Importin β Can Mediate the Nuclear Import of an Arginine-Rich Nuclear Localization Signal in the Absence of Importin α

Palmeri, Diana; Malim, Michael H.

doi:10.1128/mcb.19.2.1218

Cited by 198 publications

(188 citation statements)

References 77 publications

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“…However, our analyses showed that SPHK2 possesses a putative NLS (RGRRGGRRR) with an arginine cluster, which is highly similar to a type of monopartite NLS enriched in arginine residues identified in the Tat and Rev proteins of human immunodeficiency virus type 1 (19), the Rex protein of human T-cell leukemia virus type 1 (20), and the atypical protein kinase C (21) (Fig. 3B).…”

Section: Nls Is Localized In the N-terminalmentioning

confidence: 99%

Sphingosine Kinase 2 Is a Nuclear Protein and Inhibits DNA Synthesis

Igarashi

Okada

Hayashi

et al. 2003

Journal of Biological Chemistry

388

346

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Sphingosine kinase-1 (SPHK1) is a key enzyme catalyzing the formation of an important bioactive lipid messenger, sphingosine 1-phosphate, and is implicated in the regulation of cell proliferation and antiapoptotic processes. Biological features of another isozyme SPHK2, however, remain unclear. The present studies were undertaken to characterize SPHK2 by comparison with SPHK1. When SPHK2 was transiently expressed in various cell lines, it was localized in the nuclei as well as in the cytosol, whereas SPHK1 was distributed in the cytosol but not in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal region of SPHK2. We have observed that the expression of SPHK2 in various cell types causes inhibition of DNA synthesis, resulting in the cell cycle arrest at G 1 /S phase. We have also demonstrated that an NLS mutant of SPHK2, SPHK2R93E/R94E, failed to enter the nucleus and to inhibit DNA synthesis. Moreover, a fusion protein, NLS-SPHK1, where SPHK1 was fused to the NLS sequence of SPHK2 acquired the ability to enter nuclei and inhibited DNA synthesis. These results indicate that SPHK2 localizes in the nuclei and causes inhibition of DNA synthesis, and this may affect subsequent cellular events.Sphingosine 1-phosphate (SPP) 1 is a bioactive lipid that regulates diverse biological processes such as calcium mobilization, cell growth, differentiation, survival, motility, and cytoskeletal reorganization, acting both inside and outside the cells (1, 2). Recently, SPP was identified as the ligand for a family of G protein-coupled receptors known as the endothelial differentiation gene-1 family, now collectively renamed SPP receptors (3-6), supporting a role for SPP as an extracellular ligand. However, the intracellular targets of SPP have not yet been identified.Sphingosine kinase (SPHK), the enzyme that catalyzes the phosphorylation of sphingosine, regulates the intracellular levels of SPP. Two isoforms of mammalian SPHK (SPHK1 and SPHK2) have been cloned and characterized (7,8). SPHK1 predominantly localizes in the cytosol, and its overexpression induces cell proliferation by promoting the G 1 to S transition of the cell cycle as well as by inhibiting the apoptotic response to serum deprivation or ceramide treatment (9). Several cellular proteins have recently been identified as SPHK1-interacting molecules, namely TRAF2 (10), RPK118 (11), and AKAP-related protein (12), which should help facilitate the understanding of the regulation and intracellular site of action of SPHK1.In contrast to SPHK1, little is known about the cellular actions of the other isozyme, SPHK2. In the present studies, we investigated the biological features of SPHK2. We have discovered that SPHK2 localizes in the nuclei of cells through its novel nuclear localization signal (NLS) sequence, depending on cell type and cell density. We have also demonstrated that nuclear localization of SPHK2 causes inhibition of DNA synthesis in various cell types.

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Section: Nls Is Localized In the N-terminalmentioning

confidence: 99%

Sphingosine Kinase 2 Is a Nuclear Protein and Inhibits DNA Synthesis

Igarashi

Okada

Hayashi

et al. 2003

Journal of Biological Chemistry

388

346

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“…It was also reported that some cargoes can interact directly with importin␤. The sequences recognized by importin␤ are less well understood and may vary, but in some cases, they are reminiscent of classical NLSs in that they are comprised of regions rich in basic residues (17)(18)(19). Several nuclear receptors, including various steroid receptors, RXR, VDR, and thyroid hormone receptor, were found to contain an NLS, termed NL1, between the two zinc fingers of their DBD (20,21).…”

mentioning

confidence: 99%

Nuclear Import of the Retinoid X Receptor, the Vitamin D Receptor, and Their Mutual Heterodimer

