Protein kinase A regulates resumption of meiosis by phosphorylation of Cdc25B in mammalian oocytes

In mammalian oocytes, meiosis arrests at prophase I. Meiotic resumption requires activation of Maturation-Promoting Factor (MPF), comprised of a catalytic Cyclin-dependent kinase-1 (Cdk1) and a regulatory subunit cyclin B, and results in germinal vesicle breakdown (GVBD). Cyclic AMP (cAMP)-mediated Protein Kinase A (PKA) activity sustains prophase arrest by inhibiting Cdk1. However, the link between PKA activity and MPF inhibition remains unclear. Cdc25 phosphatases can activate Cdks by removing inhibitory phosphates from Cdks. Thus one method for sustaining prophase arrest could be inhibition of the activity of the Cdc25 protein required for MPF activation. Indeed, studies in Xenopus identify Cdc25C as a target of PKA activity in meiosis. However, in mice, studies suggest that Cdc25B is the phosphatase essential for GVBD and, therefore, the likely target of PKA activity. To assess these questions, we targeted a potential PKA substrate, a highly conserved serine 321 residue of Cdc25B and evaluated the effect on oocyte maturation. A Cdc25B-Ser321Ala point mutant mRNA induces GVBD when injected into prophase-arrested oocytes more rapidly than wild type mRNA. Using fluorescently-tagged proteins we also determined that the mutant protein enters the nucleus more rapidly than its wildtype counterpart. These data suggest that phosphorylation of the Ser321 residue plays a key role in the negative regulation and localization of Cdc25B during prophase arrest. PKA also phosphorylates a wildtype Cdc25B protein but not a Ser321Ala mutant protein in vitro. Mutation of Ser321 in Cdc25B also affects its association with a sequestering protein, 14-3-3. Our studies suggest that Cdc25B is a direct target of PKA in prophase-arrested oocytes and that Cdc25B phosphorylation results in its inhibition and sequestration by the 14-3-3 protein.     

Cdc14 phosphatases preferentially dephosphorylate a subset of cyclin-dependent kinase (Cdk) sites containing phosphoserine

Mitotic cell division is controlled by cyclin-dependent kinases (Cdks), which phosphorylate hundreds of protein substrates responsible for executing the division program. Cdk inactivation and reversal of Cdk-catalyzed phosphorylation are universal requirements for completing and exiting mitosis and resetting the cell cycle machinery. Mechanisms that define the timing and order of Cdk substrate dephosphorylation remain poorly understood. Cdc14 phosphatases have been implicated in Cdk inactivation and are thought to be generally specific for Cdk-type phosphorylation sites. We show that budding yeast Cdc14 possesses a strong and unusual preference for phosphoserine over phosphothreonine at Pro-directed sites in vitro. Using serine to threonine substitutions in the Cdk consensus sites of the Cdc14 substrate Acm1, we demonstrate that phosphoserine specificity exists in vivo. Furthermore, it appears to be a conserved property of all Cdc14 family phosphatases. An invariant active site residue was identified that sterically restricts phosphothreonine binding and is largely responsible for phosphoserine selectivity. Optimal Cdc14 substrates also possessed a basic residue at the +3 position relative to the phosphoserine, whereas substrates lacking this basic residue were not effectively hydrolyzed. The intrinsic selectivity of Cdc14 may help establish the order of Cdk substrate dephosphorylation during mitotic exit and contribute to roles in other cellular processes.     

The energetic network of hotspot residues between Cdc25B phosphatase and its protein substrate

We have investigated the functional network of hotspot residues at the remote docking site of two cell cycle regulators, namely Cdc25B phosphatase and its native protein substrate Cdk2-pTpY/CycA. Specifically, we have studied the roles of energetically important residues (Arg488, Arg492, Tyr497 on Cdc25B and Asp206 and Asp210 on Cdk2-pTpY/CycA) by generating a diverse set of substitutions and performing double and triple mutant cycle analyses. This transient protein-protein interaction is particularly well-suited for this mutagenic approach because various control experiments ensure that the effect of each mutation is limited to the interaction of interest. We find binary coupling energies for ion pairs and hydrogen bonds ranging from 0.7 kcal/mol to 3.9 kcal/mol and ternary coupling energies of 1.9 kcal/mol and 2.8 kcal/mol. Overall our biochemical analyses are in good agreement with the docked structure of the complex and suggest the following roles for the individual hotspot residues on Cdc25B. The most important contributor, Arg492, forms a specific and tight bidentate interaction with Asp206 and a weaker interaction with Asp210 that cannot be replaced by a Lys. Although Tyr497 does not directly participate in this ionic network, it is important for buttressing Arg492 using both its hydrophobic (aromatic ring) and hydrophilic characteristics (hydrogen bonding). Arg488 participates less specifically in the electrostatic network with Asp206 and Asp210 of the protein substrate as it can partially be replaced by Lys. Our data provide insight how a cluster of residues in a docking site remote from the site of the chemical reaction can bring about efficient and specific substrate recognition.     

