PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation

It has been known for over a decade that inhibition of protein phosphatase 1 (PP1) activity prevents entry into M phase, but the relevant substrate has not been identified. We report here that PP1 is required for dephosphorylation of the Cdc2-directed phosphatase Cdc25 at Ser287 (of Xenopus Cdc25; Ser216 of human Cdc25C), a site that suppresses Cdc25 during interphase. Moreover, PP1 recognizes Cdc25 directly by interacting with a PP1-binding motif in the Cdc25 N-terminus. We have also found that 14-3-3 binding to phospho-Ser287 protects Cdc25 from premature dephosphorylation. Upon entry into M phase, 14-3-3 removal from Cdc25 precedes Ser287 dephosphorylation, suggesting the existence of a phosphatase- independent pathway for 14-3-3 removal from Cdc25. We show here that this dissociation of 14-3-3 from Cdc25 requires the activity of the cyclin-dependent kinase Cdk2, providing a molecular explanation for the previously reported requirement for Cdk2 in promoting mitotic entry. Collectively, our data clarify several steps important for Cdc25 activation and provide new insight into the role of PP1 in Cdc2 activation and mitotic entry.     

Phosphorylation of Xenopus Cdc25C at Ser285 interferes with ability to activate a DNA damage replication checkpoint in pre-midblastula embryos

We have recently demonstrated that negative regulation of human Cdc25 protein phosphatases by phosphorylation at their 14-3-3 site can be antagonized through phosphorylation at an adjacent site in the -2 position.1 Based on structural homology for different Cdc25 phosphatases, a similar regulatory pathway also could be conserved in Xenopus embryos, where cell cycle checkpoints are not operational prior to the Midblastula Transition (MBT). Here, we demonstrate that before MBT, XeCdc25C is phosphorylated on Ser285, an analogous site to Ser214 in human Cdc25C or Ser307 Cdc25B.(1) Phosphorylation of Ser285 prevents subsequent inhibitory phosphorylation of XeCdc25C on Ser287, thus maintaining XeCdc25C in an active form. Mutation of Ser285 to alanine allows the reconstitution of a DNA damage replication checkpoint. This effect is completely dependent on Ser287 phosphorylation as additional mutation of Ser287 to alanine fully reversed the cell cycle inhibitory effect of Ser285A XeCdc25C. We propose that phosphorylation of XeCdc25C Ser285 may account for the lack of a DNA replication checkpoint in cleaving Xenopus embryos prior to the MBT.     

Phosphorylation of Xenopus Cdc25C at Ser285 Interferes with Ability to Activate a DNA Damage Replication Checkpoint in the Pre-Midblastula Embryos

We have recently demonstrated that negative regulation of human Cdc25 protein phosphatases by phosphorylation at their 14-3-3 site can be antagonized through phosphorylation at an adjacent site in the -2 position.1 Based on structural homology for different Cdc25 phosphatases, a similar regulatory pathway also could be conserved in Xenopus embryos, where cell cycle checkpoints are not operational prior to the Midblastula Transition (MBT). Here, we demonstrate that before MBT, XeCdc25C is phosphorylated on Ser285, an analogous site to Ser214 in human Cdc25C or Ser307 in Cdc25B.1 Phosphorylation of Ser285 prevents subsequent inhibitory phosphorylation of XeCdc25C on Ser287, thus maintaining XeCdc25C in an active form. Mutation of Ser285 to alanine allows the reconstitution of a DNA damage replication checkpoint. This effect is completely dependent on Ser287 phosphorylation as additional mutation of Ser287 to alanine fully reversed the cell cycle inhibitory effect of Ser285A XeCdc25C. We propose that phosphorylation of XeCdc25C Ser285 may account for the lack of a DNA replication checkpoint in cleaving Xenopus embryos prior to the MBT.     

