Nuclear Accumulation Of Cyclin B1 Research Articles

G2/M checkpoint regulation and apoptosis facilitate the nuclear egress of parvoviral capsids.

The nuclear export factor CRM1-mediated pathway is known to be important for the nuclear egress of progeny parvovirus capsids in the host cells with virus-mediated cell cycle arrest at G2/M. However, it is still unclear whether this is the only pathway by which capsids exit the nucleus. Our studies show that the nuclear egress of DNA-containing full canine parvovirus. capsids was reduced but not fully inhibited when CRM1-mediated nuclear export was prevented by leptomycin B. This suggests that canine parvovirus capsids might use additional routes for nuclear escape. This hypothesis was further supported by our findings that nuclear envelope (NE) permeability was increased at the late stages of infection. Inhibitors of cell cycle regulatory protein cyclin-dependent kinase 1 (Cdk1) and pro-apoptotic caspase 3 prevented the NE leakage. The change in NE permeability could be explained by the regulation of the G2/M checkpoint which is accompanied by early mitotic and apoptotic events. The model of G2/M checkpoint activation was supported by infection-induced nuclear accumulation of cyclin B1 and Cdk1. Both NE permeability and nuclear egress of capsids were reduced by the inhibition of Cdk1. Additional proof of checkpoint function regulation and promotion of apoptotic events was the nucleocytoplasmic redistribution of nuclear transport factors, importins, and Ran, in late infection. Consistent with our findings, post-translational histone acetylation that promotes the regulation of several genes related to cell cycle transition and arrest was detected. In conclusion, the model we propose implies that parvoviral capsid egress partially depends on infection-induced G2/M checkpoint regulation involving early mitotic and apoptotic events.

Targeted inactivation of nuclear interaction partner of ALK disrupts meiotic prophase

NIPA (nuclear interaction partner of ALK) is an F-box-like protein that monitors the timing of mitotic entry. Constitutively active NIPA delays mitotic entry by preventing accumulation of nuclear cyclin B1. Here, we have investigated the consequences of Nipa inactivation by using a conditional knockout strategy. Nipa-deficient animals are viable but show a lower birth rate and reduced body weight. Furthermore, Nipa-deficient males are sterile owing to a block of spermatogenesis during meiotic prophase. Whereas Nipa-/- mouse embryonic fibroblasts show no severe phenotype, Nipa-/- spermatocytes arrest during stage IV of the epithelial cycle with subsequent TUNEL-positive apoptosis resulting from improper synapsis, defects in the repair of DNA double-stranded breaks and synaptonemal complex formation. Moreover, we show nuclear accumulation of cyclin B1 with a subsequent premature increase in G2/M kinase activity in Nipa-/- spermatocytes. Together, these results reveal a novel role for NIPA in meiosis.

KX-01, a novel Src kinase inhibitor directed toward the peptide substrate site, synergizes with tamoxifen in estrogen receptor α positive breast cancer

KX-01 is the first clinical Src inhibitor of the novel peptidomimetic class that targets the peptide substrate site of Src providing more specificity toward Src kinase. The present study was designed to evaluate the effects of KX-01 as a single agent and in combination with tamoxifen (TAM) on cell growth and apoptosis of ERα positive breast cancer in vitro and in vivo. Flow cytometry demonstrated that KX-01 induced cell cycle arrest in G2/M phase. Immunofluorescent staining for mitotic phase markers and TUNEL staining indicated that cells had arrested in the mitotic phase and mitotic arrested cells were undergoing apoptosis. KX-01 induced nuclear accumulation of cyclin B1, and activation of CDK1, MPM2, and Cdc25C that is required for progression past the G2/M checkpoint. Apoptosis resulted from activation of caspases 6, 7, 8, and 9. Combinational index analysis revealed that combinations of KX-01 with TAM resulted in synergistic growth inhibition of breast cancer cell lines. KX-01 combined with TAM resulted in decreased ERα phosphorylation at Src-regulated phosphorylation sites serines 118 and 167 that were associated with reduced ERα transcriptional activity. Orally administered KX-01 resulted in a dose dependent growth inhibition of MCF-7 tumor xenografts, and in combination with TAM exhibited synergistic growth inhibition. Immunohistochemical analysis revealed that combinational treatment reduced angiogenesis, and ERα signaling in tumors compared to either drug alone that may underlie the synergistic tumor growth inhibition. Combinations of KX-01 with endocrine therapy present a promising new strategy for clinical management of ERα positive breast cancer.

