Nuclear Localization Signal Activity Research Articles

Characterization of nuclear localization signal in Ostrinia furnacalis Masculinizer protein.

Bombyx mori Masculinizer protein (BmMasc) is essential for both masculinization and dosage compensation in B. mori. We previously identified a bipartite nuclear localization signal (NLS) of BmMasc and two essential residues (lysine at 274 [K274] and arginine at 275 [R275]) implicated in its function. Sequence comparison showed the presence of putative NLSs in lepidopteran Masc proteins, but their functional properties and critical residues are unknown. Here we characterized a putative NLS of Ostrinia furnacalis Masc (OfMasc) using B. mori ovary-derived BmN-4 cell line. Deletion and alanine scanning mutagenesis revealed that a putative NLS is required for nuclear localization of OfMasc. However, mutations at both K227 and R228, which correspond to K274 and R275 of BmMasc, respectively, do not greatly abolish the NLS activity. Additional mutagenesis analysis revealed that triple mutations at K227, R228, and K240 almost completely inhibited OfMasc nuclear localization. These results suggest that lepidopteran Masc proteins possess a common functional NLS, but the critical residues for its activity are different. Moreover, we examined the masculinizing activity of OfMasc derivatives and found that nuclear localization is not required for the masculinizing activity of OfMasc. The results from our studies indicate that lepidopteran Masc proteins function in the cytoplasm to drive masculinizing cascade.

Hsp105α suppresses Adriamycin‐induced cell death via nuclear localization signal‐dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.

Contribution of the residue at position 4 within classical nuclear localization signals to modulating interaction with importins and nuclear targeting

Nuclear import involves the recognition by importin (IMP) superfamily members of nuclear localization signals (NLSs) within protein cargoes destined for the nucleus, the best understood being recognition of classical NLSs (cNLSs) by the IMPα/β1 heterodimer. Although the cNLS consensus [K-(K/R)-X-(K/R) for positions P2–P5] is generally accepted, recent studies indicated that the contribution made by different residues at the P4 position can vary. Here, we apply a combination of microscopy, molecular dynamics, crystallography, in vitro binding, and bioinformatics approaches to show that the nature of residues at P4 indeed modulates cNLS function in the context of a prototypical Simian Virus 40 large tumor antigen-derived cNLS (KKRK, P2–5). Indeed, all hydrophobic substitutions in place of R impaired binding to IMPα and nuclear targeting, with the largest effect exerted by a G residue at P4. Substitution of R with neutral hydrophobic residues caused the loss of electrostatic and van der Waals interactions between the P4 residue side chains and IMPα. Detailed bioinformatics analysis confirmed the importance of the P4 residue for cNLS function across the human proteome, with specific residues such as G being associated with low activity. Furthermore, we validate our findings for two additional cNLSs from human cytomegalovirus (HCMV) DNA polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2–3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.

A novel nuclear localization signal spans the linker of the two DNA-binding subdomains in the conserved paired domain of Pax6.

Paired box (Pax) 6, a member of the Pax family of transcription factors, contains two DNA-binding domains, called the paired domain (PD) and the homeodomain (HD), and plays pivotal roles in development of structures such as the eye, central nervous system and pancreas. Pax6 is a major developmental switching molecule because, for example, ectopic expression of the Pax6 gene can induce ectopic whole eye development. Intensive research has been devoted to elucidating the molecular mechanism(s) involved in the function(s) of Pax6, but many issues remain unexplained. One of the important issues is to identify the nuclear localization signal (NLS) in the PD of Pax6, which is predicted to have a stronger NLS activity than that in the HD. We produced expression plasmid constructs that encode the chick Pax6 protein modified to delete the entire PD except for fragments containing putative NLS sequences, and electroporated them in ovo into the developing chick midbrain to define the NLS of the PD. The results show that the NLS in the PD of chick Pax6 consists of an unusually long sequence of 36 amino acid residues. Within this long NLS motif, the central 18 amino acids comprising two consecutive nine-residue segments showed highest NLS activity; this central area corresponds to the C-terminal half of the third α-helix of the PAI subdomain and the subsequent 11 amino acids of a 16-residue linker between PAI and the adjacent RED subdomain. This information helps to elucidate the molecular mechanism by which Pax6 plays a pivotal role during ontogeny.

