OxLDL stimulates Id1 nucleocytoplasmic shuttling in endothelial cell angiogenesis via PI3K pathway

Angiogenesis plays remarkable roles in the development of atherosclerotic rupture plaques. However, its essential mechanism remains unclear. The purpose of the study was to investigate whether inhibitor of DNA binding-1 or inhibitor of differentiation 1 (Id1) promoted angiogenesis when exposed to oxidised low-density lipoprotein (oxLDL), and to determine the molecular mechanism involved. Using aortic ring assay and tube formation assay as a model system, a low concentration of oxLDL was found to induce angiogenic sprouting and capillary lumen formation of endothelial cell. But the Id1 expression was significantly upregulated by oxLDL at low and high concentrations. The Id1 was localised in the nuclei of the human umbilical vein endothelial cells in the control group and in the high-concentration oxLDL group. Id1 was translocated to the cytoplasm at low oxLDL concentrations. The nucleocytoplasmic shuttling at low oxLDL concentration was inhibited by treatment with the nuclear export inhibitor leptomycin B. Protein kinase A (PKA) inhibitor H89 promoted nuclear export of Id1, and phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 reduced the nuclear export of Id1. PI3K inhibition blocked oxLDL-induced angiogenesis. Low concentrations of oxLDL promoted angiogenic sprouting and capillary formation. And this process depends on nuclear export of Id1, which in turn is controlled by the PI3K pathway. This report presents a new link between oxLDL and Id1 localisation, and may provide a new insight into the interactions of ox-LDL and Id1 in the context of atherosclerosis.     

ZAP is a CRM1-dependent nucleocytoplasmic shuttling protein

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MMLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. In this report, we demonstrate that ZAP is predominantly localized in the cytoplasm at steady state but shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. Two nuclear localization sequences (NLS) and one nuclear export sequence (NES) were identified. One NLS was mapped to amino acids 68-RARVCRRK-75 and the other mapped to a region including amino acids K405 and K406. The NES was mapped to amino acids 284-LEDVSVDV-291. These findings help to understand why ZAP specifically prevents the accumulation of viral RNA in the cytoplasm. These findings also suggest possible functions of ZAP in the nucleus.     

Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1

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 beta. 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.     

Features in the N and C termini of the MAPK-interacting kinase Mnk1 mediate its nucleocytoplasmic shuttling

Eukaryotic initiation factor eIF4E binds to the 5'-cap structure of the mRNA and also to the molecular scaffold protein eIF4G. eIF4E is a phosphoprotein, and the kinases that act on it have been identified as the MAPK-interacting kinases Mnk1 and Mnk2. Mnk1/2 also bind to the scaffold protein eIF4G. The N-terminal region of Mnk1 has previously been shown to bind to importin alpha, a component of the nuclear transport machinery, although Mnk1 itself is cytoplasmic. Here we identify a CRM1-type nuclear export motif in the C-terminal part of Mnk1. Substitution of hydrophobic residues in this motif results in Mnk1 becoming nuclear. This has allowed us to study the features of Mnk1 that are involved in its transport to the nucleus. This process requires part, but not all, of a polybasic region near the N terminus of Mnk1. Residues required for nuclear transport are also required for its interaction with importin alpha. This polybasic region also serves a second function in that it is required for the binding of Mnk1 to eIF4G, although the residues involved in this interaction are not identical to those involved in the binding of Mnk1 to importin alpha. Interaction of Mnk1 with eIF4G promotes the phosphorylation of eIF4E. Mutations that reduce the binding of Mnk1 to eIF4G in vivo and in vitro also decrease the ability of Mnk1 to enhance eIF4E phosphorylation in vivo, underlining the importance of the eIF4G-Mnk1 interaction in this process.     

ERK1 nucleocytoplasmic shuttling rate depends on specific N-terminal aminoacids

Despite ERK1 and ERK2 were considered interchangeable isoforms for a long time, their roles are now emerging as only partially overlapping. We recently reported that the nucleocytoplasmic trafficking of GFP-tagged ERK1 is slower than that of ERK2, this difference being caused by a unique domain of ERK1 located at its N-terminus (ERK1-Nt). In the present report we further investigated this issue by asking which were the specific aminoacids involved in such process. By photobleaching strategy, we demonstrated that ERK1-Nt is a domain capable to slow down the nucleocytoplasmic shuttling rate even of a small cargo protein. ERK1-Nt was then dissected into three regions as follows: 1 (aa 1-9), 2 (aa 10-29) and 3, (aa 30-39) that were deleted or mutated at specific sites. Dynamic imaging assessment of the role played by each region in determining the shuttling rate revealed that: region 1 has no significant role, region 2 and specific aminoacids of region 3 (V₃₁, K_33, P₃₆) are critical, but singularly do not totally account for the difference in the shuttling rate between ERK1 and 2. Finally, we demonstrated that the nucleocytoplasmic shuttling rate of a passively diffusing protein (mRED) is inversely related to ERK1-Nt-GFP concentrations inside the cell, thus suggesting that ERK1-Nt-GFP occupies the nuclear pore perhaps because of an important affinity of ERK1-Nt for nucleoporins.In conclusion, ERK1-Nt is a domain able per se to confer a slower shuttling rate to a cargo protein. Specific regions within this domain were identified as responsible for this biophysical property.     