The C-terminal tail of the dual-specificity Cdc25B phosphatase mediates modular substrate recognition.

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases (Cdk/cyclins), thus triggering initiation and progression of successive phases of the cell cycle. In our efforts to elucidate the interaction between Cdc25B and the natural substrate, bis-phosphorylated Cdk2/CycA (Cdk2-pTpY/CycA), we have previously found that the 17 residues of the C-terminal tail mediate a factor of 10 in substrate recognition. In the studies reported here, we localize the majority of this interaction using site-directed mutagenesis to two arginine residues (Arg556 and Arg562) located within this C-terminal region. We also show that the catalytic domain of Cdc25C, which differs most significantly from Cdc25B in this tail region, has a 100-fold lower activity toward Cdk2-pTpY/CycA. We further demonstrate that the proper presentation of the C-terminal tail of Cdc25B can be achieved in a "gain-of-function" chimeric protein consisting of the C-terminal tail of Cdc25B fused onto the catalytic core of Cdc25C. The >10-fold increase in activity seen only in the chimeric protein containing the two critical arginine residues demonstrates that the modular C-terminal tail of Cdc25B is the basis for most of the catalytic advantage of Cdc25B versus Cdc25C toward the Cdk2-pTpY/CycA substrate.     

Catalytic mechanism of Cdc25

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases, thus causing initiation and progression of successive phases of the cell cycle. Although it is not significantly homologous in sequence or structure to other dual-specificity phosphatases, Cdc25 belongs to the class of well-studied cysteine phosphatases as it contains their active site signature motif. Like other dual-specificity phosphatases, Cdc25 contains an active site cysteine whose pK(a) of 5.9 can be measured in pH-dependent kinetics using both small molecule and protein substrates such as Cdk2-pTpY/CycA. We have previously shown that the catalytic acid expected in phosphatases of this family and apparent in kinetics with the natural protein substrate does not appear to lie within the known structure of Cdc25 [Chen, W., et al. (2000) Biochemistry 39, 10781]. Here we provide experimental evidence for a novel mechanism wherein Cdc25 uses as its substrate a monoprotonated phosphate in contrast to the more typical bisanionic phosphate. Our pH-dependent studies, including one-turnover kinetics, solvent kinetic isotope effects, equilibrium perturbation, substrate depletion, and viscosity measurements, show that the monoprotonated phosphate of the protein substrate Cdk2-pTpY/CycA provides the critical proton to the leaving group. Additionally, we provide evidence that Glu474 on the Cdc25 enzyme serves an important role as a base in the transfer of the proton from the phosphate to the leaving group. Because of its greater intrinsic reactivity, the use of a monoprotonated phosphate as a phosphatase substrate is a chemically attractive solution and suggests the possibility of designing inhibitors specific for the Cdc25 dual-specificity phosphatase, an important anticancer target.     

Dual-specific Cdc25B phosphatase: in search of the catalytic acid

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases, thus causing initiation and progression of successive phases of the cell cycle. Although it is not significantly structurally homologous to other well-characterized members, Cdc25 belongs to the class of well-studied cysteine phosphatases as it contains their active site signature motif. However, the catalytic acid needed for protonation of the leaving group has yet to be identified. To elucidate the role and identity of this key catalytic residue, we have performed a detailed pH-dependent kinetic analysis of Cdc25B. The pK(a) of the catalytic cysteine was found to be 5.6-6.3 in steady state and one-turnover burst experiments using the small molecule substrates p-nitrophenyl phosphate and 3-O-methylfluorescein phosphate. Interestingly, Cdc25B does not exhibit the typical bell-shaped pH-rate profile with small molecule substrates seen in other cysteine phosphatases and indicative of the catalytic acid because it lacks pH dependence between 6.5 and 9. Reactions of Cdc25B with the natural substrate Cdk2-pTpY/CycA, however, did yield a bell-shaped pH-rate profile with a pK(a) of 6.1 for the catalytic acid residue. Recent structural studies of Cdc25 have suggested that Glu474 [Fauman, E. B., et al. (1998) Cell 93, 617-625] or Glu478 [Reynolds, R. A., et al. (1999) J. Mol. Biol. 293, 559-568] could function as the catalytic acid in Cdc25B. Using site-directed mutagenesis and truncation experiments, however, we found that neither of these residues, nor the unstructured C-terminus, is responsible for the observed pH dependence. These results indicate that the catalytic acid does not appear to lie within the known structure of Cdc25B and may lie on its protein substrate.     

