Mutations at multiple CDK phosphorylation consensus sites on Cdt2 increase the affinity of CRL4Cdt2 for PCNA and its ubiquitination activity in S phase.

CRL4Cdt2 ubiquitin ligase plays an important role maintaining genome integrity during the cell cycle. A recent report suggested that Cdk1 negatively regulates CRL4Cdt2 activity through phosphorylation of its receptor, Cdt2, but the involvement of phosphorylation remains unclear. To address this, we mutated all CDK consensus phosphorylation sites located in the C-terminal half region of Cdt2 (Cdt2-18A) and examined the effect on substrate degradation. We show that both cyclinA/Cdk2 and cyclinB/Cdk1 phosphorylated Cdt2 in vitro and that phosphorylation was reduced by the 18A mutation both in vitro and in vivo. The 18A mutation increased the affinity of Cdt2 to PCNA, and a high amount of Cdt2-18A was colocalized with PCNA foci during S phase in comparison with Cdt2-WT. Poly-ubiquitination activity to Cdt1 was concomitantly enhanced in cells expressing Cdt2-18A. Other CRL4Cdt2 substrates, Set8 and thymine DNA glycosylase, begin to accumulate around late S phase to G2 phase, but the accumulation was prevented in Cdt2-18A cells. Furthermore, mitotic degradation of Cdt1 after UV irradiation was induced in these cells. Our results suggest that CDK-mediated phosphorylation of Cdt2 inactivates its ubiquitin ligase activity by reducing its affinity to PCNA, an important strategy for regulating the levels of key proteins in the cell cycle.

Significance of ACADM mutations identified through newborn screening of MCAD deficiency in Japan.

BACKGROUND: Since the first case was detected in 2000, there has been a remarkable increase in Japanese patients diagnosed with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. Genetic analysis has revealed a spectrum of mutations that is quite different from those observed in Caucasian populations. In 2014, Japan initiated nationwide newborn screening (NBS) for MCAD using tandem mass spectrometry (MS/MS). It is an urgent issue to assess the risk of acute metabolic decompensation from the respective novel mutations found thus far. METHODS: To evaluate the pathogenic effect of each mutation, we established a eukaryotic cell expression system and prepared 11 mutant proteins identified in five symptomatic patients and eight MS/MS-NBS-positive newborns, as well as two common Caucasian mutations, p.K329E (c.985G>A) and p.Y67H (c.157C>T) for comparison. RESULTS: The expression of four mutant proteins (p.Q45R, p.P92L, p.P128X and p.Y397N) were severely impaired, whereas the others expressed normally, as did p.K329E and p.Y67H. Based on their dehydrogenase activities toward n-octanoyl-CoA, we determined three mutations (p.R53C, p.R281S and p.G362E) to be disease-causing, two mutations having (p.R17H and p.M274V) to be of marginal risk, and two mutations (p.K271E and p.I416T) as benign. Their allele-specific activities were as a whole in accordance with those estimated from the results of measurement in peripheral blood mononuclear cells. CONCLUSION: As most of the mutations detected in the Japanese population are unique, prudent genetic and enzymatic analysis is essential to precisely evaluate the latent risk of clinical onset for screening-positive newborns.

Hoxb4 transduction down-regulates Geminin protein, providing hematopoietic stem and progenitor cells with proliferation potential.

Retrovirus-mediated transduction of Hoxb4 enhances hematopoietic stem cell (HSC) activity and enforced expression of Hoxb4 induces in vitro development of HSCs from differentiating mouse embryonic stem cells, but the underlying molecular mechanism remains unclear. We previously showed that the HSC activity was abrogated by accumulated Geminin, an inhibitor for the DNA replication licensing factor Cdt1 in mice deficient in Rae28 (also known as Phc1), which encodes a member of Polycomb-group complex 1. In this study we found that Hoxb4 transduction reduced accumulated Geminin in Rae28-deficient mice, despite increasing the mRNA, and restored the impaired HSC activity. Supertransduction of Geminin suppressed the HSC activity induced by Hoxb4 transduction, whereas knockdown of Geminin promoted the clonogenic and replating activities, indicating the importance of Geminin regulation in the molecular mechanism underlying Hoxb4 transduction-mediated enhancement of the HSC activity. This facilitated our investigation of how transduced Hoxb4 reduced Geminin. We showed in vitro and in vivo that Hoxb4 and the Roc1 (also known as Rbx1)-Ddb1-Cul4a ubiquitin ligase core component formed a complex designated as RDCOXB4, which acted as an E3 ubiquitin ligase for Geminin and down-regulated Geminin through the ubiquitin-proteasome system. Down-regulated Geminin and the resultant E2F activation may provide cells with proliferation potential by increasing a DNA prereplicative complex loaded onto chromatin. Here we suggest that transduced Hoxb4 down-regulates Geminin protein probably by constituting the E3 ubiquitin ligase for Geminin to provide hematopoietic stem and progenitor cells with proliferation potential.

