The abscopal effect of radiation therapy.

Radiation therapy (RT) in some cases results in a systemic anticancer response known as the abscopal effect. Multiple hypotheses support the role of immune activation initiated by RT-induced DNA damage. Optimal radiation dose is necessary to promote the cGAS-STING pathway in response to radiation and initiate an IFN-1 signaling cascade that promotes the maturation and migration of dendritic cells to facilitate antigen presentation and stimulation of cytotoxic T cells. T cells then exert a targeted response throughout the body at areas not subjected to RT. These effects are further augmented through the use of immunotherapeutic drugs resulting in increased T-cell activity. Tumor-infiltrating lymphocyte presence and TREX1, KPNA2 and p53 signal expression are being explored as prognostic biomarkers.

KPNA2 expression is a potential marker for differential diagnosis between osteosarcomas and other malignant bone tumor mimics.

BACKGROUND: Karyopherin α2 (KPNA2), a member of the karyopherin α family, has been studied in several cancers but has not yet been substantially investigated in malignant bone tumors. The purpose of the current study was to evaluate the KPNA2 expression level and its utility as a novel diagnostic biomarker in osteosarcomas and malignant bone tumor mimics, such as chondrosarcomas and Ewing sarcomas (ESs). METHOD: We investigated the expression of KPNA2 protein by immunohistochemistry on paraffin-embedded surgical specimens from 223 patients with malignant and benign bone tumors, including 81 osteosarcomas, 42 chondrosarcomas, 15 ESs, 28 osteoid osteomas, 20 osteochondromas and 37 chondroblastomas. Immunoreactivity was scored semiquantitatively based on staining extent and intensity. RESULTS: Sixty-seven of 81 (82.7%) osteosarcoma, zero of 42 (0%) chondrosarcoma and one of 15 (6.7%) ES samples showed immunoreactivity for KPNA2. Negative KPNA2 expression was observed in all benign bone tumors. The expression of KPNA2 in osteosarcoma samples was much higher than that in chondrosarcoma and ES samples (P < 0.001). The sensitivity and specificity of KPNA2 immunoexpression for detecting osteosarcoma were 82.7 and 100%, respectively. Several subtypes of osteosarcoma were analyzed, and immunostaining of KPNA2 was frequent in osteoblastic samples (90.9%), with 39 samples (70.9%) showing strong-intensity staining. KPNA2 positivity was observed in ten of 13 (76.9%) chondroblastic, two of 6 (33.3%) fibroblastic, three of 4 (75%) telangiectatic and two of 3 (66.7%) giant cell-rich osteosarcoma samples. The strongest intensity staining was observed in osteoblastic osteosarcoma. CONCLUSION: KPNA2 is frequently expressed in osteosarcomas, particularly in osteoblastic and chondroblastic tumors, but is rarely positive in chondrosarcomas and ESs. This feature may aid in distinguishing between osteosarcoma and other bone sarcoma mimics. This report supports KPNA2 as a novel marker for the diagnosis of osteosarcoma.

High expression of karyopherin-α2 and stathmin 1 is associated with proliferation potency and transformation in the bile duct and gall bladder epithelia in the cases of pancreaticobiliary maljunction.

BACKGROUNDS AND OBJECTIVES: Pancreaticobiliary maljunction (PBM) may be associated with an increased frequency of gall bladder cancer with no bile duct dilation. Karyopherin-α2 (KPNA2) and stathmin 1 (STMN1) were reported to play important roles in carcinogenesis and cancer progression. METHODS: Fifteen patients with PBM who underwent surgical resection between 1999 and 2014 were included in this study. Using immunohistochemistry, we investigated the expression of p53, Ki-67, KPNA2, and STMN1 in normal biliary tract epithelium, hyperplastic epithelium, and cholangiocarcinoma (CC) tissues. RESULTS: Nuclear expression of KPNA2, p53, and Ki-67 expression was detected in hyperplastic epithelium and CC tissues. High KPNA2 expression was significantly associated with gender (P = 0.04), p53 nuclear accumulation (P = 0.00435), and Ki-67 expression (P = 0.0443) in the gall bladder and bile duct of PBM. On the other hand, STMN1 was only expressed in CC tissues and was not observed in normal bile duct and hyperplastic epithelia. CONCLUSIONS: KPNA2 might be a useful marker of hyperplasia, dysplasia, and carcinogenicity in patients with PBM. STMN1 evaluation might be a cancer-specific marker for CC patients with PBM similar as that for other cancers. J. Surg. Oncol. 2016;114:462-468. © 2016 Wiley Periodicals, Inc.

