scholarly journals Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights

2013 ◽  
Vol 69 (12) ◽  
pp. 2495-2505 ◽  
Author(s):  
Gergely Róna ◽  
Mary Marfori ◽  
Máté Borsos ◽  
Ildikó Scheer ◽  
Enikő Takács ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nucleocytoplasmic Transport ◽  
Wild Type ◽  
Post Translational Modification ◽  
Nuclear Localization Signals ◽  
M Phase ◽  
Importin Α ◽  
Localization Signal

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-α, the karyopherin molecule responsible for `classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-α–wild-type and the importin-α–hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the post-translational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme.

Mutations of the SBDS Gene Result in Nuclear Mislocalization.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2034-2034
Author(s):  
Masafumi Yamaguchi ◽  
Kingo Fujimura ◽  
Hanae Toga-Yamaguchi ◽  
Valentina Svetic ◽  
Naoki Okamura ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Exocrine Pancreatic Insufficiency ◽  
Expression Level ◽  
Wild Type ◽  
Nuclear Localization Signals ◽  
Localization Signal ◽  
Domain I

Abstract Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported that Shwachman-Bodian- Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of granulocyte precursor cells. (Exp Hematol35; 579, 2007). A wide variety of mutations in SBDS gene has been identified, and almost of all patients show truncated immature proteins, p.K62X (c.183_184TA>CT) or p.C84fsX3 (c.258+2T>C). However, it is not yet clear how these truncated proteins affect cellular processes that result in the SDS phenotype. The SBDS protein is localized to the nucleoli but does not have the canonical nuclear localization signal. In order to clarify the molecular basis of pathogenicity of mutated SBDS proteins, we explored the subcellular distribution of normal and mutant SBDS proteins in Hela and 32Dcl3 cells. Using various N-terminal and C-terminal deletion constructs, we found N-terminal region, domain I (1-87 amino acid residue) in particular, was necessary to localize to the nucleus. The disease related mutations (C31W, K33E, N34I, L71P) and the mutations which are conserved among the species in the domain I (E44K, K62E, D70N, E82K) were generated. C31W and N34I mutants failed to localize SBDS to the nuclei. The SV40 derived nuclear localization signal was fused to these mutated SBDS protein, and these proteins were clearly localized to the nuclei. In addition to the mislocalization, the protein expression level of these mutants showed a dramatic decrease compared to the wild type. We also established SBDS wild type and domain I overexpressed 32Dcl3 cell. SBDS wild type overexpressed cells could differentiate to normal neutrophils in the presence of mG-CSF, however domain I overexpressed cells did not differentiate. Almost of all cells showed apoptosis in this domain I overexpressed cells in the presence of mG-CSF, and this was very similar like SBDS RNAi knockdown cells. The localization of endogenous SBDS protein was also analyzed in this domain I overexpressed cells. The domain I was concentrated to nuclei, however endogenous SBDS protein was diffused to cytosol. Conclusions: The present findings enable us to document the nuclear localization signals in SBDS domain I, and that the shuttling protein would promote SBDS to nuclei. These results also showed that mislocalization and/or low expression level of mutated SBDS protein would cause SDS.

scholarly journals Human mRNA Cap Methyltransferase: Alternative Nuclear Localization Signal Motifs Ensure Nuclear Localization Required for Viability

2005 ◽  
Vol 25 (7) ◽  
pp. 2644-2649 ◽  
Author(s):  
Beth Shafer ◽  
Chun Chu ◽  
Aaron J. Shatkin
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Localization Signals ◽  
Truncation Mutant ◽  
Capping Enzyme ◽  
Importin Α ◽  
Localization Signal ◽  
Targeting Signals ◽  
Sirna Knockdown ◽  
Interfering Rna

