Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway (original) (raw)
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NUP214 in Leukemia: It’s More than Transport
Cells
NUP214 is a component of the nuclear pore complex (NPC) with a key role in protein and mRNA nuclear export. Chromosomal translocations involving the NUP214 locus are recurrent in acute leukemia and frequently fuse the C-terminal region of NUP214 with SET and DEK, two chromatin remodeling proteins with roles in transcription regulation. SET-NUP214 and DEK-NUP214 fusion proteins disrupt protein nuclear export by inhibition of the nuclear export receptor CRM1, which results in the aberrant accumulation of CRM1 protein cargoes in the nucleus. SET-NUP214 is primarily associated with acute lymphoblastic leukemia (ALL), whereas DEK-NUP214 exclusively results in acute myeloid leukemia (AML), indicating different leukemogenic driver mechanisms. Secondary mutations in leukemic blasts may contribute to the different leukemia outcomes. Additional layers of complexity arise from the respective functions of SET and DEK in transcription regulation and chromatin remodeling, which may drive malignan...
Journal of Biological Chemistry, 2016
Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A) ؉ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A) ؉ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors.
Nucleo-cytoplasmic transport as a therapeutic target of cancer
Journal of Hematology & Oncology, 2014
Shuttling of specific proteins out of the nucleus is essential for the regulation of the cell cycle and proliferation of both normal and malignant tissues. Dysregulation of this fundamental process may affect many other important cellular processes such as tumor growth, inflammatory response, cell cycle, and apoptosis. It is known that XPO1 (Exportin-1/Chromosome Region Maintenance 1/CRM1) is the main mediator of nuclear export in many cell types. Nuclear proteins exported to the cytoplasm by XPO1 include the drug targets topoisomerase IIα (topo IIα) and BCR-ABL and tumor suppressor proteins such as Rb, APC, p53, p21, and p27. XPO1 can mediate cell proliferation through several pathways: (i) the sub-cellular localization of NES-containing oncogenes and tumor suppressor proteins, (ii) the control of the mitotic apparatus and chromosome segregation, and (iii) the maintenance of nuclear and chromosomal structures. The XPO1 protein is elevated in ovarian carcinoma, glioma, osteosarcoma, pancreatic and cervical cancer. There is a growing body of research indicating that XPO1 may have an important role as a prognostic marker in solid tumors. Because of this, nuclear export inhibition through XPO1 is a potential target for therapeutic intervention in many cancers. The best understood XPO1 inhibitors are the small molecule nuclear export inhibitors (NEIs; Leptomycin B and derivatives, ratjadones, PKF050-638, valtrate, ACA, CBS9106, selinexor/KPT-330, and verdinexor/KPT-335). Selinexor and verdinexor are orally bioavailable, highly potent, small molecules that are classified as Selective Inhibitors of Nuclear Export (SINE). KPT-330 is the only NEI currently in Phase I/II human clinical trials in hematological and solid cancers. Of all the potential targets in nuclear cytoplasmic transport, the nuclear export receptor XPO1 remains the best understood and most advanced therapeutic target for the treatment of cancer.
Journal of Biological Chemistry, 2010
NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wildtype NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34؉ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.
Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export
Journal of Biological Chemistry, 2006
The nuclear pore complex (NPC) conducts macromolecular transport to and from the nucleus and provides a kinetic/hydrophobic barrier composed of phenylalanine-glycine (FG) repeats. Nuclear transport is achieved through permeation of this barrier by transport receptors. The transport receptor CRM1 facilitates export of a large variety of cargoes. Export of the preribosomal 60 S subunit follows this pathway through the adaptor protein NMD3. Using RNA interference, we depleted two FG-containing cytoplasmically oriented NPC complexes, Nup214-Nup88 and Nup358, and investigated CRM1-mediated export. A dramatic defect in NMD3-mediated export of preribosomes was found in Nup214-Nup88-depleted cells, whereas only minor export defects were evident in other CRM1 cargoes or upon depletion of Nup358. We show that the large C-terminal FG domain of Nup214 is not accessible to freely diffusing molecules from the nucleus, indicating that it does not conduct 60 S preribosomes through the NPC. Consistently, derivatives of Nup214 lacking the FG-repeat domain rescued the 60 S export defect. We show that the coiled-coil region of Nup214 is sufficient for 60 S nuclear export, coinciding with recruitment of Nup88 to the NPC. Our data indicate that Nup214 plays independent roles in NPC function by participating in the kinetic/ hydrophobic barrier through its FG-rich domain and by enabling NPC gating through association with Nup88.
Molecular and Cellular Biology, 2004
Nuclear pore complexes (NPCs) traverse the nuclear envelope (NE), providing a channel through which nucleocytoplasmic transport occurs. Nup358/RanBP2, Nup214/CAN, and Nup88 are components of the cytoplasmic face of the NPC. Here we show that Nup88 localizes midway between Nup358 and Nup214 and physically interacts with them. RNA interference of either Nup88 or Nup214 in human cells caused a strong reduction of Nup358 at the NE. Nup88 and Nup214 showed an interdependence at the NPC and were not affected by the absence of Nup358. These data indicate that Nup88 and Nup214 mediate the attachment of Nup358 to the NPC. We show that localization of the export receptor CRM1 at the cytoplasmic face of the NE is Nup358 dependent and represents its empty state. Also, removal of Nup358 causes a distinct reduction in nuclear export signal-dependent nuclear export. We propose that Nup358 provides both a platform for rapid disassembly of CRM1 export complexes and a binding site for empty CRM1 recy...
