p21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775

The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21^-/-, RPE p21^-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.     

Proliferating human cells hypomorphic for origin recognition complex 2 and pre-replicative complex formation have a defect in p53 activation and Cdk2 kinase activation

The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Delta/-) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G1 to S-phase transition. Cdk2 kinase activation in G1 is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G1 cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2.     

Residual Cdk1/2 activity after DNA damage promotes senescence

In response to DNA damage, a cell can be forced to permanently exit the cell cycle and become senescent. Senescence provides an early barrier against tumor development by preventing proliferation of cells with damaged DNA. By studying single cells, we show that Cdk activity persists after DNA damage until terminal cell cycle exit. This low level of Cdk activity not only allows cell cycle progression, but also promotes cell cycle exit at a decision point in G2 phase. We find that residual Cdk1/2 activity is required for efficient p21 production, allowing for nuclear sequestration of Cyclin B1, subsequent APC/C^Cdh1‐dependent degradation of mitotic inducers and induction of senescence. We suggest that the same activity that triggers mitosis in an unperturbed cell cycle enforces senescence in the presence of DNA damage, ensuring a robust response when most needed.     

Dual Phosphorylation of Cdk1 Coordinates Cell Proliferation with Key Developmental Processes in Drosophila

Eukaryotic organisms use conserved checkpoint mechanisms that regulate Cdk1 by inhibitory phosphorylation to prevent mitosis from interfering with DNA replication or repair. In metazoans, this checkpoint mechanism is also used for coordinating mitosis with dynamic developmental processes. Inhibitory phosphorylation of Cdk1 is catalyzed by Wee1 kinases that phosphorylate tyrosine 15 (Y15) and dual-specificity Myt1 kinases found only in metazoans that phosphorylate Y15 and the adjacent threonine (T14) residue. Despite partially redundant roles in Cdk1 inhibitory phosphorylation, Wee1 and Myt1 serve specialized developmental functions that are not well understood. Here, we expressed wild-type and phospho-acceptor mutant Cdk1 proteins to investigate how biochemical differences in Cdk1 inhibitory phosphorylation influence Drosophila imaginal development. Phosphorylation of Cdk1 on Y15 appeared to be crucial for developmental and DNA damage-induced G2-phase checkpoint arrest, consistent with other evidence that Myt1 is the major Y15-directed Cdk1 inhibitory kinase at this stage of development. Expression of non-inhibitable Cdk1 also caused chromosome defects in larval neuroblasts that were not observed with Cdk1(Y15F) mutant proteins that were phosphorylated on T14, implicating Myt1 in a novel mechanism promoting genome stability. Collectively, these results suggest that dual inhibitory phosphorylation of Cdk1 by Myt1 serves at least two functions during development. Phosphorylation of Y15 is essential for the premitotic checkpoint mechanism, whereas T14 phosphorylation facilitates accumulation of dually inhibited Cdk1–Cyclin B complexes that can be rapidly activated once checkpoint-arrested G2-phase cells are ready for mitosis.     

Mechanisms underlying the pro-survival pathway of p53 in suppressing mitotic death induced by adriamycin

The p53 tumor suppressor responds to chemotherapeutic stress by triggering apoptosis or eliciting pro-survival pathway through arresting cell cycle progression for DNA damage repair. Here we examined the pro-survival activity of p53 on the adriamycin-induced stress using H1299 cells stably expressing tsp53 V143A, a temperature-sensitive mutant activating only the subset of p53 target genes related to growth arrest and DNA repair, but not apoptosis. At 38 degrees C, cells evaded from adriamycin-induced G2 arrest and died of apoptosis and mitotic catastrophe, which could be inhibited by Cdk inhibitors. Activation of functional tsp53 V143A at 32 degrees C led to suppression of Cdk1/2 activities and Cyclin B1/Cdk1 expression, cells exhibited prolonged G2 arrest, regained reproductive potential and were protected from mitotic catastrophe induced by adriamycin. Inhibition of mitotic catastrophe and Cyclin B1/Cdk1 expression was ablated upon silencing p21 Waf1 expression in tsp53 V143A-H1299 cells or in HCT116 cells. Together we show that p21 Waf1 is a key component of G2 checkpoint necessary and sufficient for protecting tumor cells against adriamycin-induced mitotic catastrophe.     