The conserved basic residues and the charged amino acid residues at the α-helix of the zinc finger motif regulate the nuclear transport activity of triple C2H2 zinc finger proteins.

Zinc finger (ZF) motifs on proteins are frequently recognized as a structure for DNA binding. Accumulated reports indicate that ZF motifs contain nuclear localization signal (NLS) to facilitate the transport of ZF proteins into nucleus. We investigated the critical factors that facilitate the nuclear transport of triple C2H2 ZF proteins. Three conserved basic residues (hot spots) were identified among the ZF sequences of triple C2H2 ZF proteins that reportedly have NLS function. Additional basic residues can be found on the α-helix of the ZFs. Using the ZF domain (ZFD) of Egr-1 as a template, various mutants were constructed and expressed in cells. The nuclear transport activity of various mutants was estimated by analyzing the proportion of protein localized in the nucleus. Mutation at any hot spot of the Egr-1 ZFs reduced the nuclear transport activity. Changes of the basic residues at the α-helical region of the second ZF (ZF2) of the Egr-1 ZFD abolished the NLS activity. However, this activity can be restored by substituting the acidic residues at the homologous positions of ZF1 or ZF3 with basic residues. The restored activity dropped again when the hot spots at ZF1 or the basic residues in the α-helix of ZF3 were mutated. The variations in nuclear transport activity are linked directly to the binding activity of the ZF proteins with importins. This study was extended to other triple C2H2 ZF proteins. SP1 and KLF families, similar to Egr-1, have charged amino acid residues at the second (α2) and the third (α3) positions of the α-helix. Replacing the amino acids at α2 and α3 with acidic residues reduced the NLS activity of the SP1 and KLF6 ZFD. The reduced activity can be restored by substituting the α3 with histidine at any SP1 and KLF6 ZFD. The results show again the interchangeable role of ZFs and charge residues in the α-helix in regulating the NLS activity of triple C2H2 ZF proteins.

Nuclear localization of a putative Phytophthora sojae bZIP1 transcription factor is mediated by multiple targeting motifs.

Oomycetes are fungal-like eukaryotic microbes in the kingdom Stramenopila. We recently found that the oomycete plant pathogen Phytophthora sojae uses nuclear localization signals (NLSs) for translocation of proteins into the nucleus that differ from conventional well-characterized NLSs from mammals and yeast. Here, we have characterized in depth the NLSs of a P. sojae basic leucine zipper transcription factor, PsbZIP1. Nuclear localization of PsbZIP1 was determined by a central conserved region overlapping the DNA binding domain. Mutational analysis of this region identified four distinct elements that contributed multiplicatively to nuclear localization, but the conserved DNA binding residues were not required. Three of the elements showed autonomous NLS activity and the fourth served as a nuclear localization enhancer. Sequences within two of the nuclear localization elements defined a new form of bipartite NLS consisting of a triplet of basic residues followed by a tail of scattered basic amino acids.

Mapping of the Sequences Directing Localization of the Drosophila Germ Cell-Expressed Protein (GCE).

Drosophila melanogaster germ cell-expressed protein (GCE) belongs to the family of bHLH-PAS transcription factors that are the regulators of gene expression networks that determine many physiological and developmental processes. GCE is a homolog of D. melanogaster methoprene tolerant protein (MET), a key mediator of anti-metamorphic signaling in insects and the putative juvenile hormone receptor. Recently, it has been shown that the functions of MET and GCE are only partially redundant and tissue specific. The ability of bHLH-PAS proteins to fulfill their function depends on proper intracellular trafficking, determined by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). Nevertheless, until now no data has been published on the GCE intracellular shuttling and localization signals. We performed confocal microscopy analysis of the subcellular distribution of GCE fused with yellow fluorescent protein (YFP) and YFP-GCE derivatives which allowed us to characterize the details of the subcellular traffic of this protein. We demonstrate that GCE possess specific pattern of localization signals, only partially consistent with presented previously for MET. The presence of a strong NLS in the C-terminal part of GCE, seems to be unique and important feature of this protein. The intracellular localization of GCE appears to be determined by the NLSs localized in PAS-B domain and C-terminal fragment of GCE, and NESs localized in PAS-A, PAS-B domains and C-terminal fragment of GCE. NLSs activity can be modified by juvenile hormone (JH) and other partners, likely 14-3-3 proteins.