Dominant-negative STAT1 SH2 domain mutations in unrelated patients with Mendelian susceptibility to mycobacterial disease.

Patients carrying two loss-of-function (or hypomorphic) alleles of STAT1 are vulnerable to intracellular bacterial and viral diseases. Heterozygosity for loss-of-function dominant-negative mutations in STAT1 is responsible for autosomal dominant (AD) Mendelian susceptibility to mycobacterial disease (MSMD), whereas heterozygosity for gain-of-function loss-of-dephosphorylation mutations causes AD chronic mucocutaneous candidiasis (CMC). The two previously reported types of AD MSMD-causing STAT1 mutations are located in the tail segment domain (p.L706S) or in the DNA-binding domain (p.E320Q and p.Q463H), whereas the AD CMC-causing mutations are located in the coiled-coil domain. We identified two cases with AD-STAT1 deficiency in two unrelated patients from Japan and Saudi Arabia carrying heterozygous missense mutations affecting the SH2 domain (p.K637E and p.K673R). p.K673R is a hypomorphic mutation that impairs STAT1 tyrosine phosphorylation, whereas the p.K637E mutation is null and affects both STAT1 phosphorylation and DNA-binding activity. Both alleles are dominant negative and result in impaired STAT1-mediated cellular responses to interferon (IFN)-γ and IL-27. In contrast, STAT1-mediated cellular responses against IFN-α and IFN-λ1 were preserved at normal levels in patients' cells. We describe here the first dominant mutations in the SH2 domain of STAT1, revealing the importance of this domain for tyrosine phosphorylation and DNA binding, as well as for antimycobacterial immunity.

Overexpression of BMI-1 correlates with drug resistance in B-cell lymphoma cells through the stabilization of survivin expression.

The expression of BMI-1 is correlated with disease progression in cancer patients. We showed that ectopic expression of BMI-1 in B-cell lymphoma cell lines, HT and RL, conferred resistance to etoposide and oxaliplatin, known to enhance sensitivity by targeting the survivin gene, but not to irinotecan, which is not relevant to the downregulation of survivin expression. The expression of survivin was not only augmented in cells transduced with BMI-1, but persisted in the presence of etoposide in cells overexpressing BMI-1. By contrast, the mock-transduced cells succumbed in the medium with anticancer drugs, with an accompanying decrease in BMI-1 and survivin expression. BMI-1 overexpression stabilized survivin post-translationally without an accompanying rise in the mRNA, suggesting survivin as a potential target for BMI-1. Knockdown of either BMI-1 or survivin restored sensitivity to etoposide in the BMI-1-overexpressing lymphoma cells. An analysis of six patients with B-cell lymphoma showed that in the drug-resistant patients, levels of BMI-1 and survivin were maintained even after drug administration. However, downregulation of both BMI-1 and survivin expression was observed in the drug-sensitive patients. Therefore, BMI-1 might facilitate drug resistance in B-cell lymphoma cells through the regulation of survivin. BMI-1 could be an important prognostic marker as well as a future therapeutic target in the treatment of drug-resistant lymphomas.

Decreased expression in nuclear factor-κB essential modulator due to a novel splice-site mutation causes X-linked ectodermal dysplasia with immunodeficiency.

X-linked ectodermal dysplasia with immunodeficiency (XL-ED-ID) is caused by hypomorphic mutations in NEMO, which encodes nuclear factor-kappaB (NF-κB) essential modulator. We identified a novel mutation, 769-1 G>C, at the splicing acceptor site of exon 7 in NEMO in a Japanese patient with XL-ED-ID. Although various abnormally spliced NEMO messenger RNAs (mRNAs) were observed, a small amount of wild-type (WT) mRNA was also identified. Decreased NEMO protein expression was detected in various lineages of leukocytes. Although one abnormally spliced NEMO protein showed residual NF-κB transcription activity, it did not seem to exert a dominant-negative effect against WT-NEMO activity. CD4(+) T cell proliferation was impaired in response to measles and mumps, but not rubella. These results were consistent with the clinical and laboratory findings of the patient, suggesting the functional importance of NEMO against specific viral infections. The 769-1 G>C mutation is responsible for decreased WT-NEMO protein expression, resulting in the development of XL-ED-ID.