Inactivation of Cdk1/Cyclin B in metaphase-arrested mouse FT210 cells induces exit from mitosis without chromosome segregation or cytokinesis and allows passage through another cell cycle

It is well known that inactivation of Cdk1/Cyclin B is required for cells to exit mitosis. The work reported here tests the hypothesis that Cdk1/Cyclin B inactivation is not only necessary but also sufficient to induce mitotic exit and reestablishment of the interphase state. This hypothesis predicts that inactivation of Cdk1 in metaphase-arrested cells will induce the M to G1-phase transition. It is shown that when mouse FT210 cells (in which Cdk1 is temperature-sensitive) are arrested in metaphase and then shifted to their non-permissive temperature, they rapidly exit mitosis as evidenced by reassembly of interphase nuclei, decondensation of chromosomes, and dephosphorylation of histones H1 and H3. The resulting interphase cells are functionally normal as judged by their ability to progress through another cell cycle. However, they have double the normal number of chromosomes because they previously bypassed anaphase, chromosome segregation, and cytokinesis. These results, taken together with other observations in the literature, strongly suggest that in mammalian cells, inactivation of Cdk1/cyclin B is the trigger for mitotic exit and reestablishment of the interphase state.     

Inhibitory phosphorylation of PP1alpha catalytic subunit during the G(1)/S transition.

We have shown earlier that, in cells expressing the retinoblastoma protein (pRB), a protein phosphatase (PP) 1alpha mutant (T320A) resistant to inhibitory phosphorylation by cyclin-dependent kinases (Cdks) causes G(1) arrest. In this study, we examined the cell cycle-dependent phosphorylation of PP1alpha in vivo using three different antibodies. PP1alpha was phosphorylated at Thr-320 during M-phase and again in late G(1)- through early S-phase. Inhibition of Cdk2 led to a small increase in PP1 activity and also prevented PP1alpha phosphorylation. In vitro, PP1alpha was a substrate for Cdk2 but not Cdk4. In pRB-deficient cells, phosphorylation of PP1alpha occurred in M-phase but not at G(1)/S. G(1)/S phosphorylation was at least partially restored after reintroduction of pRB into these cells. Consistent with this result, PP1alpha phosphorylated at Thr-320 co-precipitated with pRB during G(1)/S but was found in extracts immunodepleted of pRB in M-phase. In conjunction with earlier studies, these results indicate that PP1alpha may control pRB function throughout the cell cycle. In addition, our new results suggest that different subpopulations of PP1alpha regulate the G(1)/S and G(2)/M transitions and that PP1alpha complexed to pRB requires inhibitory phosphorylation by G(1)-specific Cdks in order to prevent untimely reactivation of pRB and permit transition from G(1)- to S-phase and/or complete S-phase.     

A double-assurance mechanism controls cell cycle exit upon terminal differentiation in Drosophila

Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. We examined the process of cell cycle exit in Drosophila wings and eyes and discovered that cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation.     

Mechanism of Cdk2/Cyclin E inhibition by p27 and p27 phosphorylation.

The biochemical interactions between the Cdk2/Cyclin E kinase and its inhibitor p27, were investigated using purified, recombinant p27 and CAK-phosphorylated Cdk2/Cyclin E. From kcat/Km determinations using either histone H1 or pRb as substrates, we found that Cdk2/Cyclin E has 60-fold higher specificity for pRb than for histone H1. The IC50 value of p27 increased with increasing Cdk2/Cyclin E concentrations while it remained constant at various ATP and histone H1 concentrations, suggesting that p27 acts as a tight binding inhibitor of Cdk2/Cyclin E. We also found that p27 could be phosphorylated by Cdk2/Cyclin E only at high enzyme concentrations, and that p27 forms a stable interaction with Cdk2/Cyclin E regardless of its phosphorylation state. Our results further indicate that the Cdk2/Cyclin E/p27 ternary complex is kinetically inactive as an enzyme; instead it serves as a substrate for Cdk2/Cyclin E. These results suggest that if phosphorylation of p27 by Cdk2/Cyclin E is involved in its ubiquitin-dependent degradation, as previously suggested, then the target for such event is the phosphorylated p27 bound to Cdk2/Cyclin E and not free p27.     

