C-terminal Nuclear Export Signal Research Articles

The acidic stretch and the C-terminal nuclear export signal motif of NPM1 mutant: are they druggable in AML?

The acidic stretch and the C-terminal nuclear export signal motif of NPM1 mutant: are they druggable in AML?

Acute Myeloid Leukemia with Mutated NPM1 Is Dependent on the Cytoplasmic Localization of NPM1c

▪NPM1 mutations are among the most common mutations in acute myeloid leukemia (AML), being found in about 30% of de novo AML in adults. NPM1 is a multifunctional nucleolar chaperone involved in genomic stability and ribosome biogenesis. All the mutations in NPM1 described so far result in cytoplasmic protein localization (NPM1c) through the acquisition of a nuclear export signal (NES) at the C-terminus. Based on these observations, we hypothesized that cytoplasmic localization of NPM1c is necessary for AML pathogenesis and maintenance. To test our hypothesis, we edited the C-terminus of the NPM1 mutant allele in order to remove the NES and re-localize NPM1c to the nucleus.Specifically, using our recently optimized CRISPR-Cas9 approach (Gundry, et al. Cell Reports 2016), we sought to introduce indels to disrupt the C-terminal NES in AML cells bearing NPM1 mutation A (heterozygous 4bp insertion at the C-terminus), thereby creating novel edited alleles encoding for a mutant NPM1 with nuclear localization. Cell lines, primary derived xenografts (PDX) and primary AML samples harboring NPM1 mutation A were successfully edited with an sgRNA spanning the 4bp insertion.While the NPM1 wild-type allele remained intact, the mutant allele was edited with up to 90% efficiency. The novel edited alleles could direct nuclear localization of a GFP-NPM1 fusion construct. Additionally, re-localization of cytoplasmic NPM1 in edited cells was confirmed by immunofluorescence. Return of NPM1 protein to the nucleus resulted in terminal differentiation, and significantly impaired cell growth, colony forming ability and engraftment in xenograft models. Transcriptome analysis on two cell lines with mutated NPM1 revealed that upon nuclear re-localization of NPM1c, only 22 genes were down regulated more than 2 fold in both cell lines. Strikingly, 11 of these 22 genes were HOX genes. Altogether these data indicate that the cytoplasmic localization of NPM1c is necessary to maintain the transcriptional signature and leukemic phenotype of AML cells with mutated NPM1 .To extend our study further, we used homology-directed repair (HDR) to successfully correct NPM1 mutations to the wild type sequence in AML cell lines (Fig.1). We simultaneously fused GFP to the C-terminus of the protein to verify its appropriate localization (Fig.1). Moreover, to assess whether the phenotype of AML cells depends on the amount of mutant NPM1 in the cytoplasm, we inserted a series of NPM1 mutant alleles (a, b and c) that resulted in different proportions of cytoplasmic versus nuclear localization (i.e. a - no cytoplasmic mutant NPM1, b - ~50% cytoplasmic/50% nuclear mutant NPM1, c - ~100% cytoplasmic mutant NPM1; Fig.1) in two different AML cell lines with mutated NPM1 . Cells with high levels of nuclear NPM1 tended to differentiate rapidly, while cells with a greater proportion of cytoplasmic NPM1 remained less differentiated and continued to proliferate (Fig.1). This established an ongoing competition in which cells with higher nuclear NPM1 were rapidly outcompeted by unedited cells, while cells with higher cytoplasmic NPM1 were not. Thus, these experiments clearly demonstrate the dependence of the leukemic phenotype on the cytoplasmic localization of NPM1.Recently, compounds able to inhibit the nuclear exporter CRM1/XPO1 have been developed and are currently being tested in clinical trials (e.g. selinexor). Since NPM1 is shuttled to the cytoplasm by CRM1/XPO1, its inhibition re-localizes NPM1c to the nucleus. We therefore treated cell lines, PDX and primary samples with selinexor with the aim to reproduce the nuclear re-localization observed using CRISPR-Cas9. As expected, selinexor treatment resulted in clear growth arrest, differentiation, and NPM1c re-localization with dynamics similar to what was seen with gene editing. Importantly, after only 3 days of treatment selinexor produced a dramatic drop of HOXA, HOXB and MEIS1 expression.In conclusion, allele-specific editing is a powerful tool to probe the mechanistic aspects of oncogenic dependencies. By achieving nuclear re-localization of mutant NPM1, we demonstrated that cytoplasmic localization of NPM1c is necessary for NPM1-mutant AML cells to maintain their leukemic phenotype. Drugs promoting mutant NPM1 nuclear localization are therefore attractive candidates for clinical treatment of AML with mutated NPM1 . [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nuclear relocalization of mutant NPM1 induces downregulation of Hox/Meis1, terminal differentiation, and cell cycle arrest