Activation of cyclin B1–Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis

The cyclin B-Cdk1 kinase triggers mitosis in most eukaryotes. In animal cells, cyclin B shuttles between the nucleus and cytoplasm in interphase before rapidly accumulating in the nucleus at prophase, which promotes disassembly of the nuclear lamina and nuclear envelope breakdown (NEBD). What triggers the nuclear accumulation of cyclin B1 is presently unclear, although the prevailing view is that the Plk1 kinase inhibits its nuclear export. In this study, we use a biosensor specific for cyclin B1-Cdk1 activity to show that activating cyclin B1-Cdk1 immediately triggers its rapid accumulation in the nucleus through a 40-fold increase in nuclear import that remains dependent on Cdk1 activity until NEBD. Nevertheless, a substantial proportion of cyclin B1-Cdk1 remains in the cytoplasm. The increase in nuclear import is driven by changes in the nuclear import machinery that require neither Plk1 nor inhibition of nuclear export. Thus, the intrinsic link between cyclin B1-Cdk1 activation and its rapid nuclear import inherently coordinates the reorganization of the nucleus and the cytoplasm at mitotic entry.

Abstract 2535: KX-01, a novel Src kinase inhibitor directed towards the peptide substrate site, induces robust apoptosis and synergizes with tamoxifen and chemotherapy in breast cancer

Abstract New therapeutic regimens are needed to increase efficacy and reduce resistance of endocrine therapy and chemotherapy in breast cancer. c-Src is an oncogenic non-receptor tyrosine kinase that is up-regulated in approximately half of all breast cancer. However the efficacy of existing multi-kinase src inhibitors in breast cancer has been limited. KX-01 (Kinex Pharmaceuticals) is a novel class of clinical non-ATP src inhibitor that targets the peptide binding site. In a panel of breast cancer cell lines, KX-01 resulted in dose dependent inhibition of growth and induction of apoptosis that was independent of p53 status, and was preceded by rapid inhibition of src activity. KX-01 induced apoptosis in MDA-MB-468 cells and BT-549 cell lines reported to be resistant to multi-kinase src inhibitors. KX-01 (50 nM, 6 hours) resulted in significant accumulation of MDA-MB-231 cells (ERα−/PR−/HER2/neu−) and MCF-7 cells (ERα+) in G2/M phase. Immunofluorescent staining for mitotic phase marker phospho-histone 3 indicated that cells had arrested in mitotic phase and many of the mitotic arrested cells were undergoing apoptosis (TUNEL), a novel cell death for a small molecule tyrosine kinase inhibitor. KX-01 induced nuclear accumulation of cyclin B1 and activation of CDK1, MPM2 and Cdc25C that is required for progression past the G2/M checkpoint. KX-01 resulted in cytochrome C release and activation of caspases 6, 7, 8 and 9. Combinations of KX-01 (5-75 nM) with tamoxifen (TAM), paclitaxel (PAC) or doxorubicin (DOX) resulted in synergistic growth inhibition of MCF-7 cells (KX-01 + TAM) and MDA-MB-231 cells (KX-01 + DOX or PAC). In addition, synergistic induction of apoptosis was achieved with combination of KX-01 with DOX, PAC or TAM. c-Src induces phosphorylation of ERα at serines 118 and 167, sites required for full ERα activity. KX-01 combined with TAM resulted in decreased serine 167 phosphorylation and reduced ERα transcriptional activity. In tumor xenografts, KX-01 resulted in a dose dependent inhibition of MDA-MB-231 and MCF-7 tumor growth (1, 2.5 or 5 mg/kg body weight, twice daily by oral gavage). KX-01 exhibited synergistic growth inhibition of MCF-7 tumor xenografts when combined with tamoxifen and caused regression of MDA-MB-231 tumor xenografts when combined with paclitaxel or doxorubicin. Immunohistochemical (IHC) analysis in MCF-7 xenografts revealed that KX-01 + TAM induced robust apoptosis and inhibited neovascularization. KX-01 reduced metastasis to bone (femur) and lung as measured by PCR for detection of human chromosome 17. Interestingly, KX-01 resulted in reexpression of ERα and E-cadherin in MDA-MB-231 xenografts. These data define KX-01 as a potent and unique src kinase inhibitor that synergizes with endocrine therapy and chemotherapy to induce robust cell death, tumor growth inhibition/regression and inhibit metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2535.

