TNFalpha release from peripheral blood leukocytes depends on a CRM1-mediated nuclear export

Tumor necrosis factor-alpha (TNFalpha) is a potent pro-inflammatory cytokine that plays a major role in the pathogenesis of acute and chronic inflammatory disorders such as septic shock and arthritis, respectively. Leukocytes stimulated with inflammatory signals such as lipopolysaccharide (LPS) are the predominant producers of TNFalpha, and thus control of TNFalpha release from stimulated leukocytes represents a potential therapeutic target. Here, we report that leptomycin B (LMB), a specific inhibitor of CRM1-dependent nuclear protein export, inhibits TNFalpha release from LPS-stimulated human peripheral blood neutrophils and mononuclear cells. In addition, immunofluorescence confocal microscopy and immunoblotting analysis indicate that TNFalpha is localized in the nucleus of human neutrophils and mononuclear cells. This study demonstrates that the cellular release of TNFalpha from stimulated leukocytes is mediated by the CRM1-dependent nuclear export mechanism. Inhibition of CRM1-dependent cellular release of TNFalpha could thus provide a novel therapeutic approach for disorders involving excessive TNFalpha release.     

An allosteric mechanism to displace nuclear export cargo from CRM1 and RanGTP by RanBP1

The karyopherin CRM1 mediates nuclear export of proteins and ribonucleoproteins bearing a leucine‐rich nuclear export signal (NES). To elucidate the precise mechanism by which NES‐cargos are dissociated from CRM1 in the cytoplasm, which is important for transport directionality, we determined a 2.0‐Å resolution crystal structure of yeast CRM1:RanBP1:RanGTP complex, an intermediate in the disassembly of the CRM1 nuclear export complex. The structure shows that on association of Ran‐binding domain (RanBD) of RanBP1 with CRM1:NES‐cargo:RanGTP complex, RanBD and the C‐terminal acidic tail of Ran induce a large movement of the intra‐HEAT9 loop of CRM1. The loop moves to the CRM1 inner surface immediately behind the NES‐binding site and causes conformational rearrangements in HEAT repeats 11 and 12 so that the hydrophobic NES‐binding cleft on the CRM1 outer surface closes, squeezing out the NES‐cargo. This allosteric mechanism accelerates dissociation of NES by over two orders of magnitude. Structure‐based mutagenesis indicated that the HEAT9 loop also functions as an allosteric autoinhibitor to stabilize CRM1 in a conformation that is unable to bind NES‐cargo in the absence of RanGTP.     

Topoisomerase II binds importin alpha isoforms and exportin/CRM1 but does not shuttle between the nucleus and cytoplasm in proliferating cells

Resistance to anticancer drugs that target DNA topoisomerase II (topo II) isoforms alpha and/or beta is associated with decreased nuclear and increased cytoplasmic topo IIalpha. Earlier studies have confirmed that functional nuclear localization and export signal sequences (NLS and NES) are present in both isoforms. In this study, we show that topo II alpha and beta bind and are imported into the nucleus by importin alpha1, alpha3, and alpha5 in conjunction with importin beta. Topo IIalpha also binds exportin/CRM1 in vitro. However, wild-type topo IIalpha has only been observed in the cytoplasm of cells that are entering plateau phase growth. This suggests that topo IIalpha may shuttle between the nucleus and the cytoplasm with the equilibrium towards the nucleus in proliferating cells but towards the cytoplasm in plateau phase cells. The CRM1 inhibitor Leptomycin B increases the nuclear localization of GFP-tagged topo IIalpha with a mutant NLS, suggesting that its export is being inhibited. However, homokaryon shuttling experiments indicate that fluorescence-tagged wild-type topo II alpha and beta proteins do not shuttle in proliferating Cos-1 or HeLa cells. We conclude that topo II alpha and beta nuclear export is inhibited in proliferating cells so that these proteins do not shuttle.     

CRM1 mediates nuclear export of HDAC7 independently of HDAC7 phosphorylation and association with 14-3-3s

CRM1, 14-3-3 proteins, and CaMK play important roles in trafficking of HDAC7, but the interplay between these proteins in this process is not clearly understood. Here, we show that CRM1 is capable of promoting cytoplasmic localization of wild-type and mutant HDAC7 (S178A/S344A/S479A), which is normally found in the nucleus. Using phospho-specific antibodies to HDAC7, we demonstrate that CaMK I promotes phosphorylation of S178, S344, and S479 of HDAC7. We also show that endogenous S178-phosphorylated HDAC7 is localized in both the nucleus and the cytoplasm, whereas S344- and S479-phosphorylated HDAC7 are exclusively localized in the nucleus. An HDAC7 mutant, S178E/S344E/S479E, which lost the ability to bind 14-3-3s, is localized in both the nucleus and the cytoplasm. Furthermore, the nuclear export of S178E/S344E/S479E is inhibited by LMB, but is enhanced by the CRM1. Taken together, these results strongly suggest that CRM1 mediated-nuclear export of HDAC7 is independent of HDAC7 phosphorylation and its association with 14-3-3s.     

Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358

Leucine-rich nuclear export signals (NESs) mediate rapid nuclear export of proteins via interaction with CRM1. This interaction is stimulated by RanGTP but remains of a relatively low affinity. In order to identify strong signals, we screened a 15-mer random peptide library for CRM1 binding, both in the presence and absence of RanGTP. Under each condition, strikingly similar signals were enriched, conforming to the NES consensus sequence. A derivative of an NES selected in the absence of RanGTP exhibits very high affinity for CRM1 in vitro and stably binds without the requirement of RanGTP. Localisation studies and RNA interference demonstrate inefficient CRM1-mediated export and accumulation of CRM1 complexed with the high-affinity NES at nucleoporin Nup358. These results provide in vivo evidence for a nuclear export reaction intermediate. They suggest that NESs have evolved to maintain low affinity for CRM1 to allow efficient export complex disassembly and release from Nup358.     

Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

The study of Hutchinson–Gilford progeria syndrome (HGPS) has provided important clues to decipher mechanisms underlying aging. Progerin, a mutant lamin A, disrupts nuclear envelope structure/function, with further impairment of multiple processes that culminate in senescence. Here, we demonstrate that the nuclear protein export pathway is exacerbated in HGPS, due to progerin‐driven overexpression of CRM1, thereby disturbing nucleocytoplasmic partitioning of CRM1‐target proteins. Enhanced nuclear export is central in HGPS, since pharmacological inhibition of CRM1 alleviates all aging hallmarks analyzed, including senescent cellular morphology, lamin B1 downregulation, loss of heterochromatin, nuclear morphology defects, and expanded nucleoli. Exogenous overexpression of CRM1 on the other hand recapitulates the HGPS cellular phenotype in normal fibroblasts. CRM1 levels/activity increases with age in fibroblasts from healthy donors, indicating that altered nuclear export is a common hallmark of pathological and physiological aging. Collectively, our findings provide novel insights into HGPS pathophysiology, identifying CRM1 as potential therapeutic target in HGPS.     

Homodimerization antagonizes nuclear export of survivin

Survivin plays separate roles during different phases of the cell cycle. In mitosis, Survivin is a key regulator of cell division, while in interphase, Survivin is able to protect cells from apoptosis. Survivin shuttles between nucleus and cytoplasm under the influence of one or more nuclear export signals (NESs). Paradoxically, our data show that Survivin poorly binds CRM1 in vitro because hydrophobic residues of the NES are occupied in homodimer contacts. We show that NES-preserving dimerization mutants behave as monomers in solution, show dramatically increased CRM1 binding and are more efficiently exported in vivo than wild-type Survivin. These data indicate that Survivin contains a monomer-specific NES and that dimerization modulates cytoplasmic access of the protein. Our findings have implications for both the mitotic and interphase roles of survivin.     

Exportin 1-independent nuclear export of GAPDH

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme of the glycolytic pathway. Recent studies have demonstrated an additional role in apoptosis: GAPDH is targeted to the nucleus during apoptotic signalling. This nuclear transport has also been observed in serum-depleted cells, but it is reversible in fibroblasts, in contrast to apoptotic-induced transport (Eur J Cell Biol 80 (2001) 419). Here, we analyse the serum depletion-induced transport processes of GAPDH in NIH 3T3 cells. Prolonged serum depletion did not cause cell death, nuclear fragmentation (hoechst staining) or a significant increase in DNA strand-breaks (comet assay). Using cells expressing green fluorescent protein (GFP)-tagged GAPDH allowed us to monitor its intracellular localisation by confocal laser scanning microscopy (CLSM). Treatment of cells with the exportin1 inhibitor leptomycin B (LMB) did not influence cytoplasmic localisation of GFP-GAPDH, indicating that nuclear targeting of GAPDH is not constitutive and may be altered via a serum-dependent regulatory export process. Suprisingly, the export of nuclear GFP-GAPDH after re-addition of serum to starved cells was not prevented by LMB. Thus, nuclear export of GAPDH upon serum depletion is not mediated by exportin1.     

Protracted nuclear export of glucocorticoid receptor limits its turnover and does not require the exportin 1/CRM1-directed nuclear export pathway