Importin α1 Mediates Yorkie Nuclear Import via an N-terminal Non-canonical Nuclear Localization Signal.

The Hippo signaling pathway controls organ size by orchestrating cell proliferation and apoptosis. When the Hippo pathway was inactivated, the transcriptional co-activator Yorkie translocates into the nucleus and forms a complex with transcription factor Scalloped to promote the expression of Hippo pathway target genes. Therefore, the nuclear translocation of Yorkie is a critical step in Hippo signaling. Here, we provide evidence that the N-terminal 1-55 amino acids of Yorkie, especially Arg-15, were essential for its nuclear localization. By mass spectrometry and biochemical analyses, we found that Importin α1 can directly interact with the Yorkie N terminus and drive Yorkie into the nucleus. Further experiments show that the upstream component Hippo can inhibit Importin α1-mediated Yorkie nuclear import. Taken together, we identified a potential nuclear localization signal at the N-terminal end of Yorkie as well as a critical role for Importin α1 in Yorkie nuclear import.

KPNA2 over-expression is a potential marker of prognosis and therapeutic sensitivity in colorectal cancer patients.

BACKGROUND: Karyopherin α 2 (KPNA2) is a member of the Karyopherin α family and has recently been reported to play an important role in tumor progression. The aim of the current study was to elucidate the clinicopathological significance of KPNA2 over-expression in colorectal cancer (CRC). PATIENTS AND METHODS: KPNA2 expression was evaluated by immunohistochemistry in 122 surgically resected CRC and 13 biopsy specimens obtained at colonoscopy during screening for preoperative hyperthermochemoradiation therapy (HCRT). The association between KPNA2 expression and clinicopathological features and preoperative HCRT efficacy were examined. RESULTS: The high and low KNPA2 expression groups were comprised of 91 (74.6%) and 31 CRC patients, respectively. A significant association was observed between high expression and lymphatic invasion (P = 0.0245). KPNA2 high expression group had decreased overall survival (P = 0.00374). Multivariate analysis demonstrated high KPNA2 expression was independently associated with poor prognosis. Histological examinations revealed 11 (84.6%) and 2 (15.4%) of cases were KPNA2 positive and negative, respectively. Pathological complete response (pCR) was observed in 9.1% of KPNA2-positive cases and 100% of KPNA2-negative cases. CONCLUSION: High KPNA2 expression was found to be associated with poor prognosis and resistance to HCRT.

Low cytoplasmic and nuclear KPNA2 expression in radiotherapy-treated head and neck squamous cell cancer is associated with an adverse outcome.

BACKGROUND: KPNA2 has effects on carcinogenesis, cell differentiation and transcriptional regulation. KPNA2 has been linked to DNA damage repair by its role to import the DNA double strand break repair complex MRN into the nucleus. The aim of our study was to evaluate the prognostic value of KPNA2 expression in both cytoplasmic and nuclear location in patients with HNSCC treated with radio(chemo)therapy. MATERIAL AND METHODS: 225 patients with HNSCC treated with neoadjuvant, definitive or adjuvant radio(chemo)therapy were included. Immunohistochemical staining was performed on tissue micro arrays to evaluate nuclear and cytoplasmic KPNA2 expression. RESULTS: The median fraction of tumor cells with nuclear KPNA2 expression was 15%. 47% of tumor samples showed positive cytoplasmic staining. Patients with low nuclear as well as negative cytoplasmic expression tended to have an unfavorable prognosis. There was no correlation between nuclear and cytoplasmic KPNA2 expression. Low nuclear combined with negative cytoplasmic KPNA2 had a clearly unfavorable prognostic effect in local failure-free survival (P=0.014), metastasis-free survival (P=0.001) and no evidence of disease (P=0.008). A combination of low nuclear/negative cytoplasmic with high nuclear/high cytoplasmic KPNA2 expression was prognostically unfavorable with regard to tumor specific survival (P=0.021) and to a lower extent to overall survival (P=0.18). In multivariate analysis low nuclear/negative cytoplasmic versus any high KPNA2 (P=0.008) and T-category (P=0.002) proved as independent prognostic variables. CONCLUSION: The combination of nuclear and cytoplasmic KPNA2 expression is a potential excellent prognostic parameter in HNSCC treated with radio(chemo)therapy.