NPM1 mutated acute myeloid leukemia (AML) is a distinct entity in the WHO classification of hematopoietic cancers. It displays a specific phenotype characterized by favorable prognosis and upregulation of HOX cluster genes. NPM1 is a multifunctional nucleolar chaperone. All the mutations in NPM1 described so far in result in cytoplasmic protein localization (NPM1c) through the acquisition of a nuclear export signal (NES) at the C-terminus. We hypothesized that NPM1 mutA, the most frequent NPM1 mutation, could be specifically targeted using CRISPR to disrupt the C-terminal NES. After transfection of two NPM1 mutated cell lines (OCI-AML3 and IMS-M2) with NPM1c sgRNA, we found that the NPM1mutA allele was edited in 70-90% of cells, while the NPM1wt allele was not targeted. We cloned the edited alleles into a GFP-NPM1 fusion construct, observing nuclear localization in all tested alleles. Nuclear re-localization was followed by significant impairment of cell growth (3.7-4 fold decrease), colony forming ability (16-20 fold reduction in colonies) and engraftment in xenograft models (significant loss of indels at NPM1mutA allele in engrafted cells). The observed immunophenotype and growth defects were preceded by significant downregulation (5 fold) of HOXA genes, HOXB genes and MEIS1. Chromatin profiling revealed that the mechanism of downregulation appeared to involve loss of active promoters and enhancers, rather than the deposition of repressive chromatin marks. We reasoned that the dependency of NPM1-mutated cells on the cytoplasmic localization of NPM1c would sensitize them to nuclear export inhibitors. Indeed, treatment of OCI-AML3 cells with 50nM Selinexor (KPT-330) induced both morphologic and immunophenotypic differentiation beginning as early as day 5. Furthermore, the transcriptional and chromatin changes phenocopied those observed with CRISPR-targeting of the mutant allele. By achieving nuclear re-localization of mutant NPM1, we demonstrated that cytoplasmic localization of NPM1 is necessary for maintenance of the leukemic phenotype. Drugs promoting mutant NPM1 nuclear localization are attractive candidates for clinical success in NPM1-mutated AML.

Abstract 1031: Nuclear relocalization of NPM1c induces terminal differentiation and cell growth arrest