A role for PP1 in the Cdc2/Cyclin B-mediated positive feedback activation of Cdc25

The Cdc25 phosphatase promotes entry into mitosis through the removal of inhibitory phosphorylations on the Cdc2 subunit of the Cdc2/CyclinB complex. During interphase, or after DNA damage, Cdc25 is suppressed by phosphorylation at Ser287 (Xenopus numbering; Ser216 of human Cdc25C) and subsequent binding of the small acidic protein, 14-3-3. As reported recently, at the time of mitotic entry, 14-3-3 protein is removed from Cdc25 and S287 is dephosphorylated by protein phosphatase 1 (PP1). After the initial activation of Cdc25 and consequent derepression of Cdc2/CyclinB, Cdc25 is further activated through a Cdc2-catalyzed positive feedback loop. Although the existence of such a loop has been appreciated for some time, the molecular mechanism for this activation has not been described. We report here that phosphorylation of S285 by Cdc2 greatly enhances recruitment of PP1 to Cdc25, thereby accelerating S287 dephosphorylation and mitotic entry. Moreover, we show that two other previously reported sites of Cdc2-catalyzed phosphorylation on Cdc25 are required for maximal biological activity of Cdc25, but they do not contribute to PP1 regulation and do not act solely through controlling S287 phosphorylation. Therefore, multiple mechanisms, including enhanced recruitment of PP1, are used to promote full activation of Cdc25 at the time of mitotic entry.     

Regulation of Cdc25C Activity During the Meiotic G2/M Transition

The Cdc25C phosphatase is a key activator of Cdc2/cyclin B that controls M-phase entry in eukaryotic cells. Here we discuss the regulation of Cdc25C by phosphorylation during the meiotic maturation of Xenopus oocytes. In G2 arrested oocytes, Cdc25C is phosphorylated on Ser287 and associated with 14-3-3 proteins. Entry of the oocytes into M-phase of meiosis is triggered by progesterone, which activates a signaling pathway leading to the dephosphorylation of Ser287, probably mediated by the PP1 phosphatase. The activation of Cdc25C during oocyte maturation correlates also with its phosphorylation on multiple sites. These phosphorylations involve several signaling pathways, including Polo kinases and MAP kinases, and might require also the inhibition of the PP2A phosphatase. Finally, Cdc25C is further phosphorylated by its substrate Cdc2/cyclin B, as part of an auto-amplification loop that ensures the high Cdc2/cyclin B activity level required to drive the oocyte through the meiotic cell cycle.     

When the Checkpoints Have Gone: Insights into Cdc25 Functional Activation

DNA-responsive checkpoints operate at the G2/M transition to prevent premature mitosis in the presence of incompletely replicated or damaged DNA. These pathways prevent mitotic entry, at least in part, by suppressing Cdc25, the phosphatase that activates Cdc2/Cyclin B. To gain insight into how checkpoint signaling controls Cdc25 function, we have carefully examined the individual steps in Cdc25 activation. We found that removal of the regulatory protein, 14-3-3, that binds to phosphorylated Cdc25 during interphase is one of the early steps in mitotic activation. Moreover, our studies unexpectedly implicated the phosphatase PP1 and the G1/S kinase Cdk2 in the process of Cdc25 activation. Here we integrate our findings and those of others to propose a model for Cdc25 activation in an effort to provide insight into novel loci of DNA-responsive checkpoint control of mitotic entry.     

When the checkpoints have gone: insights into Cdc25 functional activation

DNA-responsive checkpoints operate at the G(2)/M transition to prevent premature mitosis in the presence of incompletely replicated or damaged DNA. These pathways prevent mitotic entry, at least in part, by suppressing Cdc25, the phosphatase that activates Cdc2/Cyclin B. To gain insight into how checkpoint signaling controls Cdc25 function, we have carefully examined the individual steps in Cdc25 activation. We found that removal of the regulatory protein, 14-3-3, that binds to phosphorylated Cdc25 during interphase is one of the early steps in mitotic activation. Moreover, our studies unexpectedly implicated the phosphatase PP1 and the G(1)/S kinase Cdk2 in the process of Cdc25 activation. Here we integrate our findings and those of others to propose a model for Cdc25 activation in an effort to provide insight into novel loci of DNA-responsive checkpoint control of mitotic entry.     