The α-importome of mammalian germ cell maturation provides novel insights for importin biology.

Importin α proteins function as adaptors to connect a cargo protein and importin ß1 in the classical nuclear import pathway. Here we measure for the first time the stoichiometry of importins α2, α3, α4, and ß1 in primary cells corresponding to 2 successive stages of rat spermatogenesis: meiotic spermatocytes and haploid round spermatids. Importin α2 levels were more than 2-fold higher in spermatocytes than in spermatids, while importins α4 and ß1 levels did not differ significantly. We performed a comprehensive proteomics analysis to identify binding proteins in spermatocytes and spermatids using recombinant importin α2 and α4 proteins. Among the 100 candidate partners, 42 contained a strong classical nuclear localization signal (cNLS; score of>6 by cNLS Mapper), while 8 nuclear proteins lacked any cNLS. In addition, we developed a new strategy to predict which cargoes bind to importin α through the conserved C-terminal acidic domain (ARM repeats 9-10), and provided functional validation of a predicted importin α C-terminal binding segment in Senataxin and Smarca4. Evaluation of this set of candidate binding partners from spermatogenic cells using several bioinformatics approaches provides new evidence that individual importin αs may serve unique and nonredundant roles in mediating cellular differentiation.

High nuclear karyopherin α 2 expression is a strong and independent predictor of biochemical recurrence in prostate cancer patients treated by radical prostatectomy.

Increased levels of karyopherin α2 (KPNA2) expression have been described to be linked to poor prognosis in a variety of malignancies. This study was undertaken to evaluate the clinical impact of KPNA2 expression and its association with key genomic alterations in prostate cancers. A tissue microarray containing samples from 11 152 prostate cancers was analyzed for KPNA2 expression by immunohistochemistry. Results were compared with oncological follow-up data and genomic alterations such as TMPRSS2-ERG fusions and deletions of PTEN, 5q21, 6q15 or 3p13. KPNA2 expression was absent or weak in benign prostatic glands and was found to be in weak, moderate or strong intensities in 68.4% of 7964 interpretable prostate cancers. KPNA2 positivity was significantly linked to the presence of ERG rearrangement (P<0.0001). In ERG-negative and -positive prostate cancers, KPNA2 immunostaining was significantly associated with advanced pathological tumor stage (pT3b/pT4), high Gleason grade and early biochemical recurrence (P<0.0001 each). Multivariate analysis including all established prognostic criteria available after surgery revealed that the prognostic role of KPNA2 (P=0.001) was independent of high Gleason grade, advanced pathological tumor stage, high preoperative prostate-specific antigen level and positive surgical margin status (P<0.0001 each). The comparison of KPNA2 expression with deletions of PTEN, 5q21, 6q15 and 3p13 in ERG-positive and -negative cancers revealed a strong link to PTEN deletions in both subgroups (P<0.0001). In conclusion, the strong independent prognostic impact of KPNA2 expression raises the possibility that measurement of KPNA2 expression alone or in combination with other molecular parameters might possibly result in clinically useful information. The data also emphasize a critical role of the functionality of the nuclear import machinery for prostate cancer biology.

Nuclear karyopherin a2: a novel biomarker for infiltrative astrocytomas.

The karyopherin (KPNA) protein family is involved in nucleocytoplasmic trafficking. Increased KPNA levels have been found to predict poor prognosis for a variety of solid tumors, including breast, ovarian, cervical, and prostate cancer, and melanoma. The purpose of this study was to evaluate karyopherin a2 as novel biomarker for astrocytic gliomas of WHO grades II-IV. We semiquantitatively measured nuclear expression of karyopherin a2 and the MIB1 labeling index, by immunohistochemical analysis, for 94 primary (23 astrocytomas WHO grade II, 24 astrocytomas WHO grade III, 47 glioblastomas) and 12 recurrent gliomas. In addition, IDH1 mutation status and Nijmegen breakage syndrome 1 protein expression were assessed, by immunohistochemical analysis, for all 71 malignant (WHO grade III and IV) and all 94 primary gliomas, respectively. Statistical analysis was performed by use of standard techniques. Karyopherin a2 expression correlated significantly with histological grade (p < 0.001), with proliferative activity as assessed by the MIB1 index (p < 0.001), with IDH1 mutation status (p = 0.032), and with Nijmegen breakage syndrome 1 protein expression (p = 0.001). Recurrent tumors expressed significantly higher levels of karyopherin a2 (p = 0.045) than primary growths. Multivariate analysis of the overall series identified low karyopherin a2 expression (defined as less than 5 %) as an independent prognostic predictor of overall (p = 0.041) and progression-free survival (p = 0.004). Survival of glioblastoma patients >5 years was seen only in those with KPNA2 expression levels ≤1 % (p = 0.014). KPNA2 expression may have potential as a novel diagnostic and prognostic biomarker for astrocytic gliomas.