Glucocorticoid receptors (GRs) are shuttling proteins, yet they preferentially accumulate within either the cytoplasmic or nuclear compartment when overall rates of nuclear import or export, respectively, are limiting. Hormone binding releases receptors from stable heteromeric complexes that restrict their interactions with soluble nuclear import factors and contribute to their cytoplasmic retention. Although hormone dissociation leads to the rapid release of GRs from chromatin, unliganded nuclear receptors are delayed in their export. We have used a chimeric GR that contains a heterologous, leucine-rich nuclear export signal sequence (NES) to assess the consequences of accelerated receptor nuclear export. Leucine-rich NESs utilize the exportin 1/CRM1-dependent nuclear export pathway, which can be blocked by leptomycin B (LMB). The fact that rapid nuclear export of the NES-GR chimera, but not the protracted export of wild-type GR, is sensitive to LMB, suggests that GR does not require the exportin 1/CRM1 pathway to exit the nucleus. Despite its more rapid export, the NES-GR chimera appears indistinguishable from wild-type GR in its transactivation activity in transiently transfected cells. However, accelerated nuclear export of the NES-GR chimera is associated with an increased rate of hormone-dependent down-regulation. The increase in NES-GR down-regulation is overcome by LMB treatment, thereby confirming the connection between receptor nuclear export and down-regulation. Given the presence of a nuclear recycling pathway for GR, the protracted rate of receptor nuclear export may increase the efficiency of biological responses to secondary hormone challenges by limiting receptor down-regulation and hormone desensitization.     

G2E3 is a nucleo-cytoplasmic shuttling protein with DNA damage responsive localization

G2E3 was originally described as a G2/M-specific gene with DNA damage responsive expression. The presence of a conserved HECT domain within the carboxy-terminus of the protein indicated that it likely functions as a ubiquitin ligase or E3. Although HECT domains are known to function in this capacity for many proteins, we demonstrate that a portion of the HECT domain from G2E3 plays an important role in the dynamic subcellular localization of the protein. We have shown that G2E3 is a nucleo-cytoplasmic shuttling protein with nuclear export mediated by a novel nuclear export domain that functions independently of CRM1. In full-length G2E3, a separate region of the HECT domain suppresses the function of the NES. Additionally, G2E3 contains a nucleolar localization signal (NoLS) in its amino terminus. Localization of G2E3 to the nucleolus is a dynamic process, and the protein delocalizes from the nucleolus rapidly after DNA damage. Cell cycle phase-specific expression and highly regulated subcellular localization of G2E3 suggest a possible role in cell cycle regulation and the cellular response to DNA damage.     

CRM1-dependent nuclear export and dimerization with hMSH5 contribute to the regulation of hMSH4 subcellular localization

MSH4 and MSH5 are members of the MutS homolog family, a conserved group of proteins involved in DNA mismatch correction and homologous recombination. Although several studies have provided compelling evidences suggesting that MSH4 and MSH5 could act together in early and late stages of meiotic recombination, their precise roles are poorly understood and recent findings suggest that the human MSH4 protein may also exert a cytoplasmic function. Here we show that MSH4 is present in the cytoplasm and the nucleus of both testicular cells and transfected somatic cells. Confocal studies on transfected cells provide the first evidence that the subcellular localization of MSH4 is regulated, at least in part, by an active nuclear export pathway dependent on the exportin CRM1. We used deletion mapping and mutagenesis to define two functional nuclear export sequences within the C-terminal part of hMSH4 that mediate nuclear export through the CRM1 pathway. Our results suggest that CRM1 is also involved in MSH5 nuclear export. In addition, we demonstrate that dimerization of MSH4 and MSH5 facilitates their nuclear localization suggesting that dimerization may regulate the intracellular trafficking of these proteins. Our findings suggest that nucleocytoplasmic traffic may constitute a regulatory mechanism for MSH4 and MSH5 functions.     

Nuclear export signal in CDC25B

CDC25B is a dual-specificity phosphatase that activates CDK1/cyclin B. The nuclear exclusion of CDC25B is controlled by the binding of 14-3-3 to the nuclear export signal (NES) of CDC25B, which was reported to be amino acids H28 to L40 in the N-terminal region of CDC25B. In studying the subcellular localization of CDC25B, we found a functional NES at V52 to L65, the sequence of which is VTTLTQTMHDLAGL, where bold letters are leucine or hydrophobic amino acids frequently seen in an NES. The deletion of this NES sequence caused the mutant protein to locate exclusively in nuclei, while NES-fused GFP was detected in the cytoplasm. Moreover, the introduction of point mutations at some of the critical amino acids impaired cytoplasmic localization. Treatment with leptomycin B, a potent inhibitor of CRM1/exportin1, disrupted the cytoplasmic localization of both Flag-tagged CDC25B and NES-fused GFP. From these results, we concluded that the sequence we found is a bona fide NES of CDC25B.     

Identification of functional nuclear export sequences in human sphingosine kinase 1

Sphingosine kinase (SPHK) is an enzyme that phosphorylates sphingosine to form sphingosine 1-phosphate (S1P). Human SPHK1 (hSPHK1) was localized predominantly in the cytoplasm when transiently expressed in Cos7 cells. In this study, we have found two functional nuclear export signal (NES) sequences in the middle region of hSPHK1. Deletion and mutagenesis studies revealed that the cytoplasmic localization of SPHK1 depends on its nuclear export, directed by the NES. Furthermore, upon treatment with leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, a marked nuclear accumulation of hSPHK1 was observed, indicating that hSPHK1 shuttles between the cytoplasm and the nucleus. Our results provide the first evidence of the active nuclear export of SPHK1 and suggest it is mediated by a CRM1-dependent pathway.