Abstract NPM1 mutated acute myeloid leukemia (AML) is a distinct entity in the WHO classification of hematopoietic cancers. It displays a specific phenotype characterized by favorable prognosis and upregulation of HOX cluster genes. NPM1 is a multifunctional nucleolar chaperone. Mutations in NPM1 result in cytoplasmic protein localization (NPM1c+) through the acquisition of a nuclear export signal (NES) at the C-terminus. The most frequent NPM1 mutation is a heterozygous 4bp insertion in exon 12 (mutA). The role of NPM1 in leukemogenesis is still a matter of debate and there is no direct proof that cytoplasmic localization of mutant NPM1 is necessary for maintenance of leukemia. We recently developed a CRISPR strategy for highly efficient gene editing in hematopoietic cells. We hypothesized that NPM1 mutA could be specifically targeted due to its 4bp insertion, avoiding the WT allele. Using an sgRNA spanning the insertion site (NPM1c sgRNA), we aimed to introduce indels adjacent to the mutation and disrupt the C-terminal NES. To test our hypothesis, we used the NPM1 mutated OCI-AML3 cell line. After transfection of OCI-AML3 with NPM1c sgRNA, while NPM1mutA allele showed 70-90% indel frequencies, the NPM1wt allele was intact. To determine whether the novel edited alleles generated NPM1 that was re-localized to the nucleus, we cloned the alleles into a GFP-NPM1 fusion construct and observed nuclear localization after transfection. Consistent with this finding, immunofluorescence revealed that edited OCI-AML3 cells had lost nearly all cytoplasmic NPM1. Return of NPM1 protein to the nucleus was followed by terminal differentiation and cell cycle arrest in G1 phase (controls 45±3%, NPM1c sgRNA 68±1.5%). Cell growth (3.7-4 fold decrease in cell counts in NPM1c sgRNA samples), colony forming ability (16-20 fold reduction in colonies in NPM1c sgRNA samples) and engraftment in xenograft models (significant loss of indels at NPM1mutA allele in engrafted cells) were also significantly impaired after NPM1mutA targeting. Furthermore, RNA sequencing on NPM1mutA-targeted and control OCI-AML3 cells revealed almost complete loss of expression of the HOXA and HOXB cluster genes as well as MEIS1 in treated cells (4.5 average fold reduction of HOXA9-A13; 5.6 average fold reduction of HOXB7-B13; 5 fold reduction of MEIS1). Allele-specific editing is a powerful tool to probe mechanistic aspects of oncogene dependencies. By achieving nuclear re-localization of mutant NPM1, we demonstrated that cytoplasmic localization of NPM1c+ is necessary for OCI-AML3 cells to maintain their leukemic phenotype. Drugs promoting mutant NPM1 nuclear localization such as CRM1 inhibitors are attractive candidates for clinical success in NPM1-mutated AML. Citation Format: Lorenzo Brunetti, Michael C. Gundry, Anna G. Guzman, Ilaria Gionfriddo, Federica Mezzasoma, Francesca Milano, Maria Paola Martelli, Brunangelo Falini, Margaret A. Goodell. Nuclear relocalization of NPM1c induces terminal differentiation and cell growth arrest [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1031. doi:10.1158/1538-7445.AM2017-1031

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.

Mutagenesis by AID: Being in the Right Place at the Right Time

Mutagenesis by AID: Being in the Right Place at the Right Time

Axotrophin/MARCH7 acts as an E3 ubiquitin ligase and ubiquitinates tau protein in vitro impairing microtubule binding

Tau is the major microtubule-associated protein in neurons involved in microtubule stabilization in the axonal compartment. Changes in tau gene expression, alternative splicing and posttranslational modification regulate tau function and in tauopathies can result in tau mislocalization and dysfunction, causing tau aggregation and cell death. To uncover proteins involved in the development of tauopathies, a yeast two-hybrid system was used to screen for tau-interacting proteins. We show that axotrophin/MARCH7, a RING-variant domain containing protein with similarity to E3 ubiquitin ligases interacts with tau. We defined the tau binding domain to amino acids 552–682 of axotrophin comprising the RING-variant domain. Co-immunoprecipitation and co-localization confirmed the specificity of the interaction. Intracellular localization of axotrophin is determined by an N-terminal nuclear targeting signal and a C-terminal nuclear export signal. In AD brain nuclear localization is lost and axotrophin is rather associated with neurofibrillary tangles. We find here that tau becomes mono-ubiquitinated by recombinant tau-interacting RING-variant domain, which diminishes its microtubule-binding. In vitro ubiquitination of four-repeat tau results in incorporation of up to four ubiquitin molecules compared to two molecules in three-repeat tau. In summary, we present a novel tau modification occurring preferentially on 4-repeat tau protein which modifies microtubule-binding and may impact on the pathogenesis of tauopathies.

An intrabody specific for the nucleophosmin carboxy-terminal mutant and fused to a nuclear localization sequence binds its antigen but fails to relocate it in the nucleus

The cytoplasmic accumulation of NPM1 (NPMc+) is found in acute myeloid leukemia (AML) with NPM1 mutation. NPM1 must shuttle between nucleus and cytoplasm to assure physiological protein synthesis and, therefore, the elimination of NPMc+ is not a suitable therapeutic option. We isolated, characterized, and produced a functional scFv intrabody fused to nuclear localization signal(s) (NLS) that does not recognize NPM1 but binds to the mutant-specific C-terminal NES (nuclear export signal) of NPMc+, responsible for its cytoplasmic accumulation. The scFv-NLS fusion accumulated in the nuclei of wild type cells and strongly bound to its antigen in the cytoplasm of NPMc+ expressing cells. However, it failed to relocate the majority of NPMc+ in the nucleus, even when fused to four NLS. Our results show the technical feasibility of producing recombinant intrabodies with defined sub-cellular targeting and nuclear accumulation but the lack of information concerning the features that confer variable strength to the signal peptides impairs the development of biomolecules able to counteract pathological sub-cellular distribution of shuttling proteins.

