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.     

Mechanisms regulating the nuclear translocation of p38 MAP kinase

p38 mitogen‐activated protein kinase (MAPK) is of fundamental importance in a cell's response to environmental stresses, cytokines and DNA damage. p38 resides in the cytoplasm of resting cells, and translocates into the nucleus upon activation, yet the exact mechanisms remain largely unclear. We show here that the phosphorylation‐dependent nuclear translocation of p38 is a common phenomenon when cells are stimulated with various stresses. On the other hand, the nuclear export of p38 requires its dephosphorylation, and it is exported both in a MK2‐dependent and a nuclear export signal (NES)‐independent manner. Although different p38‐regulated/activated protein kinase (PRAK) mutants all dictate the intracellular localization of p38, results from a PRAK‐deficient cell line indicate that it plays no role in this process. Microtubule depolymerizing reagent nocodazole and dynein inhibitor EHNA both block the nuclear translocation of p38, demonstrating roles for microtubules and dynein in p38 transport. Taken together, stress‐induced nuclear accumulation of p38 is a phosphorylation‐dependent, microtubule‐ and dynein‐associated process. J. Cell. Biochem. 110: 1420–1429, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

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.     

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.     

Both nuclear-cytoplasmic shuttling of the dual specificity phosphatase MKP-3 and its ability to anchor MAP kinase in the cytoplasm are mediated by a conserved nuclear export signal

MAP kinase phosphatase (MKP)-3 is a cytoplasmic dual specificity protein phosphatase that specifically binds to and inactivates the ERK1/2 MAP kinases in mammalian cells. However, the molecular basis of the cytoplasmic localization of MKP-3 or its physiological significance is unknown. We have used MKP-3-green fluorescent protein fusions in conjunction with leptomycin B to show that the cytoplasmic localization of MKP-3 is mediated by a chromosome region maintenance-1 (CRM1)-dependent nuclear export pathway. Furthermore, the nuclear translocation of MKP-3 seen in the presence of leptomycin B is mediated by an active process, indicating that MKP-3 shuttles between the nucleus and cytoplasm. The amino-terminal noncatalytic domain of MKP-3 is both necessary and sufficient for nuclear export of the phosphatase and contains a single functional leucine-rich nuclear export signal (NES). Even though this domain of the protein also mediates the binding of MKP-3 to MAP kinase, we show that mutations of the kinase interaction motif which abrogate ERK2 binding do not affect MKP-3 localization. Conversely, mutation of the NES does not affect either the binding or phosphatase activity of MKP-3 toward ERK2, indicating that the kinase interaction motif and NES function independently. Finally, we demonstrate that the ability of MKP-3 to cause the cytoplasmic retention of ERK2 requires both a functional kinase interaction motif and NES. We conclude that in addition to its established function in the regulated dephosphorylation and inactivation of MAP kinase, MKP-3 may also play a role in determining the subcellular localization of its substrate. Our results reinforce the idea that regulatory proteins such as MKP-3 may play a key role in the spatio-temporal regulation of MAP kinase activity.     

Antiviral properties of combination peptides of HIV-1 Rev NLS and NES

Nuclear translocation signal has been identified as a mediator of protein shuttling between nuclear and cytoplasm. Here we report that the combination of peptides from nuclear localization signal (NLS) and nuclear export signal (NES) of HIV-1 Rev have an antiviral activity against the Herpes virus of turkey and Marek's disease virus serotype 1.     

Identification of intracellular localization signals and of mechanisms underlining the nucleocytoplasmic shuttling of human aryl hydrocarbon receptor repressor

Two members of the ‘AhR family’ (a family which is part of the bHLH-PAS superfamily), aryl hydrocarbon receptor (AhR) and AhR repressor (AhRR), originated from a common ancestor and form a regulatory circuit in xenobiotic signal transduction. AhRR is a nucleocytoplasmic shuttle protein, harboring both a nuclear localization signal (NLS) and a nuclear export signal (NES). Because NLS is dominant over NES, AhRR resides predominantly in the nuclear compartment. The NES of AhRR resembles that of AhR in sensitivity to leptomycin B, whereas the NLS of AhRR is monopartite and is, therefore, distinguished from the reported bipartite NLS of AhR. The NLS deletion mutant of GFP-AhRR was transported into the nuclear compartment in the presence of AhR nuclear translocator (Arnt), suggesting the assembly of an AhRR/Arnt heterodimer complex in the cytoplasmic compartment and Arnt-dependent nuclear translocation of this complex.     

Nucleocytoplasmic transport of proteins

In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)--a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.     