Cdc25 phosphatases: structure, specificity, and mechanism

Cdc25 phosphatases, as activators of the Cdk/cyclins, play critical roles in the regulation of the eukaryotic cell cycle. Because of their overexpression and correlation with poor prognosis in many diverse cancers, Cdc25 phosphatases are attractive targets for anticancer drug development. Over the past few years, much knowledge of the basic enzymology of the Cdc25 phosphatases that may aid in the development of specific inhibitors has been gained. We review herein the structure, specificity, and mechanism of the Cdc25 phosphatases with a special focus on the activity of Cdc25 phosphatases with native protein substrates.     

Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr¹⁶¹ by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr¹⁴ and Tyr¹⁵ phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G₂/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr¹⁵. In addition, Tyr¹⁵-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G₂ and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr¹⁶¹ phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G₂/M is tightly coupled and regulated by Cdc25 phosphatases.     

Kinetic and structural studies of specific protein-protein interactions in substrate catalysis by Cdc25B phosphatase

Using a combination of steady-state and single-turnover kinetics, we probe substrate association, dissociation, and chemistry for the reaction of Cdc25B phosphatase with its Cdk2-pTpY/CycA protein substrate. The rate constant for substrate association for the wild-type enzyme is 1.3 x 10(6) M(-1) s(-1). The rate constant for dissociation is slow compared to the rate constant for phosphate transfer to form the phospho-enzyme intermediate (k2 = 1.1 s(-1)), making Cdk2-pTpY/CycA a sticky substrate. Compared to the wild type, all hotspot mutants of residues at the remote docking site that specifically affect catalysis with the protein substrate (Arg488, Arg492, and Tyr497 on Cdc25B and Asp206 on Cdk2) have greatly slowed rate constants of association (70- to 4500-fold), and some mutants have decreased k2 values compared to that of the wild type. Most dramatically, R492L, despite showing no significant changes in a crystal structure at 2.0 A resolution, has an approximately 100-fold decrease in k2 compared to that of wild-type Cdc25B. The active site C473S mutant binds tightly to and dissociates slowly from Cdk2-pTpY/CycA (Kd = 10 nM, k(off) = 0.01 s(-1)). In contrast, the C473D mutant, despite showing only localized perturbations in the active site at 1.6 A resolution, has a much weaker affinity and dissociates rapidly (Kd of 2 microM, k(off) > 2 s(-1)) from the protein substrate. Overall, we demonstrate that the association of Cdc25B with its Cdk2-pTpY/CycA substrate is governed to a significant extent by the interactions of the remote hotspot residues, whereas dissociation is governed by interactions at the active site.     

A Cdc25A antagonizing K vitamin inhibits hepatocyte DNA synthesis in vitro and in vivo

Thioalkyl containing K vitamin analogs have been shown to be potent inhibitors of hepatoma cell growth and antagonizers of protein tyrosine phosphatase activity. We now show that they inhibit the activity of specific protein tyrosine phosphatases (PTP) in cell-free conditions in vitro, particularly the dual specificity phosphatase Cdc25A. Using primary cultures of adult rat hepatocytes that are in G0/G1 phase until stimulated into DNA synthesis by epidermal growth factor, we found that 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5) inhibited hepatocyte DNA synthesis and PTP activity in cell culture and in vivo after a two-thirds partial hepatectomy. We found a selective inhibition of Cdc25A activity in vitro, using both synthetic substrates and authentic cellular substrate, immunoprecipitated phospho-Cdk4. Intact Cpd 5-treated cells had decreased cellular Cdc25A activity and increased tyrosine phosphorylation of Cdk4, resulting in decreased phosphorylation of retinoblastoma (Rb). Loss of Cdk4 activity was confirmed using Cdk4 immunoprecipitates from either Cpd 5-treated or untreated cells and measuring its kinase activity using GST-Rb as target. We found a similar order of activity for inhibition of growth and Cdc25A activity using several thiol-containing analogs. Cdc25A inhibitors may thus be useful for defining biochemical pathways involving protein tyrosine phosphorylation that mediate cell growth inhibition.     

Dual G1 and G2 phase inhibition by a novel,selective Cdc25 inhibitor 6-chloro-7-[corrected](2-morpholin-4-ylethylamino)-quinoline-5,8-dione

