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Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob. 相似文献
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PTEN is involved in the signal transduction pathway of contact inhibition in endometrial cells 总被引:2,自引:0,他引:2
Uegaki K Kanamori Y Kigawa J Kawaguchi W Kaneko R Naniwa J Takahashi M Shimada M Oishi T Itamochi H Terakawa N 《Cell and tissue research》2006,323(3):523-528
PTEN is involved in the regulation of normal cellular functions in addition to its well–known role as a tumor suppressor.
In the present study, we have shown that stable transfection of the PTEN gene into PTEN–mutated endometrial carcinoma cells leads to contact inhibition accompanied by a decreased level of phosphorylated–Akt (p–Akt)
expression, an increase in p27Kip1, and a decrease in β–catenin. PTEN–induced cells with contact inhibition exhibit G0–G1 cell-cycle arrest, and the Ki–67 labeling
index is reduced. These changes are canceled by transfection of a double–stranded short–interfering RNA against the PTEN gene. Normal endometrial stromal cells increase their PTEN expression when reaching confluence; this is followed by changes
in the expression of Akt–related proteins in the same way as in tumor cells. These results indicate that PTEN, p–Akt, p27,
and β–catenin are involved in the signal transduction of contact inhibition and suggest that PTEN may, in part, control the
proliferation of endometrial carcinoma cells through the induction of contact inhibition. 相似文献
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The human cytomegalovirus protein pUL69 belongs to a family of regulatory factors that is conserved within the Herpesviridae and includes the proteins ICP27 of herpes simplex virus type 1 and EB2 of Epstein–Barr virus. ICP27 and EB2 have been shown to facilitate the nuclear export of viral mRNAs via interacting with the cellular mRNA export factor REF. Furthermore, direct RNA-binding of these proteins was found to be essential for their stimulating effects on mRNA export. Recently, we demonstrated that pUL69 shares common features with ICP27 and EB2 such as (i) nucleocytoplasmic shuttling and (ii) stimulation of nuclear RNA export via binding to the cellular mRNA export machinery. Here, we demonstrate that pUL69 can also interact with RNA both in vivo and in vitro via a complex N-terminal RNA-binding domain consisting of three arginine-rich motifs. Interestingly, the RNA-binding domain of pUL69 overlaps with both the NLS and the binding site of the cellular mRNA export factors UAP56 and URH49. While the deletion of the UAP56/URH49-binding site abolished pUL69-mediated RNA export, an RNA-binding deficient pUL69 mutant which still interacts with UAP56/URH49 retained its RNA export activity. This surprising finding suggests that, in contrast to its homologues, RNA-binding is not a prerequisite for pUL69-mediated nuclear RNA export. 相似文献
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The cell cycle inhibitor p57Kip2 induces cell cycle arrest by inhibiting the activity of cyclin-dependent kinases. p57, although active as a cyclin A-CDK2 inhibitor, is largely unfolded or intrinsically disordered as shown by circular dichroism and fluorescence spectra characteristic of an unfolded protein and a hydrodynamic radius consistent with an unfolded structure. In addition, the N-terminal domain of p57 is both functionally independent as a cyclin A-CDK2 inhibitor and unstructured, as demonstrated by circular dichroism and fluorescence spectra indicative of unfolded proteins, a lack of 1H chemical shift dispersion and a hydrodynamic radius consistent with a highly unfolded structure. The amino acid compositions of full-length p57 and the excised QT domain of p57 exhibit significant deviations from the average composition of globular proteins that are consistent with the observed intrinsic disorder. However, the amino acid composition of the CDK inhibition domain of p57 does not exhibit such a striking deviation from the average values observed for proteins, implying that a general low level of hydrophobicity, rather than depletion or enrichment in specific amino acids, contributes to the intrinsic disorder of the excised p57 CDK inhibition domain. 相似文献
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Kim M. Huffman Salvatore J. Arrigo Michael G. Schmidt 《Journal of biomedical science》1999,6(3):194-205
