Hierarchical order of phosphorylation events commits Cdc25A to betaTrCP-dependent degradation

We have recently demonstrated that regulation of Cdc25A protein abundance during S phase and in response to DNA damage is mediated by SCF(betaTrCP) activity. Based on sequence homology of known betaTrCP substrates, we found that Cdc25A contains a conserved motif (DSG), phosphorylation of which is required for interaction with betaTrCP.1 Here, we show that phosphorylation at Ser 82 within the DSG motif anchors Cdc25A to betaTrCP and that Chk1-dependent phosphorylation at Ser 76 affects this interaction as well as betaTrCP-dependent degradation. We propose that a hierarchical order of phosphorylation events commits Cdc25A to betaTrCP-dependent degradation. According to our model, phosphorylation at Ser 76 is a "priming" step required for Ser 82 phosphorylation, which in turn allows recruitment of Cdc25A by betaTrCP and subsequent betaTrCP-dependent degradation.     

CK1ε targets Cdc25A for ubiquitin-mediated proteolysis under normal conditions and in response to checkpoint activation

Cdc25A phosphatase, which is essential in cell cycle progression, is degraded by the proteasome throughout interphase and in response to genotoxic stress. Phosphorylation of Cdc25A on Ser82 in the DSG motif is important in the recognition by β-TrCP, resulting in targeting of Cdc25A for ubiquitination. Chk1 is known to phosphorylate Cdc25A on Ser76, and NEK11 or CK1α relays phosphorylation of Cdc25A to Ser82 in a hierarchical manner. In this study, we found that CK1ε has unique enzymatic activity on the serine residue in the DSG motif using a β-catenin N-terminal region as a substrate. We then examined whether CK1ε has activity on the DSG motif of Cdc25A. We found CK1ε directly phosphorylated Ser82 without any prior phosphorylation of Cdc25A, and depletion of CK1ε stabilized the cellular Cdc25A in 293 cells. Moreover, we found that CK1ε also has activity as a relaying kinase like NEK11 or CK1α when the cell is exposed to DNA damage. Taken together, our results indicate that CK1ε regulates the cellular levels of Cdc25A in parallel with Chk1-dependent Cdc25A degradation, contributing to the precise control of cell division.     

mTOR generates an auto-amplification loop by triggering the βTrCP- and CK1α-dependent degradation of DEPTOR

DEPTOR is a recently identified inhibitor of the mTOR?kinase that is highly regulated at the posttranslational level. In response to mitogens, we found that DEPTOR was rapidly phosphorylated on three serines in a conserved degron, facilitating binding and ubiquitylation by the F box protein βTrCP, with consequent proteasomal degradation of DEPTOR. Phosphorylation of the βTrCP degron in DEPTOR is executed by CK1α after a priming phosphorylation event mediated by either the mTORC1 or mTORC2 complexes. Blocking the βTrCP-dependent degradation of DEPTOR via βTrCP knockdown or expression of a stable DEPTOR mutant that is unable to bind βTrCP results in mTOR inhibition. Our findings reveal that mTOR cooperates with CK1α and βTrCP to generate an auto-amplification loop to promote its own full activation. Moreover, our results suggest that pharmacologic inhibition of CK1 may be?a viable therapeutic option for the treatment of cancers characterized by activation of mTOR-signaling pathways.     

A human cancer-predisposing polymorphism in Cdc25A is embryonic lethal in the mouse and promotes ASK-1 mediated apoptosis

Background

Failure to regulate the levels of Cdc25A phosphatase during the cell cycle or during a checkpoint response causes bypass of DNA damage and replication checkpoints resulting in genomic instability and cancer. During G1 and S and in cellular response to DNA damage, Cdc25A is targeted for degradation through the Skp1-cullin-β-TrCP (SCF^β-TrCP) complex. This complex binds to the Cdc25A DSG motif which contains serine residues at positions 82 and 88. Phosphorylation of one or both residues is necessary for the binding and degradation to occur.