KX-01, a Novel Src Kinase Inhibitor Directed towards the Peptide Substrate Site, Induces Robust Apoptosis and Synergizes with Tamoxifen and Chemotherapy in Breast Cancer.

Abstract New therapeutic regimens that increase efficacy or reduce onset of resistance for endocrine therapy and chemotherapy are needed for better clinical management of breast cancer. c-Src is an oncogenic non-receptor tyrosine kinase that is up-regulated in approximately half of all breast cancer. However the efficacy of existing multi-kinase src inhibitors in breast cancer has been limited. KX-01 (Kinex Pharmaceuticals) is a novel class of non-ATP src inhibitor that targets the peptide binding site of src and is currently completing Phase-1 testing for solid tumors. In a panel of breast cancer cell lines, KX-01 resulted in dose dependent inhibition of growth and induction of apoptosis that was independent of p53 status, and was preceded by rapid inhibition of src activity. KX-01 induced apoptosis in two cell lines reported to be resistant to multi-kinase src inhibitors, MDA-MB-468 cells and BT-549. Cell cycle analysis revealed that KX-01 (50 nM, 6 hours) resulted in significant accumulation of MDA-MB-231 cells (ERα/PR/HER2/neu negative) and MCF-7 cells (ERα positive) in G2/M phase. Immunofluorescent staining for mitotic phase marker phospho-histone 3 indicated that cells had arrested in mitotic phase and many of the mitotic arrested cells were undergoing apoptosis (TUNEL), a novel cell death for a small molecule tyrosine kinase inhibitor. KX-01 induced nuclear accumulation of cyclin B1, and activation of CDK1, MPM2 and Cdc25C that is required for progression past the G2/M checkpoint. KX-01 resulted in cytochrome C release and activation of caspases 3, 6, 7, 8 and 9. A matrix design using the median-effect principle to delineate the interaction between two drugs was applied for KX-01 alone and in combination tamoxifen (TAM), paclitaxel (PAC) or doxorubicin (DOX). Combinations of KX-01 (5-75 nM) with each of these agents resulted in synergistic growth inhibition of MCF-7 cells (KX-01 + TAM) and MDA-MB-231 cells (KX-01 + DOX or PAC). In addition, synergistic induction of apoptosis was achieved by combining low doses of KX-01 with DOX, PAC or TAM. c-Src induces phosphorylation of ERα at serines 118 and 167, sites required for full receptor activity. KX-01 combined with TAM resulted in decreased phosphorylation at serine 167 that was associated with reduced transcriptional activity of ERα. In tumor xenograft models, KX-01 resulted in a dose dependent inhibition of MDA-MB-231 and MCF-7 tumor growth after 30 days (1, 2.5 or 5 mg/kg body weight, twice daily by oral gavage). In MDA-MB-231 xenografts, KX-01 reduced metastasis to bone (femur) and lung as measured by PCR for detection of human chromosome 17.These data define KX-01 as a potently active src kinase inhibitor that induces robust cell death, tumor growth inhibition and anti-metastatic effects. Combinations of KX-01 with endocrine therapy and chemotherapy present a promising new strategy for clinical management of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6104.

The Mechanism by Which Fizzy-Related (Fzr1) Controls Prophase I Arrest in Mouse Oocytes.