A functional dissection of PTEN N-terminus: implications in PTEN subcellular targeting and tumor suppressor activity.

Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations.

Evolutionary diversification of MCM3 genes in Xenopus laevis and Danio rerio

Embryonic cell cycles of amphibians are rapid and lack zygotic transcription and checkpoint control. At the mid-blastula transition, zygotic transcription is initiated and cell divisions become asynchronous. Several cell cycle-related amphibian genes retain 2 distinct forms, maternal and zygotic, but little is known about the functional differences between these 2 forms of proteins. The minichromosome maintenance (MCM) 2–7 complex, consisting of 6 MCM proteins, plays a central role in the regulation of eukaryotic DNA replication. Almost all eukaryotes retain just a single MCM gene for each subunit. Here we report that Xenopus and zebrafish have 2 copies of MCM3 genes, one of which shows a maternal and the other a zygotic expression pattern. Phylogenetic analysis shows that the Xenopus and zebrafish zygotic MCM3 genes are more similar to their mammalian MCM3 ortholog, suggesting that maternal MCM3 was lost during evolution in most vertebrate lineages. Maternal MCM3 proteins in these 2 species are functionally different from zygotic MCM3 proteins because zygotic, but not maternal, MCM3 possesses an active nuclear localization signal in its C-terminal region, such as mammalian MCM3 orthologs do. mRNA injection experiments in zebrafish embryos show that overexpression of maternal MCM3 impairs proliferation and causes developmental defects, whereas zygotic MCM3 has a much weaker effect. This difference is brought about by the difference in their C-terminal regions, which contain putative nuclear localization signals; swapping the C-terminal region between maternal and zygotic genes diminishes the developmental defects. This study suggests that evolutionary diversification has occurred in MCM3 genes, leading to distinct functions, possibly as an adaption to the rapid DNA replication required for early development of Xenopus and zebrafish.

Functional analysis of the nuclear localization signal of the POU transcription factor Skn-1a in epidermal keratinocytes

POU domain proteins are a family of critical regulators of development and differentiation due to their transcriptional activity in the nucleus. Skn‑1a, a member of the POU domain protein family, appears to be expressed predominantly in epidermal keratinocytes and is thought to play a critical role in keratinocyte differentiation and proliferation. In this study, we examined the mechanisms involved in the nuclear localization of Skn‑1a. We transiently expressed enhanced green fluorescent protein (EGFP) reporter constructs encoding EGFP fusions with Skn‑1a deletion and mutation proteins in normal human epidermal keratinocytes (NHEKs). The experiments clearly demonstrated that Skn‑1a contained a functional nuclear localization signal (NLS) domain, and that the smallest domain necessary for Skn‑1a nuclear transport was the GRKRKKR sequence located within amino acids 279‑285. Previous studies have shown that the phosphorylation of specific amino acids neighboring the NLS may regulate nuclear transport and that the amino acid residues threonine (Thr) and serine (Ser) have the potential to undergo phosphorylation. We examined whether the amino acids Thr286 and Ser287, which reside adjacent to the NLS at the carboxy‑terminal side, play a role in Skn‑1a nuclear localization. For this purpose, we generated three EGFP‑Skn‑1a mutation constructs, in which Thr286, Ser287, or both Thr286 and Ser287 residues were replaced with alanine, respectively. The results showed that the Thr286 and Ser287 residues were involved in the regulation of nuclear localization as well as epidermal differentiation. These results suggested that the epidermal differentiation signaling pathway, involving kinase and phosphatase activation, may regulate the NLS activity of Skn‑1a in keratinocytes. Collectively, these data contribute to understanding the mechanisms of nuclear translocation of POU domain proteins and epidermal differentiation.