Results

We now show that mutation of serine 88 to phenylalanine, which is a cancer-predisposing polymorphic variant in humans, leads to early embryonic lethality in mice. The mutant protein retains its phosphatase activity both in vitro and in cultured cells. It fails to interact with the apoptosis signal-regulating kinase 1 (ASK1), however, and therefore does not suppress ASK1-mediated apoptosis.

Conclusions

These data suggest that the DSG motif, in addition to its function in Cdc25A-mediated degradation, plays a role in cell survival during early embyogenesis through suppression of ASK1-mediated apoptosis.

     

Transforming growth factor beta facilitates beta-TrCP-mediated degradation of Cdc25A in a Smad3-dependent manner

Ubiquitin-dependent degradation of Cdc25A is a major mechanism for damage-induced S-phase checkpoint. Two ubiquitin ligases, the Skp1-cullin-beta-TrCP (SCFbeta-TrCP) complex and the anaphase-promoting complex (APCCdh1), are involved in Cdc25A degradation. Here we demonstrate that the transforming growth factor beta (TGF-beta)-Smad3 pathway promotes SCF(beta-TrCP)-mediated Cdc25A ubiquitination. Cells treated with TGF-beta, as well as cells transfected with Smad3 or a constitutively active type I TGF-beta receptor, exhibit increased ubiquitination and markedly shortened half-lives of Cdc25A. Furthermore, Cdc25A is stabilized in cells transfected with Smad3 small interfering RNA (siRNA) and cells from Smad3-null mice. TGF-beta-induced ubiquitination is associated with Cdc25A phosphorylation at the beta-TrCP docking site (DS82G motif) and physical association of Cdc25A with Smad3 and beta-TrCP. Cdc25A mutant proteins deficient in DS82G phosphorylation are resistant to TGF-beta-Smad3-induced degradation, whereas a Cdc25A mutant protein defective in APCCdh1 recognition undergoes efficient degradation. Smad3 siRNA inhibits beta-TrCP-Cdc25A interaction and Cdc25A degradation in response to TGF-beta. beta-TrCP2 siRNA also inhibits Smad3-induced Cdc25A degradation. In contrast, Cdh1 siRNA had no effect on Cdc25A down-regulation by Smad3. These data suggest that Smad3 plays a key role in the regulation of Cdc25A ubiquitination by SCFbeta-TrCP and that Cdc25A stabilization observed in various cancers could be associated with defects in the TGF-beta-Smad3 pathway.     

Degradation of Cdc25A phosphatase in HeLa cells under normal and stress conditions

Cdc25A phosphatase regulates cell cycle progression by removing the inhibitory phosphates from cyclin-dependent kinases. Activity of Cdc25A depends on its phosphorylation status. During normal cell cycle progression and after DNA damage phosphorylation by Chk1 (or Chk2) triggers Cdc25A degradation via ubiquitin-proteasome pathway. In this study we investigate the role of various phosphorylation sites (Ser123, Ser75, Ser17 and Ser115) in the regulation of Cdc25A stability. We have shown that only S75A mutation abrogates Cdc25A degradation both in normal and stress conditions. We also studied the influence of stable form of Cdc25A on checkpoint progression after DNA damage. We have found out that delay in DNA synthesis after UV and IR does not depend on Cdc25A activity. However, the presence of stable Cdc25A increases the number of mitotic cells after these stresses.     

