Inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus

Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.     

Inhibition of nuclear import mediated by the Rev-arginine rich motif by RNA molecules

The HIV-1 Rev protein plays a pivotal role in viral replication, and therefore, inhibition of its function should block the progression of the virus-induced immune deficiency syndrome (AIDS). Here, RNA molecules have been shown to inhibit import of the HIV-1 Rev protein into nuclei of permeabilized cells. Nuclear uptake of biotinylated recombinant His-tagged Rev-GFP was assessed in nuclear extracts from digitonin-permeabilized cells by binding to either importin beta-receptors or nickel molecules immobilized on a microtiter plate. Using this method together with fluorescence microscopy, we determined that nuclear import of Rev is inhibited by the addition of a reticulocyte lysate which routinely is used as a source of nuclear import receptors. This inhibition was released by treatment with the RNase enzyme. Also t-RNA molecules and the oligoribonucleotide RRE IIB, namely, the second stem structure of the Rev responsive element (RRE) of the viral RNA, inhibit Rev nuclear import. Similar results were obtained when BSA molecules with covalently attached Rev-arginine rich motif (ARM) peptides were used as a nuclear transport substrate, indicating that the nuclear import inhibition of the Rev protein is due to the presence of the ARM domain. Binding experiments revealed that the RNA molecules inhibit the interaction between the ARM region and importin beta, implying that the RNA prevents the formation of the import complex. The implication of our results for the regulation of the nuclear import of Rev as well as for the use of RNA molecules as antiviral drugs is discussed.     

Ceramide regulation of nuclear protein import

Nucleocytoplasmic trafficking is an essential and responsive cellular mechanism that directly affects cell growth and proliferation, and its potential to address metabolic challenge is incompletely defined. Ceramide is an antiproliferative sphingolipid found within vascular smooth muscle cells in atherosclerotic plaques, but its mechanism of action remains unclear. The hypothesis that ceramide inhibits cell growth through nuclear transport regulation was tested. In smooth muscle cells, exogenously supplemented ceramide inhibited classical nuclear protein import that involved the activation of cytosolic p38 mitogen-activated protein kinase (MAPK). After application of SB 202190, a specific and potent pharmacological antagonist of p38 MAPK, sphingolipid impingement on nuclear transport was corrected. Distribution pattern assessments of two essential nuclear transport proteins, importin-alpha and Cellular Apoptosis Susceptibility, revealed ceramide-mediated relocalization that was reversed upon the addition of SB 202190. Furthermore, cell counts, nuclear cyclin A, and proliferating cell nuclear antigen expression, markers of cellular proliferation, were diminished after ceramide treatment and effectively rescued by the addition of inhibitor. Together, these data demonstrate, for the first time, the sphingolipid regulation of nuclear import that defines and expands the adaptive capacity of the nucleocytoplasmic transport machinery.     

Molecular mechanism of the nuclear protein import cycle

The nuclear import of proteins through nuclear pore complexes (NPCs) illustrates how a complex biological function can be generated by a spatially and temporally organized cycle of interactions between cargoes, carriers and the Ran GTPase. Recent work has given considerable insight into this process, especially about how interactions are coordinated and the basis for the molecular recognition that underlies the process. Although considerable progress has been made in identifying and characterizing the molecular interactions in the soluble phase that drive the nuclear protein import cycle, understanding the precise mechanism of translocation through NPCs remains a major challenge.     

Studying nuclear protein import in yeast

The yeast Saccharomyces cerevisiae is a common model organism for biological discovery. It has become popularized primarily because it is biochemically and genetically amenable for many fundamental studies on eukaryotic cells. These features, as well as the development of a number of procedures and reagents for isolating protein complexes, and for following macromolecules in vivo, have also fueled studies on nucleo-cytoplasmic transport in yeast. One limitation of using yeast to study transport has been the absence of a reconstituted in vitro system that yields quantitative data. However, advances in microscopy and data analysis have recently enabled quantitative nuclear import studies, which, when coupled with the significant advantages of yeast, promise to yield new fundamental insights into the mechanisms of nucleo-cytoplasmic transport.     

Inhibition of nuclear import of LIMK2 in endothelial cells by protein kinase C-dependent phosphorylation at Ser-283

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-delta isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-delta-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G1-to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.     

Effects of mitogen-activated protein kinases on nuclear protein import

ERK-2 MAP kinase activation induces inhibitory effects on nuclear protein import in vascular smooth muscle cells. The mechanism and characteristics of this effect of ERK-2 were investigated. An unusual dose-dependent effect of ERK-2 on nuclear protein import was identified. At higher concentrations (1 microg/mL) of ERK-2, nuclear protein import was stimulated, whereas lower concentrations (0.04 microg/mL) inhibited import. Intermediate concentrations exerted intermediate effects. The stimulatory and inhibitory effects at the 2 different ERK-2 concentrations were observed in both conventional, permeabilized cell assays of nuclear protein import and with in situ microinjection of smooth muscle cells. The biphasic effects of ERK-2 on import were also found for the other 2 members of the MAPK family, p38 and JNK. RanGAP was identified by structural analysis as a candidate target protein responsible for mediating the effects of ERK-2. After pretreatment with high concentrations of ERK-2, RanGAP activity was significantly increased by approximately 50%. In contrast, low concentrations of ERK-2 significantly attenuated RanGAP activity. These results demonstrate that all 3 members of the MAPK family can alter nuclear protein import in opposite directions depending upon the concentration of ERK-2 used. RanGAP represents the MAP kinase target whereby nuclear transport can be stimulated or inhibited.     

