Cyclin A- and cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm

Cyclins A and E and their partner cyclin-dependent kinases (Cdks) are key regulators of DNA synthesis and of mitosis. Immunofluorescence studies have shown that both cyclins are nuclear and that a proportion of cyclin A is localized to sites of DNA replication. However, recently, both cyclin A and cyclin E have been implicated as regulators of centrosome replication, and it is unclear when and where these cyclin-Cdks can interact with cytoplasmic substrates. We have used live cell imaging to study the behavior of cyclin/Cdk complexes. We found that cyclin A and cyclin E are able to regulate both nuclear and cytoplasmic events because they both shuttle between the nucleus and the cytoplasm. However, we found that there are marked differences in their shuttling behavior, which raises the possibility that cyclin/Cdk function could be regulated at the level of nuclear import and export. In the course of these experiments, we have also found that, contrary to published results, mutations in the hydrophobic patch of cyclin A do affect Cdk binding and nuclear import. This has implications for the role of the hydrophobic patch as a substrate selection motif.     

Two Distinct Controls of Mitotic Cdk1/Cyclin B1 Activity Requisite for Cell Growth Prior to Cell Division

Cell growth prior to cell division is restricted by the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complexes. Recently, we identified that the death-effector domain (DED) containing protein, DEDD, acts as a novel inhibitor of mitotic Cdk1/cyclin B1, influencing cell size. Like cyclin B1, DEDD protein levels specifically peak during the G2/M phase. In the nucleus, DEDD associates with Cdk1/cyclin B1 complexes, via direct binding to cyclin B1, and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD-/-) embryonic fibroblasts is increased compared to that in DEDD+/+ cells. This accelerates mitotic progression in DEDD-/- cells, with a shortened G2/M phase, reduced rRNA, and diminished cell volume. Likewise, DEDD-/- mice show decreased body and organ weights relative to DEDD+/+ mice. Interestingly, the DED domain is not involved in the association of DEDD with Cdk1/cyclin B1, but is indispensable for the cell sizing function of DEDD. Together, in addition to the well-established machinery for activation of Cdk1 through dephosphorylation of its inhibitory-residues, we propose a novel mechanism for impeditive regulation of mitotic Cdk1/cyclin B1 mediated by DEDD within the nucleus, which allows sufficient cell growth prior to cell division.     

p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G₁. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21^Waf1/Cip1 enter S phase with a ≥4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G₁/S and G₂/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21^+/+ and p21^−/− cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21^−/− cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G₁-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21^+/+ cells paralleled the onset of endoreduplication in HCT116 p21^−/− cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.     

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.     

Dissecting the Signaling Events That Impact Classical Nuclear Import and Target Nuclear Transport Factors

Background

Signaling through MEK→ERK1/2 and PI3 kinases is implicated in many aspects of cell physiology, including the survival of oxidant exposure. Oxidants play a role in numerous physiological and pathophysiological processes, many of which rely on transport in and out of the nucleus. However, how oxidative stress impacts nuclear trafficking is not well defined.

Methodology/Principal Findings

To better understand the effect of stress on nucleocytoplasmic trafficking, we exposed cells to the oxidant diethyl maleate. This treatment activated MEK→ERK1/2 as well as PI3 kinase→Akt cascades and triggered the inhibition of classical nuclear import. To define the molecular mechanisms that regulate nuclear transport, we examined whether MEK and PI3 kinase signaling affected the localization of key transport factors. Using recently developed tools for image acquisition and analysis, the subcellular distributions of importin-α, CAS, and nucleoporins Nup153 and Nup88 were quantified in different cellular compartments. These studies identified specific profiles for the localization of transport factors in the nucleus and cytoplasm, and at the nuclear envelope. Our results demonstrate that MEK and PI3 kinase signaling as well as oxidative stress control nuclear trafficking and the localization of transport components. Furthermore, stress not only induced changes in transport factor distribution, but also upregulated post-translational modification of transport factors. Our results are consistent with the idea that the phosphorylation of importin-α, CAS, Nup153, and Nup88, and the O-GlcNAc modification of Nup153 increase when cells are exposed to oxidant.

Conclusions/Significance

Our studies defined the complex regulation of classical nuclear import and identified key transport factors that are targeted by stress, MEK, and PI3 kinase signaling.     