Yasmin

Williams

et al. 2005

Journal of Biological Chemistry

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The nuclear receptor retinoid X receptor (RXR) can regulate transcription through homotetramers, homodimers, and heterodimers with other nuclear receptors such as the vitamin D receptor (VDR). The mechanisms that underlie the nuclear import of RXR, VDR, and RXR-VDR heterodimers were investigated. We show that RXR and VDR translocate into the nucleus by distinct pathways. RXR strongly bound to importin␤ and was predominantly nuclear in the absence of ligand. Importin binding and nuclear localization of RXR were modestly enhanced by its ligand, 9-cis-retinoic acid. On the other hand, VDR selectively associated with importin␣. Importin association and correspondingly nuclear import of VDR were markedly augmented by 1,25(OH) 2 D 3 . RXR-VDR dimerization inhibited the ability of RXR to bind importin␤ and to mobilize into the nucleus using its own nuclear localization signal. In contrast, VDR recruited RXR-VDR heterodimers to importin␣ and mediated nuclear import of the heterodimers in response to 1,25(OH) 2 D 3 . Hence nuclear import of RXR-VDR heterodimers is mediated preferentially by VDR and is controlled by the VDR ligand. The observations reveal a novel mechanism by which an RXR heterodimerization partner dominates the activity of the heterodimers.The retinoid X receptor (RXR) 3 is a member of the superfamily of nuclear hormone receptors that is activated by the vitamin A metabolite 9-cis-retinoic acid (9cRA). Like other nuclear receptors, RXR is comprised of several distinct functional domains: an amino-terminal domain, involved in ligand-independent basal transcriptional activity; a DNA-binding domain (DBD) containing two "zinc finger" motifs; a flexible hinge region; and a carboxyl-terminal region, termed the ligandbinding domain (LBD). The LBD contains the ligand-binding pocket as well as regions that mediate multiple protein-protein interactions including association with transcriptional co-regulators, formation of dimers, and, in the case of RXR, formation of tetramers. The LBD of nuclear receptors, including RXR, thus coordinates their liganddependent transcriptional activities (1).In the absence of its cognate ligand, RXR forms high affinity homotetramers. These tetramers are transcriptionally silent, but they rapidly dissociate upon ligand binding (2-4). Hence RXR tetramers appear to serve as an inactive storage pool from which active species can be liberated in response to 9cRA. An additional role for the RXR tetramers was recently suggested by the observations that these oligomers act as DNA architectural factors. It was thus demonstrated that binding of RXR tetramers to promoter regions that contain two RXR response elements in tandem results in a dramatic DNA looping, thereby enabling transcriptional regulation by factors placed far upstream from start sites of target genes (5). Hence by modulating DNA geometry, RXR tetramers can regulate gene expression in a manner that is responsive to 9cRA but is independent of the intrinsic transcriptional activity of the receptor (5). Although RXR can activate ...

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“…These FG-Nups form a permeable and selective barrier in the NPC that inhibits the efficient translocation of large molecules (>40 kDa) unless they are chaperoned by transport receptors. Importin β1 (Imp β1), one of the major transport receptors, recognizes a cargo molecule to form a transport complex either directly or indirectly (i.e., via Importin α) (8)(9)(10). Imp β1 promotes the movement of the transport complex through the NPC by a series of transient interactions with the FG-Nups.…”

mentioning

confidence: 99%

Three-dimensional distribution of transient interactions in the nuclear pore complex obtained from single-molecule snapshots

Yang²

2010

Proc. Natl. Acad. Sci. U.S.A.

115

239

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The translocation of large macromolecules through the nuclear pore complex (NPC) of eukaryotic cells is hindered by the phenylalanine-glycine (FG) nucleoporin (Nup) barrier unless molecules are chaperoned by transport receptors. The precise mechanism of facilitated translocation remains unclear due to the challenges of measuring the series of transient interactions between a transport receptor and the FG-Nups. This study developed single-point edgeexcitation subdiffraction microscopy to obtain a three-dimensional density map of the transient interactions with a spatiotemporal resolution of 9 nm and 400 μs. Three unique features were observed under real-time trafficking conditions that have escaped detection by conventional electron microscopy: (i) the spatial density of interaction sites between Importin β1 (Imp β1, a major transport receptor) and the FG-Nups gradually increases from both sides of the NPC and is highest in the central pore region; (ii) cargofree or cargo-bound Imp β1 rarely occupies an axial channel with a diameter of approximately 10-20 nm at its narrowest point through the NPC; and (iii) the pathway of facilitated translocation through the NPC depends more on the interaction sites of the FG-Nups than on the NPC architecture. N uclear pore complexes (NPCs) span the nuclear envelope (NE) and enable bidirectional transport between the cytoplasm and nucleus of eukaryotic cells. Proteins destined for nuclear functions, such as nucleic acid polymerases, histones, and splicing and transcription factors, must transit into the nucleus after synthesis on cytoplasmic ribosomes. The major cellular RNAs generated by transcription in the nucleus (mRNAs, tRNAs, and rRNAs) are exported to the cytoplasm. Small molecules (less than ≈20-40 kDa) can transit NPCs without specific recognition (passive diffusion). Large molecules or molecular complexes (up to ≈25-50 MDa) transit through a carrier-mediated, signal-dependent process (facilitated translocation) (1-3).Electron microscopy has revealed that the pore itself is approximately 40-90 nm in length and about 40-75 nm wide at its narrowest point. Flexible filaments extend out from the pore approximately 50 nm into the cytoplasm, and a filamentous open basket structure extends about 75 nm into the nucleoplasm (3-5). The NPC consists of approximately 30 different nucleoporins (Nups), about one-third of which lack an ordered secondary structure and contain domains rich in phenylalanine-glycine (FG) repeats (3, 6-7). These FG-Nups form a permeable and selective barrier in the NPC that inhibits the efficient translocation of large molecules (>40 kDa) unless they are chaperoned by transport receptors. Importin β1 (Imp β1), one of the major transport receptors, recognizes a cargo molecule to form a transport complex either directly or indirectly (i.e., via Importin α) (8-10). Imp β1 promotes the movement of the transport complex through the NPC by a series of transient interactions with the FG-Nups. Thousands of FG repeats are distributed within an NPC, and Imp β1 is pr...

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Importin β Can Mediate the Nuclear Import of an Arginine-Rich Nuclear Localization Signal in the Absence of Importin α

Cited by 198 publications

References 77 publications

Sphingosine Kinase 2 Is a Nuclear Protein and Inhibits DNA Synthesis

Sphingosine Kinase 2 Is a Nuclear Protein and Inhibits DNA Synthesis

Nuclear Import of the Retinoid X Receptor, the Vitamin D Receptor, and Their Mutual Heterodimer

Three-dimensional distribution of transient interactions in the nuclear pore complex obtained from single-molecule snapshots

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