Recently we discovered that Fzr1/Cdh1 activity is essential to maintain prophase I stage arrest in fully-grown mouse oocytes by mediating degradation of cyclin B1. Fzr1 is an activator on the Anaphase-Promoting Complex (APC), a multisubunit E3 ubiquitin ligase responsible for polyubiquitinating substrates, such as cyclin B1, that in so being are earmarked for degradation through the 26S proteasome. Cyclin B1 is the regulatory subunit of Maturation-Promoting Factor, forming a heterodimer with CDK1, whose kinase activity requires cyclin B1 binding and is essential for dissolution of the nuclear envelope and condensation of chromatin associated with entry into M-phase. APCFZR1 activity would therefore maintain prophase I arrest by degrading cyclin B1 and in so doing prevent a rise in CDK1 kinase activity. However, with respect to oocytes we currently do not understand how Fzr1 activity is fine tuned, such that cyclin B1 is maintained at sufficient levels to allow oocytes to resume meiosis but not at too high a level to resume meiosis prematurely. Here we describe how intracellular partitioning would explain how this regulation is achieved. Using an oocyte cDNA library, generated from prophase I oocytes in Swiss Quackenbush mice, we found only one splice variant, Fzr1α/Cdh1α, of the single-copy mammalian fzr1 gene, in contrast to various splice variants we found in other tissues. This gene product contained a predicted nuclear localization signal. By immunofluorescence we confirmed this nuclear location, both in isolated oocytes from mice and in oocytes contained in antral follicles from ovarian sections. In fact all the degradation machinery appeared enhanced in the nucleus, as observed by immunofluorescence of APC6, an essential APC subunit and PSMD11, an essential 26S proteasome subunit. In contrast, cyclin B1 was predominantly cytoplasmic. In all systems studied it has been demonstrated that cyclin B1 shuttles between the cytoplasm and nucleus, with nuclear localization occurring just before nuclear envelope breakdown. We reasoned therefore that the nuclear localization of the APCFZR1 and 26S proteasome would aid in maintaining low nuclear levels of cyclin B1. To test this idea further we used two cyclin B mutants, coupled to GPF, which differed in their intracellular location. F146A-cyclin B1, which accumulates in the nucleus due to a single point mutation in the nuclear export signal, and 5A-cyclin B1 in which 5 S/T residues, phosphorylated on cyclin B1 entry into the nucleus, are alanine substituted. We found that nuclear accumulation of cyclin B1 was necessary in order for it to promote meiotic resumption because over-expression of F146A-cyclin B1 accelerated entry into meiosis, whereas 5A-cyclin B1 did not despite the same level of expression. However, in oocytes maintained in prophase I arrested through addition of milrinone, rates of F146A-cyclin B1 degradation were 5 fold (6% vs 32%) that of 5A-cyclin B1. We conclude that in oocytes, an increase in the nuclear-cytoplasmic ratio of cyclin B1 is an essential step in meiotic resumption. During prophase I the constant shuttling of cyclin B1 between nucleus and cytoplasm presents the possibility of precocious meiotic entry, but we argue that this is physiologically prevented by nuclear APCFZR1 activity. Research supported by the NHMRC (Grant 569202) to KTJ. (platform)

133. SPATIAL REGULATION OF APCCdh1 INDUCED CYCLIN B1 DEGRADATION MAINTAINS GV ARREST IN MOUSE OOCYTES

Within the mammalian ovary, oocytes remain prophase I arrested until a hormonal cue triggers meiotic resumption. The E3 ubiquitin ligase, Anaphase-Promoting Complex with its co-activator Cdh1 (APCCdh1) is known to be essential for this process by promoting cyclin B1 degradation via the 26S proteasome. Cyclin B1 is the regulatory subunit of Maturation-Promoting Factor, forming a heterodimer with CDK1, which is essential for nuclear envelope breakdown (NEB). Here we describe the intracellular partitioning that would explain how Cdh1 activity is fine-tuned, such that cyclin B1 is maintained at sufficient levels to allow oocytes to resume meiosis but not so high as to cause premature meiosis re-entry. Using RT-PCR we detected only one splice variant in mouse oocytes, Cdh1α, which possessed a nuclear localisation signal. By immunofluorescence we confirmed the nuclear location of Cdh1 and degradation machinery components including essential subunits of the APC and 26S proteasome. In all systems studied, cyclin B1 shuttles between the cytoplasm and nucleus, with nuclear localisation occurring just before NEB. We reasoned therefore that the nuclear localisation of the APCCdh1 and 26S proteasome would aid in maintaining low nuclear levels of cyclin B1. Using two GFP-coupled cyclin B1 mutants, which differed in their intracellular location we found that nuclear accumulation of cyclin B1 was necessary in order for it to promote meiotic resumption because over-expression of nuclear-cyclin B1 accelerated entry into meiosis, whereas cytoplasmic-cyclin B1 did not. However, in milrinone-arrested GV oocytes rates of nuclear-cyclin B1 degradation were 5 fold higher than cytoplasmic-cyclin B1. Therefore we conclude that in oocytes, an increase in the nuclear-cytoplasmic ratio of cyclin B1 is an essential step in meiotic resumption, and that nuclear APCCdh1 activity guards against early meiotic resumption, until the degradation machinery is overwhelmed by cyclin B1 translocation.

Cyclin A2 Regulates Nuclear-Envelope Breakdown and the Nuclear Accumulation of Cyclin B1

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.