Promotion of importin alpha-mediated nuclear import by the phosphorylation-dependent binding of cargo protein to 14-3-3

14-3-3 proteins are phosphoserine/threonine-binding proteins that play important roles in many regulatory processes, including intracellular protein targeting. 14-3-3 proteins can anchor target proteins in the cytoplasm and in the nucleus or can mediate their nuclear export. So far, no role for 14-3-3 in mediating nuclear import has been described. There is also mounting evidence that nuclear import is regulated by the phosphorylation of cargo proteins, but the underlying mechanism remains elusive. Myopodin is a dual-compartment, actin-bundling protein that functions as a tumor suppressor in human bladder cancer. In muscle cells, myopodin redistributes between the nucleus and the cytoplasm in a differentiation-dependent and stress-induced fashion. We show that importin alpha binding and the subsequent nuclear import of myopodin are regulated by the serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3. These results establish a novel paradigm for the promotion of nuclear import by 14-3-3 binding. They provide a molecular explanation for the phosphorylation-dependent nuclear import of nuclear localization signal-containing cargo proteins.     

The nuclear import of protein kinase D3 requires its catalytic activity

The protein kinase D (PKD) family consists of three serine/threonine protein kinases termed PKD, PKD2, and PKD3, which are similar in overall structure and primary amino acid sequence. However, each isozyme displays a distinctive intracellular localization. Taking advantage of the structural homology and opposite nuclear localization of PKD2 and PKD3, we generated an extensive set of chimeric proteins between both isozymes to determine which PKD3 domain(s) mediates its nuclear localization. We found that the C-terminal region of PKD3, which contains its catalytic domain, is necessary but not sufficient for its nuclear localization. Real time imaging of a photoactivatable green fluorescent protein fused to PKD3 revealed that point mutations that render PKD3 catalytically inactive completely prevented its nuclear import despite its interaction with importin alpha and beta. We also found that activation loop phosphorylation of PKD3 did not require its nuclear localization, and it was not sufficient to promote the nuclear import of PKD3. These results identify a novel function for the kinase activity of PKD3 in promoting its nuclear entry and suggest that the catalytic activity of PKD3 may regulate its nuclear import through autophosphorylation and/or interaction with another protein(s).     

Recent developments in the understanding of nuclear protein import

The process of nucleocytoplasmic exchange of various macromolecules and metabolites between the nuclear and cytoplasmic compartment is crucial for cell function and survival. It is also closely involved with several pathogeneses including cancer and viral infections. Here, we will discuss the current understanding of the classical nuclear import pathway and the factors that are essential for this process.     

Phosphorylation of the nuclear transport machinery down-regulates nuclear protein import in vitro

We have examined whether signal-mediated nucleocytoplasmic transport can be regulated by phosphorylation of the nuclear transport machinery. Using digitonin-permeabilized cell assays to measure nuclear import and export, we found that the phosphatase inhibitors okadaic acid and microcystin inhibit transport mediated by the import receptors importin beta and transportin, but not by the export receptor CRM1. Several lines of evidence, including the finding that transport inhibition is partially reversed by the broad specificity protein kinase inhibitor staurosporine, indicate that transport inhibition is due to elevated phosphorylation of a component of the nuclear transport machinery. The kinases and phosphatases involved in this regulation are present in the permeabilized cells. A phosphorylation-sensitive component of the nuclear transport machinery also is present in permeabilized cells and is most likely a component of the nuclear pore complex. Substrate binding by the importin alpha.beta complex and the association of the complex with the nucleoporins Nup358/RanBP2 and Nup153 are not affected by phosphatase inhibitors, suggesting that transport inhibition by protein phosphorylation does not involve these steps. These results suggest that cells have mechanisms to negatively regulate entire nuclear transport pathways, thus providing a means to globally control cellular activity through effects on nucleocytoplasmic trafficking.     