Requirement of Cyclin/Cdk2 and protein phosphatase 1 activity for chromatin assembly factor 1-dependent chromatin assembly during DNA synthesis

The influence of reversible protein phosphorylation on nucleosome assembly during DNA replication was analyzed in extracts from human cells. Inhibitor studies and add-back experiments indicated requirements of cyclin A/Cdk2, cyclin E/Cdk2, and protein phosphatase type 1 (PP1) activities for nucleosome assembly during DNA synthesis by chromatin assembly factor 1 (CAF-1). The p60 subunit of CAF-1 is a molecular target for reversible phosphorylation by cyclin/Cdk complexes and PP1 during nucleosome assembly and DNA synthesis in vitro. Purified p60 can be directly phosphorylated by purified cyclin A/Cdk2, cyclin E/Cdk2, and cyclin B1/Cdk1, but not by cyclin D/Cdk4 complexes in vitro. Cyclin B1/Cdk1 triggers hyperphosphorylation of p60 in the presence of additional cytosolic factors. CAF-1 containing hyperphosphorylated p60 prepared from mitotic cells is inactive in nucleosome assembly and becomes activated by dephosphorylation in vitro. These data provide functional evidence for a requirement of the cell cycle machinery for nucleosome assembly by CAF-1 during DNA replication.     

Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression

Activation of cyclin B1–cyclin-dependent kinase 1 (Cdk1), triggered by a positive feedback loop at the end of G2, is the key event that initiates mitotic entry. In metaphase, anaphase-promoting complex/cyclosome–dependent destruction of cyclin B1 inactivates Cdk1 again, allowing mitotic exit and cell division. Several models describe Cdk1 activation kinetics in mitosis, but experimental data on how the activation proceeds in mitotic cells have largely been lacking. We use a novel approach to determine the temporal development of cyclin B1–Cdk1 activity in single cells. By quantifying both dephosphorylation of Cdk1 and phosphorylation of the Cdk1 target anaphase-promoting complex/cyclosome 3, we disclose how cyclin B1–Cdk1 continues to be activated after centrosome separation. Importantly, we discovered that cytoplasmic cyclin B1–Cdk1 activity can be maintained even when cyclin B1 translocates to the nucleus in prophase. These experimental data are fitted into a model describing cyclin B1–Cdk1 activation in human cells, revealing a striking resemblance to a bistable circuit. In line with the observed kinetics, cyclin B1–Cdk1 levels required to enter mitosis are lower than the amount of cyclin B1–Cdk1 needed for mitotic progression. We propose that gradually increasing cyclin B1–Cdk1 activity after centrosome separation is critical to coordinate mitotic progression.     

Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence ⁴¹⁸KRPR⁴²¹, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin β/α1,3,7 whereas hMSH2 specifically recognizes importin β/α3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.     

Conserved Arginines of Bovine Adenovirus-3 33K Protein Are Important for Transportin-3 Mediated Transport and Virus Replication

The L6 region of bovine adenovirus (BAdV)-3 encodes a spliced protein designated 33K. The 33K specific sera detected five major proteins and three minor proteins in transfected or virus infected cells, which could arise by internal initiation of translation and alternative splicing. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells. The 33K nuclear transport utilizes both classical importin-α/-β and importin-β dependent nuclear import pathways and preferentially binds to importin-α5 and transportin-3 receptors, respectively. Analysis of mutant 33K proteins demonstrated that amino acids 201–240 of the conserved C-terminus of 33K containing RS repeat are required for nuclear localization and, binding to both importin-α5 and transportin-3 receptors. Interestingly, the arginine residues of conserved RS repeat are required for binding to transportin-3 receptor but not to importin-α5 receptor. Moreover, mutation of arginines residues of RS repeat proved lethal for production of progeny virus. Our results suggest that arginines of RS repeat are required for efficient nuclear transport of 33K mediated by transportin-3, which appears to be essential for replication and production of infectious virion.     

