In eukaryotic cells, control mechanisms of cell-cycle progression have evolved to accurately monitor the integrity of genetic information to be transferred to the progeny. Cdc25A phosphatase is an essential activator of cell-cycle progression and is targeted by checkpoint signals. Ubiquitylation regulates Cdc25A activity through fine tuning of its protein levels. Two different ubiquitin ligases (APC/C and SCF complex) are involved in Cdc25A turnover. While APC/C is involved in regulating Cdc25A at the exit of mitosis, SCF regulates the abundance of Cdc25A in S phase and G2. In response to DNA damage or to stalled replication, the activation of the ATM and ATR protein kinases leads to Chk1 and Chk2 activation and to Cdc25A hyperphosphorylation. These events stimulate SCF-mediated ubiquitylation of Cdc25A and its proteolysis. This contributes to delaying cell-cycle progression, thereby preventing genomic instability. Based on recent findings, we discuss the role of Cdc25A ubiquitylation and degradation in cell-cycle progression and in response to DNA damage. Moreover, we discuss the role of phosphorylation at multiple sites in triggering ubiquitylation signals.
A mammalian cell must tightly regulate each of its cell-cycle phase transitions to accurately transmit a copy of its genome to daughter cells. Cyclin-dependent kinases (Cdks) regulate these cell-cycle transitions and their activity is in turn controlled by various positive and negative upstream mechanisms.
Two key Cdks, Cdk1 and Cdk2 are expressed at constant levels during cell cycle. To be active, they require association with cyclin subunits and phosphorylation on a threonine residue located in a conserved domain, the T-loop. In contrast, the phosphorylation of two specific amino-acid residues (Tyr15 and Thr14) located within the ATP binding loop leads to Cdk1 and Cdk2 inactivation. The Wee1/Mik1/Myt1 protein kinases are known to mediate these inhibitory phosphorylation events, while the Cdc25 dual specificity phosphatases are responsible for the activating dephosphorylation of these same sites (see, for a review, Pines, 1999).
Cdc25 was first identified in fission yeast as a factor required for entry into mitosis (Russell and Nurse, 1986). Subsequently, three mammalian genes were identified that complement the G2 arrest phenotype of a yeast cdc25ts strain: cdc25A, -B and -C (Galaktionov and Beach, 1991). All three genes encode phosphatases that can dephosphorylate phosphotyrosine and phosphothreonine residues, in order to activate their Cdk substrates (Dunphy and Kumagai, 1991). However, despite these common features, the different Cdc25 phosphatases are known to have distinct roles in regulating cell-cycle transition (see, for a review, Donzelli and Draetta, 2003). Whereas Cdc25B and -C regulate only the G2/M transition, Cdc25A is thought to play a more general role, being involved in both early (G1/S) and late (G2/M) cell-cycle transitions. Cdc25A is tightly regulated at the protein level, being periodically synthesized and degraded via ubiquitin-mediated proteolysis (Donzelli et al., 2002). In this review, we discuss the role of ubiquitylation in regulating Cdc25A levels during normal cell-cycle progression and in the DNA-damage response.
Cdc25A function and regulation in the cell cycle
The finding that the microinjection of anti-Cdc25A antibodies causes a G1 arrest in serum-stimulated cells and the observation that the overexpression of the protein causes accelerated S phase entry and Cdk2 activation, suggested that Cdc25A functions to regulate S phase entry, as a result of its dephosphorylating action on Cdk2 (Hoffmann et al., 1994; Jinno et al., 1994; Blomberg and Hoffmann, 1999; Sexl et al., 1999). The levels of Cdc25A protein are known to increase from the G1/S transition until mitosis (Bernardi et al., 2000; Molinari et al., 2000; Donzelli et al., 2002). In addition, Cdc25A overexpression induces unscheduled mitosis while its siRNA-mediated silencing causes a decrease in the percentage of mitotic cells (Molinari et al., 2000; Zhao et al., 2002). These data suggest a broader role for Cdc25A in cell-cycle control, including regulation of the G2/M transition through the activation of Cdk1 complexes, as shown by Mailand et al. (2002).