Regulation of p53 Nuclear Export through Sequential Changes in Conformation and Ubiquitination

Wild-type p53 is a conformationally labile protein that undergoes nuclear-cytoplasmic shuttling. MDM2-mediated ubiquitination promotes p53 nuclear export by exposing or activating a nuclear export signal (NES) in the C terminus of p53. We observed that cancer-derived p53s with a mutant (primary antibody 1620-/pAb240+) conformation localized in the cytoplasm to a greater extent and displayed increased susceptibility to ubiquitination than p53s with a more wild-type (primary antibody 1620+/pAb240-) conformation. The cytoplasmic localization of mutant p53s required the C-terminal NES and an intact ubiquitination pathway. Mutant p53 ubiquitination occurred at lysines in both the DNA-binding domain (DBD) and C terminus. Interestingly, Lys to Arg mutations that inhibited ubiquitination restored nuclear localization to mutant p53 but had no apparent effect on p53 conformation. Further studies revealed that wild-type p53, like mutant p53, is ubiquitinated by MDM2 in both the DBD and C terminus and that ubiquitination in both regions contributes to its nuclear export. MDM2 binding can induce a conformational change in wild-type p53, but this conformational change is insufficient to promote p53 nuclear export in the absence of MDM2 ubiquitination activity. Taken together, these results support a stepwise model for mutant and wild-type p53 nuclear export. In this model, the conformational change induced by either the cancer-derived mutation or MDM2 binding precedes p53 ubiquitination. The addition of ubiquitin to DBD and C-terminal lysines then promotes nuclear export via the C-terminal NES.

Reciprocal Interaction between NPM Leukemic Mutants and Arf: Structural Basis and Functional Consequences.

Mutations at exon-12 of nucleophosmin (NPM) gene are the most common gene lesion in Acute Myeloid Leukemia (AML) (Falini et al., NEJM, 352:254, 2005). They result indisruption of tryptophan (W) 288 and/or 290, constituting the Nucleolar Localization Signal (NoLS), andcreation of a new C-terminal Nuclear Export Signal (NES) motif, with 6 variations observed to date.Both alterations are necessary for aberrant NPM accumulation in leukemic cell cytoplasm (NPMc+ AML, Falini et al., Blood , 107:4514, 2006).In NPM mutants, there is a correlation between NES sequence and the type of NoLS disruption: the most common NES motif (LxxxVxxVxL) always associates with loss of both W288 and W290, e.g. mutant A; when retained, W288 always associates with rare NES variant sequences, such as LxxxLxxVxL in NPM mutant E. We demonstrated this correlation is explained by efficiency differences between NES motifs: NPM mutant A is not efficiently exported in cytoplasm upon artificial W288 reinsertion; the same happens when LxxxLxxVxL NES motif in mutant E is artificially replaced with LxxxVxxVxL, typical of mutant A. This suggest LxxxVxxVxL NES motif is weaker than the others, being unable to efficiently export mutant NPM to cytoplasm when artificially coupled with W288 (repesenting a partial NoLS).Such changes at NPM C-terminus also influence NPM-Arf binding and subcellular distribution. Tumor suppressor Arf is a major NPM interactor in the nucleolus, and its deregulation would be strongly implicated in leukemogenesis. In co-transfected cells, NPM leukemic mutants partially relocates Arf from nucleolus to nucleoplasm and cytoplasm and, in turn, Arf partially relocates NPM mutants A and E from cytoplasm to nucleus in a dose-related manner. Artificial NPM mutants carrying W288 coupled with LxxxVxxVxL NES motif show higher degree of co-localization with Arf, and lower doses of Arf are required for them to be completely relocated to nucleoli as compared to NPM leukemic mutants A and E. Co-immunoprecipitation studies show that leukemic mutants A and E bind less Arf than wild-type NPM or artificial NPM mutants. This obervation suggest that NPM leukemic mutants and Arf reciprocally interact and influence each otheri n a dose-related manner, with C-terminal NPM mutated sequence playing a role in Arf binding.Cytofluorimetric analysis of Arf-transfected NIH-3T3 cells shows G1 cell cycle arrest. Upon co-transfection, neither wild-type NPM nor leukemic or artificial NPM mutants are able to relieve Arf-induced cell cycle arrest.In conclusion, we suggest there is a “determinism” in the combination of NES motif/NoLS disruption: NPM mutants are selected for efficient cytoplasmic export and reduced Arf binding as compared to wild-type NPM. Both mechanisms may contribute to Arf dislocation/degradation, thus having the same functional consequences as NPM silencing; moreover, it is tempting to speculate that NPM leukemic mutants could be themselves less sensitive to ARF induced inhibition. Nevertheless, in our experimental model, changes in Arf subcellular distribution didn't result in functional alterations. To clarify wether these findings will be relevant to the pathogenesis of NPMc+ AML, future studies should address in deeper detail the relationship between NPM and Arf doses and function in specific subcellular compartments.