Chromatin decondensation in S-phase involves recruitment of Cdk2 by Cdc45 and histone H1 phosphorylation

Cdc45 is required for initiation of DNA replication and fork progression, but its function in these processes remains unknown. We show that targeting Cdc45 to specific chromosomal sites in mammalian cells results in large-scale chromatin decondensation that strongly correlates with histone H1 phosphorylation. Cdk2 is recruited to sites of Cdc45 decondensation, and Cdk2 inhibitors reduce the level of decondensation. Targeting wild-type Cdk2, but not kinase-defective Cdk2, to chromatin is also effective at inducing decondensation involving phospho-H1. Cdc45, Cdk2, Cyclin A, and phospho-H1 associate with chromatin during S-phase, and Cdc45, Cdk2, and an active H1 kinase physically interact. Replicating DNA and phospho-H1 foci colocalize in vivo, and S-phase progression and H1 phosphorylation are directly related and Cdk2 dependent. Because Cdk2 colocalizes with replication foci and H1 regulates higher-order chromatin, we suggest a model in which Cdc45 recruits Cdk2 to replication foci, resulting in H1 phosphorylation, chromatin decondensation, and facilitation of fork progression.     

Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

A phosphatase threshold sets the level of Cdk1 activity in early mitosis in budding yeast

Entry into mitosis is initiated by synthesis of cyclins, which bind and activate cyclin-dependent kinase 1 (Cdk1). Cyclin synthesis is gradual, yet activation of Cdk1 occurs in a stepwise manner: a low level of Cdk1 activity is initially generated that triggers early mitotic events, which is followed by full activation of Cdk1. Little is known about how stepwise activation of Cdk1 is achieved. A key regulator of Cdk1 is the Wee1 kinase, which phosphorylates and inhibits Cdk1. Wee1 and Cdk1 show mutual regulation: Cdk1 phosphorylates Wee1, which activates Wee1 to inhibit Cdk1. Further phosphorylation events inactivate Wee1. We discovered that a specific form of protein phosphatase 2A (PP2A(Cdc55)) opposes the initial phosphorylation of Wee1 by Cdk1. In vivo analysis, in vitro reconstitution, and mathematical modeling suggest that PP2A(Cdc55) sets a threshold that limits activation of Wee1, thereby allowing a low constant level of Cdk1 activity to escape Wee1 inhibition in early mitosis. These results define a new role for PP2A(Cdc55) and reveal a systems-level mechanism by which dynamically opposed kinase and phosphatase activities can modulate signal strength.     

Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase

Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C^Cdh1. However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C^Cdh1-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.     

Hyperoxia Induces S-Phase Cell-Cycle Arrest and p21-Independent Cdk2 Inhibition in Human Carcinoma T47D-H3 Cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O₂, 40–64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21^Cip1. The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21^Cip1 or p27^Kip1 in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21^Cip1/p27^Kip1-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Wee1 kinase as a target for cancer therapy

Wee1, a protein kinase, regulates the G₂ checkpoint in response to DNA damage. Preclinical studies have elucidated the role of wee1 in DNA damage repair and the stabilization of replication forks, supporting the validity of wee1 inhibition as a viable therapeutic target in cancer. MK-1775, a selective and potent small-molecule inhibitor of wee1, is under clinical development as a potentiator of DNA damage caused by cytotoxic chemotherapies. We present a review of the role of wee1 in the cell cycle and DNA replication and summarize the clinical development to date of this novel class of anticancer agents.     