Nuclear localization signals in prototype foamy viral integrase for successive infection and replication in dividing cells.

We identified four basic amino acid residues as nuclear localization signals (NLS) in the C-terminal domain of the prototype foamy viral (PFV) integrase (IN) protein that were essential for viral replication. We constructed seven point mutants in the C-terminal domain by changing the lysine and arginine at residues 305, 308, 313, 315, 318, 324, and 329 to threonine or proline, respectively, to identify residues conferring NLS activity. Our results showed that mutation of these residues had no effect on expression assembly, release of viral particles, or in vitro recombinant IN enzymatic activity. However, mutations at residues 305 (R → T), 313(R → T), 315(R → P), and 329(R → T) lead to the production of defective viral particles with loss of infectivity, whereas non-defective mutations at residues 308(R → T), 318(K → T), and 324(K → T) did not show any adverse effects on subsequent production or release of viral particles. Sub-cellular fractionation and immunostaining for viral protein PFV-IN and PFV-Gag localization revealed predominant cytoplasmic localization of PFV-IN in defective mutants, whereas cytoplasmic and nuclear localization of PFV-IN was observed in wild type and non-defective mutants. However sub-cellular localization of PFV-Gag resulted in predominant nuclear localization and less presence in the cytoplasm of the wild type and non-defective mutants. But defective mutants showed only nuclear localization of Gag. Therefore, we postulate that four basic arginine residues at 305, 313, 315 and 329 confer the karyoplilic properties of PFV-IN and are essential for successful viral integration and replication.

Androgen Induces a Switch from Cytoplasmic Retention to Nuclear Import of the Androgen Receptor

The androgen receptor (AR) has critical functions as a transcription factor in both normal and cancer cells, but the specific mechanisms that regulate its nuclear localization are not well defined. We found that an AR mutation commonly reported in prostate cancer generates an androgen-independent gain of function for nuclear import. The substitution, Thr877Ala, is within the ligand-binding domain, but the nuclear import gain of function is mediated by the bipartite nuclear localization signal (NLS) spanning the DNA-binding domain (DBD) and hinge region. Bipartite NLS activity depends on the structure provided by the DBD, and protein interactions with the bipartite NLS are repressed by the hinge region. The bipartite NLS is recognized by importin 7, a nuclear import receptor for several proteins. Importin 7 binding to AR, however, inhibits import by shielding the bipartite NLS. Androgen binding relieves the inhibition by inducing a switch that promotes exchange of importin 7 for karyopherin alpha import receptors. Importin 7 contributes to the regulation of AR import by restraining import until androgen is detected in the cytoplasm.

The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

Dicer is a key player in microRNA (miRNA) and RNA interference (RNAi) pathways, processing miRNA precursors and double-stranded RNA into ∼21-nt-long products ultimately triggering sequence-dependent gene silencing. Although processing of substrates in vertebrate cells occurs in the cytoplasm, there is growing evidence suggesting Dicer is also present and functional in the nucleus. To address this possibility, we searched for a nuclear localization signal (NLS) in human Dicer and identified its C-terminal double-stranded RNA binding domain (dsRBD) as harboring NLS activity. We show that the dsRBD-NLS can mediate nuclear import of a reporter protein via interaction with importins β, 7, and 8. In the context of full-length Dicer, the dsRBD-NLS is masked. However, duplication of the dsRBD localizes the full-length protein to the nucleus. Furthermore, deletion of the N-terminal helicase domain results in partial accumulation of Dicer in the nucleus upon leptomycin B treatment, indicating that CRM1 contributes to nuclear export of Dicer. Finally, we demonstrate that human Dicer has the ability to shuttle between the nucleus and the cytoplasm. We conclude that Dicer is a shuttling protein whose steady-state localization is cytoplasmic.

