Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences

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.     

Nuclear translocation of plk1 mediated by its bipartite nuclear localization signal

Polo-like kinase 1 (Plk1), a mammalian ortholog of Drosophila Polo, is a serine-threonine protein kinase implicated in the regulation of multiple aspects of mitosis. The protein level, activity, and localization of Plk1 change during the cell cycle, and its proper subcellular localization is thought to be crucial for its function. Although localization of Plk1 to the centrosome has been established, nuclear localization or nucleocytoplasmic translocation of Plk1 has not been fully addressed. Here we show that Plk1 accumulates in both the nucleus and the cytoplasm in addition to its localization to the centrosome during S and G(2) phases. Our results identify a conserved region in the kinase domain of Plk1 (residues 134-146) as a functional bipartite nuclear localization signal (NLS) sequence that regulates nuclear translocation of Plk1. The identified NLS is necessary and sufficient for directing nuclear localization of Plk1. This bipartite NLS has an unusually short spacer sequence between two clusters of basic amino acids but is sensitive to RanQ69L, a dominant negative form of Ran, similar to ordinary bipartite NLS. Remarkably, the expression of an NLS-disrupted mutant of Plk1 during S phase was found to arrest the cells in G(2) phase. These results suggest that the bipartite NLS-dependent nuclear localization of Plk1 before mitosis is important for ensuring normal cell cycle progression.     

A bipartite nuclear localization signal is required for p53 nuclear import regulated by a carboxyl-terminal domain.

Abnormal p53 cellular localization has been considered to be one of the mechanisms that could inactivate p53 function. To understand the regulation of p53 cellular trafficking, we have previously identified two p53 domains involved in its localization. A basic domain, Lys(305)-Arg(306), is required for p53 nuclear import, and a carboxyl-terminal domain, namely the cytoplasmic sequestration domain (CSD) from residues 326-355, could block the nuclear import of Lys(305) or Arg(306) mutated p53. To characterize further the function of these two domains, we demonstrate in this report that the previously described major nuclear localization signal works together with Lys(305)-Arg(306) to form a bipartite and functional nuclear localization sequence (NLS) for p53 nuclear import. The CSD could block the binding of p53 to the NLS receptor, importin alpha, and reduce the efficiency of p53 nuclear import in MCF-7, H1299, and Saos-2 cells. The blocking effect of the CSD is not due to the enhancement of nuclear export or oligomerization of the p53. These results indicate that the CSD can regulate p53 nuclear import by controlling access of the NLS to importin alpha binding.     

Characterization of the human herpesvirus 6 U69 gene product and identification of its nuclear localization signal

To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin alpha/beta in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-alpha subtype, NPI2/importin-alpha7 elicited more efficient transport activity than did Rch1/importin-alpha1 or Qip1/importin-alpha3. These results suggest a relationship between the localization of NPI2/importin-alpha7 and the cell tropism of HHV-6.     

Importin alpha binds to an unusual bipartite nuclear localization signal in the heterogeneous ribonucleoprotein type I.

The heterogeneous nuclear ribonucleoprotein (hnRNP) type I, a modulator of alternative splicing, localizes in the nucleoplasm of mammalian cells and in a discrete perinucleolar structure. HnRNP I contains a novel type of bipartite nuclear localization signal (NLS) at the N-terminus of the protein that we have previously named nuclear determinant localization type I (NLD-I). Recently, a neural counterpart of hnRNP I has been identified that contains a putative NLS with two strings of basic amino acids separated by a spacer of 30 residues. In the present study we show that the neural hnRNP I NLS is necessary and sufficient for nuclear localization and represents a variant of the novel bipartite NLS present in the NLD-I domain. Furthermore, we demonstrate that the NLD-I is transported into the nucleus by cytoplasmic factor(s) with active transport modality. Binding assays using recombinant importin alpha show an interaction with NLD-I similar to that of SV40 large T antigen NLS. Deletion analysis indicates that both stretches of basic residues are necessary for binding to importin alpha. The above experimental results lead to the conclusion that importin alpha acts as cytoplasmic receptor for proteins characterized by a bipartite NLS signal that extends up to 37 residues.     

