Evolutionary development of redundant nuclear localization signals in the mRNA export factor NXF1

In human cells, the mRNA export factor NXF1 resides in the nucleoplasm and at nuclear pore complexes. Karyopherin β2 or transportin recognizes a proline-tyrosine nuclear localization signal (PY-NLS) in the N-terminal tail of NXF1 and imports it into the nucleus. Here biochemical and cellular studies to understand the energetic organization of the NXF1 PY-NLS reveal unexpected redundancy in the nuclear import pathways used by NXF1. Human NXF1 can be imported via importin β, karyopherin β2, importin 4, importin 11, and importin α. Two NLS epitopes within the N-terminal tail, an N-terminal basic segment and a C-terminal R-X(2-5)-P-Y motif, provide the majority of binding energy for all five karyopherins. Mutation of both NLS epitopes abolishes binding to the karyopherins, mislocalized NXF1 to the cytoplasm, and significantly compromised its mRNA export function. The understanding of how different karyopherins recognize human NXF1, the examination of NXF1 sequences from divergent eukaryotes, and the interactions of NXF1 homologues with various karyopherins reveals the evolutionary development of redundant NLSs in NXF1 of higher eukaryotes. Redundancy of nuclear import pathways for NXF1 increases progressively from fungi to nematodes and insects to chordates, potentially paralleling the increasing complexity in mRNA export regulation and the evolution of new nuclear functions for NXF1.     

Apoptin nucleocytoplasmic shuttling is required for cell type-specific localization, apoptosis, and recruitment of the anaphase-promoting complex/cyclosome to PML bodies

The chicken anemia virus protein Apoptin selectively induces apoptosis in transformed cells while leaving normal cells intact. This selectivity is thought to be largely due to cell type-specific localization: Apoptin is cytoplasmic in primary cells and nuclear in transformed cells. The basis of Apoptin cell type-specific localization and activity remains to be determined. Here we show that Apoptin is a nucleocytoplasmic shuttling protein whose localization is mediated by an N-terminal nuclear export signal (NES) and a C-terminal nuclear localization signal (NLS). Both signals are required for cell type-specific localization, since Apoptin fragments containing either the NES or the NLS fail to differentially localize in transformed and primary cells. Significantly, cell type-specific localization can be conferred in trans by coexpression of the two separate fragments, which interact through an Apoptin multimerization domain. We have previously shown that Apoptin interacts with the APC1 subunit of the anaphase-promoting complex/cyclosome (APC/C), resulting in G(2)/M cell cycle arrest and apoptosis in transformed cells. We found that the nucleocytoplasmic shuttling activity is critical for efficient APC1 association and induction of apoptosis in transformed cells. Interestingly, both Apoptin multimerization and APC1 interaction are mediated by domains that overlap with the NES and NLS sequences, respectively. Apoptin expression in transformed cells induces the formation of PML nuclear bodies and recruits APC/C to these subnuclear structures. Our results reveal a mechanism for the selective killing of transformed cells by Apoptin.     

Analysis of nuclear targeting activities of transport signals in the human immunodeficiency virus Rev protein

The human immunodeficiency Rev protein shuttles between the nucleus and cytoplasm, while accumulating to high levels in the nucleus. Rev has a nuclear localization signal (NLS; AA 35-50) with an arginine-rich motif (ARM) that interacts with importin beta and a leucine-rich nuclear export signal (NES; AA 75-84) recognized by CRM1/exportin 1. Here we explore nuclear targeting activities of the transport signals of Rev. GFP tagging and quantitative fluorescence microscopy were used to study the localization behavior of Rev NLS/ARM mutants under conditions inhibiting the export of Rev. Rev mutant M5 was actively transported to the nucleus, despite its known failure to bind importin beta. Microinjection of transport substrates with Rev-NES peptides revealed that the Rev-NES has both nuclear import and export activities. Replacement of amino acid residues "PLER" (77-80) of the NES with alanines abolished bidirectional transport activity of the Rev-NES. These results indicate that both transport signals of Rev have nuclear import capabilities and that the Rev NLS has more than one nuclear targeting activity. This suggests that Rev is able to use various routes for nuclear entry rather than depending on a single pathway.     

Characterization of the nuclear localization and nuclear export signals of bovine herpesvirus 1 VP22

The bovine herpesvirus 1 (BHV-1) tegument protein VP22 is predominantly localized in the nucleus after viral infection. To analyze subcellular localization in the absence of other viral proteins, a plasmid expressing BHV-1 VP22 fused to enhanced yellow fluorescent protein (EYFP) was constructed. The transient expression of VP22 fused to EYFP in COS-7 cells confirmed the predominant nuclear localization of VP22. Analysis of the amino acid sequence of VP22 revealed that it does not have a classical nuclear localization signal (NLS). However, by constructing a series of deletion derivatives, we mapped the nuclear targeting domain of BHV-1 VP22 to amino acids (aa) 121 to 139. Furthermore, a 4-aa motif, 130PRPR133, was able to direct EYFP and an EYFP dimer (dEYFP) or trimer (tEYFP) predominantly into the nucleus, whereas a deletion or mutation of this arginine-rich motif abrogated the nuclear localization property of VP22. Thus, 130PRPR133 is a functional nonclassical NLS. Since we observed that the C-terminal 68 aa of VP22 mediated the cytoplasmic localization of EYFP, an analysis was performed on these C-terminal amino acid sequences, and a leucine-rich motif, 204LDRMLKSAAIRIL216, was detected. Replacement of the leucines in this putative nuclear export signal (NES) with neutral amino acids resulted in an exclusive nuclear localization of VP22. Furthermore, this motif was able to localize EYFP and dEYFP in the cytoplasm, and the nuclear export function of this NES could be blocked by leptomycin B. This demonstrates that this leucine-rich motif is a functional NES. These data represent the first identification of a functional NLS and NES in a herpesvirus VP22 homologue.     

Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation.

To study the intracellular localization of MAPKAP kinase 2 (MK2), which carries a putative bipartite nuclear localization signal (NLS), we constructed a green fluorescent protein-MAPKAP kinase 2 fusion protein (GFP-MK2). In transfected cells, this protein is located predominantly in the nucleus; unexpectedly, upon stress, it rapidly translocates to the cytoplasm. This translocation can be blocked by the p38 MAP kinase inhibitor SB203580, indicating its regulation by phosphorylation. Molecular mimicry of MK2 phosphorylation at T317 in GFP-MK2 led to a mutant which is located almost exclusively in the cytoplasm of the cell, whereas the mutant T317A shows no stress-induced redistribution. Since leptomycin B, which inhibits the interaction of exportin 1 with the Rev-type leucine-rich nuclear export signal (NES), blocks stress-dependent translocation of GFP-MK2, it is supposed that phosphorylation-induced export of the protein causes the translocation. We have identified the region responsible for nuclear export in MK2 which is partially overlapping with and C-terminal to the autoinhibitory motif. This region contains a cluster of hydrophobic amino acids in the characteristic spacing of a leucine-rich Rev-type NES which is necessary to direct GFP-MK2 to the cytoplasm. However, unlike the Rev-type NES, this region alone is not sufficient for nuclear export. The data obtained indicate that MK2 contains a constitutively active NLS and a stress-regulated signal for nuclear export. Keywords: nuclear export/nuclear import/protein phosphorylation/signal transduction/stress response     

Histone deacetylase 4 possesses intrinsic nuclear import and export signals

Nucleocytoplasmic trafficking of histone deacetylase 4 (HDAC4) plays an important role in regulating its function, and binding of 14-3-3 proteins is necessary for its cytoplasmic retention. Here, we report the identification of nuclear import and export sequences of HDAC4. While its N-terminal 118 residues modulate the nuclear localization, residues 244 to 279 constitute an authentic, strong nuclear localization signal. Mutational analysis of this signal revealed that three arginine-lysine clusters are necessary for its nuclear import activity. As for nuclear export, leucine-rich sequences located in the middle part of HDAC4 do not function as nuclear export signals. By contrast, a hydrophobic motif (MXXLXVXV) located at the C-terminal end serves as a nuclear export signal that is necessary for cytoplasmic retention of HDAC4. This motif is required for CRM1-mediated nuclear export of HDAC4. Furthermore, binding of 14-3-3 proteins promotes cytoplasmic localization of HDAC4 by both inhibiting its nuclear import and stimulating its nuclear export. Unlike wild-type HDAC4, a point mutant with abrogated MEF2-binding ability remains cytoplasmic upon exogenous expression of MEF2C, supporting the notion that direct MEF2 binding targets HDAC4 to the nucleus. Therefore, HDAC4 possesses intrinsic nuclear import and export signals for its dynamic nucleocytoplasmic shuttling, and association with 14-3-3 and MEF2 proteins affects such shuttling and thus directs HDAC4 to the cytoplasm and the nucleus, respectively.     

Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences

Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob.     

Difference in nucleocytoplasmic shuttling sequences of rat and human constitutive active/androstane receptor

Fluorescence recovery after photobleaching (FRAP) in spontaneous multinuclear cells shows that both rat and human constitutive active/androstane receptors (CARs) are shuttling proteins with both nuclear localization signals (NLSs) and nuclear export signals (NESs). We previously identified two NLSs in rat CAR: NLS1 in the hinge region (residues 100-108) and NLS2 in the ligand-binding domain (residues 111-320). In the present study, we compared the intracellular localization signals between rat and human CARs. There was a marked difference in their intracellular localization in COS-7 cells because, unlike rat CAR, human CAR does not contain NLS1 due to an amino acid change at position 106. A CRM1-dependent leucine-rich NES, which is sensitive to an inhibitory effect of leptomycin B, was found in the cytoplasmic retention region previously identified within the ligand-binding domain of rat CAR (residues 220-258). We found that human CAR instead has a NES in the ligand-binding domain between residues 170 and 220. Also, we detected CRM1-independent C-terminal NESs between residues 317-358 of rat and human CARs. Removal of NLS1 by N-terminal truncation and mutation of xenochemical response signal caused rat CAR to localize in the cytoplasm of COS-7 cells, which we suspect is due to the masking of NLS2.     

A novel nuclear trafficking module regulates the nucleocytoplasmic localization of the rabies virus interferon antagonist, p protein

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.