Calcium/calmodulin-dependent protein kinase (CaMK) IV mediates nucleocytoplasmic shuttling and release of HMGB1 during lipopolysaccharide stimulation of macrophages

The chromatin-binding factor high-mobility group box 1 (HMGB1) functions as a proinflammatory cytokine and late mediator of mortality in murine endotoxemia. Although serine phosphorylation of HMGB1 is necessary for nucleocytoplasmic shuttling before its cellular release, the protein kinases involved have not been identified. To investigate if calcium/calmodulin-dependent protein kinase (CaMK) IV serine phosphorylates and mediates the release of HMGB1 from macrophages (Mphi) stimulated with LPS, RAW 264.7 cells or murine primary peritoneal Mphi were incubated with either STO609 (a CaMKIV kinase inhibitor), KN93 (a CaMKIV inhibitor), or we utilized cells from which CaMKIV was depleted by RNA interference (RNAi) before stimulation with LPS. We also compared the LPS response of primary Mphi isolated from CaMKIV(+/+) and CaMKIV(-/-) mice. In both cell types LPS induced activation and nuclear translocation of CaMKIV, which preceded HMGB1 nucleocytoplasmic shuttling. However, Mphi treated with KN93, STO609, or CaMKIV RNAi before LPS showed reduced nucleocytoplasmic shuttling of HMGB1 and release of HMGB1 into the supernatant. Additionally, LPS induced serine phosphorylation of HMGB1, which correlated with an interaction between CaMKIV and HMGB1 and with CaMKIV phosphorylation of HMGB1 in vitro. In cells, both HMGB1 phosphorylation and interaction with CaMKIV were inhibited by STO609 or CaMKIV RNAi. Similarly, whereas CaMKIV(+/+) Mphi showed serine phosphorylation of HMGB1 in response to LPS, this phosphorylation was attenuated in CaMKIV(-/-) Mphi. Collectively, our results demonstrate that CaMKIV promotes the nucleocytoplasmic shuttling of HMGB1 and suggest that the process may be mediated through CaMKIV-dependent serine phosphorylation of HMGB1.     

Involvement of nucleocytoplasmic shuttling of yeast Nap1 in mitotic progression

Nucleosome assembly protein 1 (Nap1) is widely conserved from yeasts to humans and facilitates nucleosome formation in vitro as a histone chaperone. Nap1 is generally localized in the cytoplasm, except that subcellular localization of Drosophila melanogaster Nap1 is dynamically regulated between the cytoplasm and nucleus during early development. The cytoplasmic localization of Nap1 is seemingly incompatible with the proposed role of Nap1 in nucleosome formation, which should occur in the nucleus. Here, we have examined the roles of a putative nuclear export signal (NES) sequence in yeast Nap1 (yNap1). yNap1 mutants lacking the NES-like sequence were localized predominantly in the nucleus. Deletion of NAP1 in cells harboring a single mitotic cyclin gene is known to cause mitotic delay and temperature-sensitive growth. A wild-type NAP1 complemented these phenotypes while nap1 mutant genes lacking the NES-like sequence or carboxy-terminal region did not. These and other results suggest that yNap1 is a nucleocytoplasmic shuttling protein and that its shuttling is important for yNap1 function during mitotic progression. This study also provides a possible explanation for Nap1's involvement in nucleosome assembly and/or remodeling in the nucleus.     

Mouse Disabled1 (DAB1) is a nucleocytoplasmic shuttling protein

Disabled1 (DAB1) is an intracellular mediator of the Reelin-signaling pathway and essential for correct neuronal positioning during brain development. So far, DAB1 has been considered a cytoplasmic protein. Here, we show that DAB1 is subject to nucleocytoplasmic shuttling. In its steady state, DAB1 is mainly located in the cytoplasm. However, treatment with leptomycine B, a specific inhibitor of the CRM1 (chromosomal region maintenance 1)-RanGTP-dependent nuclear export, resulted in nuclear accumulation of DAB1. By using deletion or substitutional mutants of DAB1 fused with enhanced green fluorescent protein, we have mapped a bipartite nuclear localization signal and two CRM1-dependent nuclear export signals. These targeting signals were functional in both Neuro2a cells and primary cerebral cortical neurons. Using purified recombinant proteins, we have shown that CRM1 binds to DAB1 directly in a RanGTP-dependent manner. We also show that tyrosine phosphorylation of DAB1, which is indispensable for the layer formation of the brain, by Fyn tyrosine kinase or Reelin stimulation did not affect the subcellular localization of DAB1 in vitro. These results suggest that DAB1 is a nucleocytoplasmic shuttling protein and raise the possibility that DAB1 plays a role in the nucleus as well as in the cytoplasm.     

