Glycogen synthase kinase-3 enhances nuclear export of a Dictyostelium STAT protein

Extracellular cAMP stimulates the rapid tyrosine phosphorylation and nuclear translocation of the DICTYOSTELIUM: STAT protein Dd-STATa. Here we show that it also induces serine phosphorylation by GskA, a homologue of glycogen synthase kinase-3 (GSK-3). Tyrosine phosphorylation occurs within 10 s of stimulation, whereas serine phosphorylation takes 5 min, matching the kinetics observed for the cAMP regulation of GskA. Phosphorylation by GskA enhances nuclear export of Dd-STATa. The phosphorylated region, however, is not itself a nuclear export signal and we identify a region elsewhere in the protein that mediates nuclear export. These results suggest a biphasic regulation of Dd-STATa, in which extracellular cAMP initially directs nuclear import and then, via GskA, promotes its subsequent export. It also raises the possibility of an analogous regulation of STAT nuclear export in higher eukaryotes.     

The human tap nuclear RNA export factor contains a novel transportin-dependent nuclear localization signal that lacks nuclear export signal function.

The human Tap protein mediates the sequence-specific nuclear export of RNAs containing the constitutive transport element and is likely also critical for general mRNA export. Here, we demonstrate that a previously defined arginine-rich nuclear localization signal (NLS) present in Tap acts exclusively via the transportin import factor. Previously, transportin has been shown to mediate the nuclear import of several heterogeneous nuclear ribonucleoproteins, including heterogeneous nuclear ribonucleoprotein (hnRNP) A1, by binding to a sequence element termed M9. Although the Tap NLS and the hnRNP A1 M9 element are shown to compete for transportin binding, they show no sequence homology, and the Tap NLS does not conform to the recently defined M9 consensus. The Tap NLS also differs from M9 in that only the latter is able to act as a nuclear export signal. The Tap NLS is therefore the first member of a novel class of transportin-specific NLSs that lack nuclear export signal function.     

Characterization of nuclear localization and nuclear export signals of yeast actin-binding protein Pan1

Pan1 is an actin patch-associated protein involved in endocytosis. Our studies revealed that in oleate-grown cells Pan1 is located in the nucleus as well as in patches. One of three putative nuclear localization signals (NLS) of Pan1, NLS2, directed beta-galactosidase (beta-gal) to the nucleus. However, GFP-Pan1(886-1219), containing NLS2, was found in the cytoplasm indicating that it may contain a nuclear export signal (NES). A putative Pan1 NES, overlapping with NLS3, re-addressed NLS(H2B)-NES/NLS3-beta-gal from the nucleus to the cytoplasm. Inactivation of the NES allowed NLS3 to be effective. Thus, Pan1 contains functional NLSs and a NES and appears to shuttle in certain circumstances.     

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.     

CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin

Survivin is a member of the inhibitor of apoptosis (IAP) family of negative regulators of programmed cell death that is frequently overexpressed in human tumors. Survivin is not only involved in the regulation of apoptosis, but is also known to play a role in the control of cell cycle progression at the G2/M phase. Survivin is a predominantly cytoplasmic protein expressed in a cell cycle-dependent manner, but the mechanism(s) that determine its nuclear-cytoplasmic localization have not been described. In this study, we report that Survivin is a nuclear shuttling protein that is actively exported from the nucleus via the CRM1-dependent pathway. Nuclear export of Survivin is independent of the export of other shuttling proteins that control the G2/M phase transition, such as cyclin B1 and cdc25. The carboxy-terminal domain of Survivin is both necessary and sufficient for its nuclear export, although this region does not contain a functional leucine-rich nuclear export signal. Differences in the amino acid sequence of this region determine the dramatically different localization of Survivin (in the cytoplasm) and its splicing variant Survivin-DeltaEx3 (in the nucleus). The carboxy-terminal end of Survivin-DeltaEx3 contains a bipartite nuclear localization signal, not present in Survivin, which mediates its strong nuclear accumulation. These data suggest that active transport between the nucleus and cytoplasm may constitute an important regulatory mechanism for Survivin function.     

Phosphorylation and nuclear localization of the varicella-zoster virus gene 63 protein.

The protein encoded by varicella-zoster virus open reading frame 63 and carboxy-terminal deletions of the same were expressed either as fusion proteins at the carboxy terminus of the maltose-binding protein in Escherichia coli or independently in transfected mammalian cells. The truncations contained amino acids 1 to 142 (63 delta N) or 1 to 210 (63 delta K) of the complete 278-amino-acid primary sequence. Recombinant casein kinase II phosphorylated the 63F and 63 delta KF fusion proteins in vitro but did not phosphorylate the 63 delta NF fusion protein, implying that phosphorylation occurred between amino acids 142 and 210. Immunoprecipitation of 35S- or 32P-labelled extracts of cells transfected with plasmids expressing 63, 63 delta N, or 63 delta K also indicated that in situ phosphorylation most likely occurred between amino acids 142 and 210. These combined results suggest that casein kinase II plays a significant role in the phosphorylation of the varicella-zoster virus 63 protein. Indirect immunofluorescence of transfected cells indicated nuclear localization of the 63 protein and cytoplasmic localization of 63 delta K and 63 delta N, implying a requirement for sequences between amino acids 210 and 278 for efficient nuclear localization.     

Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.

Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.     

