrest. Failure to regulate Cdc25A levels compromises checkpoint arrest and can result in enhanced DNA damage [3–5, 14, 15]. Overexpression of Cdc25A, which frequently occurs in multiple tumor types , leads to accelerated entry of cells into S-phase  and mitosis .
The ubiquitinylation-mediated degradation of Cdc25A is associated with phosphorylation of Cdc25A at two residues within the β-TrCP docking site (DSG motif). The DSG docking site of Cdc25A is comprised of serines 79, 82 and 88, and the absence of their phosphorylation is sufficient to abolish β-TrCP binding and interfere with Cdc25A degradation . Several kinases have been reported to phosphorylate the DSG motif and to target Cdc25A for SCFβ-TrCP-mediated degradation [18–20]. The NIMA related kinase 11 (NEK11) has been identified as a kinase that specifically phosphorylates Cdc25A on serines 82 and 88 in cultured cells. This phosphorylation is important for subsequent ubiquitinylation and proteasome-mediated degradation . The existence of an S88F polymorphic variant in humans  that elevates the risk for cancer  underscores the importance of serine 88 in cell function, behavior and disease. Its importance appears to have been conserved in evolution since the same serine to phenylalanine mutation in C. elegans at a serine equivalent to human serine 88 is a gain of function mutation that causes deregulated intestinal cell hyper-proliferation and hyperplasia .
To better understand the significance of Cdc25A phosphorylation within the DSG domain in cell cycle regulation and DNA damage response, we generated a knock-in mouse model with a substitution of phenylalanine for serine at residue 88. The expectation was that cells from mice harboring this Cdc25A variant would be less susceptible to SCFβ-TrCP-mediated degradation. Inhibition of Cdc25A degradation in turn should lead to its accumulation, accompanied by bypass of DNA damage and replication checkpoints, enhanced DNA damage and increased risk of cancer. In the mouse, we find that this mutation, when homozygous, produces early fetal lethality, indicating that the Cdc25A DSG motif and serine 88 phosphorylation may also have a role in early embryogenesis. Homozygous mutant embryos that persist display an altered morphology with extensive cellular degeneration and die at or prior to embryonic day 3.5. Circular dichroism (CD) studies revealed that the S88F substitution in Cdc25A protein had no major effect on protein secondary structure that would account for the observed phenotype. The mutant protein retains phosphatase activity but, unlike the wildtype Cdc25A, the S88F mutant fails to interact with the apoptosis signal-regulating kinase-1 (ASK1) facilitating apoptosis mediated by ASK1. These data demonstrate that the DSG domain of Cdc25A has a role in cell survival during early embryogenesis that is separate from its role in Cdc25A ubiquitin-mediated degradation in response to DNA damage.
Serines 82 and 88 are required for efficient degradation of Cdc25A following DNA damage
Cdc25A S88F induces early embryonic lethality in mice
Genotype distribution in the pups and embryos from Cdc25A heterozygous crosses
Expression of Cdc25A S88F is lethal to early embryos and cultured cells
The S88F substitution confers only subtle conformational changes on Cdc25A
Deconvolution of Cdc25A far-UV CD spectra using CDSSTR.
Cdc25A S88F retains phosphatase activity in vitro and in cultured cells
Cdc25A S88F loses its interaction with ASK1 and fails to suppress the stress-inducible ASK1-mediated apoptotic pathway
Cdc25A protein levels are tightly regulated by multiple, apparently redundant, mechanisms. The importance of its tight regulation is underscored by the observation that defects in maintaining steady state levels of Cdc25A translate into increased cell proliferation that can lead to cancer. Indeed, Cdc25A is frequently overexpressed in multiple types of cancer . Ubiquitin-dependent degradation of Cdc25A is a major effector of the DNA-damage-induced S-phase checkpoint. Ubiquitinylation is mediated by the SCFβ-TrCP complex which binds to a DSG consensus sequence in Cdc25A that is dependent on phosphorylation of serines 82 and 88 within the DSG motif [10, 12].
To begin to dissect the functions of Cdc25A and to better understand the role of ubiquitinylation and degradation in Cdc25A function, we have generated a knock-in mouse (S88F) in which serine 88, one of two key serine residues in the DSG domain, is substituted with a phenylalanine. This serine residue is important for β-TrCP binding and for ubiquitin-dependent degradation of Cdc25A protein. The expectation was that mice expressing Cdc25A S88F would have increased amounts of the protein due to a reduction in its degradation and that cells would undergo unscheduled replication, display genomic instability and early tumor onset in the progeny. No mice that were homozygous for Cdc25A S88F, however, were born. The Cdc25A -/- mice exhibit very early embryonic lethality, which is consistent with a critical Cdc25A function in cell cycle regulation . Analysis of Cdc25A S88F mutant embryos showed that the latest embryonic stage at which homozygous embryos were found was the 3.5 day blastocyst. By this stage, the very few homozygous Cdc25A S88F embryos that were present, contained cells that had lost their spherical shape and looked shrunken with blebbed membranes consistent with an apoptotic phenotype. The effect of the Cdc25A S88F mutation was apparent at the morula stage and even at the oocyte stage. The rapid cell death that ensued raised a concern that the S88F mutation in Cdc25A produces major conformational changes in the protein or eliminates its phosphatase activity. Analysis of Cdc25A protein structure by CD and assessment of its phosphatase activity in vivo and in vitro showed that the mutation has only a very minor effect on Cdc25A structure and that its activity is not significantly different than that of wildtype, indicating that neither a change in protein conformation nor a change in enzymatic activity accounts for its lethal phenotype.