NIPA Defines an SCF-Type Mammalian E3 Ligase that Regulates Mitotic Entry

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-box subunit of the SCF specifically recruiting a given substrate to the SCF core. Here we identify NIPA (nuclear interaction partner of ALK) as a human F-box-containing protein that defines an SCF-type E3 ligase (SCF(NIPA)) controlling mitotic entry. Assembly of this SCF complex is regulated by cell-cycle-dependent phosphorylation of NIPA, which restricts substrate ubiquitination activity to interphase. We show nuclear cyclin B1 to be a substrate of SCF(NIPA). Inactivation of NIPA by RNAi results in nuclear accumulation of cyclin B1 in interphase, activation of cyclin B1-Cdk1 kinase activity, and premature mitotic entry. Thus, SCF(NIPA)-based ubiquitination may regulate S-phase completion and mitotic entry in the mammalian cell cycle.

Cdk5 Activator-binding Protein C53 Regulates Apoptosis Induced by Genotoxic Stress via Modulating the G2/M DNA Damage Checkpoint

In response to DNA damage, the cellular decision of life versus death involves an intricate network of multiple factors that play critical roles in regulation of DNA repair, cell cycle, and cell death. DNA damage checkpoint proteins are crucial for maintaining DNA integrity and normal cellular functions, but they may also reduce the effectiveness of cancer treatment. Here we report the involvement of Cdk5 activator p35-binding protein C53 in regulation of apoptosis induced by genotoxic stress through modulating Cdk1-cyclin B1 function. C53 was originally identified as a Cdk5 activator p35-binding protein and a caspase substrate. Importantly, our results demonstrated that C53 deficiency conferred partial resistance to genotoxic agents such as etoposide and x-ray irradiation, whereas ectopic expression of C53 rendered cells susceptible to multiple genotoxins that usually trigger G(2)/M arrest. Furthermore, we found that Cdk1 activity was required for etoposide-induced apoptosis of HeLa cells. Overexpression of C53 promoted Cdk1 activity and nuclear accumulation of cyclin B1, whereas C53 deficiency led to more cytoplasmic retention of cyclin B1, suggesting that C53 acts as a pivotal player in modulating the G(2)/M DNA damage checkpoint. Finally, C53 and cyclin B1 co-localize and associate in vivo, indicating a direct role of C53 in regulating the Cdk1-cyclin B1 complex. Taken together, our results strongly indicate that in response to genotoxic stress, C53 serves as an important regulatory component of the G(2)/M DNA damage checkpoint. By overriding the G(2)/M checkpoint-mediated inhibition of Cdk1-cyclin B1 function, ectopic expression of C53 may represent a novel approach for chemo- and radio-sensitization of cancer cells.

NIPA Targets Nuclear Cyclin B1 in a Cell-Cycle Dependent Manner.

Ubiquitin-mediated destruction of regulatory proteins marks the vital means of controlling cell cycle progresssion. The E3 ubiquitin-ligases are prominent in this process, as they allow the transfer of ubiquitin to the target protein and mediate substrate binding specificity. Recently, a new class of E3 ligases referred to as SCF complexes has been identified that consists of four subunits:SKP1, Cul1, Roc1 and an F-box protein, the latter of which determines substrate specifity.We previously reported the cloning of NIPA (nuclear interaction partner of ALK) in complex with constitutively-active oncogenic fusions of ALK, which contribute to the development of certain lymphomas and sarcomas. Subsequently we characterized NIPA as a human F-box protein that determines a novel SCF complex (SCFNIPA) whose cell cycle regulated activity is restricted to interphase to allow for substrate expression at G2/M and mitosis. Phosphorylation of NIPA in late S-phase was found to be the underlying mechanism of SCFNIPA inactivation.We have now identified the key mitotic regulator cyclin B1 to serve as the relevant substrate of the SCFNIPA complex. This targeting process is restricted to interphase and directed towards the nuclear pool of cyclin B1. Inactivation of NIPA by siRNAs results in nuclear accumulation of cyclin B1 in interphase and an elevation of cells in S-phase and mitosis. In contrast, expression of a phosphorylation deficient NIPA mutant that retains constitutive SCFNIPA activity throughout the cell cycle arrests cells at early prophase thus delaying mitotic entry. Both effects are likely attributable to either cyclin B1 accumulation in the case of NIPA inactivation by siRNA or untimely cyclin B degradation at G2/M upon expression of the constitutively active SCFNIPA complex. Cyclin B1 is physiologically kept cytoplasmic during interphase and premature nuclear accumulation has been associated with untimely mitotic entry, loss of checkpoint control and genomic instability. Our data provides a mechanism to inhibit premature nuclear accumulation of cyclin B1 in the mammalian cell cycle. NIPAs association with NPM-ALK of ALCL has been shown to be associated with NIPA phosphorylation and thus to the inactivation of the SCFNIPA complex. The mechanism described above may therefore provide a framework for understanding how this oncogene interferes with the physiologic regulation of cyclin B - a potential mechanism by which NPM-ALK transforms hematopoietic cells.

