Summary of known phosphorylation sites on Cdc25 family members and the kinases responsible
Dual specificity phosphatases of the Cdc25 family are critically important regulators of the cell cycle. They activate cyclin-dependent kinases (CDKs) at key cell cycle transitions such as the initiation of DNA synthesis and mitosis. They also represent key points of regulation for pathways monitoring DNA integrity, DNA replication, growth factor signaling and extracellular stress. Since their mis-regulation allows cells to function in a genetically unstable state, it is not surprising that these phosphatases are involved in transformation to a cancerous state. Cdc25 phosphatases are heavily regulated by phosphorylation. Many regulatory phosphorylation sites on Cdc25 influence catalytic activity, substrate specificity, subcellular localization and stability. This chapter summarizes the current literature on the phospho-regulation of these proteins.
2. Yeast genetics and Xenopus oocyte maturation – Setting the stage
The study of cell division in eukaryotes was dramatically changed with the isolation of temperature sensitive “cell division cycle” (
Concurrently, Maturation Promoting Factor (MPF) was discovered through a series of elegant cytoplasmic transfer experiments conducted on frog, starfish and sea urchin oocytes [8-10]. Immature
The cytoplasmic transfer experiments showed that MPF activity correlates with increased protein phosphorylation in donor oocytes , targeting a large number of nuclear proteins [27,28]. MPF is a histone H1 kinase, an activity which is still used today to measure CDK function . MPF induces many of the cytological changes associated with mitosis such as nuclear envelope breakdown, chromosome condensation and mitotic spindle formation [30-32]. Mass spectrometry has shown that the single Cdk1 homologue in budding yeast (CDC28) phosphorylates 547 sites on 308 proteins
3. Taxonomic distribution, duplication and divergence of Cdc25 homologues
Cdc25 is present in all eukaryotic cells. The yeasts,
4. Regulation of cell cycle transitions by Cdc25
4.1. The fission yeast G2/M transition – The prototype Cdc25/cyclin-CDK circuit
Regulation of the transition from G2 to mitosis in
4.2. Vertebrate cell cycle
While fission yeast Cdc25 is solely involved in the G2/M transition, Cdc25 orthologues in vertebrates also play a role in G1 and S-phase progression. Vertebrates have several CDK-cyclin complexes which participate in these transitions through positive feedback loops with Cdc25. In addition to associating with cyclin B, Cdk1 associates with cyclin A during late S-phase and G2 . A second CDK, Cdk2, was discovered as a cDNA which could complement the loss of the budding yeast Cdk1 homologue, CDC28 . Cdk2 functions early in the cell cycle and is likewise negatively regulated by phosphorylation of Y15 [78,79]. It forms a complex with Cyclin A and Cyclin E [80,81]. Cdk4 and Cdk6 operate early in G1 in association with D-type cyclins . In many cases a particular cyclin class (ie. A, B, D, E) has multiple members. For the sake of clarity, cyclins will be referred to by their subtype only. Furthermore, only the CDKs and cyclins directly responsible for cell cycle transitions in concert with Cdc25 orthologues will be discussed. For instance, Cdk1/cyclin B is activated by a CDK-Activating Kinase (CAK), a complex of Cdk7 and cyclin H , but as CAKs are not activated by Cdc25 they are outside the scope of this review.
4.2.1. Cell cycle re-entry from Go
Most somatic cells spend their time in Go. Cells in Go may commit to entry into the cell cycle when they receive stimuli in the form of growth factors The cells then deactivate the cell cycle repressors which have kept them in Go and transcribe the positive regulators of the next cell cycle transition, G1/S.
