Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors

Shah, Sundeep; Forbes, Douglass J.

doi:10.1016/s0960-9822(98)00018-9

Cited by 117 publications

(91 citation statements)

References 55 publications

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“…To test whether importin-b is involved in 53BP1 nuclear import, we took advantage of previous reports showing that overexpression of the Nterminal and C-terminal part of NUP153 inhibits transportin-1 and importin-b-mediated transport, respectively. 24 We expressed these fragments in U2OS cells and assayed their impact on 53BP1 localization. Although the N-terminal fragment of NUP153 did not affect 53BP1 localization, cells overexpressing the C-terminal fragment showed a predominantly cytoplasmic 53BP1 (Figure 6a).…”

Section: Resultsmentioning

confidence: 99%

Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1

et al. 2011

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53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-b interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-b pathway and implicate this mechanism in the maintenance of genome integrity. Cell Death and Differentiation (2012) 19, 798-807; doi:10.1038/cdd.2011; published online 11 November 2011The cellular DNA damage response (DDR) machinery serves as a biological barrier against accumulation of genetic changes associated with aging, and guards against neurodegenerative and immunodeficiency disorders and cancer. 1 The proximal DDR kinases ATM, ATR and DNA-PK have a central role in response to various DNA lesions including DNA double strand breaks (DSBs), in part by phosphorylating histone H2AX (gH2AX) and its sensor MDC1, which in turn coordinates recruitment of signaling and repair factors to DNA damage foci. 1-4 These foci include 53BP1 5,6 whose retention at the DSB-flanking chromatin requires histone ubiquitylation by E3-ubiquitin ligases RNF8, RNF168 and HERC2, SUMOylation by the PIAS4/UBC9 complex, and histone methylation by MMSET and SET8 methyltransferases. [1][2][3]7 Apart from the role of 53BP1 in telomere maintenance, cell cycle checkpoints and DNA repair by non-homologous end joining of DSBs, 2,3,5,6 53BP1 is also involved in response to viruses, 8 and contributes to replication stress responses by protecting under-replicated genomic loci in G1 phase of the cell cycle. 9,10 Trafficking across the nuclear membrane occurs through nuclear pore complexes (NPCs), large channels consisting of over 30 nucleoporins (NUPs). The central channel of NPC is filled with hydrophobic phenylalanine-glycine (FG) repeats contained in many NUPs, which mediate mostly low-affinity interactions with nuclear transport factors. NUP153 is also an FG-repeat containing NUP, but contains a high-affinity site for importin-b. 11,12 NUP15...

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Section: Resultsmentioning

confidence: 99%

Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1

et al. 2011

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“…Tpr appears to be required neither for nuclear protein import and export nor for bulk export of mRNAs (66) 3 but might play a role in quality control of export cargoes (67). Nup153 in turn appears to play a role in the import of a subset of nuclear proteins (38,68) and in different pathways of protein and mRNA export (69). Nup50 again has been reported to be involved in both nuclear protein import and export (40,41).…”

Section: Discussionmentioning

confidence: 99%

Caspases Target Only Two Architectural Components within the Core Structure of the Nuclear Pore Complex

Patre

Tabbert

Hermann

et al. 2006

Journal of Biological Chemistry

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Caspases were recently implicated in the functional impairment of the nuclear pore complex during apoptosis, affecting its dual activity as nucleocytoplasmic transport channel and permeability barrier. Concurrently, electron microscopic data indicated that nuclear pore morphology is not overtly altered in apoptotic cells, raising the question of how caspases may deactivate nuclear pore function while leaving its overall structure largely intact. To clarify this issue we have analyzed the fate of all known nuclear pore proteins during apoptotic cell death. Our results show that only two of more than 20 nuclear pore core structure components, namely Nup93 and Nup96, are caspase targets. Both proteins are cleaved near their N terminus, disrupting the domains required for interaction with other nucleoporins actively involved in transport and providing the permeability barrier but dispensable for maintaining the nuclear pore scaffold. Caspase-mediated proteolysis of only few nuclear pore complex components may exemplify a general strategy of apoptotic cells to efficiently disable huge macromolecular machines.Caspases are a family of highly specific cysteine proteases that play a central role in programmed cell death by apoptosis (1). By cleavage of a limited number of structural and regulatory proteins, they produce changes in cellular morphology and metabolism that are hallmarks of the apoptotic process. In the nucleus such changes include pronounced chromatin condensation, massive nucleosomal DNA fragmentation, and alterations in nuclear shape (2, 3). Recent reports also indicate that the regulated exchange of macromolecules between the nucleus and cytoplasm is impaired during apoptosis (4 -8).Regulated nucleocytoplasmic transport across the nuclear envelope occurs via nuclear pore complexes (NPCs), 2 channels of elaborate architecture that consist of multiple copies of about 30 different nucleoporins (9). Many nucleoporins are components of distinct subcomplexes that are arranged around the central pore channel in 8-fold rotational symmetry. The vertebrate Nup93 subcomplex, embedded within the NPC core, harbors Nup205, Nup188,. This subcomplex is believed to be an anchor site for the Nup62 subcomplex that resides close to the pore channel mid-axis and consists of Nup62, Nup58, and Nup54 (13,14). The Nup93 subcomplex furthermore is flanked on both sides by the Nup160 subcomplex, consisting of nine proteins, i.e. Nup160, Nup133, Nup107, Nup96, Nup75/85, Nup43, Nup37, Sec13, and Seh1 (12,[15][16][17][18]. Another nucleoporin, Nup98 (19,20), is not stably integrated in the Nup160 subcomplex but interacts with one of its components, Nup96 (16,21). Whether other NPC core components, namely Nup155 (22), NLP1/CG1 (23), Nup35, and Aladin (9) directly interact with any known subcomplex or might be part of yet another still needs to be investigated.These subcomplexes are thought to be anchored to the pore wall by direct or indirect interaction with transmembrane proteins. Two such pore wall transmembrane proteins, gp210 (2...

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“…Nup153 associates with soluble components of the importin b and Ran GTPase nuclear transport machineries. The NTD of Nup153 contains binding sites for Tpr, Nup50, and import receptor transportin 1, which is proposed to be required for Nup153 localization to the nucleus [128][129][130][131]. Cleavage of Nup153, probably at site DITD 349 ↓F, produces a 130-kDa fragment that is released into the cytosol [118,124,125] and loss of the N terminus impairs its localization to the NPC as well as that of other nucleoporins (Nup50, Tpr, Nup93, Nup98, and Nup214) [127,132].…”

Section: Nup50 Nup153 and Tprmentioning

confidence: 99%

Caspases rule the intracellular trafficking cartel

2017

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During apoptosis, caspases feast on several hundreds of cellular proteins to orchestrate rapid cellular demise. Indeed, caspases are known to get a taste of every cellular process in one way or another, activating some, but most often shutting them down. Thus, it is not surprising that caspases proteolyze proteins involved in intracellular trafficking with particularly devastating consequences for this important process. This review article focuses on how caspases target the machinery responsible for smuggling goods within and outside the cell.

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Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors

Abstract: Two nuclear import pathways, mediated by importin beta and transportin, converge on a single nucleoporin, Nup153. Dominant-negative fragments of Nup153 can now be used to distinguish different nuclear import pathways and, potentially, to dissect nuclear export.

Cited by 117 publications

References 55 publications

Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1

Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1

Caspases Target Only Two Architectural Components within the Core Structure of the Nuclear Pore Complex

Caspases rule the intracellular trafficking cartel

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