Proteomic analysis of importin α-interacting proteins in adult mouse brain.

Many transport factors, such as importins and exportins, have been identified, and the molecular mechanisms underlying nucleocytoplasmic transport have been characterized. The specific molecules that are carried by each transport factor and the temporal profiles that characterize the movements of various proteins into or out of the nucleus, however, have yet to be elucidated. Here, we used a proteomic approach to identify molecules that are transported into the nuclei of adult mouse brain cells via importin α5. We identified 48 proteins in total, among which we chose seven to characterize more extensively: acidic (leucine-rich) nuclear phosphoprotein 32 family member A (Anp32a), far upstream element binding protein 1 (FUBP1), thyroid hormone receptor ß1 (TRß1), transaldolase 1, CDC42 effector protein 4 (CDC42-ep4), Coronin 1B, and brain-specific creatine kinase (CK-B). Analyses using green fluorescent protein (GFP)-fused proteins showed that Anp32a, FUBP1, and TRß1 were localized in the nucleus, whereas transaldolase 1, CDC42-ep4, CK-B, and Coronin 1B were distributed in both the cytoplasm and nucleus. Using a digitonin-permeabilized in vitro transport assay, we demonstrated that, with the exception of CK-B, these proteins were transported into the nucleus by importin α5 together with importin ß and Ran. Further, we found that leptomycin B (LMB) treatment increased nuclear CK-B-GFP signals, suggesting that CK-B enters the nucleus and is then exported in a CRM1-dependent manner. Thus, we identified a comprehensive set of candidate proteins that are transported into the nucleus in a manner dependent on importin α5, which enhances our understanding of nucleocytoplasmic signaling in neural cells.

KPNA2 expression is an independent adverse predictor of biochemical recurrence after radical prostatectomy.

PURPOSE: To analyze rates of expression of karyopherin alpha 2 (KPNA2) in different prostate tissues and to evaluate the prognostic properties for patients with primary prostate cancer. EXPERIMENTAL DESIGN: Tissue microarrays (TMA) contained 798 formalin-fixed, paraffin-embedded prostate tissue cores from two different institutes of pathology. TMAs were stained immunohistochemically for KPNA2 and NBS1. SiRNA technologies were used to inhibit KPNA2 expression in vitro, and the effect of this inhibition on cellular viability was determined. Efficiency of knockdown experiments was determined by Western blot analysis. RESULTS: KPNA2 expression was significantly upregulated in carcinomas of the prostate, especially in metastatic and castration-resistant prostate cancer samples. Positive nuclear KPNA2 immunoreactivity was identified as a novel predictor of biochemical recurrence after radical prostatectomy (n = 348), and was independent of the well-established predictive factors preoperative PSA value, Gleason score, tumor stage, and surgical margin status. These results were validated by analyzing a second and independent prostate cancer cohort (n = 330). Further, in vitro experiments showed that the cell proliferation and viability of PC3 cells was significantly reduced when KPNA2 expression was inhibited. KPNA2 knockdown did not induce PARP cleavage as marker for apoptosis. No significantly increased sub-G(1) fraction could be found by FACS analysis. CONCLUSIONS: KPNA2 is a novel independent prognostic marker for disease progression after radical prostatectomy. This allows to identify patients who need more aggressive treatment. It can moreover be speculated that patients not suited for surveillance regimens might be identified at initial biopsy by a positive KPNA2 immunohistochemistry.

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities.

Identifying and characterizing protein interactions are fundamental steps toward understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the colocalization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin alpha (Imp alpha), and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified colocalization assay is sensitive enough to detect protein interactions with K(d) values that span over four orders of magnitude (1 nM to 15 microM). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris.