In non-dividing cells, the Retinoblastoma protein (Rb) binds to E2F thus preventing transcription of genes required for cell cycle progression including cyclin D, cyclin A and Cdc25A [84-87]. (Figure 1) After exposure to growth factors, cells in Go re-enter the cell cycle through activation of the Ras pathway via the Raf/MAP (Mitogen Activated Protein) kinase pathway . This leads to the degradation of the Cdk4 inhibitors p15INK4B and p16INK4A and the Cdk2 inhibitors p27KIP, p21CIP and induction of cyclin D. Cdk4 and Cdk6 bound to cyclin D inhibit the Rb protein  allowing transcription of cyclin E and cyclin A [89,90]. Cdc25A activates Cdk4-cyclin D but not Cdk6-cyclin D
4.2.2. The G1/S transition
Cdc25A is transcribed following relief of Rb-mediated transcriptional repression, reaching its maximal level at the end of G1 and dephosphorylating Y15 on Cdk2 [87,91]. Cdk2 is phosphorylated on Y15 by Wee1 . Dephosphorylation of Cdk2 takes place via both Cdc25A and Cdc25B [94-97]. Cdk2 immunoprecipitated from cell lysates where Cdc25A has been overexpressed has high histone H1 kinase activity and low levels of Y15 phosphorylation . Such overexpression accelerates entry into S-phase through activation of Cdk2-cyclin E [91,98]. DNA-synthesis can be blocked in these cells by injecting them with anti-Cdc25A antibodies . Cdk2-cyclin E and Cdc25A are mutually activated by a positive feedback loop allowing passage of the G1/S boundary . Depletion of Cdk2 or cyclin E prevents phosphorylation of Cdc25A and recombinant Cdc25A can be activated by Cdk2-cyclin E
S-phase initiation requires activation of DNA replication proteins by Cdk2-cyclin A. Injecting G1 cells with anti-cyclin A antibodies stops entry into S-phase . Phosphorylation of the essential DNA replication initiator Cdc6 by Cdk2-cyclin A leads to its nuclear import [101,102]. Cdk2 is recruited to chromatin by the replication initiation factor Cdc45 where it phosphorylates histone H1 and induces chromosome de-condensation . As S-phase progresses high Cdk2-cyclin A activity induces degradation of Cdc6, preventing re-initiation of DNA synthesis at origins which have already fired .
Cdc25B has a role late in S-phase as Cdc25B immunoprecipitated from late S-phase HeLa cell extracts is phosphorylated, activated, and able to dephosphorylate Cdk2-cyclin A . The murine homologue of Cdc25B purified from S-phase extracts promotes cyclin A and cyclin E associated histone H1 kinase activity
4.2.3. The G2/M transition
Unlike the simple circuit of Cdc25-Cdc2 activation in fission yeast, the vertebrate G2/M transition involves a series of interconnected loops with positive and negative inputs from a variety of pathways. This led to what could be considered the “traditional model” of G2/M transition in vertebrates with respect to Cdc25 regulation by CDK-cyclin complexes. In reality things may be more complex. Cdc25C is not explicitly required for mitotic entry; siRNA knockdown of Cdc25C does not prevent the G2/M transition . In addition, mouse lines which lack Cdc25B and/or Cdc25C are viable with the only obvious phenotype being a defect in oocyte maturation observed in
4.2.4. Activation of Cdc25B and Cdk1/2-cyclinA
Phosphorylation of human Cdc25B by Cdk1-cyclin A during G2 causes Cdc25B activation but also destabilizes the protein [112,113]. Cdc25B activates Cdk2-cyclinA in a positive feedback loop . Cdk2-cyclin A mediated destabilization of Cdc25B has not been reported, although the Cdk1 and Cdk2 kinase complexes modify Cdc25B to approximately the same degree and have a similar set of substrates [113,115]. However, Cdk2 is the preferred binding partner of cyclin A and is more active than Cdk1-cyclin A during G2 [80,100]. Cdk2 cyclin A has two peaks of activation, one during S-phase and one prior to G2/M. [80,116]. Inhibition of Cdk2-cyclin A delays mitotic entry . Depletion of Cdk1-cyclin A, activation of the CDK inhibitor p21cip, or addition of an inhibitory ATP analogue destabilizes Cdc25B in
Cyclin B accumulates throughout G2 but Cdc25A and Cdc25B are required to induce formation of the Cdk1-cyclin B complex at G2/M . Cdk1-cyclin B interaction occurs earlier in G2 when Cdc25A or Cdc25B are overexpressed. Cdk1 and cyclin B are almost exclusively cytoplasmic during interphase with a small portion of the complex associating with the centrosome at G2/M [119-121]. A population of Cdk2-cyclinA is likewise localized to the centrosomes prior to prophase . In