Visual and Both Non-visual Arrestins in Their “Inactive” Conformation Bind JNK3 and Mdm2 and Relocalize Them from the Nucleus to the Cytoplasm

Arrestins bind active phosphorylated G protein-coupled receptors, terminating G protein activation. Receptor-bound non-visual arrestins interact with numerous partners, redirecting signaling to alternative pathways. Arrestins also have nuclear localization and nuclear exclusion signals and shuttle between the nucleus and the cytoplasm. Constitutively shuttling proteins often redistribute their interaction partners between the two compartments. Here we took advantage of the nucleoplasmic shuttling of free arrestins and used a "nuclear exclusion assay" to study their interactions with two proteins involved in "life-and-death" decisions in the cell, the kinase JNK3 and the ubiquitin ligase Mdm2. In human embryonic kidney 293 cells green fluorescent protein (GFP)-JNK3 and GFP-Mdm2 predominantly localize in the nucleus, whereas visual arrestin, arrestin2(Q394L) mutant equipped with the nuclear exclusion signal, and arrestin3 localize exclusively to the cytoplasm. Coexpression of arrestins moves both GFP-JNK3 and GFP-Mdm2 to the cytoplasm. Arrestin mutants "frozen" in the basal conformation are the most efficacious. Thus, arrestins in their basal state interact with JNK3 and Mdm2, suggesting that arrestins are likely "preloaded" with their interaction partners when they bind the receptor. Robust interaction of free arrestins with JNK3 and Mdm2 and their ability to regulate subcellular localization of these proteins may play an important role in the survival of photoreceptors and other neurons, as well as in retinal and neuronal degeneration.

A short Id2 protein fragment containing the nuclear export signal forms amyloid-like fibrils

The negative regulator of DNA-binding/cell-differentiation Id2 is a small protein containing a central helix-loop-helix (HLH) motif and a C-terminal nuclear export signal (NES). Whereas the former is essential for Id2 dimerization and nuclear localization, the latter is responsible for the transport of Id2 from the nucleus to the cytoplasm. Whereas the isolated Id2 HLH motif is highly helical, large C-terminal Id2 fragments including the NES sequence are either unordered or aggregation-prone. To study the conformational properties of the isolated NES region, we synthesized the Id2 segment 103–124. The latter was insoluble in water and only temporarily soluble in water/alcohol mixtures, where it formed quickly precipitating β-sheets. Introduction of a positively charged N-terminal tail prevented aggressive precipitation and led to aggregates consisting of long fibrils that bound thioflavin T. These results show an interesting structural aspect of the Id2 NES region, which might be of significance for both protein folding and function.

Cytoplasmic nucleophosmin in myeloid sarcoma occurring 20 years after diagnosis of acute myeloid leukaemia

Falini and colleagues1 reported that 35% of adults with acute myeloid leukaemia (AML) carry mutations at exon 12 of the nucleophosmin gene (NPM1), which leads to the formation of a C-terminal nuclear-export signal and mutations at tryptophans 288 and 2901 that cause aberrant accumulation of nucleophosmin (NPM) in the cytoplasm.2 Mutation of NPM1 at exon 12 is the genetic lesion that arises most specifically and frequently in adults1 and children3 with AML who have a normal karyotype. These findings have been confirmed in about 1000 patients with AML.

