Calcium-calmodulin kinase I cooperatively regulates nucleocytoplasmic shuttling of CCTα by accessing a nuclear export signal

We identified a new calmodulin kinase I (CaMKI) substrate, cytidyltransferase (CCTα), a crucial enzyme required for maintenance of cell membranes. CCTα becomes activated with translocation from the cytoplasm to the nuclear membrane, resulting in increased membrane phospholipids. Calcium-activated CCTα nuclear import is mediated by binding of its C-terminus to 14-3-3 ζ, a regulator of nuclear trafficking. Here CaMK1 phosphorylates residues within this C-terminus that signals association of CCTα with 14-3-3 ζ to initiate calcium-induced nuclear entry. CaMKI docks within the CCTα membrane-binding domain (residues 290–299), a sequence that displays similarities to a canonical nuclear export signal (NES) that also binds CRM1/exportin 1. Expression of a CFP-CCTα mutant lacking residues 290–299 in cells results in cytosolically retained enzyme. CRM1/exportin 1 was required for CCTα nuclear export, and its overexpression in cells was partially sufficient to trigger CCTα nuclear export despite calcium stimulation. An isolated CFP-290-299 peptide remained in the nucleus in the presence of leptomycin B but was able to target to the cytoplasm with farnesol. Thus CaMKI vies with CRM1/exportin 1 for access to a NES, and assembly of a CaMKI–14-3-3 ζ–CCTα complex is a key effector mechanism that drives nuclear CCTα translocation.

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Identification of nuclear localization signal and nuclear export signal of VP1 from the chicken anemia virus and effects on VP2 shuttling in cells

Virology Journal ◽

10.1186/s12985-019-1153-5 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 2

Author(s):

Jai-Hong Cheng ◽

Guan-Hua Lai ◽

Yi-Yang Lien ◽

Fang-Chun Sun ◽

Shan-Ling Hsu ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Chicken Anemia Virus ◽

Localization Signal ◽

Anemia Virus ◽

Export Signal ◽

In Cells

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Localized RanGTP Accumulation Promotes Microtubule Nucleation at Kinetochores in Somatic Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e07-10-1050 ◽

2008 ◽

Vol 19 (5) ◽

pp. 1873-1882 ◽

Cited By ~ 56

Author(s):

Liliana Torosantucci ◽

Maria De Luca ◽

Giulia Guarguaglini ◽

Patrizia Lavia ◽

Francesca Degrassi

Keyword(s):

Nuclear Export ◽

Mammalian Cells ◽

Nuclear Export Signal ◽

Spindle Assembly ◽

Microtubule Nucleation ◽

Leptomycin B ◽

Data Support ◽

Assembly Pathways ◽

Export Signal ◽

In Cells

Centrosomes are the major sites for microtubule nucleation in mammalian cells, although both chromatin- and kinetochore-mediated microtubule nucleation have been observed during spindle assembly. As yet, it is still unclear whether these pathways are coregulated, and the molecular requirements for microtubule nucleation at kinetochore are not fully understood. This work demonstrates that kinetochores are initial sites for microtubule nucleation during spindle reassembly after nocodazole. This process requires local RanGTP accumulation concomitant with delocalization from kinetochores of the hydrolysis factor RanGAP1. Kinetochore-driven microtubule nucleation is also activated after cold-induced microtubule disassembly when centrosome nucleation is impaired, e.g., after Polo-like kinase 1 depletion, indicating that dominant centrosome activity normally masks the kinetochore-driven pathway. In cells with unperturbed centrosome nucleation, defective RanGAP1 recruitment at kinetochores after treatment with the Crm1 inhibitor leptomycin B activates kinetochore microtubule nucleation after cold. Finally, nascent microtubules associate with the RanGTP-regulated microtubule-stabilizing protein HURP in both cold- and nocodazole-treated cells. These data support a model for spindle assembly in which RanGTP-dependent abundance of nucleation/stabilization factors at centrosomes and kinetochores orchestrates the contribution of the two spindle assembly pathways in mammalian cells. The complex of RanGTP, the export receptor Crm1, and nuclear export signal-bearing proteins regulates microtubule nucleation at kinetochores.