A kinetic model of the cyclin E/Cdk2 developmental timer in Xenopus laevis embryos

Early cell cycles of Xenopus laevis embryos are characterized by rapid oscillations in the activity of two cyclin-dependent kinases. Cdk1 activity peaks at mitosis, driven by periodic degradation of cyclins A and B. In contrast, Cdk2 activity oscillates twice per cell cycle, despite a constant level of its partner, cyclin E. Cyclin E degrades at a fixed time after fertilization, normally corresponding to the midblastula transition. Based on published data and new experiments, we constructed a mathematical model in which: (1) oscillations in Cdk2 activity depend upon changes in phosphorylation, (2) Cdk2 participates in a negative feedback loop with the inhibitory kinase Wee1; (3) cyclin E is cooperatively removed from the oscillatory system; and (4) removed cyclin E is degraded by a pathway activated by cyclin E/Cdk2 itself. The model's predictions about embryos injected with Xic1, a stoichiometric inhibitor of cyclin E/Cdk2, were experimentally validated.     

Probing the Specificity of Binding to the Major Nuclear Localization Sequence-binding Site of Importin-α Using Oriented Peptide Library Screening

Importin-α is the nuclear import receptor that recognizes the classic monopartite and bipartite nuclear localization sequences (cNLSs), which contain one or two clusters of basic amino acids, respectively. Different importin-α paralogs in a single organism are specific for distinct repertoires of cargos. Structural studies revealed that monopartite cNLSs and the C-terminal basic clusters of the bipartite cNLSs bind to the same site on importin-α, termed the major cNLS-binding site. We used an oriented peptide library approach with five degenerate positions to probe the specificity of the major cNLS-binding site in importin-α. We identified the sequences KKKRR, KKKRK, and KKRKK as the optimal sequences for binding to this site for mouse importin-α2, human importin-α1, and human importin-α5, respectively. The crystal structure of mouse importin-α2 with its optimal peptide confirmed the expected binding mode resembling the binding of simian virus 40 large tumor-antigen cNLS. Binding assays confirmed that the peptides containing these sequences bound to the corresponding proteins with low nanomolar affinities. Nuclear import assays showed that the sequences acted as functional cNLSs, with specificity for particular importin-αs. This is the first time that structural information has been linked to an oriented peptide library screening approach for importin-α; the results will contribute to understanding of the sequence determinants of cNLSs, and may help identify as yet unidentified cNLSs in novel proteins.     

Centrosomal localization of cyclins E and A: Structural similarities and functional differences

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G₁/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.Key words: cyclin A, cyclin E, Cdk2, centrosome, CLS, PSTAIRE, DNA synthesis     

Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53^+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53^−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53^−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.     

Structural Characterisation of the Nuclear Import Receptor Importin Alpha in Complex with the Bipartite NLS of Prp20

The translocation of macromolecules into the nucleus is a fundamental eukaryotic process, regulating gene expression, cell division and differentiation, but which is impaired in a range of significant diseases including cancer and viral infection. The import of proteins into the nucleus is generally initiated by a specific, high affinity interaction between nuclear localisation signals (NLSs) and nuclear import receptors in the cytoplasm, and terminated through the disassembly of these complexes in the nucleus. For classical NLSs (cNLSs), this import is mediated by the importin-α (IMPα) adaptor protein, which in turn binds to IMPβ to mediate translocation of nuclear cargo across the nuclear envelope. The interaction and disassembly of import receptor:cargo complexes is reliant on the differential localisation of nucleotide bound Ran across the envelope, maintained in its low affinity, GDP-bound form in the cytoplasm, and its high affinity, GTP-bound form in the nucleus. This in turn is maintained by the differential localisation of Ran regulating proteins, with RanGAP in the cytoplasm maintaining Ran in its GDP-bound form, and RanGEF (Prp20 in yeast) in the nucleus maintaining Ran in its GTP-bound form. Here, we describe the 2.1 Å resolution x-ray crystal structure of IMPα in complex with the NLS of Prp20. We observe 1,091 Ų of buried surface area mediated by an extensive array of contacts involving residues on armadillo repeats 2-7, utilising both the major and minor NLS binding sites of IMPα to contact bipartite NLS clusters ¹⁷RAKKMSK²³ and ³KR⁴, respectively. One notable feature of the major site is the insertion of Prp20NLS Ala¹⁸ between the P0 and P1 NLS sites, noted in only a few classical bipartite NLSs. This study provides a detailed account of the binding mechanism enabling Prp20 interaction with the nuclear import receptor, and additional new information for the interaction between IMPα and cargo.     