4.2.5. Everybody into the nucleus
Cyclin B has a cytoplasmic retention sequence which is sufficient to induce cytoplasmic localization of the normally nuclear protein and contains a nuclear export signal (NES) . Nuclear export is blocked by phosphorylation of S126 at the end of prophase [127-129]. Cyclin B is phosphorylated by Cdk1-Cyclin B as starfish oocytes pass the prophase II to metaphase II arrest . Human cyclin B S126 is followed by a proline residue suggesting Cdk1-cyclin B autophosphorylation. Cyclin B is phosphorylated by the
The activated cytoplasmic pool of Cdk1-cyclin B phosphorylates Cdc25B on S160 inducing its nuclear import  (All phosphorylated residues on human Cdc25B are numbered according to the sequence of the longest splice variant Cdc25B3). Overexpression of human Cdc25B causes an increase in cells with condensed chromatin, whereas overexpression of Cdc25B-S160G does not induce mitotic entry. S160 phosphorylation does not affect the
Plk1 is involved in human Cdc25B nuclear import following the initial activation of Cdk1-cyclin B. After addition of the Plk1 inhibitor thiophene benzimidazole, nuclear accumulation of GFP-Cdc25B is reduced. Conversely, expression of a constitutively active Plk1 mutant enhances Cdc25 nuclear localization . Co-overexpression of Plk1 and Cdc25B in U2OS osteosarcoma cells induces chromosome condensation to a greater degree compared to cells expressing Plk1 or Cdc25B alone. This is partially dependent on the presence of a functional nuclear localization signal (NLS) in Cdc25B. Plk1 docking to Cdc25B requires prior phosphorylation of S50 by Cdk1/cyclin B . Mass spectrometry identified thirteen phosphorylated Plk1 sites on Cdc25B
4.2.6. Full activation of Cdk1-CyclinB and Cdc25C
Activated Cdk1-cyclin B phosphorylates human Cdc25C on T48, T67, S122, T130, S168 and S214
4.2.7. Mitotic exit
Following chromosome alignment on the metaphase plate a cascade of APC mediated degradation events occurs to reset conditions for the start of the next cell cycle . The APC regulates two important processes required for completion of mitosis. First, it targets Securin, the inhibitory subunit of Separase, which is responsible for Cohesin cleavage and chromosome separation. Second, it targets cyclin A and cyclin B for destruction, inactivating Cdk1. Cyclin B is degraded after the metaphase-anaphase transition while cyclin A is degraded during metaphase. . Cdk1 mediated phosphorylation of Cdc25 paralogues is reversed by Cdc14 family phosphatases. Cdk1-cyclin B phosphorylates human Cdc25A S18, S40, S88, S116, S261 and S283
5. Cdc25 phosphorylation by the DNA damage and replication checkpoint
DNA damage causes activation of checkpoints which delay cell cycle transitions to allow sufficient time for repair. Stalling of replication forks causes a similar cell cycle arrest, with additional need for stabilizing replication forks and/or modulating replication origin firing until the cause of the stalling is eliminated. Checkpoint effector kinases impinge on the central cell cycle machinery by phosphorylating Cdc25. This modification variously inhibits Cdc25 phosphatase activity, induces degradation or creates binding sites for 14-3-3 proteins which modify localization of the protein.
5.1. Fission yeast
Cell cycle arrest following DNA damage requires that Cdc2 is kept in a Y15- phosphorylated, inhibited state . Cdc25 is inhibited through phosphorylation by Chk1 and Cds1 kinases in response to DNA damage and replication fork arrest, respectively [155-157]. Cells over-expressing Cdc25 or expressing a Y15F phospho-mimetic mutation of Cdc2 fail to arrest cell cycle progression after exposure to ionizing radiation . In the absence of Cds1, Chk1 can cause cell cycle arrest following stalling of replication forks by hydroxyurea (HU) exposure. Cells lacking both kinases are unable to arrest . Cds1 also phosphorylates multiple substrates to stabilize stalled replication forks and prevent the occurrence of inappropriate recombination events . Upstream regulation of the DNA damage and replication checkpoint pathway occurs through activation of the ATM (Ataxia-telangiectasia mutated) homologue Rad3 through a well conserved signaling cascade .