Cdc25A is an unstable protein whose cellular levels are regulated by periodic synthesis and by ubiquitin-mediated proteolysis. In late G1 phase, Cdc25A accumulates as a result of E2F-1- and c-Myc-mediated transcriptional activation (Jinno et al., 1994; Galaktionov et al., 1996; Vigo et al., 1999). Cdc25A then dephosphorylates Cdk2, activating Cdk2/Cyclin E complex which, in turn, creates an autoamplification loop further phosphorylating and activating Cdc25A (Hoffmann et al., 1994). This apparently contributes to S phase progression by recruiting the essential replication factor Cdc45 and DNA polymerase on replication origins (Zou and Stillman, 1998). Recent findings seem to challenge the notion that Cdk2/Cyclin E complex is essential to trigger cell-cycle progression, since both cyclin E- and Cdk2-deficient MEFs can apparently proceed normally through the cell cycle (Geng et al., 2003; Ortega et al., 2003; Parisi et al., 2003). Therefore, it could be speculated that Cdc25A regulation of the G1/S transition also occurs through the dephosphorylation of substrates distinct from Cdk2, but still involved in controlling this cell-cycle transition.
Cdc25A is constantly turned over in cycling cells. From the time it first appears in late G1, and until mitosis, continuous de novo Cdc25A synthesis is counterbalanced by its degradation through the ubiquitin–proteasome pathway, thus maintaining a fixed activation threshold for Cdk2 dephosphorylation. Cdc25A ubiquitylation is mediated by two ubiquitin ligase complexes, APC/CCdh1 and SCFβTrCP, each acting at distinct stages of the cell cycle (Donzelli et al., 2002; Busino et al., 2003). Interaction with these complexes requires specific recognition motifs in Cdc25A. Binding of Cdc25A to SCFβTrCP requires the phosphorylation of serine residues within a so-called DSG motif, while interaction with APC/CCdh1 is dependent upon a KEN motif without apparent requirement for post-translational modifications (Figure 1). Throughout the text, we have numbered amino-acid residues according to the human Cdc25A protein sequence described in the NCBI database (accession number BC007401). We have verified this sequence by analysing cDNA samples from different human tissue sources.
Mitotic stabilization and APC/CCdh1-mediated degradation of Cdc25A upon exit from mitosis
In mitosis, Cdc25A is phosphorylated by Cdk1/Cyclin B on two specific serine residues (Ser 18 and Ser 116). This results in Cdc25A stabilization, uncoupling it from its ubiquitin-mediated turnover (Figure 1 and Table 1), thus creating a positive feedback loop that allows Cdc25A to dephosphorylate and further activate Cdk1 complexes (Mailand et al., 2002). At mitotic exit and in early G1, Cdc25A levels rapidly decrease and remain almost undetectable for all of G1 phase: this postmitotic degradation is carried out by APC/CCdh1-mediated ubiquitylation, as shown in Figure 2a (Donzelli et al., 2002).
The vertebrate anaphase-promoting complex/cyclosome (APC/C) is a large multiprotein complex that functions as an ubiquitin ligase (E3) (Hochstrasser, 1996). To be fully active, APC/C requires two additional subunits that regulate its target specificity, Cdc20 and Cdh1, thus forming two distinct complexes (APC/CCdc20 and APC/CCdh1) that play a specific role in separate cell-cycle phases (see, for a review, Peters, 2002). Target specificity is due to Cdc20 and Cdh1 ability to recognize two specific destruction sequences on APC/C subtrates, namely D-box and KEN-box motifs. (Glotzer et al., 1991; Pfleger and Kirschner, 2000). At mitotic exit, Cdh1 is dephosphorylated and can therefore bind and activate APC/C. Once formed, APC/CCdh1 is ready to degrade Cdc20, thus inhibiting the APC/CCdc20 activity that peaks at mitosis (Zachariae et al., 1998; Kramer et al., 2000; Listovsky et al., 2000). APC/CCdh1 activation at the end of mitosis is part of the program for inactivating Cdk1. The specific function of APC/CCdh1 in G1 phase acts to prevent the accumulation of cyclins and other S phase activators. Thus APC/CCdh1 must be inactivated at G1/S transition in order to achieve the accumulation of replication factors, such as S-phase Cdks/cyclin complexes (Lukas et al., 1999).