RanGAP1*SUMO1 is phosphorylated at the onset of mitosis and remains associated with RanBP2 upon NPC disassembly.

The RanGTPase activating protein RanGAP1 has essential functions in both nucleocytoplasmic transport and mitosis. In interphase, a significant fraction of vertebrate SUMO1-modified RanGAP1 forms a stable complex with the nucleoporin RanBP2/Nup358 at nuclear pore complexes. RanBP2 not only acts in the RanGTPase cycle but also is a SUMO1 E3 ligase. Here, we show that RanGAP1 is phosphorylated on residues T409, S428, and S442. Phosphorylation occurs before nuclear envelope breakdown and is maintained throughout mitosis. Nocodazole arrest leads to quantitative phosphorylation. The M-phase kinase cyclin B/Cdk1 phosphorylates RanGAP1 efficiently in vitro, and T409 phosphorylation correlates with nuclear accumulation of cyclin B1 in vivo. We find that phosphorylated RanGAP1 remains associated with RanBP2/Nup358 and the SUMO E2–conjugating enzyme Ubc9 in mitosis, hence mitotic phosphorylation may have functional consequences for the RanGTPase cycle and/or for RanBP2-dependent sumoylation.

Initial activation of cyclin-B1-cdc2 kinase requires phosphorylation of cyclin B1.

At the G(2)/M transition of the cell cycle, the cdc25c phosphatase dephosphorylates inhibitory residues of cdc2, and cyclin-B-cdc2 kinase (MPF) is activated. Phosphorylation of cyclin B1 induces its nuclear accumulation, and, since cdc25c is also believed to accumulate and activate shortly before G(2)/M in the nucleus, it has been proposed that this induces cyclin-B1-cdc2 kinase activation. We demonstrate that cyclin B1 phosphorylation has another essential function in vivo: it is required for cdc25c and MPF activation, which does not require nuclear accumulation of cyclin B1, and occurs in the cytoplasm.

Nuclear accumulation of cyclin B1 in mouse two-cell embryos is controlled by the activation of Cdc2.

In the present study, the sequential expression and cellular localization of cyclin B1 was examined in two-cell mouse embryos to elucidate the mechanism of the two-cell block. One-cell embryos derived from in vitro fertilization were cultured with oviductal tissue (nonblocking condition) or without oviductal tissue (blocking condition) to establish the experimental conditions in which the embryos either overcome the two-cell block or do not. The amount of cyclin B1 gradually increased through the second cell cycle (through S to G2 phase). However, the difference was not observed between culture conditions. This showed that even embryos exhibiting the two-cell block normally synthesize cyclin B1 through the cell cycle. Cyclin B1 in embryos cultured under nonblocking condition accumulates in the nucleus during the transition from the G2 to the M phase, whereas that in embryos cultured in blocking condition localizes in the cytoplasm throughout the cell cycle. These data indicate that two-cell embryos cultured in blocking condition are able to normally synthesize cyclin B1 but have defects in nuclear accumulation of the protein. However, when two-cell blocked embryos were treated with okadaic acid, an activator of Cdc2 kinase, part of cyclin B1 in the embryos translocated into the nucleus. Moreover, treatment with butyrolactone I, a specific inhibitor of Cdc2 kinase, inhibits nuclear translocation of cyclin B1 in those embryos. These results suggest that Cdc2 kinase regulates the nuclear accumulation of cyclin B1 in mouse two-cell embryos.

Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1.

Activation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. We show here that the previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. We propose that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, we found that treatment of cells with leptomycin B, which disrupted Cyclin B1-CRM1 interactions, led to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export were affected by mutation of these phosphorylation sites; thus, we propose that Cyclin B1 phosphorylation at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation.

Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.

Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition.

Cell cycle arrest in G1 in response to ionizing radiation or senescence is believed to be provoked by inactivation of G1 cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(Cip1/Waf1/Sdi1). We provide evidence that in addition to exerting negative control of the G1/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G2/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21-/- mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G2 that may contribute to the implementation of late cell cycle checkpoint controls.