In order to investigate the mechanism of stat3 nuclear import. positioned a characterized NLS of the SV40 large T antigen into Stat3-GFP, Dstat3-GFP respectively between the C-terminus of Stat3 and the N-terminus of GFP to create Stat3-NLS-GFP and Dstat3-NLS-GFP. With NLS-GFP as the positive control, Expression of the Stat3-NLS-GFP without IL-6 stimulation and Stat3-GFP with IL-6 stimulation resulted in a predominantly nuclear localization in 293T cell. Expression of Stat3-GFP and Dstat3-NLS-GFP without IL-6 stimulation resulted in predominantly cytoplasm localization in 293T cell. The results suggest that latent Stat3 is not anchored in the cytoplasm, and that nuclear localization in response to IL-6 is facilitated by gain of an NLS function.
Active Transport, Cell Nucleus ; Cell Nucleus ; metabolism ; Nuclear Localization Signals ; Plasmids ; metabolism ; STAT3 Transcription Factor ; metabolism

p53 is a well-known multi-functional transcription regulator and is critical in the induction of apoptosis in response to various stresses. Human cytomegalovirus (HCMV) infection induced the accumulation of p53, which was partly relocalized in cytoplasm, but no apparent cell death in human fibroblasts. p53 in HCMV-infected cells was mainly mono-ubiquitinated, which might be resulted from the decreased expression of MDM2 in the course of HCMV infection. Ubiquitinated p53 was also phosphorylated at serine 20. CRM1 increased in the cytoplasm of HCMV-infected cells. It was found that p53 and its mutant in nuclear export sequences were localized in the cytoplasm of cells when co-expressed with CRM1. Collectively, our data suggest that HCMV infection modifies p53 into a stable and exportable form and accumulates it in the cytoplasm, but does not result in apoptotic death of cells.
Active Transport, Cell Nucleus ; Apoptosis ; Cell Death ; Cytomegalovirus Infections* ; Cytomegalovirus* ; Cytoplasm* ; Fibroblasts ; Humans* ; Serine ; Ubiquitin

Abnormal localization of tumor suppressor proteins is a common feature of renal cancer. Nuclear export of these tumor suppressor proteins is mediated by chromosome region maintenance-1 (CRM1). Here, we investigated the antitumor eff ects of a novel reversible inhibitor of CRM1 on renal cancer cells. We found that S109 inhibits the CRM1-mediated nuclear export of RanBP1 and reduces protein levels of CRM1. Furthermore, the inhibitory eff ect of S109 on CRM1 is reversible. Our data demonstrated that S109 signifi cantly inhibits proliferation and colony formation of renal cancer cells. Cell cycle assay showed that S109 induced G1-phase arrest, followed by the reduction of Cyclin D1 and increased expression of p53 and p21. We also found that S109 induces nuclear accumulation of tumor suppressor proteins, Foxo1 and p27. Most importantly, mutation of CRM1 at Cys528 position abolished the eff ects of S109. Taken together, our results indicate that CRM1 is a therapeutic target in renal cancer and the novel reversible CRM1 inhibitor S109 can act as a promising candidate for renal cancer therapy.
Active Transport, Cell Nucleus ; Cell Cycle Checkpoints* ; Cell Cycle* ; Cell Proliferation* ; Cyclin D1 ; Kidney Neoplasms* ; Tumor Suppressor Proteins

OBJECTIVETo determine the role of serine residues at position 63-84 of CITED1 in the nuclear translocation of CITED1 and osteoblast differentiation.

METHODSWe engineered all the 9 phosphorylated serine residues of CITED1 with a serine-to-alanine mutation at position 63-84. MC3T3E1 cells transfected with pCDNA3-CFP-CITED1 63-84 (9S>A), pCDNA3-CFP-CITED1, and vehicle plasmid were examined with confocal laser scanning microscopy before and after treatment with 100 nmol/L parathyroid hormone [PTH(1-34)] to observe the changes in the intracellular localization of CITED1. The transfected cells were induced for osteoblastic differentiation with mineralized solution in the absence or presence of 10 nmol/L PTH(1-34), and the changes in ALP activity and Ca(2+) concentration were measured; RT-PCR was used to detect the changes in ALP2, RUNX2, and OC gene expressions after the treatments.

RESULTSs PTH(1-34) promoted the nuclear translocation of CITED1 in MC3T3-E1 cells. The (63-84) 9S>A mutation of CITED1 obviously suppressed its translocation and increased ALP activity and Ca(2+) levels in the cells, which led to enhanced mineralization in the cells with also increased expressions of ALP2, RUNX2, and OC.

CONCLUSIONThe serine residues at position 63-84 of CITED1 play a vital role in the nuclear translocation of CITED1 and osteoblast differentiation.

Active Transport, Cell Nucleus ; Animals ; Cell Differentiation ; Cell Line ; Cell Nucleus ; Mice ; Mice, Inbred C57BL ; Mutation ; Nuclear Proteins ; metabolism ; Osteoblasts ; cytology ; Plasmids ; Serine ; metabolism ; Trans-Activators ; metabolism

3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, produces rapid antidepressant-like effects in animal models of depression. However, the molecular mechanisms underlying these behavioral actions remain unknown. Here, we demonstrate that CPP rapidly stimulates histone deacetylase (HDAC) 5 phosphorylation and nuclear export in rat hippocampal neurons. These effects are accompanied by calcium/calmodulin kinase II (CaMKII) and protein kinase D (PKD) phosphorylation. Behavioral experiments revealed that viral-mediated hippocampal knockdown of HDAC5 blocked the antidepressant effects of CPP in stressed animals. Taken together, our results imply that CPP acts via HDAC5 and suggest that HDAC5 is a common regulator contributing to the antidepressant actions of NMDA receptor antagonists such as CPP.
Active Transport, Cell Nucleus ; Animals ; Depression ; Hippocampus ; Histone Deacetylases* ; Histones* ; Models, Animal ; N-Methylaspartate ; Neurons ; Phosphorylation* ; Phosphotransferases ; Protein Kinases ; Rats

OBJECTIVETo establish a new method for studying the mechanism of nuclear localization signal (NLS)-mediated nuclear translocation in living cells.