Our laboratory (Donzelli et al. 2002) has contributed experimental evidence demonstrating that at mitotic exit Cdc25A is degraded via APC/CCdh1 ubiquitylation: Cdh1 (but not Cdc20) overexpression results in a significant decrease of the steady-state level of Cdc25A, while siRNA-mediated silencing of Cdh1 causes an increase in the amount of endogenous Cdc25A; moreover, APC/CCdh1, but not APC/CCdc20, is able to ubiquitylate Cdc25A in vitro. Cdc25A contains in its N-terminal part a KEN-box motif essential for recognition by Cdh1. Mutation of this KEN-box abolishes APC/CCdh1-mediated ubiquitylation of Cdc25A (Figure 1 and Table 1).
Cdc25A degradation at mitotic exit and in early G1 could be required to interrupt Cdk1 dephosphorylation on Thr14/Tyr15. This helps to reset the system until the next G1/S transition when, in response to transcriptional induction, Cdc25A protein starts to accumulate again.
Cdc25A and checkpoints
The ability of cells to respond to DNA damage involves a complex network of mechanisms that have evolved to maintain the integrity of genetic information. The arrest of the cell cycle and the activation of DNA repair networks minimize the chance of transmitting mutations to the cells' progeny. In eukaryotic cells, the upstream factors responsible for initiating a checkpoint response are the ATM and ATR protein kinases. These two enzymes are key components of the DNA damage response that phosphorylate the Chk1 and Chk2 protein kinases. This ATM/ATR–Chk1/Chk2 pathway is ubiquitous to many cell types and cell-cycle phases. However, its downstream checkpoint effectors differ among the various cell-cycle phases and cell types. This results in a broad range of substrates acting at multiple cell-cycle levels in the checkpoint response (Falck et al., 2001; see, for a review, Zhou and Elledge, 2000). Here, we will consider the role of this pathway in regulating Cdc25A turnover in unperturbed cells and in cells exposed to DNA damage during S and G2 phases.
Cdc25A is rapidly degraded in response to DNA damage or stalled replication and is known to be a crucial substrate in the checkpoint response (Mailand et al., 2000; Molinari et al., 2000). Ultraviolet (UV) and ionizing radiation (IR) treatments are known to rapidly activate the ATM/ATR–Chk1/2 pathway (Mailand et al., 2000; Falck et al., 2001), leading to phosphorylation of Cdc25A and triggering the signal for its degradation by the proteasome (Mailand et al., 2000). This has been proposed to result in the inactivation of the Cdk2/cyclin E complex that involves a DNA replication arrest.
The induction of Cdc25A phosphorylation and degradation represents a rapid cellular response that imposes a DNA synthesis block prior to the activation of the p53–p21 pathway, which ensures a more sustained proliferation arrest (Di Leonardo et al., 1994). Chk1 is a labile protein whose activity is restricted to S and G2 phases (Kaneko et al., 1999; Lukas et al., 2001) even in the absence of DNA damage (Zhao et al., 2002; Sorensen et al., 2003). UV and hydroxyurea (HU) treatment causes phosphorylation of Chk1 on Ser 345, and its consequent activation. IR also appear to stimulate Chk1 phosphorylation and activation, but to a lesser extent (Liu et al., 2000). Contrary to Chk1, Chk2 is apparently only active in cells exposed to DNA damage (UV, HU and IR) through ATM-mediated phosphorylation (on Thr 68) (Melchionna et al., 2000).
Chk1 phosphorylates Cdc25A during S phase and G2, contributing to its rapid turnover in unperturbed cells (Zhao et al., 2002; Xiao et al., 2003). Cdc25A turnover requires phosphorylation by Chkl on four different residues (Ser 124, 178, 279, 293) (Falck et al., 2001; Sorensen et al., 2003). The basal turnover of Cdc25A is accelerated upon IR treatment, when an increase in phosphorylation at Ser 124 is detected. Moreover, Sorensen et al. (2003) showed that IR-induced activation of Chk2 cooperates with Chk1 in phosphorylating Cdc25A on serine 124, 178, 293 (Figure 1 and Table 1). The contribution of Chk2 to the maintenance of DNA-damage response is partially redundant with Chk1, since genetic and chemical ablation of Chk1 is sufficient to cause radioresistant DNA synthesis (RDS). On the other hand, Chk1 is an essential gene whose loss cannot be complemented by overexpressing Chk2, and while Chk1 ablation is lethal for embryonic development (Liu et al., 2000; Takai et al., 2000), Chk2 remains dispensable for it. Consistent with this, the activation of Chk1 in response to DNA damage or to stalled DNA replication is thought to be sufficient for Cdc25A degradation (see, for a review, Zhou and Elledge, 2000).