Regulation of PRAK subcellular location by p38 MAP kinases

The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. p38 regulated/activated protein kinase (PRAK, also known as mitogen-activated protein kinase activated protein kinase 5 [MAPKAPK5]) functions downstream of p38alpha and p38beta in mediating the signaling of the p38 pathway. Immunostaining revealed that endogenous PRAK was predominantly localized in the cytoplasm. Interestingly, ectopically expressed PRAK was localized in the nucleus and can be redistributed by coexpression of p38alpha or p38beta to the locations of p38alpha and p38beta. Mutations in the docking groove on p38alpha/p38beta, or the p38-docking site in PRAK, disrupted the PRAK-p38 interaction and impaired the ability of p38alpha and p38beta to redistribute ectopically expressed PRAK, indicating that the location of PRAK could be controlled by its docking interaction with p38alpha and p38beta. Although the majority of PRAK molecules were detected in the cytoplasm, PRAK is consistently shuttling between the cytoplasm and the nucleus. A sequence analysis of PRAK shows that PRAK contains both a putative nuclear export sequence (NES) and a nuclear localization sequence (NLS). The shuttling of PRAK requires NES and NLS motifs in PRAK and can be regulated through cellular activation induced by stress stimuli. The nuclear content of PRAK was reduced after stimulation, which resulted from a decrease in the nuclear import of PRAK and an increase in the nuclear export of PRAK. The nuclear import of PRAK is independent from p38 activation, but the nuclear export requires p38-mediated phosphorylation of PRAK. Thus, the subcellular distribution of PRAK is determined by multiple factors including its own NES and NLS, docking interactions between PRAK and docking proteins, phosphorylation of PRAK, and cellular activation status. The p38 MAPKs not only regulate PRAK activity and PRAK activation-related translocation, but also dock PRAK to selected subcellular locations in resting cells.     

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.     

Transport of galectin-3 between the nucleus and cytoplasm. II. Identification of the signal for nuclear export

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. Previous studies have shown that incubation of fibroblasts with leptomycin B resulted in the accumulation of galectin-3 in the nucleus, suggesting that the export of galectin-3 from the nucleus may be mediated by the CRM1 receptor. A candidate nuclear export signal fitting the consensus sequence recognized by CRM1 can be found between residues 240 and 255 of the murine galectin-3 sequence. This sequence was engineered into the pRev(1.4) reporter system, in which candidate sequences can be tested for nuclear export activity in terms of counteracting the nuclear localization signal present in the Rev(1.4) protein. Rev(1.4)-galectin-3(240-255) exhibited nuclear export activity that was sensitive to inhibition by leptomycin B. Site-directed mutagenesis of Leu247 and Ile249 in the galectin-3 nuclear export signal decreased nuclear export activity, consistent with the notion that these two positions correspond to the critical residues identified in the nuclear export signal of the cAMP-dependent protein kinase inhibitor. The nuclear export signal activity was also analyzed in the context of a full-length galectin-3 fusion protein; galectin-3(1-263; L247A) showed more nuclear localization than wild-type, implicating Leu247 as critical to the function of the nuclear export signal. These results indicate that residues 240-255 of the galectin-3 polypeptide contain a leucine-rich nuclear export signal that overlaps with the region (residues 252-258) identified as important for nuclear localization.     

Nuclear import and export signals in control of Nrf2

Nrf2 binds to the antioxidant response element and regulates expression and antioxidant induction of a battery of chemopreventive genes. In this study, we have identified nuclear import and export signals of Nrf2 and show that the nuclear import and export of Nrf2 is regulated by antioxidants. We demonstrate that Nrf2 contains a bipartite nuclear localization signal (NLS) and a leucine-rich nuclear export signal, which regulate Nrf2 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nrf2 accumulates in the nucleus within 15 min of antioxidant treatment and is exported out of nucleus by 8 h after treatment. Nrf2 mutant lacking the NLS failed to enter the nucleus and displayed diminished expression and induction of the downstream NAD(P)H:quinone oxidoreductase 1 gene. The Nrf2 NLS sequence, when fused to green fluorescence protein, resulted in the nuclear accumulation of green fluorescence protein, indicating that this signal sequence was sufficient to direct nuclear localization of Nrf2. A nuclear export signal (NES) was characterized in the C terminus of Nrf2, the deletion of which caused Nrf2 to accumulate predominantly in the nucleus. The Nrf2 NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Further studies demonstrate that NES-mediated nuclear export of Nrf2 is required for degradation of Nrf2 in the cytosol. These results led to the conclusion that Nrf2 localization between cytosol and nucleus is controlled by both nuclear import and export of Nrf2, and the overall distribution of Nrf2 is probably the result from a balance between these two processes. Antioxidants change this balance in favor of nuclear accumulation of Nrf2, leading to activation of chemopreventive proteins. Once this is achieved, Nrf2 exits the nucleus for binding to INrf2 and degradation.