The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells.

Here we show that the VirD2 protein of A. tumefaciens functions as a nuclear localizing protein in plant cells. The nuclear localization signal of VirD2 consists of two regions containing 4-5 basic amino acids (KRPR and RKRER), located within the C-terminal 34 amino acids. These regions conform to the KR/KXR/K motif required for numerous nuclear localized nonplant eukaryotic proteins. Each region independently directs a beta-glucuronidase reporter protein to the nucleus; however, both regions are necessary for maximum efficiency. VirD2 has been shown to be tightly bound to the 5' end of the single-stranded DNA transfer intermediate, T-strand, transferred from Agrobacterium to the plant cell genome. The present results imply that T-strand transport to the plant nucleus is mediated by the tightly attached VirD2 protein via an import pathway common to higher eukaryotes.     

The human poly(ADP-ribose) polymerase nuclear localization signal is a bipartite element functionally separate from DNA binding and catalytic activity.

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA-binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site-directed mutagenesis, its DNA-binding domain fused to the N-terminus of beta-galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK-X(11)-KKKSKK (residues 207-226). In vitro, both the DNA-binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure-function relationship of the second cluster of basic residues is discussed.     

核定位信号肽用于基因转移的研究进展

核定位信号(nuclear localization signal,NLS)是一段富含Arg、Lys的氨基酸序列,它存在于真核细胞核蛋白和病毒蛋白中,并具有引导它们趋向定位核区的功能。近年来发展的利用含核定位信号肽的非病毒载体为基因转移提供了一个崭新的途径。     

Identification of a bipartite nuclear localization sequence necessary for nuclear import of 5-lipoxygenase.

5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. This enzyme can reside either in the cytoplasm or the nucleus; its subcellular distribution is influenced by extracellular factors, and its nuclear import correlates with changes in leukotriene synthetic capacity. To identify sequences responsible for the nuclear import of 5-lipoxygenase, we transfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors encoding various 5-lipoxygenase constructs fused to green fluorescent protein. Overexpression of wild type 5-lipoxygenase with or without fusion to green fluorescent protein resulted in a predominantly intranuclear pattern of fluorescence, similar to the distribution of native 5-lipoxygenase in primary alveolar macrophages. Within the 5-lipoxygenase protein is a sequence (Arg(638)-Lys(655)) that closely resembles a bipartite nuclear localization signal. Studies using deletion mutants indicated that this region was necessary for nuclear import of 5-lipoxygenase. Analysis of mutants containing specific amino acid substitutions within this sequence confirmed that it was this sequence that was necessary for nuclear import of 5-lipoxygenase and that a specific arginine residue was critical for this function. As nuclear import of 5-lipoxygenase may regulate leukotriene production, natural or induced mutations in this bipartite nuclear localization sequence may also be important in affecting leukotriene synthesis.     

Molecular characterization and nuclear localization of rat timeless-like gene product

Among three period genes (per1, per2, per3) in mammals, only per2 gene was shown to be involved in the core clock mechanism. To elucidate the molecular function of rat PERIOD2 (rPER2), we searched for binding proteins to the PAS domain of rPER2. We isolated a binding protein to this domain and identified it as a TIMELESS-like protein (TLP) on the basis of mass analyses. Then, we isolated a rat TLP cDNA from the rat hypothalamus library. RNA blot analysis and in situ hybridization indicates that rTLP mRNA was expressed in all rat tissues from whole brain, the suprachiasmatic nucleus, eye, lung, heart, liver, kidney, placenta, and testis. When rTLP gene product was expressed in COS-1 cells, nuclear localization of rTLP was detected in 99.6% of transfected cells. These results suggest that the interaction of rPER2 with rTLP may influence the regulation of circadian clock components in nucleus after rPER2 is translocated into the nucleus.     