Dynamic Binding of PKA Regulatory Subunit RIα

Recent crystal structures have revealed that regulatory subunit RIalpha of PKA undergoes a dramatic conformational change upon complex formation with the catalytic subunit. Molecular dynamics studies were initiated to elucidate the contributions of intrinsic conformational flexibility and interactions with the catalytic subunit in formation and stabilization of the complex. Simulations of a single RIalpha nucleotide binding domain (NBD), missing cAMP, showed that its C helix spontaneously occupies two distinct conformations: either packed against the nucleotide binding domain as in its cAMP bound structure, or extended into an intermediate form resembling that of the holoenzyme structure. C helix extension was not seen in a simulation of either RIalpha NBD. In a model complex containing both NBDs and the catalytic subunit, well-conserved residues at the interface between the NBDs in the cAMP bound form were found to stabilize the complex through contacts with the catalytic subunit. The model structure is consistent with available experimental data.

Involvement of Nuclear Export in Human Papillomavirus Type 18 E6-Mediated Ubiquitination and Degradation of p53

The E6 protein from high-risk human papillomaviruses (HPVs) targets the p53 tumor suppressor for degradation by the proteasome pathway. This ability contributes to the oncogenic potential of these viruses. However, several aspects concerning the mechanism of E6-mediated p53 degradation at the cellular level remain to be clarified. This study therefore examined the role of cell localization and ubiquitination in the E6-mediated degradation of p53. As demonstrated within, following coexpression both p53 and high-risk HPV type 18 (HPV-18) E6 (18E6) shuttle from the nucleus to the cytoplasm. Mutation of the C-terminal nuclear export signal (NES) of p53 or treatment with leptomycin B inhibited the 18E6-mediated nuclear export of p53. Impairment of nuclear export resulted in only a partial reduction in 18E6-mediated degradation, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. This was also consistent with the observation that 18E6 mediated the accumulation of polyubiquitinated p53 in the nucleus. In comparison, a p53 isoform that localizes predominantly to the cytoplasm was not targeted for degradation by 18E6 in vivo but could be degraded in vitro, arguing that nuclear p53 is the target for E6-mediated degradation. This study supports a model in which (i) E6 mediates the accumulation of polyubiquitinated p53 in the nucleus, (ii) E6 is coexported with p53 from the nucleus to the cytoplasm via a CRM1 nuclear export mechanism involving the C-terminal NES of p53, and (iii) E6-mediated p53 degradation can be mediated by both nuclear and cytoplasmic proteasomes.

Quantitation of p53 nuclear relocation in response to stress using a yeast functional assay: effects of irradiation and modulation by heavy metal ions

Many regulatory proteins undergo transient nuclear relocation under physical or chemical stress. This phenomenon is, however, difficult to assess due to the lack of sensitive and standardized biological assays. Here, we describe a new quantitative nuclear relocation assay (QNR), based on expression in yeasts of chimeric proteins in which an artificial transcription factor is fused to a target protein acting as driver for relocation. This assay combines the experimental versatility of yeast with quantitation of nuclear relocation at low levels of protein expression. We have assessed the nuclear relocation of yeast Yap1 and human p53, two transcription factors that relocate to the nucleus in response to oxidative-stress and DNA damage, respectively. We show that p53 efficiently drives the relocation of the chimeric reporter in response to irradiation and that this process requires the C-terminal nuclear export signal (NES). Cd2+ and Hg2+, two metal ions inducing DNA damage as well as conformational changes in p53, have opposite effects on p53 relocation in response to DNA damage. Whereas Hg2+ effects are synergistic to DNA damage, Cd2+ inhibits relocation and sequesters p53 into the cytoplasm. These results demonstrate the effectiveness of QNR to investigate the regulation of p53 shuttling in response to stress signals including suspected environmental carcinogens.