14-3-3 Proteins Act as Negative Regulators of the Mitotic Inducer Cdc25 in Xenopus Egg Extracts

Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2–cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3ε and to a lesser extent with 14-3-3ζ. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G₂–M transition.     

Checkpoint kinase 1 regulates diallyl trisulfide-induced mitotic arrest in human prostate cancer cells

We have shown previously that diallyl trisulfide (DATS), a constituent of processed garlic, inhibits proliferation of PC-3 and DU145 human prostate cancer cells by causing G(2)-M phase cell cycle arrest in association with inhibition of cyclin-dependent kinase 1 activity and hyperphosphorylation of Cdc25C at Ser(216). Here, we report that DATS-treated PC-3 and DU145 cells are also arrested in mitosis as judged by microscopy following staining with anti-alpha-tubulin antibody and 4',6-diamidino-2-phenylindole and flow cytometric analysis of Ser(10) phosphorylation of histone H3. The DATS treatment caused activation of checkpoint kinase 1 and checkpoint kinase 2, which are intermediaries of DNA damage checkpoints and implicated in Ser(216) phosphorylation of Cdc25C. The diallyl trisulfide-induced Ser(216) phosphorylation of Cdc25C as well as mitotic arrest were significantly attenuated by knockdown of check-point kinase 1 protein in both PC-3 and DU145 cells. On the other hand, depletion of checkpoint kinase 2 protein did not have any appreciable effect on G(2) or M phase arrest or Cdc25C phosphorylation caused by diallyl trisulfide. The lack of a role of checkpoint kinase 2 in diallyl trisulfide-induced phosphorylation of Cdc25C or G(2)-M phase cell cycle arrest was confirmed using HCT-15 cells stably transfected with phosphorylation-deficient mutant (T68A mutant) of checkpoint kinase 2. In conclusion, the results of the present study suggest existence of a checkpoint kinase 1-dependent mechanism for diallyl trisulfide-induced mitotic arrest in human prostate cancer cells.     

MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation

The cellular response to DNA damage is mediated by evolutionarily conserved Ser/Thr kinases, phosphorylation of Cdc25 protein phosphatases, binding to 14-3-3 proteins, and exit from the cell cycle. To investigate DNA damage responses mediated by the p38/stress-activated protein kinase (SAPK) axis of signaling, the optimal phosphorylation motifs of mammalian p38alpha SAPK and MAPKAP kinase-2 were determined. The optimal substrate motif for MAPKAP kinase-2, but not for p38 SAPK, closely matches the 14-3-3 binding site on Cdc25B/C. We show that MAPKAP kinase-2 is directly responsible for Cdc25B/C phosphorylation and 14-3-3 binding in vitro and in response to UV-induced DNA damage within mammalian cells. Downregulation of MAPKAP kinase-2 eliminates DNA damage-induced G2/M, G1, and intra S phase checkpoints. We propose that MAPKAP kinase-2 is a new member of the DNA damage checkpoint kinase family that functions in parallel with Chk1 and Chk2 to integrate DNA damage signaling responses and cell cycle arrest in mammalian cells.     