Phosphorylation of Cdc25 by Chk1 and Cds1 creates binding sites for the 14-3-3 homologues Rad24 and Rad25 [160,161]. Phosphorylation and 14-3-3 binding stabilizes Cdc25, a phenomena referred to as “stockpiling”, thought to allow the cell to rapidly re-enter the cell cycle once replication or DNA damage arrest has been lifted . The first Chk1/Cds1 phosphorylated Cdc25 residue identified, S99, partially impairs the replication and DNA damage checkpoint when mutated to alanine . S99 modified Cdc25 is also phosphorylated on S192 and S359 by Cds1 and Chk1
5.2. Vertebrate Cdc25 regulation by DNA damage and replication checkpoints
In vertebrate cells detection of DNA damage is relayed through ATM and ATR (ATM-Related) to two checkpoint effectors, Chk1 and Chk2. While the
5.2.1. G1/S and Intra-S checkpoints
The G1/S checkpoint prevents the start of DNA synthesis in the presence of DNA damage while the Intra-S checkpoint protects replication forks, prevents activation of late replication origins, and keeps the cell from entering mitosis until S-phase is completed. G1-S checkpoint arrest is manifested through inhibition of Cdc25A, thus preventing activation of Cdk4-cyclin D and Cdk2-cyclin E. The checkpoint also activates p53 resulting in the induction of the Cdk2 inhibitor p21CIP and the targeting of Cyclin E to the SCF complex to reinforce Cdk2 inhibition . In rat fibroblasts UV induced DNA damage during G1 results in cell cycle arrest at the G1/S transition requiring inhibition of Cdc25A and phosphorylation of Cdk4-Y14 . In U2OS osteosarcoma cells Cdk2-cyclin E kinase activity decreases and Y15 phosphorylation increases coincident with Cdc25A degradation following UV exposure . Conversely, Cdc25A overexpression in UV exposed U2OS osteosarcoma cells results in bypass of the checkpoint and dephosphorylation of Cdk2-cyclin E. Cdc25A inhibition involves a combination of destabilization and inhibition of phosphatase activity by Chk1 . Treatment with caffeine (an ATM/ATR inhibitor) or the Chk1 inhibitor UNC-01, or depletion of Chk1, stabilizes the phosphatase [167,168]. In humans, Chk1 phosphorylates Cdc25A S76, S124, S178, and T507 [168-170]. Cdc25A catalytic activity is reduced three-fold when it is phosphorylated by hChk1
Human Cdc25A S76, S124 and/or S178 are identified in several publications as “S75, S123 and S177,” respectively [168,169,174-176]. In the original cloning of human Cdc25A , there are several substitutions in the N-terminus (Accession: AAA58415.1) and a one residue gap corresponding to residue R12 in all other full length human Cdc25A sequences in the NCBI database (Accession: P30304). Residues have been re-numbered as per “P30304” for the sake of consistency.
Cdc25A S76, S124, S178 and T507 match the consensus site for 14-3-3 binding, RXXpS/T . However, only the Chk1 dependent phosphorylation of S178 and T507 results in association with 14-3-3 . Substitution of these residues to alanine results in a complete loss of 14-3-3 interaction
Defects in the Intra-S checkpoint allow replication of damaged DNA [173,176,181]. In mammalian cells, DNA damage results in destabilization of Cdc25A and inhibition of Cdk2 . Cdk2 is involved in loading the Cdc45 origin binding factor. Inhibition of Cdc25A stops further origin firing once DNA damage is detected . Cdc25A is also unstable after HU induced replication fork stalling, which unlike in fission yeast, is controlled by activation of Chk1 in mammalian cells .