Rev has been shown to promote the export of HIV-1 RNAs fromXenopus oocyte nuclei, but a system to examine the direct effect of Rev on HIV-1 RNA export in mammalian somatic cells does not exist. In this report, the development of a cell-free RNA export system using COS cells is described. This system is capable of examining the movement of RNA from nuclei of COS cells transfected with an HIV-1 proviral construct into reconstituted cytosol from nontransfected cells. A reproducible preparation of nuclei free of residual cytoplasmic RNA is demonstrated. Export of RNA from these nuclei into reconstituted cell-free extracts was saturable and dependent on temperature and energy. Further validation of the system was obtained by confirming that the nuclear export of HIV-1-unspliced and partially spliced RNAs was dependent upon the expression of HIV-1 Rev and that the presence of Rev appeared to decrease the export of an HIV-1-spliced RNA. The system was also able to demonstrate that Rev did not appear to significantly enhance the export of an HIV-1 protease-containing RNA that has been shown to be dependent upon Rev for maximal expression. Consequently, the system appears useful for the examination of parameters of nuclear export of HIV-1 and cellular RNAs. 相似文献
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Kalam Azad A Ideue T Ohshima Y Tani T 《Biochemical and biophysical research communications》2003,310(1):176-181
Fission yeast ptr4-1 is one of the mRNA transport mutants that accumulate poly(A)(+) RNA in the nuclei at the nonpermissive temperature. We cloned the ptr4(+) gene and found that it is identical with the cut1(+) gene essential for chromosome segregation during mitosis. ptr4/cut1 has no defects in nucleocytoplasmic transport of a protein, indicative of a specific blockage of mRNA export by this mutation. A mutant of Cut2p cooperating with Cut1p in sister chromatid separation also showed defective mRNA export at the nonpermissive temperature. Our results suggest a novel linkage between the cell division cycle and nuclear mRNA export in eukaryotic cells. 相似文献
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Identification of a region in the Sindbis virus nucleocapsid protein that is involved in specificity of RNA encapsidation. 总被引:14,自引:11,他引:3
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The specific encapsidation of genomic RNA by an alphavirus requires recognition of the viral RNA by the nucleocapsid protein. In an effort to identify individual residues of the Sindbis virus nucleocapsid protein which are essential for this recognition event, a molecular genetic analysis of a domain of the protein previously suggested to be involved in RNA binding in vitro was undertaken. The experiments presented describe the generation of a panel of viruses which contain mutations in residues 97 through 111 of the nucleocapsid protein. All of the viruses generated were viable, and the results suggest that, individually, the residues mutated do not play a critical role in encapsidation. However, one mutant which had lost the ability to specifically encapsidate the genomic RNA was identified. This mutant virus, which contained a deletion of residues 97 to 106, encapsidated both the genomic RNA and the subgenomic mRNA of the virus. It is proposed that the encapsidation of this second species of RNA, which is not present in wild-type virions, is the result of the loss of a domain of the nucleocapsid protein required for specific recognition of the genomic RNA packaging signal. The results suggest that this region of the protein is important in dictating specificity in the encapsidation reaction in vivo. The isolation and preliminary characterization of two independent second-site revertants to this deletion mutant are also described. 相似文献
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Izaurralde E 《European journal of cell biology》2002,81(11):577-584
Fully processed mRNAs are exported to the cytoplasm where they direct protein synthesis. A general feature of mRNA export is that it is an active, receptor-mediated process. The mRNA export receptors are thought to recognize and bind to the mRNA-export cargoes either directly or indirectly (via adaptor proteins) and facilitate their translocation across the central channel of the nuclear pore complex (NPC). On the cytoplasmic side of the NPC, the exported mRNA is released and the receptor returns to the nucleoplasm, without the cargo, to initiate additional rounds of export. Recent, studies in yeast and in higher eukaryotes have led to the elucidation of an evolutionarily conserved pathway for the export of bulk mRNA to the cytoplasm. 相似文献