Interestingly, Shimuta et al. (2002) showed that Cdc25A in Xenopus is phosphorylated on Ser 73 (Ser 76 in human Cdc25A) by an unknown kinase. This phosphorylation is required for Chk1-induced degradation of Cdc25A and primes Cdc25A for degradation both in a normal cell cycle and in response to DNA damage. Indeed, the homologous serine in human Cdc25A (Ser 76) was found by Hassepass et al. (2003) to be a Chk1 phosphorylation site in vitro. The same group demonstrated that Ser 76 mediates Cdc25A stabilization in response to UV-induced DNA damage. Moreover, Goloudina et al. (2003), using phosphosite-specific antibodies, showed that Chk1 and p38 kinase can phosphorylate both Ser 76 and 124, upon UV-mediated DNA damage and osmotic stress, respectively (Figure 1 and Table 1). However, mutations at these sites did not result in an RDS phenotype, raising the question as to whether other mechanisms acting on Cdc25A or related pathways contribute to the S-phase checkpoint.
As mentioned above, the SCFβTrCP complex contributes to Cdc25A ubiquitylation. The SCF complex is a multisubunit E3 ubiquitin ligase. Its core components consist of the Cul1, Skp1 and F-box proteins (Figure 2b). Cul1 functions as a scaffold protein that enables the correct positioning of an E2 (ubiquitin-conjugating enzyme) for efficient substrate ubiquitylation (Zheng et al., 2002). The E2 interacts with SCF through Roc1, which in turn associates with Cul1 (at the C-terminus). The N-terminus of Cul1 interacts with Skp1 that is bound to the F-box protein through a conserved domain of 40 amino acids (F-box domain). Proteins containing an F-box motif specifically recruit SCF to the substrate enabling its ubiquitylation by the E2 enzyme (Zheng et al., 2002). Yeast two-hybrid screen using Skp1 as a bait, as well as database searches revealed that there are at least 50 mammalian F-box proteins. The F-box proteins are divided in three subfamilies, those containing WD-40 domains (Fbws), those containing leucine-rich repeats (Fbls), and the remaining ones that lack any known protein-interaction domains (Fbxs) (Cenciarelli et al., 1999; Winston et al., 1999). There are known examples of SCF involvement in regulating cell-cycle events. For example, at the G1/S transition p27, when associated with Cdk2/cyclin E, is phosphorylated on Thr 187 and targeted for ubiquitin-dependent degradation by SCFSkp2 (Carrano et al., 1999; Tsvetkov et al., 1999). In this way Cdk2/cyclin E is fully active and facilitates S phase entry. Subsequently, cyclin E is phosphorylated by its associated Cdk and is targeted for ubiquitylation by SCFCdc4 (Koepp et al., 2001; Nash et al., 2001; Strohmaier et al., 2001). In mitotic cells SCFβTrCP was recently showed to degrade Emi1 (Guardavaccaro et al., 2003; Margottin-Goguet et al., 2003; Peters, 2003), an antagonist of APC/CCdc20 and APC/CCdh1 activity (Reimann et al., 2001). This event prompts the APC/C ubiquitin ligase activation and leads to mitotic progression.
Recent data suggest that SCF complex has additional roles in regulating the checkpoint response in DNA-damaged cells. Bendjennat et al. (2003) defined a novel pathway that leads to UV-induced p21 ubiquitylation and degradation. Indeed, SCFSkp2 ubiquitylate p21 only upon low doses of UV irradiation. Thus, during DNA damage-induced checkpoint response, p21 processing switches from an ubiquitin-independent to an ubiquitin-dependent proteolytic degradation. In addition, like Cdc25A, p21 is targeted by the ATM/ATR–Chk1/Chk2 pathway in a p53-independent manner. This p21 degradation is essential for PCNA recruitment to chromatin and consequently for DNA repair.