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Facilitated Nucleocytoplasmic Shuttling of the Ran Binding Protein RanBP1

Molecular and Cellular Biology ◽

10.1128/mcb.20.10.3510-3521.2000 ◽

2000 ◽

Vol 20 (10) ◽

pp. 3510-3521 ◽

Cited By ~ 51

Author(s):

Kendra Plafker ◽

Ian G. Macara

Keyword(s):

Nuclear Export ◽

Binding Protein ◽

Nuclear Export Signal ◽

Nuclear Accumulation ◽

Leptomycin B ◽

Simple Diffusion ◽

Permeabilized Cells ◽

C Terminus ◽

Transport Receptors ◽

Export Signal

ABSTRACT The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B, and collapse of the nucleocytoplasmic Ran:GTP gradient leads to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)–RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin β. These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.

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The Nup98-HoxA9 Leukemogenic Fusion Protein Blocks Xpo1 and Tap-Mediated Nuclear Export

Blood ◽

10.1182/blood.v118.21.5228.5228 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5228-5228

Author(s):

Yildirim Dogan ◽

Sutapa Sahay ◽

David C Dorn ◽

Anna Franceschino ◽

Michael Xu ◽

...

Keyword(s):

Nuclear Export ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Shuttling ◽

Nuclear Trafficking ◽

Myeloid Leukemias ◽

Nuclear Export Receptor ◽

Export Signal

Abstract Abstract 5228 In leukemia Nup98 fusion genes are found as a product of the reciprocal translocation between Nucleoporin 98 (nup98) and homeobox-cluster genes. Nup98-HoxA9 (NHA9), the product of the t(7;11) translocation, is detected in acute myeloid leukemias (AMLs) and as a secondary abnormality in blast crisis of chronic myeloid leukemia (CML). The role of NHA9 in leukemogenesis is complex and incompletely understood. Here, we show an abrogation of nucleocytoplasmic shuttling of the nuclear export receptor Xpo1 and Tap in NHA9-expressing cells by using retroviral nuclear trafficking as a model. Lentiviral Rev, the prototype for nuclear export signal (NES)-containing proteins is shuttled through the nucleopore by Xpo1. NHA9 sequestered Xpo1 from the nuclear rim into nuclear aggregates resulting in deficient Xpo1-dependent nuclear exit of Rev and its mRNA substrates. Tap is involved in mRNA nucleocytoplasmic shuttling and is also responsible for the nuclear export of D-type retrovirus CTE-mRNAs. By using Tap/CTE-mRNA nuclear export as a model we also found that Tap colocalized in NHA9 nuclear aggregates leading to impaired Tap-mediated nuclear exit of CTE-mRNA substrates. Leukemogenicity of Nup98 fusion proteins may be accounted for in part by defects in Tap and Xpo1-mediated export of their substrates. Disclosures: No relevant conflicts of interest to declare.

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Nuclear Export Signal-Interacting Protein Forms Complexes with Lamin A/C-Nups To Mediate the CRM1-Independent Nuclear Export of Large Hepatitis Delta Antigen

Journal of Virology ◽

10.1128/jvi.02357-12 ◽

2012 ◽

Vol 87 (3) ◽

pp. 1596-1604 ◽

Cited By ~ 5

Author(s):

Cheng Huang ◽

Jia-Yin Jiang ◽

Shin C. Chang ◽

Yeou-Guang Tsay ◽

Mei-Ru Chen ◽

...

Keyword(s):

Nuclear Export ◽

Chromosome Region ◽

Nuclear Export Signal ◽

Lamin A ◽

Hepatitis Delta ◽

Interacting Protein ◽

Delta Antigen ◽

C Terminus ◽

Hepatitis Delta Antigen ◽

Export Signal

ABSTRACTNuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.