A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

Sophisticated models for the regulation of mitotic entry are lacking for human cells. Inactivating human cyclin A/Cdk2 complexes through diverse approaches delays mitotic entry and promotes inhibitory phosphorylation of Cdk1 on tyrosine 15, a modification performed by Wee1. We show here that cyclin A/Cdk2 complexes physically associate with Wee1 in U2OS cells. Mutation of four conserved RXL cyclin A/Cdk binding motifs (RXL1 to RXL4) in Wee1 diminished stable binding. RXL1 resides within a large regulatory region of Wee1 that is predicted to be intrinsically disordered (residues 1 to 292). Near RXL1 is T239, a site of inhibitory Cdk phosphorylation in Xenopus Wee1 proteins. We found that T239 is phosphorylated in human Wee1 and that this phosphorylation was reduced in an RXL1 mutant. RXL1 and T239 mutants each mediated greater Cdk phosphorylation and G₂/M inhibition than the wild type, suggesting that cyclin A/Cdk complexes inhibit human Wee1 through these sites. The RXL1 mutant uniquely also displayed increased nuclear localization. RXL1 is embedded within sequences homologous to Crm1-dependent nuclear export signals (NESs). Coimmunoprecipitation showed that Crm1 associated with Wee1. Moreover, treatment with the Crm1 inhibitor leptomycin B or independent mutation of the potential NES (NESm) abolished Wee1 nuclear export. Export was also reduced by Cdk inhibition or cyclin A RNA interference, suggesting that cyclin A/Cdk complexes contribute to Wee1 export. Somewhat surprisingly, NESm did not display increased G₂/M inhibition. Thus, nuclear export of Wee1 is not essential for mitotic entry though an important functional role remains likely. These studies identify a novel bifunctional regulatory element in Wee1 that mediates cyclin A/Cdk2 association and nuclear export.Despite broad progress in studies of cell cycle control in eukaryotes, advanced models are lacking for the regulation of mitotic entry in human cells. This regulation is pivotal in cell cycle control, and a better understanding of it may be crucial to improving cytotoxic cancer chemotherapy, the mainstay of cancer treatment. Models of mitotic entry in higher eukaryotes revolve around activation of the cyclin B/Cdk1 (cyclin-dependent kinase 1 or Cdc2) complex, which drives the major events of mitosis. A rise in the cyclin B level triggers mitotic entry in Xenopus egg extracts but not in mammalian cells (15, 47). Inhibitory phosphorylation of Cdk1 on the ATP-binding site residue tyrosine 15 (Y15) has been recognized as a key constraint throughout eukaryotes (29, 42). Wee1 and Myt kinases perform this phosphorylation in vertebrate cells, where Wee1 appears to be dominant (34). Kim and Ferrell and others have recently developed an elegant model for ultrasensitive, switch-like inactivation of Wee1 by cyclin B/Cdk1 in a positive feedback loop that contributes to mitotic entry in Xenopus egg extracts (27).Although cyclin A(A2)/Cdk2 is traditionally omitted from models of mitotic entry, accumulating evidence from several different approaches suggests that cyclin A/Cdk complexes play roles. Cyclin A levels rise during S phase and peak in G₂ before falling abruptly in prometaphase of mitosis (60). Microinjection of cyclin A/Cdk2 complexes in human G₂ phase cells was observed to drive mitotic entry (14). Conversely, microinjection of antibodies directed against cyclin A in S-phase cells inhibited mitotic entry without an apparent effect on bulk DNA synthesis (45). In complementary approaches that supported biochemical analyses, cyclin A RNA interference (RNAi) or induction of a dominant negative mutant of Cdk2 (Cdk2-dn), the major cyclin A binding partner, inhibited mitotic entry (13, 15, 21, 37). In these settings, cyclin B/Cdk1 complexes accumulated in inactive, Y15-phosphorylated forms (13, 21, 37). Cdc25 phosphatases, which can reverse this phosphorylation, show reduced activity in this context (37), but increased Cdc25 activity could not readily overcome the arrest (13). RNAi-mediated knockdown of Wee1 was found capable of overriding the arrest mediated by cyclin A RNAi, suggesting that Wee1 is a key rate-limiting factor (13). However, whether and by what mechanisms cyclin A complexes might regulate Wee1 and drive Cdk1 dephosphorylation and mitotic entry have remained unclear.Recently, genetic studies in mice have reinforced these observations while providing evidence for some cell type differences (24). Although Cdk2 is not essential, in its absence Cdk1 binds more cyclin A and E and provides redundant functions (4, 25, 44). Deletion of the cyclin A gene is lethal for embryos and adults (24). Gene deletion in fibroblasts in vitro did not completely abrogate their proliferation but caused S and G₂/M delays. In this setting cyclin E was upregulated, and combined deletion of cyclin E yielded arrest in G₁, S, and G₂/M phases. Cyclin A gene deletion was alone sufficient to block proliferation of hematopoietic stem cells, suggesting that cyclin A is essential for their proliferation.Wee1 is regulated on multiple levels, including inhibitory phosphorylation in the amino-terminal regulatory domain (NRD), residues 1 to 292. This region is predicted to be intrinsically disordered (56), and few functional elements have been identified in it. The cyclin B/Cdk1 complex has been thought to be the principal or exclusive kinase responsible for NRD phosphorylation (18, 27, 28). Two sites in the Xenopus embryonic Wee1 NRD, Thr 104 and Thr 150 (referred to here by the homologous residue, T239, in human somatic Wee1), have been identified as Cdk phosphorylation sites that inhibit Wee1 activity (28). Recent studies of Xenopus somatic Wee1 suggest that T239 phosphorylation may antagonize the function of a surrounding motif, dubbed the Wee box (43). This small, conserved region appears to augment the activity of the carboxy-terminal kinase domain.We show here that cyclin A/Cdk2 complexes directly bind Wee1 as a substrate in human cells. In particular, a conserved cyclin A/Cdk binding RXL motif in the Wee1 NRD is required for efficient T239 phosphorylation. Further analysis revealed that RXL1 is located within a Crm1 binding site that mediates Wee1 export during S and G₂ phases. Cyclin A/Cdk2 activity appears to foster Wee1 export, but this export is not essential for mitotic entry. These findings further define roles of cyclin A/Cdk complexes in regulating Wee1 and mitotic entry in human cells and dissect the mechanisms and consequences of Wee1 redistribution during the run-up to mitosis.     