Polycomb-group complex 1 acts as an E3 ubiquitin ligase for Geminin to sustain hematopoietic stem cell activity.

Polycomb-group (PcG) genes encode multimeric nuclear protein complexes, PcG complex 1 and 2. PcG complex 2 was proved to induce transcription repression and to further methylate histone H3 at lysine-27 (H3K27). Subsequently PcG complex 1 is recruited through recognition of methylated H3K27 and maintains the transcription silencing by mediating monoubiquitination of histone H2A at lysine-119. Genetic evidence demonstrated a crucial role for PcG complex 1 in stem cells, and Bmi1, a member of PcG complex 1, was shown to sustain adult stem cells through direct repression of the INK4a locus encoding cyclin-dependent kinase inhibitor, p16CKI, and p19ARF. The molecular functions of PcG complex 1, however, remain insufficiently understood. In our study, deficiency of Rae28, a member of PcG complex 1, was found to impair ubiquitin-proteasome-mediated degradation of Geminin, an inhibitor of DNA replication licensing factor Cdt1, and to increase protein stability. The resultant accumulation of Geminin, based on evidence from retroviral transduction experiments, presumably eliminated hematopoietic stem cell activity in Rae28-deficient mice. Rae28 mediates recruiting Scmh1, which provides PcG complex 1 an interaction domain for Geminin. Moreover, PcG complex 1 acts as the E3 ubiquitin ligase for Geminin, as we demonstrated in vivo as well as in vitro by using purified recombinant PcG complex 1 reconstituted in insect cells. Our findings suggest that PcG complex 1 supports the activity of hematopoietic stem cells, in which high-level Geminin expression induces quiescence securing genome stability, by enhancing cycling capability and hematopoietic activity through direct regulation of Geminin.

Molecular pathogenesis of a novel mutation, G108D, in short-chain acyl-CoA dehydrogenase identified in subjects with short-chain acyl-CoA dehydrogenase deficiency.

Short-chain acyl-CoA dehydrogenase (SCAD) is a mitochondrial enzyme involved in the beta-oxidation of fatty acids. Genetic defect of SCAD was documented to cause clinical symptoms such as progressive psychomotor retardation, muscle hypotonia, and myopathy in early reports. However, clinical significance of SCAD deficiency (SCADD) has been getting ambiguous, for some variants in the ACADS gene, which encodes the SCAD protein, has turned out to be widely prevailed among general populations. Accordingly, the pathophysiology of SCADD has not been clarified thus far. The present report focuses on two suspected cases of SCADD detected through the screening of newborns by tandem mass spectrometry. In both subjects, compound heterozygous mutations in ACADS were detected. The mutated genes were expressed in a transient gene expression system, and the enzymatic activities of the obtained mutant SCAD proteins were measured. The activities of the mutant SCAD proteins were significantly lower than that of the wild-type enzyme, confirming the mechanism underlying the diagnosis of SCADD in both subjects. Moreover, the mutant SCAD proteins gave rise to mitochondrial fragmentation and autophagy, both of which were proportional to the decrease in SCAD activities. The association of autophagy with programmed cell death suggests that the mutant SCAD proteins are toxic to mitochondria and to the cells in which they are expressed. The expression of recombinant ACADS-encoded mutant proteins offers a technique to evaluate both the nature of the defective SCAD proteins and their toxicity. Moreover, our results provide insight into possible molecular pathophysiology of SCADD.

The novel IFNGR1 mutation 774del4 produces a truncated form of interferon-gamma receptor 1 and has a dominant-negative effect on interferon-gamma signal transduction.