Dual phosphorylation controls Cdc25 phosphatases and mitotic entry

Negative regulation of the Cdc25C protein phosphatase by phosphorylation on Ser 216, the 14-3-3-binding site, is an important regulatory mechanism used by cells to block mitotic entry under normal conditions and after DNA damage. During mitosis, Cdc25C is not phosphorylated on Ser 216 and ionizing radiation (IR) does not induce either phosphorylation of Ser 216, or binding to 14-3-3. Here, we show that Cdc25C is phosphorylated on Ser 214 during mitosis, which in turn prevents phosphorylation of Ser 216. Mutation of Ser 214 to Ala reconstitutes Ser 216 phosphorylation and 14-3-3 binding during mitosis. Introduction of exogenous Cdc25C(S214A) into HeLa cells depleted of endogenous Cdc25C results in a substantial delay to mitotic entry. This effect was fully reversed in a S214A/S216A double-mutant, implying that the inhibitory effect of S214A mutant was entirely dependent on Ser 216 phosphorylation. A similar regulatory mechanism may also apply to another mitotic phosphatase, Cdc25B, as well as mitotic phosphatases of other species, including Xenopus laevis. We propose that this pathway ensures that Cdc2 remains active once mitosis is initiated and is a key control mechanism for maintaining the proper order of cell-cycle transitions.     

Mitotic Phosphorylation of Cdc25B Ser321 Disrupts 14-3-3 Binding to the High Affinity Ser323 Site

Cdc25B is a key regulator of entry into mitosis, and its activity and localization are regulated by binding of the 14-3-3 dimer. There are three 14-3-3 binding sites on Cdc25B, with Ser³²³ being the highest affinity binding and is highly homologous to the Ser²¹⁶ 14-3-3 binding site on Cdc25C. Loss of 14-3-3 binding to Ser³²³ increases cyclin/Cdk substrate access to the catalytic site, thereby increasing its activity. It also affects the localization of Cdc25B. Thus, phosphorylation and 14-3-3 binding to this site is essential for down-regulating Cdc25B activity, blocking its mitosis promoting function. The question of how this inhibitory signal is relieved to allow Cdc25B activation and entry into mitosis is yet to be resolved. Here, we show that Ser³²³ phosphorylation is maintained into mitosis, but phosphorylation of Ser³²¹ disrupts 14-3-3 binding to Ser³²³, mimicking the effect of inhibiting Ser³²³ phosphorylation on both Cdc25B activity and localization. The unphosphorylated Ser³²¹ appears to have a role in stabilizing 14-3-3 binding to Ser³²³, and loss of the Ser hydroxyl group appears to be sufficient to significantly reduce 14-3-3 binding. A consequence of loss of 14-3-3 binding is dephosphorylation of Ser³²³. Ser³²¹ is phosphorylated in mitosis by Cdk1. The mitotic phosphorylation of Ser³²¹ acts to maintain full activation of Cdc25B by disrupting 14-3-3 binding to Ser³²³ and enhancing the dephosphorylation of Ser³²³ to block 14-3-3 binding to this site.     

14‐3‐3γ mediates Cdc25A proteolysis to block premature mitotic entry after DNA damage

14‐3‐3 proteins control various cellular processes, including cell cycle progression and DNA damage checkpoint. At the DNA damage checkpoint, some subtypes of 14‐3‐3 (β and ζ isoforms in mammalian cells and Rad24 in fission yeast) bind to Ser345‐phosphorylated Chk1 and promote its nuclear retention. Here, we report that 14‐3‐3γ forms a complex with Chk1 phosphorylated at Ser296, but not at ATR sites (Ser317 and Ser345). Ser296 phosphorylation is catalysed by Chk1 itself after Chk1 phosphorylation by ATR, and then ATR sites are rapidly dephosphorylated on Ser296‐phosphorylated Chk1. Although Ser345 phosphorylation is observed at nuclear DNA damage foci, it occurs more diffusely in the nucleus. The replacement of endogenous Chk1 with Chk1 mutated at Ser296 to Ala induces premature mitotic entry after ultraviolet irradiation, suggesting the importance of Ser296 phosphorylation in the DNA damage response. Although Ser296 phosphorylation induces the only marginal change in Chk1 catalytic activity, 14‐3‐3γ mediates the interaction between Chk1 and Cdc25A. This ternary complex formation has an essential function in Cdc25A phosphorylation and degradation to block premature mitotic entry after DNA damage.     