Furthermore, our group recently identified Cdc25A as an SCFβTrCP substrate, highlighting a crucial role for βTrCP in mediating the intra-S-phase checkpoint response (Busino et al., 2003).
A model of SCF-induced degradation of Cdc25A
βTrCP binds Cdc25A through a conserved DSG containing motif (DS82GXXXXS88). Phosphorylation at Ser 82 and at Ser 88 is required for efficient SCFβTrCP binding in vivo and in vitro. This phosphorylated consensus motif represents Cdc25A phosphodegron and is part of phosphorylation cluster I (Figure 3 and Table 1). Mutations at these sites affect protein degradation as well as βTrCP-induced ubiquitylation. In addition, βTrCP-depleted cells accumulate Cdc25A as they progress through the S and G2 phases. As described above, Cdc25A is targeted by checkpoint activation in S and G2, and its degradation is critical for blocking cell-cycle progression. Consistent with this, in βTrCP-depleted cells Cdc25A is not degraded upon IR. Notably, IR treatment results in an accumulation of slow-migrating, hyperphosphorylated, Cdc25A species. We also found that βTrCP depletion affects the integrity of the intra-S-phase checkpoint, resulting in an RDS phenotype. Using a phospho-specific antibody raised against the phosphorylated Ser 82 and Ser 88, we have been able to show that the DSG motif is hyperphosphorylated upon DNA damage. Moreover, the fact that a Cdc25A Ser82Ala and Ser88Ala mutant, but not a KEN-box mutant (that is not recognized by Cdh1), are not degraded upon IR treatment clearly suggests that βTrCP, but not Cdh1, is the ligase involved in inducing an acceleration of Cdc25A degradation upon DNA damage.
These new findings raise additional important questions that will need to be addressed in the future. What are the phosphorylation events that timely trigger Cdc25A to degradation? It appears that phosphorylation of Cdc25A at multiple serine clusters is essential for its efficient processing in vivo. We will discuss below two examples recently reported in literature, and propose a molecular model for Cdc25A degradation.
Sic1 is a stable G1 protein that complexes with Cdk and promotes Clb (B-type cyclin) degradation in budding yeast. Its degradation is required for the G1/S transition. Upon phosphorylation by Cln–Cdk, Sic1 is recruited by SCFcdc4 and targeted to ubiquitin-dependent degradation. (Feldman et al., 1997; Skowyra et al., 1997; Verma et al., 1997). Briefly, the presence in Sic1 of six out nine phosphorylation sites of sets a threshold that mediates the binding with SCFCdc4 (Nash et al., 2001). These are multiple low-affinity phosphorylation sites that once at a time engage a single Cdc4 binding site in a dynamic equilibrium (Orlicky et al., 2003). The overall stoichiometry of phosphorylation allows a high probability of binding with no absolute requirement for any of the phosphorylation events.
A different model for SCF-induced ubiquitylation comes from β-catenin. Briefly, CKI-alpha, GSK3 and β-catenin bind different domains of Axin. When the complex is formed CKI-alpha phosphorylates β-catenin on Ser 45. This is the primary event that triggers the subsequent phosphorylation of β-catenin on Thr 41, Ser 37 and Ser 33 by GSK3 protein kinase. Indeed, phosphorylation on Ser 37 and Ser 33 creates a recognition site for βTrCP since mutations of these serines (Ser37Ala and Ser33Ala) abolish βTrCP binding. Mutations that affect the priming site (Ser 45) abolish the subsequent GSK3 phosphorylation resulting in the loss of SCF binding (Liu et al., 2002). According to this ‘Zip’ model, both kind of serines, those involved in the physical βTrCP binding and those that prime the phosphorylation of βTrCP binding motif, are essential for the recruitment of the ligase. We have demonstrated that the Cdc25A DSG motif is sufficient to recruit βTrCP, in agreement with the structure of SCFβTrCP/β-catenin recently published (Wu et al., 2003). Moreover, the finding that mutation in the upstream Chk1 site (Ser 76) results in the loss of interaction between Cdc25A and SCFβTrCP (our unpublished results) seems to approach Cdc25A to β-catenin degradation mechanism. As in the case of β-catenin, phosphorylation events on a single cluster of Ser/Thr residues appears to be required to allow βTrCP binding. We identify two clusters of phosphorylated residues (Figure 2). Cluster I serines are absolutely required for efficient binding to the SCFβTrCP, while none of cluster II phosphorylation sites are involved in binding these SCF components (Busino et al., 2003). This would suggest that the Sic1 degradation model is not applicable to Cdc25A protein.