The mechanism by which Cdc25A is destabilized following DNA structure checkpoint activation is well understood (Figure 3). Human Cdc25A phosphorylation by Chk1 following S-phase DNA damage causes degradation by F-box protein β-TrCP associated with the Skp1-cullin-Fbox (SCF) complex [171,184]. Mutating the destruction box or KEN box (APC interaction motifs) of Cdc25A does not stabilize the protein following exposure to ionizing radiation . This indicates that SCF-mediated degradation of Cdc25A after DNA damage is independent of the cell cycle regulated APC-mediated degradation which occurs at mitotic exit. β-TrCP recognizes a degron motif of DSG(X)4S where both serine residues are phosphorylated . siRNA knockdown of β-TrCP causes stabilization of Cdc25A, and radiation-resistant DNA synthesis in cells exposed to ionizing radiation [171,184]. Human Cdc25A contains such a motif: DS82GFCLDS88. The S88A substitution does not stabilize the phosphatase, suggesting that S88 phosphorylation is not explicitly required for β-TrCP binding to Cdc25A . Following ionizing radiation, Chk1 primes Cdc25A for destruction through phosphorylation of S76 [171,186]. The NimA-related NEK11 kinase targets Cdc25A S82 and S88, within the DSG degron sequence . Depletion of NEK11 causes a marked decrease in S82 and S88 phosphorylation
Following ATM activation, Chk2 phosphorylates and activates the oncogene p53, and inhibits its negative regulator MDM2 after ionizing radiation [188-191]. p53 then induces the Cdk2-cyclin E inhibitor p21WAF . p53 also activates Glycogen Synthase Kinase (GSK-3β), which phosphorylates human Cdc25A S76 following a priming phosphorylation of S80 which can be targeted
Cdc25A S76, S124, S178 and T507 match the consensus site for 14-3-3 binding RXXpS/T . However, only the Chk1 dependent phosphorylation of S178 and T507 results in association with 14-3-3 . Substitution of these residues to alanine results in a complete loss of 14-3-3 interaction
Following exposure to ionizing radiation during G1 phosphorylation of Cdc25A on S124 by Chk2 prevents entry to S-phase . Chk2 cannot efficiently phosphorylate Cdc25A on S76 and so cannot induce Cdc25A degradation by the β-TrCP route . The mechanism by which S124 phosphorylation induces Cdc25A degradation is not clear because it is not required for degradation via β-TrCP . S124 conforms to a 14-3-3 phospho-serine binding site, but doesn’t bind 14-3-3 . In contrast to the effects of Cdc25A phosphorylation sites discussed thus far, modification of
5.2.2. G2/M DNA damage checkpoint
The response to damage in G2 is dependent on the nature of the damage signal. Exposure to UV activates p38 MAP kinase and checkpoint arrest is independent of ATM and ATR since the arrest is not caffeine sensitive . The primary target following UV irradiation is Cdc25B and although p38 can phosphorylate Cdc25C
A number of conflicting reports have appeared relating to Cdc25B regulation following UV exposure. Some groups have reported that UV has no effect on Cdc25B protein levels [208,210], but others have shown that UV causes either MAP kinase mediated Cdc25B degradation or Cdc25B accumulation [205,208,211]. Cell line specific effects have no doubt contributed to these inconsistencies as human molecular biology relies heavily on transformed cell lines and mis-regulation of Cdc25B is a common phenomenon in tumors . Cdc25B isolated from UV irradiated A2058 melanoma cells still retains a substantial portion of its Y15 phosphatase activity and is localized to the nucleus . In HeLa cells Cdc25B is localized almost exclusively to the cytoplasm as detected by cell fractionation and immunofluorescence . Variation in the apparatus used for UV irradiation