Identification of a Karyopherin β1/β2 Proline-Tyrosine Nuclear Localization Signal in Huntingtin Protein

Among the known pathways of protein nuclear import, the karyopherin β2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin β2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a β sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin β1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.

Nuclear translocation of adeno-associated virus type 2 capsid proteins for virion assembly

Adeno-associated virus (AAV) capsid assembly occurs in the nucleus. Newly synthesized capsid proteins VP1, VP2 and VP3 contain several basic regions (BRs), which may act as nuclear localization signals (NLSs). Mutation of BR2 and BR3, located at the VP1 and VP2 N termini, marginally reduced nuclear uptake of VP1 or VP2, but not of VP3, when expressed in the context of the whole AAV type 2 (AAV2) genome. Combined mutation of BR1, BR2 and BR3 resulted in capsids with slightly reduced amounts of VP1. Expression of isolated VP1/2 N termini revealed an influence of BR3 on nuclear transport, whilst BR1 or BR2 had no effect. However, deletion of an N-terminal fragment in front of the BR elements strongly reduced nuclear uptake of VP1/2 N termini. Mutation of BR4, present in all three capsid proteins, led to their retention in the cytoplasm and to the formation of speckles, resulting in a lack of capsid formation and a significant reduction in VP levels. In a VP fragment comprising BR2, BR3 and BR4, the BR4 element was not necessary for nuclear localization. Mutation of BR5 in the C-terminal part of the VPs resulted in a speckled protein distribution in the nucleus, strongly reduced capsid assembly, and low VP1 and VP2 levels. Taken together, these results showed that BR2 and BR3 have a weak influence on nuclear transport of VP1 and VP2, whilst combined mutation of BR1, BR2 and BR3 influences the stoichiometry of VPs in assembled capsids. BR4 and BR5 play a crucial role in capsid assembly but have no NLS activity.

Androgen Receptor Splice Variants Activate Androgen Receptor Target Genes and Support Aberrant Prostate Cancer Cell Growth Independent of Canonical Androgen Receptor Nuclear Localization Signal

Synthesis of truncated androgen receptor (AR) splice variants has emerged as an important mechanism of prostate cancer (PCa) resistance to AR-targeted therapy and progression to a lethal castration-resistant phenotype. However, the precise role of these factors at this stage of the disease is not clear due to loss of multiple COOH-terminal AR protein domains, including the canonical nuclear localization signal (NLS) in the AR hinge region. Despite loss of this NLS, we show that diverse truncated AR variant species have a basal level of nuclear localization sufficient for ligand-independent transcriptional activity. Whereas full-length AR requires Hsp90 and importin-β for active nuclear translocation, basal nuclear localization of truncated AR variants is independent of these classical signals. For a subset of truncated AR variants, this basal level of nuclear import can be augmented by unique COOH-terminal sequences that reconstitute classical AR NLS activity. However, this property is separable from ligand-independent transcriptional activity. Therefore, the AR splice variant core consisting of the AR NH(2)-terminal domain and DNA binding domain is sufficient for nuclear localization and androgen-independent transcriptional activation of endogenous AR target genes. Indeed, we show that truncated AR variants with nuclear as well as nuclear/cytoplasmic localization patterns can drive androgen-independent growth of PCa cells. Together, our data demonstrate that diverse truncated AR species with varying efficiencies of nuclear localization can contribute to castration-resistant PCa pathology by driving persistent ligand-independent AR transcriptional activity.

Cofilin Nuclear-Cytoplasmic Shuttling Affects Cofilin-Actin Rod Formation During Stress

Cofilin protein is involved in regulating the actin cytoskeleton during typical steady state conditions, as well as during cell stress conditions where cofilin saturates F-actin, forming cofilin-actin rods. Cofilin can enter the nucleus through an active nuclear localization signal (NLS), accumulating in nuclear actin rods during stress. Here, we characterize the active nuclear export of cofilin through a leptomycin-B-sensitive, CRM1-dependent, nuclear export signal (NES). We also redefine the NLS of cofilin as a bipartite NLS, with an additional basic epitope required for nuclear localization. Using fluorescence lifetime imaging microscopy (FLIM) and Förster resonant energy transfer (FRET) between cofilin moieties and actin, as well as automated image analysis in live cells, we have defined subtle mutations in the cofilin NLS that allow cofilin to bind actin in vivo and affect cofilin dynamics during stress. We further define the requirement of cofilin-actin rod formation in a system of cell stress by temporal live-cell imaging. We propose that cofilin nuclear shuttling is critical for the cofilin-actin rod stress response with cofilin dynamically communicating between the nucleus and cytoplasm during cell stress.