βTrCP regulates BMI1 protein turnover via ubiquitination and degradation

The polycomb group protein BMI1 has been linked to proliferation, senescence, cancer progression and stem cell phenotype. At present, very little is known about its regulation. Here, we report that BMI1 contains a functional recognition motif for the F box protein βTrCP, which regulates ubiquitination and proteasome-mediated degradation of various proteins. We show that overexpression of wild-type βTrCP but not the ΔF mutant of it promotes BMI1 ubiquitination and degradation, and knockdown of βTrCP results in increased expression of BMI1. Furthermore, a mutant of BMI1 with an altered βTrCP recognition motif is much more stable than wild-type BMI1. We also show that wild-type BMI1 but not the mutant BMI1 interacts with βTrCP. Accordingly, compared to wild-type BMI1, mutant protein exhibited increased pro-oncogenic activity. In summary, our findings suggest that βTrCP regulates turnover of BMI1 and its function relevant to oncogenesis, cellular senescence and aging.     

βTrCP controls GH receptor degradation via two different motifs

The physiological roles of GH are broad and include metabolism regulation and promotion of somatic growth. Therefore, the responsiveness of cells to GH must be tightly regulated. This is mainly achieved by a complex and well-controlled mechanism of GH receptor (GHR) endocytosis. GHR endocytosis occurs independently of GH and requires the ubiquitin ligase, SCF (βTrCP) that is recruited to the ubiquitin-dependent endocytosis (UbE) motif in the cytoplasmic tail of the GHR. In this study we report that, in addition to the UbE motif, a downstream degron, DSGRTS, binds to βTrCP. The WD40 residues on βTrCP involved in the interaction with this sequence are identical to the ones necessary for binding the classical motif, DSGxxS, in inhibitor of NFκB signalling, and β-catenin. Previously, we showed that this motif is not involved in GH-induced endocytosis. We show here that the DSGRTS sequence significantly contributes to GHR endocytosis/degradation in basal conditions, whereas the UbE motif is involved both in basal and GH-induced conditions. These findings explain the high rate of GHR degradation under basal conditions, which is important for regulating the responsiveness of cells to GH.     

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.     

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.     

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.     

Cdc6 degradation requires phosphodegron created by GSK-3 and Cdk1 for SCFCdc4 recognition in Saccharomyces cerevisiae

To ensure genome integrity, DNA replication takes place only once per cell cycle and is tightly controlled by cyclin-dependent kinase (Cdk1). Cdc6p is part of the prereplicative complex, which is essential for DNA replication. Cdc6 is phosphorylated by cyclin-Cdk1 to promote its degradation after origin firing to prevent DNA rereplication. We previously showed that a yeast GSK-3 homologue, Mck1 kinase, promotes Cdc6 degradation in a SCF^Cdc4-dependent manner, therefore preventing rereplication. Here we present evidence that Mck1 directly phosphorylates a GSK-3 consensus site in the C-terminus of Cdc6. The Mck1-dependent Cdc6 phosphorylation required priming by cyclin/Cdk1 at an adjacent CDK consensus site. The sequential phosphorylation by Mck1 and Clb2/Cdk1 generated a Cdc4 E3 ubiquitin ligase–binding motif to promote Cdc6 degradation during mitosis. We further revealed that Cdc6 degradation triggered by Mck1 kinase was enhanced upon DNA damage caused by the alkylating agent methyl methanesulfonate and that the resulting degradation was mediated through Cdc4. Thus, Mck1 kinase ensures proper DNA replication, prevents DNA damage, and maintains genome integrity by inhibiting Cdc6.     

Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. We show here that DNA-responsive checkpoints also activate PP2A/B56delta phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Our data suggest that creation of a 14-3-3 "sink," consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56delta as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis.     