Identification of karyopherin-alpha 2 as an Oct4 associated protein.

The POU domain transcription factor Oct4 is a master regulator in maintaining self-renewal and pluripotency of embryonic stem (ES) cells. To further explore the functional network of Oct4, the yeast two-hybrid system was used to search for Oct4 interacting proteins. PH domain (containing POU domain and homeodomain) of human OCT4 was used as a bait. From the human testis cDNA library, we identified a strong interaction between OCT4 and karyopherin-alpha 2 (KPNA-2). KPNA2 is involved in active nuclear import of proteins. This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. The interaction between OCT4 and KPNA-2 was further mapped to multiple regions of the two proteins. In addition, we studied nuclear localization signal (NLS) of mouse Oct4 and demonstrated that it is essential for Oct4 nuclear localization. Thus, our data suggest that Oct4 nuclear localization may be mediated by its interaction with KPNA-2.

Subcellular distribution of importins correlates with germ cell maturation.

Importin proteins regulate access to the nucleus by recognizing and transporting distinct cargo proteins. Building on studies in Drosophila and Caenorhabditis elegans, we hypothesized that regulated expression and subcellular localization of specific importins may be linked to mammalian gonadal differentiation. We identified distinct developmental and cellular localization patterns for importins beta1, alpha3, alpha4 and RanBP5 (importin beta3) in fetal and postnatal murine testes using Western blotting and immunohistochemistry. Importin beta1 protein is detected in selected germ and somatic cells in fetal gonads, with a striking perinuclear staining evident from embryonic day (E) 14.5 within testicular gonocytes. RanBP5 exhibits age- and gender-specific subcellular localization within fetal gonads. At E12.5, RanBP5 protein is cytoplasmic in gonocytes but predominantly nuclear in oogonia, but by E14.5 RanBP5 appears nuclear in gonocytes and cytoplasmic in oogonia. In postnatal testes, importin alpha3 and alpha4 in spermatocytes, spermatids, and Sertoli cells display cytoplasmic and nuclear localization, respectively.

Nuclear import of bovine papillomavirus type 1 E1 protein is mediated by multiple alpha importins and is negatively regulated by phosphorylation near a nuclear localization signal.

Papillomavirus DNA replication occurs in the nucleus of infected cells and requires the viral E1 protein, which enters the nuclei of host epithelial cells and carries out enzymatic functions required for the initiation of viral DNA replication. In this study, we investigated the pathway and regulation of the nuclear import of the E1 protein from bovine papillomavirus type 1 (BPV1). Using an in vitro binding assay, we determined that the E1 protein interacted with importins alpha3, alpha4, and alpha5 via its nuclear localization signal (NLS) sequence. In agreement with this result, purified E1 protein was effectively imported into the nucleus of digitonin-permeabilized HeLa cells after incubation with importin alpha3, alpha4, or alpha5 and other necessary import factors. We also observed that in vitro binding of E1 protein to all three alpha importins was significantly decreased by the introduction of pseudophosphorylation mutations in the NLS region. Consistent with the binding defect, pseudophosphorylated E1 protein failed to enter the nucleus of digitonin-permeabilized HeLa cells in vitro. Likewise, the pseudophosphorylation mutant showed aberrant intracellular localization in vivo and accumulated primarily on the nuclear envelope in transfected HeLa cells, while the corresponding alanine replacement mutant displayed the same cellular location pattern as wild-type E1 protein. Collectively, our data demonstrate that BPV1 E1 protein can be transported into the nucleus by more than one importin alpha and suggest that E1 phosphorylation by host cell kinases plays a regulatory role in modulating E1 nucleocytoplasmic localization. This phosphoregulation of nuclear E1 protein uptake may contribute to the coordination of viral replication with keratinocyte proliferation and differentiation.

Gene expression profiling of primary cutaneous melanoma and clinical outcome.