Since mutant Cdc25A displays no significant change in protein conformation or phosphatase activity, it is most likely that the lethal phenotype is due to an alternative Cdc25A function. It is known that Cdc25A is a transcriptional target of c-Myc and that overabundance of Cdc25A suppresses apoptotic cell death [26, 27]. One pathway to apoptosis in which Cdc25A participates involves its interaction with the apoptotic protein ASK1 . When Cdc25A is elevated, it suppresses stress-induced activation of ASK1 and downstream kinases . We therefore tested the possibility that the mutant Cdc25A S88F does not interact with ASK1 and thereby fails to suppress the pro-apoptotic activity of ASK1, promoting apoptotic cell death in early embryos and transfected cells. This scenario is supported by our finding that ectopic expression of wildtype Cdc25A effectively suppresses the expression of targets downstream of ASK1 in response to genotoxic stress while the expression of the mutant protein fails to efficiently do so. In the uterus, embryos are subjected to high levels of reactive oxygen species as a result of the hypoxic environment . Failure of the Cdc25A S88F mutant to suppress ASK1-mediated apoptosis will activate the downstream effectors leading to embryonic lethality.
Generation of Cdc25A S88F mice
Two fragments of Cdc25A genomic DNA were PCR amplified and cloned in a pBSK-TK vector. The first fragment (5030 bp) contained a point mutation GATTC to GATTT (on exon 3) at position 4780. A neomycin resistance marker flanked by loxP sites (LoxP-PGK-Neo-LoxP) was included in the intron 3 immediately after the mutation. A second homologous arm (6715 bp) was cloned after the Neo cassette using conventional molecular biology techniques. The sequence of the targeting construct was validated by direct sequencing. The vector was linearized at a unique Not I site and electroporated into 129/Sv ES cells and neomycin-resistant colonies were screened for correct targeting by Southern blots. Two faithfully targeted ES cell lines were identified, and both were injected into the blastocoel of 3.5-day-old blastocysts which were implanted into the uterus of recipient mice. Chimeras carrying the targeted allele were identified, mated with Black Swiss mice, and agouti progeny were assayed for transmission of the knock-in allele. Mice that were homozygous for the S88F variant were crossed with TgN(EIIa-Cre)C79Lmgd mice (Jackson Laboratories, Bar Harbor, ME) in an FVB/N strain background. Cre recombinase is selectively expressed in the germline of the progeny mice so that their offspring will retain the targeted allele without the neo marker but with a single remaining intronic loxP site. All animal experiments were carried out under protocols approved by the Institutional Animal Care and Use Committee of the University of Cincinnati.
Protein purification and circular dichroism
GST-tagged Cdc25A-wt and Cdc25A S88F proteins were purified using a combination of ammonium sulfate precipitation (65% cut-off), a size exclusion column fractionation, a sulphopropyl (SP) column to separate the GST tag from Cdc25A protein following GST tag removal from the fusion protein using PreScission™ Protease (Sigma), and a glutathione affinity column run according to the manufacturer's protocol (Sigma). Purified wildtype and mutant Cdc25A far-UV CD spectra were measured using an Aviv 215 circular dichroism spectrometer. Proteins were dialyzed into 10 mM Tris pH 8.0, 150 mM NaCl, 1% ethylene glycol, and 2 mM β-mercaptoethanol, and spectra were measured from 260 to 190 nm. Protein concentrations were determined using the absorbance at 280 nm and a molar extinction coefficient of 31,860 M-1cm-1, as determined by Sednterp . Data were converted to mean residue ellipticity [θ] using the equation where θ is the measured ellipticity in millidegrees, C is the concentration in molar units, l is the pathlength in cm, and n r is the number of residues. Data were analyzed using CDSSTR  in Dichroweb  with reference set 4.
Cdc25A phosphatase assay
The phosphatase activity of Cdc25A was assayed by hydrolysis of 4-nitrophenol phosphate (pNPP) (Roche Applied Science, Indianapolis, IN) as described previously  with modifications. GST-Cdc25A-wt and GST-Cdc25A-S88F mutant were purified from bacteria and then incubated in phosphatase reaction buffer (50 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, and 20 ng pNPP) for 6-10 h at 37°C. The reaction was stopped with 5N NaOH. Cdc25A activity was calculated by measuring the absorbance of p-nitrophenolate at 410 nm and subtracting the control background value. Each point is the mean ± SEM of data from two separate experiments.
Annexin V staining for flow cytometry
Apoptosis was measured using Annexin V-Cy5.5 (Invitrogen, Carlsbad, CA) and propidium iodide (PI). Briefly, harvested cells were washed in phosphate-buffered saline (PBS) and resuspended in Annexin V binding buffer (10 mM Hepes pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Cells (2 × 105) were incubated with 5 μl Annexin V-Cy5.5 and PI (1 mg/ml) for 10 min in the dark at room temperature and analyzed by flow cytometry.
NIH3T3 cells were transfected with a GFP-Cdc25A-wt, a GFP-Cdc25A-S88F or a GFP-H2B control plasmid. The cells were washed with PBS 16-18 hours post transfection and fixed with 4% paraformaldehyde. Coverslips were washed with PBS and mounted with Gelmount (Fisher Scientific, Pittsburgh, PA). Fluorescent green cells were then visualized with the use of a LSM510 laser scanning confocal or Orca microscope (Zeiss, Oberkochen, Germany).
This work was supported in part by NIH grants R03 ES015307 to EMB, R01 ES012695 and R01 ES016625 to PJS, and the Center for Environmental Genetics P30-ES006096.
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