BACKGROUND: Patients with interferon-gamma receptor 1 (IFNgammaR1) deficiency show selective susceptibility to intracellular pathogens such as mycobacteria. IFNgammaR1 deficiency is an inherited immunodeficiency disorder, which can be either recessive or dominant. Dominant forms of IFNgammaR1 deficiency are known to be associated with mutations that introduce a premature stop codon in the intracellular domain of IFNgammaR1. One such mutation, 818del4, is believed to be the most common type. Although these mutations are presumed to exert a dominant-negative effect on IFNgamma signal transduction, the underlying molecular mechanism is unresolved. OBJECTIVE: We characterised the 774del4 mutant of IFNgammaR1 using a gene-expression system to examine the effects of this mutation on IFNgamma signal transduction. RESULTS: We identified a novel dominant mutation in IFNGR1, designated 774del4, which produced a truncated form of IFNgammaR1 in a patient with recurrent mycobacterial infections. IFNgammaR1 was overexpressed on the surfaces of CD14-positive cells from the peripheral blood of this patient, and STAT1 phosphorylation in response to high doses of IFNgamma was partially deficient. We expressed two truncated forms of IFNgammaR1, 774del4 and 818del4, in HEK 293 cells using transient transfection and found that these mutants overexpressed IFNgammaR1 on the cell surface because of impaired receptor stability, which resulted in a dominant-negative effect on IFNgamma signal transduction. CONCLUSION: Like the 818del4 mutation, 774del4 produces a truncated form of IFNgammaR1, which has a dominant-negative effect on IFNgamma signal transduction through altered receptor stability.

A novel mutation (Gln433Glu) in exon 12 combined with the G insertion in exon 13 causes severe factor XI deficiency in Japanese patients.

Factor XI (FXI) deficiency is an autosomal, incompletely recessive coagulopathy. This disorder is rare in the general population worldwide, but is one of the most common inherited diseases in Ashkenazi Jews. It has been reported that a significantly higher frequency of allelic heterogeneity occurs in different ethnic groups. The study objective was to study the molecular basis of this disease in a Japanese family. Two Japanese brothers with severe FXI deficiency and three other family members were screened by direct sequencing analysis after polymerase chain reaction. We identified a novel mutation, a C-to-G transition at position 1394 in exon 12 in the FXI gene (F11 c.1394 C>G). This transition resulted in a missense mutation (Gln433Glu), which led to the disruption of the catalytic domain structure of the FXI molecule. This change, combined with a G insertion in exon 13 (501/502 ins G), led to a frameshift mutation, which has previously been reported in only one other Japanese patient. In conclusion, the compound heterozygous novel mutations that cause severe FXI deficiency were found in Japanese patients.

Role of Geminin in cell fate determination of hematopoietic stem cells (HSCs).

Geminin exerts two distinct molecular roles. Geminin negatively regulates DNA replication licensing through the direct interaction with Cdt1 to prevent re-replication in proliferating cells. Geminin also regulates chromatin remodeling through the direct interaction with Brahma/Brg1 to maintain undifferentiated states of stem cells. We previously uncovered that Polycomb-group complex 1 and Hoxb4/Hoxa9, well-known intrinsic factors that are essential for maintaining the hematopoietic stem cell (HSC) activity, alternatively act as ubiquitin-proteasome systems for Geminin protein to reduce the protein expression level, and sustain the HSC activity. Thus, Geminin is presumed to play an important role in determining cell fate, i.e., turning on and off cellular quiescence and proliferation/differentiation, in HSCs. We recently generated recombinant cell-penetrating Geminin (CP-Geminin), enabling rapid incorporation and withdraw of Geminin protein in cells. CP-Geminin may be useful in regulating the cell cycle and chromatin configuration. In this article, we summarize current information on the molecular functions of Geminin and the regulatory system for Geminin protein expression, and argue for the molecular role of Geminin in cell fate determination of HSCs, and future perspective of a new technology for manipulating the activities of HSCs and cancer stem cells (CSCs).

Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin.

Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin) was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.

Transcription of the Geminin gene is regulated by a negative-feedback loop.

Geminin performs a central function in regulating cellular proliferation and differentiation in development and also in stem cells. Of interest, down-regulation of Geminin induces gene transcription regulated by E2F, indicating that Geminin is involved in regulation of E2F-mediated transcriptional activity. Because transcription of the Geminin gene is reportedly regulated via an E2F-responsive region (E2F-R) located in the first intron, we first used a reporter vector to examine the effect of Geminin on E2F-mediated transcriptional regulation. We found that Geminin transfection suppressed E2F1- and E2F2-mediated transcriptional activation and also mildly suppressed such activity in synergy with E2F5, 6, and 7, suggesting that Geminin constitutes a negative-feedback loop for the Geminin promoter. Of interest, Geminin also suppressed nuclease accessibility, acetylation of histone H3, and trimethylation of histone H3 at lysine 4, which were induced by E2F1 overexpression, and enhanced tri-methylation of histone H3 at lysine 27 and monoubiquitination of histone H2A at lysine 119 in E2F-R. However, Geminin5EQ, which does not interact with Brahma or Brg1, did not suppress accessibility to nuclease digestion or transcription but had an overall dominant-negative effect. These findings suggest that E2F-mediated activation of Geminin transcription is negatively regulated by Geminin through the inhibition of chromatin remodeling.