BACKGROUND: Gene expression profiling data for human primary cutaneous melanomas are scarce because of the lack of retrospective collections of frozen tumors. To identify differentially expressed genes that may be involved in melanoma progression and prognosis, we investigated the relationship between gene expression profiles and clinical outcome in a cohort of patients with primary melanoma. METHODS: Labeled complementary RNA (cRNA) from each tissue sample was hybridized to a pangenomic 44K 60-mer oligonucleotide microarray. Class comparison and class prediction analyses were performed to identify genes whose expression in primary melanomas was associated with 4-year distant metastasis-free survival among 58 patients with at least 4 years of follow-up, distant metastasis, or death. Results were validated immunohistochemically at the protein level in 176 independent primary melanomas from patients with a median clinical follow-up of 8.5 years. Survival was analyzed with a Cox multivariable model and stratified log-rank test. All statistical tests were two-sided. RESULTS: We identified 254 genes that were associated with distant metastasis-free survival of patients with primary melanoma. These 254 genes include genes involved in activating DNA replication origins, such as minichromosome maintenance genes and geminin. Twenty-three of these genes were studied at the protein level; expression of five (MCM4, P = .002; MCM3, P = .030; MCM6, P = .004; KPNA2, P = .021; and geminin, P = .004) was statistically significantly associated with overall survival in the validation set. In a multivariable Cox model adjusted for tumor thickness, ulceration, age, and sex, expression of MCM4 (hazard ratio [HR] of death = 4.04, 95% confidence interval [CI] = 1.39 to 11.76; P = .010) and MCM6 (HR of death = 7.42, 95% CI = 1.99 to 27.64; P = .003) proteins was still statistically significantly associated with overall survival. CONCLUSION: We identified 254 genes whose expression was associated with metastatic dissemination of cutaneous melanomas. These genes may shed light on the molecular mechanisms underlying poor prognosis in melanoma patients.

A functional proteomics approach for the detection of nuclear proteins based on derepressed importin alpha.

The identification of functional proteomes is a major challenge in proteomic research. Here we describe a method for the detection and isolation of nuclear (localization sequence containing) proteins using a derepressed import receptor (DIRE) as a synthetic antibody. We demonstrate that the DIRE method specifically detects nuclear localization sequence containing proteins. Application to activation of primary T-lymphocytes exemplifies the potential use of DIRE for comparative proteomics and for diagnostics.

Evidence for importin alpha independent nuclear translocation of glucocorticoid receptors in Xenopus laevis oocytes.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor which resides in the cytoplasm as a complex with a number of molecular chaperones. Upon hormonal stimulation, the GR is translocated into the nucleus where it modulates transcription by binding to specific target DNA sequences. GR contains a classical basic nuclear localization signal (NLS) and a second, only poorly characterized NLS. Though the participation of the importin alpha/importin beta pathway in nuclear import of GR has been postulated, relatively little is known about its dynamics and mechanisms. We analyzed the hormone-driven nuclear import of rat GR expressed in Xenopus laevis oocytes by atomic force microscopy (AFM) and western blot analysis. AFM imaging revealed accumulation of macromolecules matching the size of GR at the nuclear envelope beginning 5 min after glucocorticoid hormone injection. In parallel, western blot analysis showed accumulation of GR over the same time scale after glucocorticoid hormone stimulation. Nuclear GR import did not trigger redistribution of importin alpha or importin beta. We conclude that hormone-driven nuclear import of rat GR expressed in Xenopus oocytes is a process with limited capacity and involves mechanisms different from the importin alpha/importin beta pathway. This represents a restriction of nuclear import downstream of import signals.

Yellow fluorescent protein-tagged and cyan fluorescent protein-tagged imaging analysis of glucocorticoid receptor and importins in single living cells.

Glucocorticoid receptor (GR) acts as a ligand-dependent transcription factor after nuclear transport from the cytoplasm in the liganded state. Importins are docking proteins for karyopherin-mediated binding of substrate in a nuclear import pathway. To investigate the spatial and temporal relation between GR and importins, we analyzed the subcellular distribution of GR and importins in response to ligand in single living cells using fusion proteins labeled with different spectral variants of green fluorescent protein. Upon activation with ligand treatment, fluorescent protein-tagged (FP-) GR was translocated from the cytoplasm to the nucleus, showing a similar time course as FP-importin-alpha in the coexpressed cells with the fusion proteins. In contrast to FP-importin-alpha, the distribution of FP-importin-beta was little changed upon ligand treatment in the coexpressed cells with FP-GR and FP-importin-beta. Analysis using fluorescence resonance energy transfer proved that GR directly interacted with importin-alpha in the whole area of the cytoplasm upon ligand treatment and detached importin-alpha shortly after nuclear import. However, direct interaction between GR and importin-beta was not detected. These studies showed visual evidence of the nuclear importing of GR in association with importin-alpha in single living cells.