ABSTRACT A characteristic feature of gene expression in eukaryotes is the addition of a 5′-terminal 7-methylguanine cap (m7GpppN) to nascent pre-mRNAs in the nucleus catalyzed by capping enzyme and cap methyltransferase. Small interfering RNA (siRNA) knockdown of cap methyltransferase in HeLa cells resulted in apoptosis as measured by terminal deoxynucleotidyltransferase-mediated dUTP-tetramethylrhodamine nick end labeling assay, demonstrating the importance of mRNA 5′-end methylation for mammalian cell viability. Nuclear localization of cap methyltransferase is mediated by interaction with importin-α, which facilitates its transport and selective binding to transcripts containing 5′-terminal GpppN. The methyltransferase 96-144 region has been shown to be necessary for importin binding, and N-terminal fusion of this sequence to nonnuclear proteins proved sufficient for nuclear localization. The targeting sequence was narrowed to amino acids 120 to 129, including a required 126KRK. Although full-length methyltransferase (positions 1 to 476) contains the predicted nuclear localization signals 57RKRK, 80KKRK, 103KKRKR, and 194KKKR, mutagenesis studies confirmed functional motifs only at positions 80, 103, and the previously unrecognized 126KRK. All three motifs can act as alternative nu clear targeting signals. Expression of N-truncated cap methyltransferase (120 to 476) restored viability of methyltransferase siRNA knocked-down cells. However, an enzymatically active 144-476 truncation mutant missing the three nuclear localization signals was mostly cytoplasmic and ineffective in preventing siRNA-induced loss of viability.

scholarly journals Kinesin-14 Family Proteins HSET/XCTK2 Control Spindle Length by Cross-Linking and Sliding Microtubules

2009 ◽  
Vol 20 (5) ◽  
pp. 1348-1359 ◽  
Author(s):  
Shang Cai ◽  
Lesley N. Weaver ◽  
Stephanie C. Ems-McClung ◽  
Claire E. Walczak
Keyword(s):  
Atpase Activity ◽  
Nuclear Localization ◽  
Hela Cells ◽  
Nuclear Localization Signal ◽  
Spindle Assembly ◽  
Cross Linking ◽  
Wild Type ◽  
Cross Link ◽  
Importin Α ◽  
Localization Signal

Kinesin-14 family proteins are minus-end directed motors that cross-link microtubules and play key roles during spindle assembly. We showed previously that the Xenopus Kinesin-14 XCTK2 is regulated by Ran via the association of a bipartite NLS in the tail of XCTK2 with importin α/β, which regulates its ability to cross-link microtubules during spindle formation. Here we show that mutation of the nuclear localization signal (NLS) of human Kinesin-14 HSET caused an accumulation of HSET in the cytoplasm, which resulted in strong microtubule bundling. HSET overexpression in HeLa cells resulted in longer spindles, similar to what was seen with NLS mutants of XCTK2 in extracts, suggesting that Kinesin-14 proteins play similar roles in extracts and in somatic cells. Conversely, HSET knockdown by RNAi resulted in shorter spindles but did not affect pole formation. The change in spindle length was not dependent on K-fibers, as elimination of the K-fiber by Nuf2 RNAi resulted in an increase in spindle length that was partially rescued by co-RNAi of HSET. However, these changes in spindle length did require microtubule sliding, as overexpression of an HSET mutant that had its sliding activity uncoupled from its ATPase activity resulted in cells with spindle lengths shorter than cells overexpressing wild-type HSET. Our results are consistent with a model in which Ran regulates the association of Kinesin-14s with importin α/β to prevent aberrant cross-linking and bundling of microtubules by sequestering Kinesin-14s in the nucleus during interphase. Kinesin-14s act during mitosis to cross-link and slide between parallel microtubules to regulate spindle length.

scholarly journals Identification and characterization of complex dual nuclear localization signals in human bocavirus NP1

2013 ◽  
Vol 94 (6) ◽  
pp. 1335-1342 ◽  
Author(s):  
Qian Li ◽  
Zhenfeng Zhang ◽  
Zhenhua Zheng ◽  
Xianliang Ke ◽  
Huanle Luo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Human Bocavirus ◽  
Structural Protein ◽  
Nuclear Localization Signals ◽  
Importin Α ◽  
Localization Signal ◽  
Bovine Parvovirus