Chromosomal Region Maintenance 1 (CRM1)-dependent Nuclear Export of Smad Ubiquitin Regulatory Factor 1 (Smurf1) Is Essential for Negative Regulation of Transforming Growth Factor-β Signaling by Smad7

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces translocation of Smad7 to the cytoplasm. Smurf1 then associates with the transforming growth factor (TGF)-beta type I receptor, TbetaR-I, enhancing turnover. However, the mechanism of nuclear export of Smad7 by Smurf1 has not been elucidated. Here we identified a functional nuclear export signal (NES) in a C-terminal region of Smurf1. In transfected cells, the Smurf1-Smad7 complex was accumulated in the cytoplasm by the nuclear export receptor, CRM1; this action was prevented by treatment with leptomycin B, a specific inactivator of CRM1 function. A green fluorescence protein fusion protein containing the C-terminal NES motif of Smurf1, located in the cytoplasm, accumulated in the nucleus following treatment with leptomycin B. Moreover, Smurf1 was shown to bind physically to CRM1 through NES, and nuclear export of the Smurf1-Smad7 complex was prevented by mutations of Smurf1 within the NES. Finally, the Smurf1 NES mutant reduced inhibition by Smad7 of the transcriptional activation induced by TGF-beta. These results thus suggest that CRM1-dependent nuclear export of Smurf1 is essential for the negative regulation of TGF-beta signaling by Smad7.

Multiple Mechanisms Regulate Subcellular Localization of Human CDC6

CDC6 is a protein essential for DNA replication, the expression and abundance of which are cell cycle-regulated in Saccharomyces cerevisiae. We have demonstrated previously that the subcellular localization of the human CDC6 homolog, HsCDC6, is cell cycle-dependent: nuclear during G(1) phase and cytoplasmic during S phase. Here we demonstrate that endogenous HsCDC6 is phosphorylated during the G(1)/S transition. The N-terminal region contains putative cyclin-dependent kinase phosphorylation sites adjoining nuclear localization sequences (NLSs) and a cyclin-docking motif, whereas the C-terminal region contains a nuclear export signal (NES). In addition, we show that the observed regulated subcellular localization depends on phosphorylation status, NLS, and NES. When the four putative substrate sites (serines 45, 54, 74, and 106) for cyclin-dependent kinases are mutated to alanines, the resulting HsCDC6A4 protein is localized predominantly to the nucleus. This localization depends upon two functional NLSs, because expression of HsCDC6 containing mutations in the two putative NLSs results in predominantly cytoplasmic distribution. Furthermore, mutation of the four serines to phosphate-mimicking aspartates (HsCDC6D4) results in strictly cytoplasmic localization. This cytoplasmic localization depends upon the C-terminal NES. Together these results demonstrate that HsCDC6 is phosphorylated at the G(1)/S phase of the cell cycle and that the phosphorylation status determines the subcellular localization.

Calcium signals in the cell nucleus. Strasbourg, France, August 20-23, 1998.

Just as the great explorations revealed a world far more diverse and rich than had been imagined before, a mass of fast accumulating data is disclosing a much more complex and sophisticated picture of the nucleus than hitherto assumed. The nucleus has long been thought of as some sort of enclosure circumscribed by a double membrane and containing the genetic material of the cell. The inner and outer nuclear membranes join periodically at the nuclear pores, forming complexes supposed to create large channels for the free diffusion of ions and small macromolecules. In this view, the flow of calcium between nucleus and cytoplasm would appear unrestricted, with nuclear Ca2+ signals originating from cytosolic Ca2+ waves merely by passive transmission through the nuclear pore complex (NPC). However, recent evidence has changed the mappa mundi of the cell. It has been shown that nuclear Ca2+ can be regulated independently of cytosolic Ca2+ changes. And, just as the Terra Incognita discovered beyond the Ocean Sea proved a fabulous New World, the nuclear envelope (NE) between the cytoplasm and nucleoplasm appears to play an essential role in the regulation of Ca2+ signals inside the nucleus. It contains proteins that regulate and respond to changes in nucleosolic Ca2+ concentration ([Ca2+]nucleosol). Furthermore, several specific Ca2+‐dependent nuclear functions have been described and the list is growing. The regulation of nuclear Ca2+ signals, and the role of nuclear Ca2+ as a specific regulator of nuclear events through Ca2+‐binding proteins, were the main themes of the recent EMBO workshop entitled ‘Calcium signals in the cell nucleus’, organized by A.N.Malviya and coworkers in Strasbourg, France. The existence of Ca2+ signals at the level of the nucleoplasm has never been a real issue, as the …