Hoxa9 transduction induces hematopoietic stem and progenitor cell activity through direct down-regulation of geminin protein.

Hoxb4, a 3'-located Hox gene, enhances hematopoietic stem cell (HSC) activity, while a subset of 5'-located Hox genes is involved in hematopoiesis and leukemogenesis, and some of them are common translocation partners for Nucleoporin 98 (Nup98) in patients with leukemia. Although these Hox gene derivatives are believed to act as transcription regulators, the molecular involvement of the Hox gene derivatives in hematopoiesis and leukemogenesis remains largely elusive. Since we previously showed that Hoxb4 forms a complex with a Roc1-Ddb1-Cul4a ubiquitin ligase core component and functions as an E3 ubiquitin ligase activator for Geminin, we here examined the E3 ubiquitin ligase activities of the 5'-located Hox genes, Hoxa9 and Hoxc13, and Nup98-Hoxa9. Hoxa9 formed a similar complex with the Roc1-Ddb1-Cul4a component to induce ubiquitination of Geminin, but the others did not. Retroviral transduction-mediated overexpression or siRNA-mediated knock-down of Hoxa9 respectively down-regulated or up-regulated Geminin in hematopoietic cells. And Hoxa9 transduction-induced repopulating and clonogenic activities were suppressed by Geminin supertransduction. These findings suggest that Hoxa9 and Hoxb4 differ from Hoxc13 and Nup98-Hoxa9 in their molecular role in hematopoiesis, and that Hoxa9 induces the activity of HSCs and hematopoietic progenitors at least in part through direct down-regulation of Geminin.

CIZ1, a p21Cip1/Waf1-interacting protein, functions as a tumor suppressor in vivo.

CIZ1 is a nuclear protein involved in DNA replication and is also implicated in human diseases including cancers. To gain an insight into its function in vivo, we generated mice lacking Ciz1. Ciz1-deficient (Ciz1(-/-)) mice grew without any obvious abnormalities, and Ciz1(-/-) mouse embryonic fibroblasts (MEFs) did not show any defects in cell cycle status, cell growth, and DNA damage response. However, Ciz1(-/-) MEFs were sensitive to hydroxyurea-mediated replication stress and susceptible to oncogene-induced cellular transformation. In addition, Ciz1(-/-) mice developed various types of leukemias by retroviral insertional mutagenesis. These results indicate that CIZ1 functions as a tumor suppressor in vivo.

Passive carriage of rabies virus by dendritic cells.

The rabies virus (RABV) is highly neurotropic and it uses evasive strategies to successfully evade the host immune system. Because rabies is often fatal, understanding the basic processes of the virus-host interactions, particularly in the initial events of infection, is critical for the design of new therapeutic approaches to target RABV. Here, we examined the possible role of dendritic cells (DCs) in the transmission of RABV to neural cells at peripheral site of exposure. Viral replication only occurred at a low level in the DC cell line, JAWS II, after its infection with either pathogenic RABV (CVS strain) or low-pathogenic RABV (ERA strain), and no progeny viruses were produced in the culture supernatants. However, both viral genomic RNAs were retained in the long term after infection and maintained their infectivity. The biggest difference between CVS and ERA was in their ability to induce type I interferons. Although the ERA-infected JAWS II cells exhibited cytopathic effect and were apparently killed by normal spleen cells in vitro, the CVS-infected JAWS II cells showed milder cytopathic effect and less lysis when cocultured with spleen cells. Strongly increased expression of major histocompatibility complex classes I, costimulatory molecules (CD80 and CD86), type I interferons and Toll- like receptor 3, and was observed only in the ERA-inoculated JAWS II cells and not in those inoculated with CVS. During the silencing of the cellular immune response in the DCs, the pathogenic CVS strain cryptically maintained an infectious viral genome and was capable of transmitting infectious RABV to permissive neural cells. These findings demonstrate that DCs may play a role in the passive carriage of RABV during natural rabies infections.