Sequences that direct subcellular traffic of the Drosophila methoprene-tolerant protein (MET) are located predominantly in the PAS domains

Methoprene-tolerant protein (MET) is a key mediator of antimetamorphic signaling in insects. MET belongs to the family of bHLH-PAS transcription factors which regulate gene expression and determine essential physiological and developmental processes. The ability of many bHLH-PAS proteins to carry out their functions is related to the patterns of intracellular trafficking, which are determined by specific sequences and indicate that a nuclear localization signal (NLS) or a nuclear export signal (NES) is present and active. Therefore, the identification of NLS and NES signals is fundamental in order to understand the intracellular signaling role of MET. Nevertheless, data on the intracellular trafficking of MET are inconsistent, and until now there hasn’t been any data on potential NLS and NES sequences. To analyze the trafficking of MET we designed a number of expression vectors encoding full-length MET, as well as various derivatives, that were fused to yellow fluorescent protein (YFP). Confocal microscopy analysis of the subcellular distribution of YFP–MET indicated that while this protein was localized mainly in the nucleus, it was also observed in the cytoplasm. This suggested the presence of both an NLS and NES in MET. Our work has shown that each of the two PAS domains of MET (PAS-A and PAS-B, respectively) contain one NLS and one NES sequence. Additional NES activity was present in the C-terminal fragment. The NLS activity located in PAS-B was dependent on the presence of juvenile hormone (JH), the potential ligand for MET. In contrast to this, JH didn’t seem to be required for the NLS in PAS-A to be active. However, on the basis of current knowledge about the function of PAS-A in other bHLH-PAS proteins, we suggest there might be other proteins that control the activity of the NLS and possibly the NES located in the PAS-A of MET. Thus, the intracellular trafficking of MET seems to be regulated by a rather complicated network of different factors.

Transactivation activity of LBP‐1 proteins and their dimerization in living cells

LBP-1 proteins form dimers and act as transcription factors that activate a number of genes related to cell growth and differentiation. LBP-1a and LBP-1c are localized in the cytoplasm when transiently expressed in cultured cells, but translocated into the nucleus after forming heterodimers with LBP-1b, which is a splicing variant of LBP-1a with an intrinsic nuclear localization signal (NLS). Here, we report that LBP-1b showed potent transactivation activity, and that forcibly expressed LBP-1a and LBP-1c in the nucleus essentially exhibited very little or no transactivation activity. Mutations in the NLS that abolished the NLS activity of LBP-1b also abrogated the transactivation activity. We have found that LBP-1 proteins contain a putative sterile alpha motif domain indispensable for their dimerization capability in the C-terminal region. To demonstrate whether homo- and heterodimers composed of LBP-1a and/or LBP-1c are generated in the nucleus, we applied the FLIM-based fluorescence resonance energy transfer imaging technique to living cells. It revealed that dimers composed of LBP-1a and LBP-1c were re-formed probably by a partner-exchange of LBP-1b-containing heterodimers.

Dbf2–Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis

Exit from mitosis is characterized by a precipitous decline in cyclin-dependent kinase (Cdk) activity, dissolution of mitotic structures, and cytokinesis. In Saccharomyces cerevisiae, mitotic exit is driven by a protein phosphatase, Cdc14, which is in part responsible for counteracting Cdk activity. Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown. In this study, we show that a MEN component, protein kinase Dbf2–Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity. Our results define a mechanism by which the MEN promotes exit from mitosis.