METHODSThe cells were treated with 67 mg/L 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), followed by incubation with 1 g/L wheat germ agglutinin (WGA), and their effects on interferon- γ (IFN-γ)-induced nuclear translocation of signal transducer and activator of transcription 1 (STAT1) were observed.

RESULTSTreatment with CHAPS alone had no effect on IFN-γ-induced nuclear translocation of STAT1, while this process was blocked by further WGA incubation.

CONCLUSIONWe established a new, simple but effective method for studying the mechanism of NLS-mediated nuclear translocation in living cells by perforating the cell membrane with CHAPS treatment.

Active Transport, Cell Nucleus ; Cell Nucleus ; metabolism ; Cholic Acids ; Cytological Techniques ; HeLa Cells ; Humans ; Interferon-gamma ; metabolism ; Nuclear Localization Signals ; metabolism ; STAT1 Transcription Factor ; metabolism ; Signal Transduction

Immuno-fluorescence technique can qualitatively determine certain nuclear translocation, of which NF-κB/ p65 implicates the activation of NF-κB signal pathways. Immuno-fluorescence analysis software with independent property rights is able to quantitatively analyze dynamic location of NF-κB/p65 by computing relative fluorescence units in nuclei and cytoplasm. We verified the quantitative analysis by Western Blot. When we applied the software to analysis of nuclear translocation in lipopolysaccharide (LPS) induced (0. 5 h, 1 h, 2 h, 4 h) primary human umbilical vein endothelial cells (HUVECs) , we found that nuclear translocation peak showed up at 2h as with calculated Western blot verification results, indicating that the inventive immuno-fluorescence analysis software can be applied to the quantitative analysis of immuno-fluorescence.
Active Transport, Cell Nucleus ; Cell Nucleus ; metabolism ; Cytoplasm ; metabolism ; Fluorescent Antibody Technique ; Human Umbilical Vein Endothelial Cells ; Humans ; NF-kappa B p50 Subunit ; metabolism ; Software

Active Transport, Cell Nucleus ; physiology ; Animals ; Cell Nucleus ; physiology ; Cilia ; physiology ; Humans ; Molecular Motor Proteins ; physiology ; beta Karyopherins ; physiology ; ran GTP-Binding Protein ; physiology

This study examined the radiation-induced ERBB2 nuclear transport in the BT474 breast cancer cell line and the relationship between caveolin-1 and radiation-induced ERBB2 nuclear transport. The BT474 cells were treated with herceptin (200 nmol/L), PP2 (a caveolin-1 inhibitor, 100 nmol/L) and irradiation combined or alone. Confocal microscopy was used to observe the nuclear import of ERBB2 and caveolin-1 after irradiation. Western blotting was employed to detect the expression of ERBB2, caveolin-1 and DNA-PKcs after irradiation, and immunoprecipitation to identify the ERBB2 and caveolin-1 complex before perinuclear ERBB2 localization. Confocal microscopy showed the transport of ERBB2 and caveolin-1 from the cell membrane to the nucleus 15 min after irradiation and the proteins accumulated at the perinuclear region within 45 min. Western blotting revealed that the expression levels of ERBB2, caveolin-1 and DNA-PKcs were increased after irradiation and reached a peak 45 min later. Both herceptin and PP2 treatments were found to decrease ERBB2 expression. An immune complex composed of ERBB2 and caveolin-1 was found in the herceptin group after irradiation. It was concluded that after irradiation, ERBB2 may be transported from the cell membrane to the nucleus and activate DNA-PKcs to trigger DNA double-strand break (DSB) repair; caveolin-1 may participate in this process. Treatments involving the downregulation of caveolin-1 may increase the radiosensitization of breast cancer cells.
Active Transport, Cell Nucleus ; physiology ; Breast Neoplasms ; metabolism ; physiopathology ; Caveolin 1 ; metabolism ; Cell Line, Tumor ; Female ; Humans ; Protein Transport ; physiology ; Radiation ; Receptor, ErbB-2 ; metabolism

The constitutive androstane receptor (CAR, NR1I3) plays a crucial role in the regulation of drug metabolism, energy homeostasis, and cancer development through modulating the transcription of its numerous target genes. Different from prototypical nuclear receptors, CAR can be activated by either direct ligand binding or ligand-independent (indirect) mechanisms both initiated with nuclear translocation of CAR from the cytoplasm. In comparison to the well-defined ligand-based activation, indirect activation of CAR appears to be exclusively involved in the nuclear translocation through mechanisms yet to be fully understood. Accumulating evidence reveals that without activation, CAR forms a protein complex in the cytoplasm where it can be functionally affected by multiple signaling pathways. In this review, we discuss recent progresses in our understanding of the signaling regulation of CAR nuclear accumulation and activation. We expect that this review will also provide greater insight into the similarity and difference between the mechanisms of direct vs. indirect human CAR activation.
Active Transport, Cell Nucleus ; genetics ; Cytoplasm ; metabolism ; Hepatocytes ; metabolism ; Humans ; Ligands ; Protein Transport ; genetics ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Signal Transduction ; genetics