2001
The nuclear pore complex (NPC) is a large proteinaceous structure through which bidirectional transport of macromolecules across the nuclear envelope (NE) takes place. Nup153 is a peripheral NPC component that has been implicated in protein and RNP transport and in the interaction of NPCs with the nuclear lamina. Here, Nup153 is localized by immunogold electron microscopy to a position on the nuclear ring of the NPC. Nuclear reconstitution is used to investigate the role of Nup153 in nucleocytoplasmic transport and NPC architecture. NPCs assembled in the absence of Nup153 lacked several nuclear basket components, were unevenly distributed in the NE and, unlike wild-type NPCs, were mobile within the NE. Importin a/b-mediated protein import into the nucleus was strongly reduced in the absence of Nup153, while transportin-mediated import was unaffected. This was due to a reduction in import complex translocation rather than to defective receptor recycling. Our results therefore reveal functions for Nup153 in NPC assembly, in anchoring NPCs within the NE and in mediating speci®c nuclear import events.
Chromosomal translocations fusing the locus of nucleoporin NUP214 each with the proto-oncogenes SET and DEK are recurrent in, largely intractable, acute leukemias. The molecular basis underlying the pathogenesis of SET-NUP214 and DEK-NUP214 are still poorly understood, but both chimeras inhibit protein nuclear export mediated by the ß-karyopherin CRM1. In this report, we show that SET-NUP214 and DEK-NUP214 both disturb the localization of proteins essential for nucleocytoplasmic transport, in particular for CRM1-mediated protein export. Endogenous and exogenous SET-NUP214 and DEK-NUP214 form nuclear bodies. These nuclear bodies disperse upon targeted inhibition of CRM1 and the two fusion proteins re-localize throughout the nucleoplasm. Moreover, SET-NUP214 and DEK-NUP214 nuclear bodies reestablish shortly after removal of CRM1 inhibitors. Likewise, cell viability, metabolism, and proliferation of leukemia cell lines harboring SET-NUP214 and DEK-NUP214 are compromised by CRM1 inhibit...
Tumor marker nucleoporin 88kDa regulates nucleocytoplasmic transport of NF-κB
Biochemical and Biophysical Research Communications, 2008
a b s t r a c t 29 Nucleoporin 88 kDa (Nup88) is a tumor marker, overexpressed in various types of cancer. In Drosophila 30 Nup88 (mbo) was reported to selectively mediate the nucleocytoplasmic transport of NF-jB, an ubiqui-31 tous transcription factor involved in immune responses, apoptosis, and cancer. We addressed the func-32 tion of Nup88 in mammalian cells. Selective depletion of Nup88 by small interfering RNA (siRNA) 33 inhibited NF-jB-dependent reporter gene activation and the nuclear translocation of NF-jB without 34 affecting the upstream activation pathway in NIH3T3 cells. In contrast, nuclear translocation of glucocor-35 ticoid receptor was not reduced by the depletion of Nup88. In metastatic melanoma cells overexpressing 36 Nup88, constitutive activation of NF-jB was found both in nucleus and cytoplasm. Nup88 depletion in 37 these cells reduced TNF-induced nuclear accumulation of NF-jB subunits. We conclude that Nup88 reg-38 ulates the activity of NF-jB at the level of nucleocytoplasmic transport. Overexpression of Nup88 in 39 tumor cells may, thus be involved in the constitutive NF-jB activation. 40 Ó 2008 Elsevier Inc. All rights reserved. 41 42 43 Nucleoporin 88 kDa (Nup88) is a component of the nuclear pore 44 complex (NPC) that mediates nucleocytoplasmic trafficking of 45 macromolecules [1]. Nup88 is reported to be located at the cyto-46 plasmic face of NPC in tight association with CAN/Nup214, another 47 nucleoporin and a proto-oncogene implicated in leukemia [2-4]. It 48 is a putative tumor marker highly expressed in cancer and embry-49 onic tissues, and is postulated to play a role in oncogenesis and 50 development [5,6]. Furthermore its expression levels highly corre-51 late with the rate of metastasis and mortality of colon cancer [7] 52 and with aggressiveness of breast cancer [8]. However, the func-53 tional consequences of Nup88 overexpression in cancer remain un-54 known to date. 55 A study carried out in Drosophila reported that members only 56 (mbo), an ortholog of Nup88 (26% homology to human Nup88), 57 selectively regulated the nuclear translocation or export of dor-58 sal, a member of the Rel protein family, which comprises NF-jB 59 [9-11]. NF-jB is a ubiquitous transcription factor mediating the 60 induction of a wide range of genes involved in immune re-61 sponse, inflammation, and apoptosis [12,13]. Constitutive activa-62 tion of NF-jB is found in cancer, and its role in cancer is 63 supported by a large body of evidence [14,15]. NF-jB, a dimer 64 composed of subunits of the Rel family of proteins, is retained 65 in the cytoplasm by its inhibitory partner, inhibitor of NF-jB 66 (IjB). The paradigm of canonical NF-jB activation involves sev-67 eral critical steps and its signaling pathway is triggered by acti-68 vation of IjB kinase (IKK) complex, subsequent phosphorylation 69 and degradation of IjB allowing the release of an active dimer 70 of NF-jB [12]. Little is known, however, about any particular 71 regulation at the nuclear membrane. We postulated that mam-72 malian Nup88 would exert a similar function as its Drosophila 73 ortholog in regulating the nuclear localization of NF-jB, and 74 that overexpression of Nup88 in cancer could affect NF-jB 75 activity. 76 In order to underscore a link between Nup88 and NF-jB signal-77 ing, we have depleted Nup88 by short interfering (si)RNA [16] and 78 evaluated the functional consequences on NF-jB activity by repor-79 ter gene assay, immunofluorescence and electrophoretic mobility 80 shift assay (EMSA). 0006-291X/$ -see front matter Ó