could also have contributed to contradictory accounts of Cdc25B regulation. A recent re-examination of the effect of UV on Cdc25B showed that after 10 J/m2 exposure, Cdc25B levels did not decrease, although following 60 J/m2 Cdc25B was clearly downregulated . Exposure of U2OS osteosarcoma cells to 10 J/m2 UV leads to Cdc25B nuclear export. Based on chemical inhibitor experiments Cdc25B downregulation is not mediated by ATM/ATR, p38 MAPK or JNK, but rather following 60 J/m2, by inhibiting Cdc25 translation. The eukaryotic initiation factor regulating Cdc25B expression, eIF2α, is phosphorylated and inhibited following UV exposure . UV mediated DNA damage during G2 involves human Cdc25B S323 phosphorylation through the p38 kinase during interphase . ATM/ATR inhibitor caffeine and the Chk1 inhibitor UNC-01 have no effect on UV mediated checkpoint arrest. Isoforms 14-3-3β and 14-3-3ε bind preferentially to Cdc25B phosphorylated S323, allowing its nuclear export . Nuclear export of Cdc25B is abolished in cells expressing the Cdc25B S323A substitution, regardless of which 14-3-3 isoform is co-expressed . Two amino-truncated Cdc25B isoforms localize to the nucleus
Cdk1 activation is prevented by UV induced checkpoint activity coincident with the appearance of a phosphorylated form of Cdc25C . In contrast with fission yeast Cdc25, which gradually accumulates in the nucleus during G2, human Cdc25C is primarily localized to the cytoplasm during interphase and only enters the nucleus at mitotic entry . Thus, nuclear export of Cdc25C is not a requirement for G2 DNA damage response, since the phosphatase is already cytoplasmic at this time. However, exposure to ionizing radiation decreases the enzymatic activity of human Cdc25C . Several research groups showed relatively early in the Cdc25 phosphorylation story that residue S216 is phosphorylated by Chk1 [219-221] and Chk2
Although Cdc25C S216A is a relatively poor substrate for Chk1 compared to the wildtype protein, Chk1 can still execute some degree of phosphorylation on the mutant phosphatase . This observation suggests the possibility that additional Cdc25C phosphorylation negatively regulates its enzymatic activity. Recent bioinformatics approaches to generate profiles from peptide binding arrays based on three diverse 14-3-3 binding sites have generated an improved 14-3-3 binding motif consensus . This helped to identify two additional phosphorylated Cdc25C residues, Ser247 and Ser263, which interact with 14-3-3. Mutation of either residue to alanine reduces 14-3-3 binding, but neither of these mutant peptides was affected by Cdc25 S216 phosphorylation when expressed in cells. S263 was previously identified in an isolated report which showed phosphorylation of this residue induces Cdc25B nuclear export . Cdc25C purified from cells treated with the topoisomerase II inhibitor etoposide is phosphorylated on S263, but S263A substitution results in enhanced nuclear localization. The kinase targeting this residue has yet to be determined experimentally. However, the residues surrounding S263 (KKTVpSLCD) conform to a Chk1 consensus site as Chk1 can tolerate a lysine (K) at the -3 position relative to the phosphorylated serine or threonine
Cdc25A is considered to regulate the G1/S transition in the “Traditional Model” of the human cell cycle but it also has a significant role in mitotic entry. As such, Cdc25A is an important target of the DNA damage checkpoint. In fact, mice lacking Cdc25B and Cdc25C do not have a G2/M checkpoint defect [106,107]. Phosphorylation by Chk1 causes degradation of Cdc25A following DNA damage during G2 ionizing radiation and exposure to the DNA intercalating agent adriamycin [172,181].