SCF acts in endosomal sorting of the GH receptor

The ubiquitin ligase SCF^TrCP is required for internalisation of the growth hormone receptor (GHR) and acts via a direct interaction with the ubiquitin-dependent endocytosis motif. Details of how the ligase communicates its information to the clathrin-mediated internalisation machinery are unknown. For the EGF receptor, c-Cbl acts both at the cell surface and in endosomes. We hypothesised that SCF^TrCP is required for GHR degradation at both sites. This was tested by truncating GHR after a di-leucine-based internalisation motif (GHR349). This receptor enters the cells via the adapter complex AP2. We show that TrCP acts in an early stage of cargo selection: both TrCP silencing and mutation of the ubiquitin-dependent endocytosis motif force the GHR to recycle between endosomes and the plasma membrane, together with the transferrin receptor. Depletion of Tsg101 (ESCRT-I) has the same effect, while silencing of Hrs (ESCRT-0) prevents GH recycling. GH passes through late endosomal vesicles, marked by Lamp1. Coexpressing GHR and EGFR demonstrates that both receptors use the same route to the lysosomes. We show for the first time that SCF^TrCP is involved in cargo-specific sorting at endosomes and that Tsg101 rather than Hrs might direct the cargo into the ESCRT machinery.     

Differential roles for checkpoint kinases in DNA damage-dependent degradation of the Cdc25A protein phosphatase

In response to DNA damage, cells activate a signaling pathway that promotes cell cycle arrest and degradation of the cell cycle regulator Cdc25A. Cdc25A degradation occurs via the SCFbeta-TRCP pathway and phosphorylation of Ser-76. Previous work indicates that the checkpoint kinase Checkpoint kinase 1 (Chk1) is capable of phosphorylating Ser-76 in Cdc25A, thereby promoting its degradation. In contrast, other experiments involving overexpression of dominant Chk2 mutant proteins point to a role for Chk2 in Cdc25A degradation. However, loss-of-function studies that implicate Chk2 in Cdc25A turnover are lacking, and there is no evidence that Chk2 is capable of phosphorylating Ser-76 in Cdc25A despite the finding that Chk1 and Chk2 sometimes share overlapping primary specificity. We find that although Chk2 can phosphorylate many of the same sites in Cdc25A that Chk1 phosphorylates, albeit with reduced efficiency, Chk2 is unable to efficiently phosphorylate Ser-76. Consistent with this, Chk2, unlike Chk1, is unable to support SCFbeta-TRCP-mediated ubiquitination of Cdc25A in vitro. In CHK2(-/-) HCT116 cells, the kinetics of Cdc25A degradation in response to ionizing radiation is comparable with that seen in HCT116 cells containing Chk2, indicating that Chk2 is not generally required for timely DNA damage-dependent Cdc25A turnover. In contrast, depletion of Chk1 by RNA interference in CHK2(-/-) cells leads to Cdc25A stabilization in response to ionizing radiation. These data support the idea that Chk1 is the primary signal transducer linking activation of the ATM/ATR kinases to Cdc25A destruction in response to ionizing radiation.     

The E3 ubiquitin ligase APC/CCdh1 degrades MCPH1 after MCPH1‐βTrCP2‐Cdc25A‐mediated mitotic entry to ensure neurogenesis

Mutations of microcephalin (MCPH1) can cause the neurodevelopmental disorder primary microcephaly type 1. We previously showed that MCPH1 deletion in neural stem cells results in early mitotic entry that distracts cell division mode, leading to exhaustion of the progenitor pool. Here, we show that MCPH1 interacts with and promotes the E3 ligase βTrCP2 to degrade Cdc25A independent of DNA damage. Overexpression of βTrCP2 or the knockdown of Cdc25A remedies the high mitotic index and rescues the premature differentiation of Mcph1‐deficient neuroprogenitors in vivo. MCPH1 itself is degraded by APC/C^Cdh1, but not APC/C^Cdc20, in late mitosis and G1 phase. Forced MCPH1 expression causes cell death, underlining the importance of MCPH1 turnover after mitosis. Ectopic expression of Cdh1 leads to premature differentiation of neuroprogenitors, mimicking differentiation defects of Mcph1‐knockout neuroprogenitors. The homeostasis of MCPH1 in association with the ubiquitin‐proteasome system ensures mitotic entry independent of cell cycle checkpoint. This study provides a mechanistic understanding of how MCPH1 controls neural stem cell fate and brain development.     

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.     

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.     

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.