A tentative model is illustrated in Figure 3. The coordinated activity of Chk1 and a still to be identified kinase determines phosphorylation on Ser 76 and on the DSG motif. These two events are essential for SCFβTrCP binding and for the subsequent ubiquitylation reactions. Furthermore, given that upon DNA damage the rate of phosphate incorporation, both on Ser 76 (Goloudina et al., 2003) and on the DSG motif (Busino et al., 2003), increases is in agreement with an accelerated SCFβTrCP-mediated proteolysis of Cdc25A during checkpoint responses.
Many questions still remain unsolved. Which is the kinase responsible of Cdc25A DSG motif phosphorylation? Does Chk1 prime phosphorylation at the DSG motif? More importantly why should Cdc25A be phosphorylated at additional sites by Chk1/2 as shown by Sorensen et al. (2003)? A possibility is that multiple phosphorylation events may influence Cdc25A folding to enhance the βTrCP ubiquitylation kinetics. A second possibility is that phosphorylation at additional sites may promote the interaction with proteins other than βTrCP that stimulate its ubiquitylation activity. A third scenario is that multiple clusters of phosphorylation drive the recruitment of different ligases. Future studies using in vitro reconstituted ubiquitylation may help to resolve this issue.
Finally, what is the physiological role of Cdc25A multiple phosphorylation? The obvious advantage of using multiple phosphorylation events, multiple kinases activity and multiple degradation mechanisms is to guarantee a flexible regulation of protein levels to impose different thresholds of signaling activity in response to the environment. These observations also suggest that multiple ways of regulating Cdc25A turnover could be affected during tumorigenesis.
Cdc25A in cancer
Cdc25A is thought to be a proto-oncogene. This is based on its capacity to transform MEFs in cooperation with Ha-Ras and loss of pRB (Galaktionov et al., 1995). In addition, several studies describe Cdc25A overexpression in many cancers, both at the mRNA and protein levels (Broggini et al., 2000; Cangi et al., 2000; Hernandez et al., 2001; Ito et al., 2002; Xu et al., 2003).
This deregulated expression may be due to anomalous E2F1/c-Myc transcriptional activity or alternatively to a reduced rate of protein degradation. Actually, deregulations at different levels of the degradation pathway of Cdc25A, both constitutive and damage induced, for example, affecting Chk1/Chk2 activities (Bartek and Lukas, 2003), can lead to a substantial accumulation of the protein, thus resulting in genomic instability and cancer predisposition. Mutations in the ubiquitin ligases that mediate Cdc25A degradation may result in increased protein levels as well, as described for cyclin E (Strohmaier et al., 2001).
Last but not least, Cdc25A could itself be mutated in specific consensus sites that mediate its degradation. This would result once again in protein stabilization. In support of this hypothesis, it has been recently reported the identification of a mutation in the DSG motif of a Caenorhabditis elegans cdc25 gene that results in a gain-of-function allele associated with deregulated proliferation of intestinal cells (Clucas et al., 2002). This scenario would be therefore similar to the one already described in several human cancers and cancer cell lines that show mutations of the proto-oncogene β-catenin in different residues of its DSGXXS βTrCP binding motif (reviewed by Polakis, 1999). Finally, due to its importance in the regulation of key components of the cell-cycle machinery (Cdc25A, Emi1, β-catenin) an in-depth analysis of βTrCP in tumors both at the genetic and functional level might offer important clues.
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We thank CL Attwooll and AP Bracken for critically reading the manuscript. Work in the authors' laboratory is supported by grants from AIRC, FIRC and Telethon.
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Busino, L., Chiesa, M., Draetta, G. et al. Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis. Oncogene 23, 2050–2056 (2004). https://doi.org/10.1038/sj.onc.1207394
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