Human bocavirus (HBoV), closely related to canine minute virus (MVC) and bovine parvovirus (BPV), is a new member of the Bocavirus genus within the Parvoviridae family. The non-structural protein NP1 of HBoV is a nuclear localized protein and plays an important role in DNA replication as well as in the evasion of host innate immunity. In the current study, we provide the first evidence that NP1 possesses a non-classical nuclear localization signal (ncNLS) (amino acids 7–50). Embedded within this ncNLS is a classical bipartite nuclear localization signal (cNLS) (amino acids 14–28), capable of transporting a heterologous cytoplasmic protein β-galactosidase fusion protein (β-gal-EGFP) to the nucleus via the classical importin α/β1-mediated pathway. Amino acids 7–50 containing the cNLS and the ncNLS of NP1 or full-length NP1 interact with importin α1, importin β1 and importin β1Δ, which lacks the importin α binding domain, indicating that the nuclear import of NP1 is through both conventional importin α/β1 heterodimer- and non-classical importinß1-mediated pathways. Given that the arrangement of a cNLS embedded within an ncNLS is unusual in viral proteins, our data together reveal a novel molecular mechanism underlying the nuclear import of HBoV NP1, providing a basis for further understanding its biological function.

scholarly journals SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

1998 ◽  
Vol 140 (3) ◽  
pp. 499-509 ◽  
Author(s):  
Michael J. Matunis ◽  
Jian Wu ◽  
Günter Blobel
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Gtpase Activating Protein ◽  
Terminal Domain ◽  
Localization Signal

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

scholarly journals Function of two discrete regions is required for nuclear localization of polymerase basic protein 1 of A/WSN/33 influenza virus (H1 N1).

1990 ◽  
Vol 10 (8) ◽  
pp. 4139-4145 ◽  
Author(s):  
S T Nath ◽  
D P Nayak
Keyword(s):  
Influenza Virus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Basic Protein ◽  
Localization Property ◽  
Nuclear Localization Signals ◽  
Chicken Muscle ◽  
Localization Signal ◽  
Cloned Cdna ◽  
Muscle Pyruvate Kinase

Polymerase basic protein 1 (PB1) of influenza virus (A/WSN/33), when expressed from cloned cDNA in the absence of other viral proteins, accumulates in the nucleus. We have examined the location and nature of the nuclear localization signal of PB1 by using deletion mutants and chimeric constructions with chicken muscle pyruvate kinase, a cytoplasmic protein. Our studies showed some novel features of the nuclear localization signal of PB1. The signal was present internally within residues 180 to 252 of PB1. Moreover, unlike most nuclear localization signals, it was not a single stretch of contiguous amino acids. Instead, it possessed two discontinuous regions separated by an intervening sequence which could be deleted without affecting its nuclear localization property. On the other hand, deletion of either of the two signal regions rendered the protein cytoplasmic, indicating that the function of both regions is required for nuclear localization and that one region alone is not sufficient. Both of these signal regions contained short stretches of basic residues. Possible ways by which this novel bipartite signal can function in nuclear localization are discussed.

scholarly journals CRM1-mediated Recycling of Snurportin 1 to the Cytoplasm

1999 ◽  
Vol 145 (2) ◽  
pp. 255-264 ◽  
Author(s):  
Efrosyni Paraskeva ◽  
Elisa Izaurralde ◽  
F. Ralf Bischoff ◽  
Jochen Huber ◽  
Ulrike Kutay ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Activation Domain ◽  
Large Domain ◽  
High Affinity ◽  
Importin Α ◽  
Localization Signal ◽  
Major Mediator ◽  
Rev Protein

Importin β is a major mediator of import into the cell nucleus. Importin β binds cargo molecules either directly or via two types of adapter molecules, importin α, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs. Both adapters have an NH2-terminal importin β–binding domain for binding to, and import by, importin β, and both need to be returned to the cytoplasm after having delivered their cargoes to the nucleus. We have shown previously that CAS mediates export of importin α. Here we show that snurportin 1 is exported by CRM1, the receptor for leucine-rich nuclear export signals (NESs). However, the interaction of CRM1 with snurportin 1 differs from that with previously characterized NESs. First, CRM1 binds snurportin 1 50-fold stronger than the Rev protein and 5,000-fold stronger than the minimum Rev activation domain. Second, snurportin 1 interacts with CRM1 not through a short peptide but rather via a large domain that allows regulation of affinity. Strikingly, snurportin 1 has a low affinity for CRM1 when bound to its m3G-capped import substrate, and a high affinity when substrate-free. This mechanism appears crucial for productive import cycles as it can ensure that CRM1 only exports snurportin 1 that has already released its import substrate in the nucleus.

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