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《Journal of molecular biology》2023,435(10):168069
The neuronal SNARE protein SNAP25a (isoform 2) forms part of the SNARE complex eliciting synaptic vesicle fusion during neuronal exocytosis. While the post-fusion cis-SNARE complex has been studied extensively, little is known about the pre-fusion conformation of SNAP25a. Here we analyze monomeric SNAP25a by NMR spectroscopy, further supported by small-angle X-ray scattering (SAXS) experiments. SAXS data indicate that monomeric SNAP25 is more compact than a Gaussian chain but still a random coil. NMR shows that for monomeric SNAP25a, before SNAP25a interacts with its SNARE partners to drive membrane fusion, only the N-terminal part (region A5 to V36) of the first SNARE motif, SN1 (L11 - L81), is helical, comprising two α-helices (ranging from A5 to Q20 and S25 toV36). From E37 onwards, SNAP25a is mostly disordered and displays high internal flexibility, including the C-terminal part of SN1, almost the entire second SNARE motif (SN2, N144-A199), and the connecting loop region. Apart from the N-terminal helices, only the C-termini of both SN1 (E73 - K79) and SN2 (region T190 - A199), as well as two short regions in the connecting loop (D99 - K102 and E123 - M127) show a weak α-helical propensity (α-helical population < 25%). We speculate that the N-terminal helices (A5 to Q20 and S25 to V36) which constitute the N-terminus of SN1 act as a nucleation site for initiating SNARE zippering. 相似文献
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Galea CA Nourse A Wang Y Sivakolundu SG Heller WT Kriwacki RW 《Journal of molecular biology》2008,376(3):827-838
p27Kip1 (p27), which controls eukaryotic cell division through interactions with cyclin-dependent kinases (Cdks), integrates and transduces promitogenic signals from various nonreceptor tyrosine kinases by orchestrating its own phosphorylation, ubiquitination and degradation. Intrinsic flexibility allows p27 to act as a “conduit” for sequential signaling mediated by tyrosine and threonine phosphorylation and ubiquitination. While the structural features of the Cdk/cyclin-binding domain of p27 are understood, how the C-terminal regulatory domain coordinates multistep signaling leading to p27 degradation is poorly understood. We show that the 100-residue p27 C-terminal domain is extended and flexible when p27 is bound to Cdk2/cyclin A. We propose that the intrinsic flexibility of p27 provides a molecular basis for the sequential signal transduction conduit that regulates p27 degradation and cell division. Other intrinsically unstructured proteins possessing multiple sites of posttranslational modification may participate in similar signaling conduits. 相似文献
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The Saccharomyces cerevisiae homologue of the linker histone H1, Hho1p, has two domains that are similar in sequence to the globular domain of H1 (and variants such as H5). It is an open question whether both domains are functional and whether they play similar structural roles. Preliminary structural studies showed that the two isolated domains, GI and GII, differ significantly in stability. In 10 mM sodium phosphate (pH 7), the GI domain, like the globular domains of H1 and H5, GH1 and GH5, was stably folded, whereas GII was largely unstructured. However, at high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate), which might mimic the charge-screening effects of DNA phosphate groups, GII was folded. In view of the potential significance of these observations in relation to the role of Hho1p, we have now determined the structures of its GI and GII domains by NMR spectroscopy under conditions in which GII (like GI) is folded. The backbone r.m.s.d. over the ordered residues is 0.43 A for GI and 0.97 A for GII. Both structures show the "winged-helix" fold typical of GH1 and GH5 and are very similar to each other, with an r.m.s.d. over the structured regions of 1.3 A, although there are distinct differences. The potential for GII to adopt a structure similar to that of GI when Hho1p is bound to chromatin in vivo suggests that both globular domains might be functional. Whether Hho1p performs a structural role by bridging two nucleosomes remains to be determined. 相似文献