5.3. Chk1 regulation of Cdc25 orthologues in unperturbed cell cycles
Cdc25A, B and C are all phosphorylated by Chk1 in the absence of externally induced DNA damage. As Cdc25 phosphatases are such potent positive regulators of cell cycle transitions it is perhaps not surprising that the cell maintains their activity at a low level until their precise point of activation. Chk1 regulates human Cdc25A stability during unperturbed cell cycles. Phosphorylation of S82 and S88 can be detected using phospho-specific antibodies in unperturbed cells . Depletion of the S82/S88 kinase NEK1 and S76 kinase CK1ε by siRNA, results in Cdc25A stabilization in the absence of DNA damage [187,199]. Cdc25A S124 phosphorylation by Chk1 also occurs in the absence of damage and destabilizes the phosphatase [168,169,200]. Inhibiting ATM/ATR, or a variety of upstream checkpoint components, also stabilizes in the absence of externally induced DNA damage which may indicate there is some basal level of spontaneous damage checkpoint signaling . Cdc25A is also phosphorylated by Casein kinase 2β (CK2β) in a damage independent manner . CK2 phosphorylates human Cdc25C T236 adjacent to the NLS
Human Cdc25B is phosphorylated
Chk1 phosphorylation of human Cdc25C and nuclear export by 14-3-3 binding keeps the phosphatase cytoplasmic during unperturbed cell cycles . Nuclear localization of Cdc25C(S216A) is enhanced, suggesting that part of the function of 14-3-3 binding is to obscure the NLS located adjacent to this residue . Overexpression of Cdc25C(S216A) induces a higher degree of premature mitotic entry .
Although Cdc25A and Cdc25B are dispensable for embryonic development, Chk1 and ATR kinases are essential [242,243]. The Cdc25B/14-3-3 interaction is important for maintaining G2 arrest, and inhibiting germinal vesicle breakdown in
5.4. Cdk1 phosphorylation of Cdc25 precludes checkpoint mediated inhibition during mitosis
If Cdc25C is phosphorylated and inactivated during interphase via 14-3-3 binding, how is it then activated at mitotic entry? Cdk1-cyclin B phosphorylation of Cdc25C causes 14-3-3 dissociation and allows removal of interphase phosphorylations. Re-phosphorylation of these residues is simultaneously blocked. Cdk1-cyclin B thus potentiates Cdc25C for its pro-mitotic function and ensures that it remains active. The region surrounding S216 in human Cdc25C and S287 in
PP1 phosphatase removes S287 phosphorylation once 14-3-3 has dissociated . Binding of PP1 to
There are no Cdc2 phosphorylation motifs (S/TP) directly upstream of any of the twelve Cds1
6. Cdc25 phosphorylation by MAP kinase cascades
In addition to regulation by DNA damage and DNA replication checkpoints, Cdc25 is the target of several MAPK cascades responding to stress and mitogenic signals. A detailed description of the variety of MAPK pathways is outside of the scope of this manuscript, but excellent reviews are available . In general, MAP kinase cascades involve the sequential activation of three kinases; a MAP kinase kinase kinase (MAPKKK) phosphorylates a MAP kinase kinase (MAPKK), which phosphorylates a MAP kinase (MAPK). There are three such cascades which are salient to our discussion of Cdc25 regulation.
The ERK1/ERK2 MAPKs are activated by Raf MAPKKKs working on MEK1/MEK2 MAPKKs. This cascade is primarily activated by extracellular signaling through receptor tyrosine kinases in response to mitogenic signals . During Cyto-Static Factor arrest in mature
6.2. p38 and JNK MAPKs
The p38 and JNK kinases activate in response to extracellular stimuli such as heatshock, oxidative stress, ionizing radiation, UV and growth factor deprivation. Both are activated by MAPKKs of the MKK family which are themselves activated by a large variety of MAPKKKs. We have already discussed the function of p38 in response to UV irradiation. p38 also phosphorylates human Cdc25A on S124 and S76 in response to osmotic stress, destabilizing the protein  and a 42 C heatshock causes p38 and Chk2 to phosphorylate S76 and S178 of human Cdc25A, respectively . MAPKAP kinase 2 (MK2) functions downstream of p38 and regulates G1 and S-phase cell cycle progression in response to UV . Downstream of p38, it phosphorylates RxxS/T motifs and activation of MK2 correlates with increased binding of 14-3-3 to Cdc25B. p38 and MK2 kinase form a tight complex and are imported into the nucleus together, so previous work showing that p38 directly phosphorylates S216 on Cdc25C and S323 on Cdc25B may in fact have been inadvertently monitoring MK2 activity . Cdc25A may also be an MK2 substrate as MK2 knockdowns ablate the G1 and S-phase checkpoints. JNK activity targets two serines within the region DAGLCMDS101PS103P of the DSG degron on human Cdc25B . Simultaneous S101A and S103A substitution prevents β-TrCP binding and Cdc25B ubiquitination. JNK also phosphorylates Cdc25C on S168 inhibiting its phosphatase activity [281,282]. This residue is transiently phosphorylated
S. pombestress activated MAP kinase pathway
In fission yeast, Cdc25 is phosphorylated by the CamKII homologue Srk1 in response to extracellular stress . Srk1 is activated downstream of the Spc1 MAPK, Wis1 MAPKK, and Win1 or Wak1MAPKKKs . This phosphorylation occurs on residues also targeted by Cds1 as Cdc25(9A) is not sensitive to Srk1 mediated inhibition . Srk1 phosphorylation of Cdc25 results in its nuclear export, similar to the response to DNA damage and replication arrest.