Scmh1 has E3 ubiquitin ligase activity for geminin and histone H2A and regulates geminin stability directly or indirectly via transcriptional repression of Hoxa9 and Hoxb4.

Polycomb-group (PcG) complex 1 acts as an E3 ubiquitin ligase both for histone H2A to silence transcription and for geminin to regulate its stability. Scmh1 is a substoichiometric component of PcG complex 1 that provides the complex with an interaction domain for geminin. Scmh1 is unstable and regulated through the ubiquitin-proteasome system, but its molecular roles are unknown, so we generated Scmh1-deficient mice to elucidate its function. Loss of Scmh1 caused derepression of Hoxb4 and Hoxa9, direct targets of PcG complex 1-mediated transcriptional silencing in hematopoietic cells. Double knockdown of Hoxb4 and Hoxa9 or transduction of a dominant-negative Hoxb4N→A mutant caused geminin accumulation. Age-related transcriptional downregulation of derepressed Hoxa9 also leads to geminin accumulation. Transduction of Scmh1 lacking a geminin-binding domain restored derepressed expression of Hoxb4 and Hoxa9 but did not downregulate geminin like full-length Scmh1. Each of Hoxb4 and Hoxa9 can form a complex with Roc1-Ddb1-Cul4a to act as an E3 ubiquitin ligase for geminin. We suggest that geminin dysregulation may be restored by derepressed Hoxb4 and Hoxa9 in Scmh1-deficient mice. These findings suggest that PcG and a subset of Hox genes compose a homeostatic regulatory system for determining expression level of geminin.

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation.

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

Nuclear export signal in CDC25B.

CDC25B is a dual-specificity phosphatase that activates CDK1/cyclin B. The nuclear exclusion of CDC25B is controlled by the binding of 14-3-3 to the nuclear export signal (NES) of CDC25B, which was reported to be amino acids H28 to L40 in the N-terminal region of CDC25B. In studying the subcellular localization of CDC25B, we found a functional NES at V52 to L65, the sequence of which is VTTLTQTMHDLAGL, where bold letters are leucine or hydrophobic amino acids frequently seen in an NES. The deletion of this NES sequence caused the mutant protein to locate exclusively in nuclei, while NES-fused GFP was detected in the cytoplasm. Moreover, the introduction of point mutations at some of the critical amino acids impaired cytoplasmic localization. Treatment with leptomycin B, a potent inhibitor of CRM1/exportin1, disrupted the cytoplasmic localization of both Flag-tagged CDC25B and NES-fused GFP. From these results, we concluded that the sequence we found is a bona fide NES of CDC25B.

Binding of 14-3-3beta but not 14-3-3sigma controls the cytoplasmic localization of CDC25B: binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B.

The dual specificity phosphatase CDC25B positively controls the G2-M transition by activating CDK1/cyclin B. The binding of 14-3-3 to CDC25B has been shown to regulate the subcellular redistribution of CDC25B from the nucleus to the cytoplasm and may be correlated with the G2 checkpoint. We used a FLAG-tagged version of CDC25B to study the differences among the binding sites for the 14-3-3 subtypes, 14-3-3beta, 14-3-3epsilon and 14-3-3sigma, and the relationship between subtype binding and the subcellular localization of CDC25B. All three subtypes were found to bind to CDC25B. Site-directed mutagenesis studies revealed that 14-3-3beta bound exclusively near serine-309 of CDC25B1, which is within a potential consensus motif for 14-3-3 binding. By contrast, 14-3-3sigma bound preferentially to a site around serine-216, and the presence of serine-137 and -309 enhanced the binding. In addition to these binding-site differences, we found that the binding of 14-3-3beta drove CDC25B to the cytoplasm and that mutation of serine-309 to alanine completely abolished the cytoplasmic localization of CDC25B. However, co-expression of 14-3-3sigma and CDC25B did not affect the subcellular localization of CDC25B. Furthermore, serine-309 of CDC25B was sufficient to produce its cytoplasmic distribution with co-expression of 14-3-3beta, even when other putative 14-3-3 binding sites were mutated. 14-3-3epsilon resembled 14-3-3beta with regard to its binding to CDC25B and the control of CDC25B subcellular localization. The results of the present study indicate that two 14-3-3 subtypes can control the subcellular localization of CDC25B by binding to a specific site and that 14-3-3sigma has effects on CDC25B other than the control of its subcellular localization.