Changes in regulatory phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during normal cell cycle progression and checkpoint arrests

Entry into mitosis is catalyzed by cdc2 kinase. Previous work identified the cdc2-activating phosphatase cdc25C and the cdc2-inhibitory kinase wee1 as targets of the incomplete replication-induced kinase Chk1. Further work led to the model that checkpoint kinases block mitotic entry by inhibiting cdc25C through phosphorylation on Ser287 and activating wee1 through phosphorylation on Ser549. However, almost all conclusions underlying this idea were drawn from work using recombinant proteins. Here, we report that in the early Xenopus egg cell cycles, phosphorylation of endogenous cdc25C Ser287 is normally high during interphase and shows no obvious increase after checkpoint activation. By contrast, endogenous wee1 Ser549 phosphorylation is low during interphase and increases after activation of either the DNA damage or replication checkpoints; this is accompanied by a slight increase in wee1 kinase activity. Blocking mitotic entry by adding the catalytic subunit of PKA also results in increased wee1 Ser549 phosphorylation and maintenance of cdc25C Ser287 phosphorylation. These results argue that in response to checkpoint activation, endogenous wee1 is indeed a critical responder that functions by repressing the cdc2-cdc25C positive feedback loop. Surprisingly, endogenous wee1 Ser549 phosphorylation is highest during mitosis just after the peak of cdc2 activity. Treatments that block inactivation of cdc2 result in further increases in wee1 Ser549 phosphorylation, suggesting a previously unsuspected role for wee1 in mitosis.     

Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding

The order and fidelity of cell cycle events in mammals is intimately linked to the integrity of the Chk1 kinase-Cdc25A phosphatase pathway. Chk1 phosphorylation targets Cdc25A for destruction and, as shown here, inhibits interactions between Cdc25A and its mitotic substrate cyclin B1-Cdk1. Phosphorylation of Cdc25A on serine 178 and threonine 507 facilitates 14-3-3 binding, and Chk1 phosphorylates both residues in vitro. Mutation of T507 to alanine (T507A) enhanced the biological activity of Cdc25A. Cdc25A(T507A) was more efficient in binding to cyclin B1, activating cyclin B1-Cdk1, and promoting premature entry into mitosis. We propose that the Chk1/Cdc25A/14-3-3 pathway functions to prevent cells from entering into mitosis prior to replicating their genomes to ensure the fidelity of the cell division process.     

Regulation of Cdc2/cyclin B activation in Xenopus egg extracts via inhibitory phosphorylation of Cdc25C phosphatase by Ca(2+)/calmodulin-dependent protein [corrected] kinase II

Activation of Cdc2/cyclin B kinase and entry into mitosis requires dephosphorylation of inhibitory sites on Cdc2 by Cdc25 phosphatase. In vertebrates, Cdc25C is inhibited by phosphorylation at a single site targeted by the checkpoint kinases Chk1 and Cds1/Chk2 in response to DNA damage or replication arrest. In Xenopus early embryos, the inhibitory site on Cdc25C (S287) is also phosphorylated by a distinct protein kinase that may determine the intrinsic timing of the cell cycle. We show that S287-kinase activity is repressed in extracts of unfertilized Xenopus eggs arrested in M phase but is rapidly stimulated upon release into interphase by addition of Ca2+, which mimics fertilization. S287-kinase activity is not dependent on cyclin B degradation or inactivation of Cdc2/cyclin B kinase, indicating a direct mechanism of activation by Ca2+. Indeed, inhibitor studies identify the predominant S287-kinase as Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII phosphorylates Cdc25C efficiently on S287 in vitro and, like Chk1, is inhibited by 7-hydroxystaurosporine (UCN-01) and debromohymenialdisine, compounds that abrogate G2 arrest in somatic cells. CaMKII delays Cdc2/cyclin B activation via phosphorylation of Cdc25C at S287 in egg extracts, indicating that this pathway regulates the timing of mitosis during the early embryonic cell cycle.     