Initiation of human DNA replication in vitro using nuclei from cells arrested at an initiation-competent state

Initiation of human DNA replication is investigated in vitro, using initiation-competent nuclei isolated from cells arrested in late G(1) phase by a 24-h treatment with 0.5 mm mimosine (Krude, T. (1999) Exp. Cell Res. 247, 148-159). Nuclei isolated from mimosine-arrested HeLa cells initiate semiconservative DNA replication upon incubation in cytosolic extracts from proliferating human cells. Initiation occurs in the absence and presence of a nuclear membrane. The cyclin-dependent kinase (Cdk) inhibitors roscovitine and olomoucine inhibit initiation of DNA replication, indicating a dependence of initiation on Cdk activity. Cell fractionation shows that cyclins A, E, and Cdk2 are bound to nuclei from mimosine-arrested cells. Exogenously added human cyclin A.Cdk2 and cyclin E.Cdk2 complexes, but not cyclin B1/Cdk1 or cyclin D2/Cdk6, can overcome inhibition of initiation by roscovitine in vitro. Depleting Cdk2 from cytosolic extract does not prevent initiation, demonstrating that cyclin.Cdk2 complexes are not required in the soluble extract, but are provided by the nuclei. Initiation depends further on an essential and soluble activity present in cytosolic extracts from proliferating cells, but not from mimosine-arrested cells, acting together with nuclear cyclin/Cdk2 activity.     

Human papillomavirus type 16 E1 E4-induced G2 arrest is associated with cytoplasmic retention of active Cdk1/cyclin B1 complexes

Human papillomavirus type 16 (HPV16) can cause cervical cancer. Expression of the viral E1 E4 protein is lost during malignant progression, but in premalignant lesions, E1 E4 is abundant in cells supporting viral DNA amplification. Expression of 16E1 E4 in cell culture causes G2 cell cycle arrest. Here we show that unlike many other G2 arrest mechanisms, 16E1 E4 does not inhibit the kinase activity of the Cdk1/cyclin B1 complex. Instead, 16E1 E4 uses a novel mechanism in which it sequesters Cdk1/cyclin B1 onto the cytokeratin network. This prevents the accumulation of active Cdk1/cyclin B1 complexes in the nucleus and hence prevents mitosis. A mutant 16E1 E4 (T22A, T23A) which does not bind cyclin B1 or alter its intracellular location fails to induce G2 arrest. The significance of these results is highlighted by the observation that in lesions induced by HPV16, there is evidence for Cdk1/cyclin B1 activity on the keratins of 16E1 E4-expressing cells. We hypothesize that E1 E4-induced G2 arrest may play a role in creating an environment optimal for viral DNA replication and that loss of E1 E4 expression may contribute to malignant progression.     

Identification of the nuclear localization signal in Xenopus cyclin E and analysis of its role in replication and mitosis

Cyclin-dependent kinase (Cdk)2/cyclin E is imported into nuclei assembled in Xenopus egg extracts by a pathway that requires importin-alpha and -beta. Here, we identify a basic nuclear localization sequence (NLS) in the N-terminus of Xenopus cyclin E. Mutation of the NLS eliminated nuclear accumulation of both cyclin E and Cdk2, and such versions of cyclin E were unable to trigger DNA replication. Addition of a heterologous NLS from SV40 large T antigen restored both nuclear targeting of Cdk2/cyclin E and DNA replication. We present evidence indicating that Cdk2/cyclin E complexes must become highly concentrated within nuclei to support replication and find that cyclin A can trigger replication at much lower intranuclear concentrations. We confirmed that depletion of endogenous cyclin E increases the concentration of cyclin B necessary to promote entry into mitosis. In contrast to its inability to promote DNA replication, cyclin E lacking its NLS was able to cooperate with cyclin B in promoting mitotic entry.     

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.     

IQGAP1 Protein Regulates Nuclear Localization of β-Catenin via Importin-β5 Protein in Wnt Signaling

In the canonical Wnt signaling pathway, the translocation of β-catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of β-catenin function via importin-β5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of β-catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-β5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and β-catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of β-catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of β-catenin in the canonical Wnt signaling pathway.