Two nuclear export signals specify the cytoplasmic localization of calcineurin B homologous protein 1

We previously showed that calcineurin B homologous protein 1 (CHP1) interacts with nuclear apoptosis-inducing protein kinase DRAK2, and that overexpression of DRAK2 induces the nuclear accumulation of CHP1, although CHP1 usually resides in the cytoplasm [Matsumoto et al. (2001) J. Biochem. 130, 217-225]. Here we show that CHP1 has two functional nuclear export signal (NES) sequences in its carboxyl-terminal region. Treatment of several cell lines with leptomycin B, a specific inhibitor of CRM1-dependent nuclear export, induces the nuclear accumulation of CHP1. Moreover, CHP1-GFP fusion proteins with deletions or point mutations affecting the two putative NES sequences accumulate in the nucleus to a greater extent than wild-type CHP1-GFP. Tagging glutathione S-transferase-GFP fusion protein with each NES sequence caused a shift in their intracellular localization from all over the cells to the cytoplasm. These results suggest that after CHP1 has entered the nucleus, it is exported to the cytoplasm in an NES-dependent manner.     

Adenovirus late gene expression does not require a Rev-like nuclear RNA export pathway

Adenovirus late mRNA export is facilitated by viral early proteins of 55 and 34 kDa. The 34-kDa protein contains a leucine-rich nuclear export signal (NES) similar to that of the human immunodeficiency virus Rev protein. It was proposed that the 34-kDa protein might facilitate the export of adenovirus late mRNA through a Rev-like NES-mediated export pathway. We have tested the role of NES-mediated RNA export during adenovirus infection, and we find that it is not essential for the expression of adenovirus late genes.     

Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1

Receptor-mediated nucleocytoplasmic transport is dependent on the GTPase Ran and Ran-binding protein 1 (RanBP1). The acidic C terminus of Ran is required for high affinity interaction between Ran and RanBP1. We found that a novel Ran mutant with four of its five acidic C-terminal amino acids modified to alanine (RanC4A) has an approximately 20-fold reduced affinity for RanBP1. We investigated the effects of RanC4A on nuclear import and export in permeabilized HeLa cells. Although RanC4A promotes accumulation of the nuclear export receptor CRM1 at the cytoplasmic nucleoporin Nup214, it strongly stimulates nuclear export of GFP-NFAT. Since RanC4A exhibits an elevated affinity for CRM1 and other nuclear transport receptors, this suggests that formation of the export complex containing CRM1, Ran-GTP, and substrate is a rate-limiting step in export, not release from Nup214. Conversely, importin alpha/beta-dependent nuclear import of bovine serum albumin, coupled to a classical nuclear localization sequence is strongly inhibited by RanC4A. Inhibition can be reversed by additional importin alpha, which promotes the formation of an importin alpha/beta complex. These results provide physiological evidence that release of Ran-GTP from importin beta by RanBP1 and importin alpha is critical for the recycling of importin beta to a transport-competent state.     

Reconstitution of nuclear protein export in isolated nuclear envelopes

Signal-dependent nuclear protein export was studied in perforated nuclei and isolated nuclear envelopes of Xenopus oocytes by optical single transporter recording. Manually isolated and purified oocyte nuclei were attached to isoporous filters and made permeable for macromolecules by perforation. Export of a recombinant protein (GG-NES) containing the nuclear export signal (NES) of the protein kinase A inhibitor through nuclear envelope patches spanning filter pores could be induced by the addition of GTP alone. Export continued against a concentration gradient, and was NES dependent and inhibited by leptomycin B and GTPgammaS, a nonhydrolyzable GTP analogue. Addition of recombinant RanBP3, a potential cofactor of CRM1-dependent export, did not promote GG-NES export at stoichiometric concentration but gradually inhibited export at higher concentrations. In isolated filter-attached nuclear envelopes, export of GG-NES was virtually abolished in the presence of GTP alone. However, a preformed export complex consisting of GG-NES, recombinant human CRM1, and RanGTP was rapidly exported. Unexpectedly, export was strongly reduced when the export complex contained RanGTPgammaS or RanG19V/Q69L-GTP, a GTPase-deficient Ran mutant. This paper shows that nuclear transport, previously studied in intact and permeabilized cells only, can be quantitatively analyzed in perforated nuclei and isolated nuclear envelopes.     

A nuclear export signal prevents Saccharomyces cerevisiae Hsp70 Ssb1p from stimulating nuclear localization signal-directed nuclear transport.

Hsp70 has been implicated in nuclear localization signal (NLS)-directed nuclear transport. Saccharomyces cerevisiae contains distinct SSA and SSB gene families of cytosolic Hsp70s. The nucleocytoplasmic localization of Ssa1p and Ssb1p was investigated using green fluorescent protein (GFP) fusions. Whereas GFP-Ssa1p localized both to the nucleus and cytoplasm, GFP-Ssb1p appeared only in the cytosol. The C-terminal domain of Ssb1p contains a leucine-rich nuclear export signal (NES) that is necessary and sufficient to direct nuclear export. The accumulation of GFP-Ssb1p in the nuclei of xpo1-1 cells suggests that Ssb1p shuttles across the nuclear envelope. Elevated levels of SSA1 but not SSB1 suppressed the NLS-GFP nuclear localization defects of nup188-Delta cells. Studies with Ssa1p/Ssb1p chimeras revealed that the Ssb1p NES is sufficient and necessary to inhibit the function of Ssa- or Ssb-type Hsp70s in nuclear transport. Thus, NES-less Ssb1p stimulates nuclear transport in nup188-Delta cells and NES-containing Ssa1p does not. We conclude that the differential function of Ssa1p and Ssb1p in nuclear transport is due to the NES-directed export of the Ssb1p and not to functional differences in their ATPase or peptide binding domains.