Inhibition of apoptosis by the retinoblastoma gene product.

Tissue homeostasis and the prevention of neoplasia require regulatory co-ordination between cellular proliferation and apoptosis. Several cellular proteins, including c-myc and E2F, as well as viral proteins such as E1A, have dual functions as positive regulators of apoptosis and proliferation. The product of the retinoblastoma tumor suppressor gene, pRb, binds these proteins and is known to function in growth suppression. To examine whether pRb may function as a negative regulator of both proliferation and apoptosis, we analyzed apoptosis induced in transfected derivatives of the human osteosarcoma cell line SAOS-2. Ionizing radiation induced apoptosis in a time- and dose-dependent manner in SAOS-2 cells, which lack pRb expression. In both a transient and stable transfection assay, SAOS-2 derivatives expressing wild-type (wt) pRb exhibited increased viability and decreased apoptosis following treatment at a variety of radiation doses. Expression in SAOS-2 of a mutant pRb that fails to complex with several known binding partners of pRb, including E1A and E2F, did not protect SAOS-2 cells from apoptosis. Radiation exposure induced a G2 arrest in SAOS-2 and in derivatives expressing pRb. Inhibition of DNA synthesis and cell cycle progression by aphidicolin treatment failed to protect SAOS-2 cells or pRb-expressing isolates from undergoing apoptosis. Our data document a novel function for pRb in suppressing apoptosis and suggest that several proteins shown to induce apoptosis, including E1A, E2F and c-myc, may do so by interfering with the protective function of pRb.     

A nuclear localization signal binding protein in the nucleolus

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine- labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.     

Molecular characterization of yeast regulatory gene CAT3 necessary for glucose derepression and nuclear localization of its product

The yeast regulatory gene CAT3 has an essential function for the depression of several glucose-repressible enzymes. Therefore, cat3 mutants are unable to grow on maltose or on non-fermentable carbon sources. Unlike the point mutants isolated previously, cat3 null allele strains also failed to utilize raffinose or galactose as sole carbon sources. Sequencing of an 1.6-kb HindIII-BglII fragment complementing cat3 mutations revealed an open reading frame of 322 codons, size of which is in good agreement with the 1.3-kb size of mRNA. No significant similarities with previously sequenced genes could be detected. CAT3-lacZ fusions confirmed the proposed reading frame. A CAT3-lacZ fusion encoding 307 amino acids of CAT3 was able to complement the growth defects of cat3 point mutants and null allele strains. Assay of beta-galactosidase activity under different growth conditions indicated a constitutive expression of the CAT3 gene product. Cellular fractionation studies showed the nuclear localization of the CAT3 protein.     

Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin alpha and beta (alpha/beta) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin alpha/beta-mediated regulation of XCTK2 microtubule binding. Our data show that importin alpha/beta directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.     

A tripartite nuclear localization signal in the PDZ-domain protein L-periaxin

The murine Periaxin gene encodes two PDZ-domain proteins in myelin-forming Schwann cells of the vertebrate peripheral nervous system (Dytrych, L., Sherman, D. L., Gillespie, C. S., and Brophy, P. J. (1998) J. Biol. Chem. 273, 5794-5800). Here we show that L-periaxin is targeted to the nucleus of embryonic Schwann cells. Subsequently, the protein redistributes to the plasma membrane processes of the myelinating Schwann cell where it is believed to function in a signaling complex. In contrast, L-periaxin remains in the nucleus when expressed ectopically in oligodendrocytes, the myelin-forming glia of the central nervous system. The nuclear localization signal (NLS) is basic and tripartite and comprises three signals that act synergistically. Nuclear targeting of L-periaxin is energy-dependent and is inhibited by cell-cell contact. These data show that L-periaxin is a member of a growing family of proteins that can shuttle between the nucleus and cortical signaling/adherence complexes.