p14ARF Triggers G2 Arrest Through ERK-Mediated Cdc25C Phosphorylation,Ubiquitination and Proteasomal Degradation

The Cdc25C phosphatase is a key regulator of mitotic entry which activity is tightly regulatedby phosphorylation. In response to DNA damage, phosphorylation at serine 216 induces thecytosolic retention of Cdc25C through 14-3-3 binding. We previously reported the ability ofthe p14ARF tumor suppressor to induce the accumulation of inactive phospho-Cdc25C(Ser216)protein as well as a decrease of Cdc25C steady state level and correlated these events with ap53-independent G2 arrest. The aim of this study was to investigate the cellular signalingpathways involved in this process. By using specific pharmacological inhibitors, wedemonstrate that activation of the ERK1/2 MAP kinases pathway is involved in the p53-independent G2 checkpoint induced by p14ARF. Moreover, we show that activated P-ERK1/2bind and phosphorylate Cdc25C on its ser216 residue following p14ARF expression, therebyidentifying Cdc25C as a new ERK1/2 target. Importantly, we further show thatphosphorylation at Ser216 by phospho-ERK1/2 promotes Cdc25C ubiquitination andproteasomal degradation, suggesting that Cdc25C proteolysis is required for a sustained G2arrest in response to p14ARF. Taken together, these results demonstrate that the MAPK ERKsignaling pathway contributes to the p53-independent antiproliferative functions of p14ARF.Furthermore, they identify a new mechanism by which phosphorylation at serine 216participates to Cdc25C inactivation.     

Coupling of Cdc20 inhibition and activation by BubR1

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.     

Regulation of G(2)/M events by Cdc25A through phosphorylation-dependent modulation of its stability

DNA replication in higher eukaryotes requires activation of a Cdk2 kinase by Cdc25A, a labile phosphatase subject to further destabilization upon genotoxic stress. We describe a distinct, markedly stable form of Cdc25A, which plays a previously unrecognized role in mitosis. Mitotic stabilization of Cdc25A reflects its phosphorylation on Ser17 and Ser115 by cyclin B-Cdk1, modifications required to uncouple Cdc25A from its ubiquitin-proteasome-mediated turnover. Cdc25A binds and activates cyclin B-Cdk1, accelerates cell division when overexpressed, and its downregulation by RNA interference (RNAi) delays mitotic entry. DNA damage-induced G(2) arrest, in contrast, is accompanied by proteasome-dependent destruction of Cdc25A, and ectopic Cdc25A abrogates the G(2) checkpoint. Thus, phosphorylation-mediated switches among three differentially stable forms ensure distinct thresholds, and thereby distinct roles for Cdc25A in multiple cell cycle transitions and checkpoints.     

Many fingers on the mitotic trigger: post-translational regulation of the Cdc25C phosphatase

The mitotic inducer Cdc25 phosphatase controls the activation of Cdc2/cyclin B protein kinase and entry into mitosis in eukaryotic cells. Cdc25C is highly regulated by multiple post-translational modifications within its N-terminal regulatory domain and site-specific protein interactions. Phosphorylation of one inhibitory site targeted by multiple kinases determines the timing of Cdc25C activation and arrests cells in G2 in response to checkpoint, stress, developmental and extracellular signals. In mitosis, phosphorylation of several Ser/Thr residues and Pin1-catalysed peptidyl-proline isomerisation produces activation. Phosphorylation of one activating site is antagonistic to the proximal inhibitory site and maintains Cdc25C activity during mitosis. Phosphorylation and interacting proteins also modulate the nuclear import and export signals on Cdc25C, inducing dramatic changes in its localisation within the cell. Thus, the regulation of Cdc25C activity and localization integrates multiple signals that govern the decision to enter mitosis.