Phosphorylation by protein kinase a inhibits nuclear import of 5-lipoxygenase

The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Protein kinase A phosphorylates 5-lipoxygenase on Ser(523), and this reduces its activity. We report here that phosphorylation of Ser(523) also shifts the subcellular distribution of 5-lipoxygenase from the nucleus to the cytoplasm. Phosphorylation and redistribution of 5-lipoxygenase could be produced by overexpression of the protein kinase A catalytic subunit alpha, by pharmacological activators of protein kinase A, and by prostaglandin E(2). Mimicking phosphorylation by replacing Ser(523) with glutamic acid caused cytoplasmic localization; replacement of Ser(523) with alanine prevented phosphorylation and redistribution in response to protein kinase A activation. Because Ser(523) is positioned within the nuclear localization sequence-518 of 5-lipoxygenase, the ability of protein kinase A to phosphorylate and alter the localization of green fluorescent protein fused to the nuclear localization sequence-518 peptide was also tested. Site-directed replacement of Ser(523) with glutamic acid within the peptide impaired nuclear accumulation; overexpression of the protein kinase A catalytic subunit alpha and pharmacological activation of protein kinase caused phosphorylation of the fusion protein at Ser(523), and the phosphorylated protein was found chiefly in the cytoplasm. Taken together, these results indicate that phosphorylation of Ser(523) inhibits the nuclear import function of a nuclear localization sequence, resulting in the accumulation of 5-lipoxygenase enzyme in the cytoplasm. As cytoplasmic localization can be associated with reduced leukotriene synthetic capacity, phosphorylation of Ser(523) serves to inhibit leukotriene production by both impairing catalytic activity and by placing the enzyme in a site that is unfavorable for action.     

Inhibition of nuclear protein kinases by adenosine analogues

Two cyclic AMP-independent protein kinases from rat liver nuclei were inhibited competitively by adenosine and a variety of its analogues: cardycepin, tubercidin, 6-mercaptopurine riboside, 6-methylaminopurine riboside, 6-dimethylaminopurine riboside, and 2'-deoxyadenosine. Neither enzyme was inhibited by 6-methoxypurine riboside. Kinase NI was more sensitive to cordycepin, tubercidin, 6-methylaminopurine riboside,, 2'-deoxyadenosine, and adenosine than was kinase NII. Although the effects of all analogues tested were reversed by increasing the concentration of ATP, kinases NII and NI exhibited marked differences in their affinities for adenosine. The results presented here suggest that 1) differences in the catalytic properties of nuclear protein kinases can be detected by inhibitor studies and 2) modifications in the purine ring and sugar moiety of an adenosine analogue can alter its ability to inhibit nuclear protein kinases.     

Monoclonal antibodies to NTF2 inhibit nuclear protein import by preventing nuclear translocation of the GTPase Ran

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.     

Inhibition of nuclear import by backbone cyclic peptidomimetics derived from the HIV-1 MA NLS sequence

In the present work we have constructed a series of backbone cyclic peptides, which differed in the amino acid residues located at the C-terminal position of the previously described BCvir peptide (A. Friedler, N. Zakai, O. Karni, Y.C. Broder, L. Baraz, M. Kotler, A. Loyter, C. Gilon, Biochemistry 37 (1998)). BCvir is a cyclic peptide, derived from the nuclear localization signal (NLS) of the human immunodeficiency virus type 1 matrix protein. The majority of the cyclic peptides described here inhibited nuclear import in vitro. The most potent inhibitors were those bearing bulky hydrophobic amino acids such as Leu, Phe or Nal (naphthyl Ala) at the C-terminus. On the other hand, peptides bearing polar amino acid residues such as Asn, Cys or a reduced amide bond were not inhibitory. The present studies demonstrate the importance of a bulky hydrophobic C-terminal side chain and an exocyclic amide bond preceding it, to the inhibitory activity of the NLS-derived BC peptides. Being only inhibitory, these BC peptides resemble classic receptor antagonists.     

Yeast cell-free nuclear protein import requires ATP hydrolysis

Saccharomyces cerevisiae nuclear proteins are shown to localize specifically to isolated yeast nuclei under conditions selective for nuclear proteins. Nuclear association is time- and temperature-dependent, requires ATP hydrolysis, and is abolished by protease pretreatment of nuclei. The nucleus-localized protein is translocated across the nuclear envelope as determined by inaccessibility to externally added immobilized protease. This cell-free system, consisting of components from an organism amenable to genetic analysis, will facilitate the study of the poorly understood mechanism of nuclear protein localization. The finding that ATP hydrolysis is required for nuclear import is the most direct evidence that nuclear localization is energy-dependent.     

Age-associated reduction of nuclear protein import in human fibroblasts

Age-dependent decreases in the protein concentrations of the nucleocytoplasmic transport factors karyopherin alpha2, CAS, and RanBP1 were found by comparing fibroblast cultures obtained from young, mature, and old human donors. Karyopherin beta1 levels do not change with age and present very little variation among donors. The decrease in the concentration of transport factors is accompanied by a reduction in the protein import rate in fibroblasts from old donors, as detected by a change in the intracellular localization of a test transport substrate that shuttles between the cytoplasm and the nucleus. Measurements of concentrations of the same import factors in organs and tissues of old mice revealed a decrease of CAS in kidney, lung, and spleen. The import reduction in old age is expected to lead to impaired activity of proteins whose functions depend on timely import into the nuclei.