7. Other kinases which phosphorylate Cdc25s
PKA prevents oocyte maturation by inhibition of Polo kinase mediated Cdc25 activation, and deactivating the Mos/MEK/ERK MAP kinase cascade which inhibits Myt1 . Progesterone exposure in
Pim1 is a serine/threonine kinase induced by the SAT3 and SAT5 transcription factors following cytokine exposure thus linking pro-proliferative signals to the cell cycle control machinery . Pim1 phosphorylates and activates Cdc25A and represses the Cdk4/6 inhibitor p21CIP to encourage the G1/S transition [289,290]. Pim1 is also able to phosphorylate and inhibit the CamKII homologue c-TAK and accelerate Cdc25C mediated mitotic entry  PAR-1/MARK (partitioning-defective 1/Microtubule affinity-Regulating Kinase) protein homologue pEG3 phosphorylates human Cdc25A S263, Cdc25B S169 and Cdc25C S216 [292,293]. Overexpressing pEg3 results in G2 arrest which can be reversed by co-expressing Cdc25B . Anti-phospho PAR1 S169 antibodies stain spindle pole and centrosome in immunofluorescence experiments . In
8. Cdc25 and disease
Cdc25 orthologues are the subject of much attention as they are commonly upregulated in human tumors . This is perhaps not surprising considering the role of Cdc25 inhibition in maintaining genomic stability and the regulation of these phosphatases by Rb, p53 and a number of other oncogenes. Cdc25A and Cdc25B themselves are oncogenes in humans . Cdc25B is overexpressed in 32% of breast cancer tissue samples, and high Cdc25B levels correlate with high incidences of recurrence and decreased 10 year survival . Overexpression of Cdc25A is similarly linked to poor clinical outcome [296,297]. Cells bearing oncogenic mutations of myc have elevated Cdc25A and Cdc25B levels . Anti-Cdc25B autoantibody has been shown to be a predictor of poor prognosis in esophageal cancer patients . Overexpression of Cdc25B has recently been shown to cause a variety of S-phase effects including increased Cdc45 recruitment to chromatin, impairment of replication fork progression DNA damage and chromosome instability .
An interesting link between Cdc25 and disease comes from the finding that the HIV-1 protein vpr causes G2/M arrest . When expressed in
It has been more than thirty five years since Cdc25 was first isolated as an elongated temperature-sensitive fission yeast mutant and twenty one years since its biochemical function was determined. The field of cell cycle research and the study of Cdc25 in particular are extremely active with numerous new manuscripts appearing each year. This research has revealed that Cdc25 is one of the most intricately regulated proteins in the cell. Cdc25 accepts input from numerous pathways and checkpoints monitoring whether conditions inside and outside the cell are permissive for cell cycle progression. When conditions warrant caution, Cdc25 is inhibited by phosphorylation leading to alterations in its catalytic activity, cellular localization, substrate recognition and stability. When the green light is given Cdc25 participates in an intricate series of interconnected positive feedback loops with the beating heart of cell cycle regulation, the Cyclin-CDK complex. When the cell loses control of Cdc25 regulation, the results are deadly.
Thank you to Silja Freitag for her critical reading of this manuscript. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to PGY.