An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export

Gle1 is required for mRNA export in yeast and human cells. Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B). The two encoded proteins are identical except for their COOH-terminal regions. hGle1A ends with a unique four-amino acid segment, whereas hGle1B has a COOH-terminal 43-amino acid span. Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex. To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP-hGle1. Both strategies show that hGle1 shuttles between the nucleus and cytoplasm. An internal 39-amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport. Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export. An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA. Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1. These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.     

Phosphorylation and dimerization regulate nucleocytoplasmic shuttling of mammalian STE20-like kinase (MST).

Mammalian STE20-like kinase (MST) is a member of the yeast STE20-related kinase family and proteolytically activated by caspase during apoptosis. However, its other cellular functions are not known, including its activation mechanism, substrate(s), and subcellular localization. In this report, using anti-MST monoclonal antibodies, we clearly show that endogenous MST is localized in cytoplasm in a leptomycin B-dependent manner. Analyses with serial deletions and point mutations show that MST has two functional nuclear export signals and, unexpectedly, another localization motif for nuclear import. When cells are treated with leptomycin, monomeric MST is accumulated more rapidly in the nucleus than dimeric MST, indicating that dimerization contributes to the cytoplasmic retention of MST. Okadaic acid, an inhibitor of phosphatase 2A, induces activation of MST and translocation into the nucleus. Using phosphopeptide-specific antibody, we directly show that okadaic acid induces phosphorylation in the activation loop of MST, and, once phosphorylated, MST is rapidly translocated to the nucleus. However, kinase-deficient MST does not enter the nucleus, indicating that phosphorylation and activation is required for okadaic acid-induced nuclear translocation. In apoptotic cells, the activation of MST does not require phosphorylation in the activation loop and occurs through the release of C-terminal regulatory domain by caspase-dependent cleavage. Kinase-deficient MST functions dominant-negatively and represses okadaic acid-induced morphological change indicating that MST plays a role in okadaic acid-induced cellular shrinkage. Our identification of cytoplasmic and nuclear localization motifs and phosphorylation-dependent translocation of MST suggests that regulation of localization is important to the biological function of MST, including its effects on cellular morphology.     

Nuclear import and export signals enable rapid nucleocytoplasmic shuttling of the atypical protein kinase C lambda

The atypical protein kinase C (PKC) isoenzymes, lambda/iota- and zetaPKC, play important roles in cellular signaling pathways regulating proliferation, differentiation, and cell survival. By using green fluorescent protein (GFP) fusion proteins, we found that wild-type lambdaPKC localized predominantly to the cytoplasm, whereas both a kinase-defective mutant and an activation loop mutant accumulated in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal part of the zinc finger domain of lambdaPKC. Leptomycin B treatment induced rapid nuclear accumulation of GFP-lambda as well as endogenous lambdaPKC suggesting the existence of a CRM1-dependent nuclear export signal (NES). Consequently, we identified a functional leucine-rich NES in the linker region between the zinc finger and the catalytic domain of lambdaPKC. The presence of both the NLS and NES enables a continuous shuttling of lambdaPKC between the cytoplasm and nucleus. Our results suggest that the exposure of the NLS in both lambda- and zetaPKC is regulated by intramolecular interactions between the N-terminal part, including the pseudosubstrate sequence, and the catalytic domain. Thus, either deletion of the N-terminal region, including the pseudosubstrate sequence, or a point mutation in this sequence leads to nuclear accumulation of lambdaPKC. The ability of the two atypical PKC isoforms to enter the nucleus in HeLa cells upon leptomycin B treatment differs substantially. Although lambdaPKC is able to enter the nucleus very rapidly, zetaPKC is much less efficiently imported into the nucleus. This difference can be explained by the different relative strengths of the NLS and NES in lambdaPKC compared with zetaPKC.     

The rules and roles of nucleocytoplasmic shuttling proteins.

The spatial separation of mRNA synthesis from translation, while providing eukaryotes with the possibility to achieve higher complexity through a more elaborate regulation of gene expression, has set the need for transport mechanisms through the nuclear envelope. In a simplistic view of nucleocytoplasmic transport, nuclear proteins are imported into the nucleus while RNAs are exported to the cytoplasm. The reality is, however, that transport of either proteins or RNAs across the nuclear envelope can be bi-directional. During the past years, an increasing number of proteins have been identified that shuttle continuously back and forth between the nucleus and the cytoplasm. The emerging picture is that shuttling proteins are key factors in conveying information on nuclear and cytoplasmic activities within the cell.