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Control of the nuclear-cytoplasmic partitioning of annexin II by a nuclear export signal and by p11 binding

Journal of Cell Science ◽

10.1242/jcs.114.17.3155 ◽

2001 ◽

Vol 114 (17) ◽

pp. 3155-3166 ◽

Cited By ~ 2

Author(s):

David A. Eberhard ◽

Larry R. Karns ◽

Scott R. VandenBerg ◽

Carl E. Creutz

Keyword(s):

Nuclear Export ◽

Consensus Sequence ◽

Nuclear Export Signal ◽

Nuclear Accumulation ◽

Annexin Ii ◽

Nuclear Entry ◽

Nuclear Exclusion ◽

Endogenous Proteins ◽

Necessary And Sufficient ◽

Export Signal

This study investigated mechanisms controlling the nuclear-cytoplasmic partitioning of annexin II (AnxII). AnxII and its ligand, p11, were localized by immunofluorescence to the cytoplasmic compartment of U1242MG cells, with minimal AnxII or p11 detected within nuclei. Similarly, GFP-AnxII and GFP-p11 chimeras localized to the endogenous proteins. Likewise, GFP-AnxII(1-22) was excluded from nuclei, whereas GFP-AnxII(23-338) and GFP alone were distributed throughout the cells. Immunoprecipitation and biochemical studies showed that GFP-AnxII did not form heteromeric complexes with endogenous p11 and AnxII. Thus, the AnxII N-tail is necessary and sufficient to cause nuclear exclusion of the GFP fusion protein but this does not involve p11 binding. A nuclear export signal consensus sequence was found in the AnxII 3-12 region. The consensus mutant GFP-AnxII(L10A/L12A) confirmed that these residues are necessary for nuclear exclusion. The nuclear exclusion of GFP-AnxII(1-22) was temperature-dependent and reversible, and the nuclear export inhibitor leptomycin B (LmB) caused GFP-AnxII or overexpressed AnxII monomer to accumulate in nuclei. Therefore, AnxII monomer can enter the nucleus and is actively exported. However, LmB had little effect on the localization of AnxII/p11 complex in U1242MG cells, indicating that the complex is sequestered in the cytoplasm. By contrast, LmB treatment of v-src-transformed fibroblasts caused endogenous AnxII to accumulate in nuclei. The LmB-induced nuclear accumulation of AnxII was accelerated by pervanadate and inhibited by genistein, suggesting that phosphorylation promotes nuclear entry of AnxII. Thus, nuclear exclusion of AnxII results from nuclear export of the monomer and sequestration of AnxII/p11 complex, and may be modulated by phosphorylation.

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Subcellular Localization of β-Arrestins Is Determined by Their Intact N Domain and the Nuclear Export Signal at the C Terminus

Journal of Biological Chemistry ◽

10.1074/jbc.m208109200 ◽

2003 ◽

Vol 278 (13) ◽

pp. 11648-11653 ◽

Cited By ~ 107

Author(s):

Ping Wang ◽

Yalan Wu ◽

Xin Ge ◽

Lan Ma ◽

Gang Pei

Keyword(s):

Subcellular Localization ◽

Nuclear Export ◽

Nuclear Export Signal ◽

C Terminus ◽

Export Signal

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The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

Molecular Biology of the Cell ◽

10.1091/mbc.12.3.539 ◽

2001 ◽

Vol 12 (3) ◽

pp. 539-549 ◽

Cited By ~ 48

Author(s):

Patrick Maurer ◽

Michael Redd ◽

Jens Solsbacher ◽

F. Ralf Bischoff ◽

Markus Greiner ◽

...

Keyword(s):

Nuclear Export ◽

Binding Protein ◽

Nuclear Export Signal ◽

Nuclear Pore ◽

Nuclear Targeting ◽

Gtp Hydrolysis ◽

C Terminus ◽

Transport Receptors ◽

Nuclear Export Receptor ◽

Export Signal

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin β-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.

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