Gauchos and ochos: a Wee1-Cdk tango regulating mitotic entry

The kinase Wee1 has been recognized for a quarter century as a key inhibitor of Cyclin dependent kinase 1 (Cdk1) and mitotic entry in eukaryotes. Nonetheless, Wee1 regulation is not well understood and its large amino-terminal regulatory domain (NRD) has remained largely uncharted. Evidence has accumulated that cyclin B/Cdk1 complexes reciprocally inhibit Wee1 activity through NRD phosphorylation. Recent studies have identified the first functional NRD elements and suggested that vertebrate cyclin A/Cdk2 complexes also phosphorylate the NRD. A short NRD peptide, termed the Wee box, augments the activity of the Wee1 kinase domain. Cdk1/2-mediated phosphorylation of the Wee box (on T239) antagonizes kinase activity. A nearby region harbors a conserved RxL motif (RxL1) that promotes cyclin A/Cdk2 binding and T239 phosphorylation. Mutation of either T239 or RxL1 bolsters the ability of Wee1 to block mitotic entry, consistent with negative regulation of Wee1 through these sites. The region in human somatic Wee1 that encompasses RxL1 also binds Crm1, directing Wee1 export from the nucleus. These studies have illuminated important aspects of Wee1 regulation and defined a specific molecular pathway through which cyclin A/Cdk2 complexes foster mitotic entry. The complexity, speed, and importance of regulation of mitotic entry suggest that there is more to be learned.     

Recruitment of cyclin G2 to promyelocytic leukemia nuclear bodies promotes dephosphorylation of γH2AX following treatment with ionizing radiation

Cyclin G2 (CycG2) and Cyclin G1 (CycG1), two members of the Cyclin G subfamily, share high amino acid homology in their Cyclin G boxes. Functionally, they play a common role as association partners of the B′γ subunit of protein phosphatase 2A (PP2A) and regulate PP2A function, and their expression is increased following DNA damage. However, whether or not CycG1 and CycG2 have distinct roles during the cellular DNA damage response has remained unclear. Here, we report that CycG2, but not CycG1, co-localized with promyelocytic leukemia (PML) and γH2AX, forming foci following ionizing radiation (IR), suggesting that CycG2 is recruited to sites of DNA repair and that CycG1 and CycG2 have distinct functions. PML failed to localize to nuclear foci when CycG2 was depleted, and vice versa. This suggests that PML and CycG2 mutually influence each other’s functions following IR. Furthermore, we generated CycG2-knockout (Ccng2^−/−) mice to investigate the functions of CycG2. These mice were born healthy and developed normally. However, CycG2-deficient mouse embryonic fibroblasts displayed an abnormal response to IR. Dephosphorylation of γH2AX and checkpoint kinase 2 following IR was delayed in Ccng2^−/− cells, suggesting that DNA damage repair may be perturbed in the absence of CycG2. Although knockdown of B′γ in wild-type cells also delayed dephosphorylation of γH2AX, knockdown of B′γ in Ccng2^−/− cells prolonged this delay, suggesting that CycG2 cooperates with B′γ to dephosphorylate γH2AX. Taken together, we conclude that CycG2 is localized at DNA repair foci following DNA damage, and that CycG2 regulates the dephosphorylation of several factors necessary for DNA repair.