The Cdc25 dual specificity phosphatases coordinate cell cycle progression, but potent and selective inhibitors have generally been unavailable. In the present study, we have examined one potential inhibitor, 6-chloro-7-(2-morpholin-4-ylethylamino)-quinoline-5,8-dione (NSC 663284), that was identified in the compound library of the National Cancer Institute [corrected]. We found that NSC 663284 arrested synchronized cells at both G(1) and G(2)/M phase, and blocked dephosphorylation and activation of Cdk2 and Cdk1 in vivo, as predicted for a Cdc25 inhibitor. Using the natural Cdc25A substrate, Tyr(15)-phosphorylated Cdk2/cyclin A, we demonstrated that NSC 663284 blocked reactivation of Cdk2/cyclin A kinase by Cdc25A catalytic domain in vitro. In-gel trypsin digestion followed by capillary liquid chromatography-electrospray ionization mass spectrometry and tandem mass spectrometry revealed the direct binding of NSC 663284 to one of the two serine residues in the active site loop HCEFSSER of the Cdc25A catalytic domain. Cdc25 binding and inhibition could contribute to the anti-proliferative activity of NSC 663284 and its ability to arrest cell cycle progression. Moreover, NSC 663284 should be a valuable reagent to probe the actions of Cdc25 phosphatases within cells and may also be useful structure for the design of more potent and selective antiproliferative agents.     

Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation

The activity of Cdk1–cyclin B1 mitotic complexes is regulated by the balance between the counteracting activities of Wee1/Myt1 kinases and Cdc25 phosphatases. These kinases and phosphatases must be strictly regulated to ensure proper mitotic timing. One masterpiece of this regulatory network is Cdk1, which promotes Cdc25 activity and suppresses inhibitory Wee1/Myt1 kinases through direct phosphorylation. The Cdk1-dependent phosphorylation of Wee1 primes phosphorylation by additional kinases such as Plk1, triggering Wee1 degradation at the onset of mitosis. Here we report that Cdc14A plays an important role in the regulation of Wee1 stability. Depletion of Cdc14A results in a significant reduction in Wee1 protein levels. Cdc14A binds to Wee1 at its amino-terminal domain and reverses CDK-mediated Wee1 phosphorylation. In particular, we found that Cdc14A inhibits Wee1 degradation through the dephosphorylation of Ser-123 and Ser-139 residues. Thus the lack of phosphorylation of these two residues prevents the interaction with Plk1 and the consequent efficient Wee1 degradation at the onset of mitosis. These data support the hypothesis that Cdc14A counteracts Cdk1–cyclin B1 activity through Wee1 dephosphorylation.     

Human Cdc25A phosphatase has a non-redundant function in G2 phase by activating Cyclin A-dependent kinases

Cdc25 phosphatases activate Cdk/Cyclin complexes by dephosphorylation and thus promote cell cycle progression. We observed that the peak activity of Cdc25A precedes the one of Cdc25B in prophase and the maximum of Cyclin/Cdk kinase activity. Furthermore, Cdc25A activates both Cdk1-2/Cyclin A and Cdk1/Cyclin B complexes while Cdc25B seems to be involved only in activation of Cdk1/Cyclin B. Concomitantly, repression of Cdc25A led to a decrease in Cyclin A-associated kinase activity and attenuated Cdk1 activation. Our results indicate that Cdc25A acts before Cdc25B - at least in cancer cells, and has non-redundant functions in late G2/early M-phase as a major regulator of Cyclin A/kinase complexes.     

Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.     

Functional homology among human and fission yeast Cdc14 phosphatases

Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.     

Cdk1 phosphorylation of fission yeast paxillin inhibits its cytokinetic ring localization

Divisions of the genetic material and cytoplasm are coordinated spatially and temporally to ensure genome integrity. This coordination is mediated in part by the major cell cycle regulator cyclin-dependent kinase (Cdk1). Cdk1 activity peaks during mitosis, but during mitotic exit/cytokinesis Cdk1 activity is reduced, and phosphorylation of its substrates is reversed by various phosphatases including Cdc14, PP1, PP2A, and PP2B. Cdk1 is known to phosphorylate several components of the actin- and myosin-based cytokinetic ring (CR) that mediates division of yeast and animal cells. Here we show that Cdk1 also phosphorylates the Schizosaccharomyces pombe CR component paxillin Pxl1. We determined that both the Cdc14 phosphatase Clp1 and the PP1 phosphatase Dis2 contribute to Pxl1 dephosphorylation at mitotic exit, but PP2B/calcineurin does not. Preventing Pxl1 phosphorylation by Cdk1 results in increased Pxl1 levels, precocious Pxl1 recruitment to the division site, and increased duration of CR constriction. In vitro Cdk1-mediated phosphorylation of Pxl1 inhibits its interaction with the F-BAR domain of the cytokinetic scaffold Cdc15, thereby disrupting a major mechanism of Pxl1 recruitment. Thus, Pxl1 is a novel substrate through which S. pombe Cdk1 and opposing phosphatases coordinate mitosis and cytokinesis.     

Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome

Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.     

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.     

Dual mode of degradation of Cdc25 A phosphatase

The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin-proteasome pathway. Here we have investigated the regulation of Cdc25 A in the cell cycle. We found that Cdc25 A degradation during mitotic exit and in early G(1) is mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase, and that a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. We propose that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment.