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PubMed2003

Translocation of N-WASP by nuclear localization and export signals into the nucleus modulates expression of HSP90.

N-WASP regulates the actin cytoskeleton through activation of the Arp2/3 complex. N-WASP localizes at the cell periphery, where it controls actin polymerization downstream of signal molecules such as adapter proteins, Cdc42, Src family kinases, and phosphoinositides. N-WASP also localizes in the nucleus; however, the role of N-WASP in the nucleus is unclear. Here, we show that localization of N-WASP is controlled through phosphorylation by Src family kinases in which phosphorylated N-WASP is exported from the nucleus in a nuclear export signal (NES) and leptomycin B-dependent manner. N-WASP had nuclear localization signal (NLS) at its basic region and NES close to the phosphorylation site by Src family kinases, indicating that phosphorylation controls the accessibility to the NES through conformational changes. Increased levels of unphosphorylated N-WASP in the nucleus suppressed expression of HSP90 and transcription from a heat shock element (HSE). N-WASP bound heat shock transcription factor (HSTF) and enhanced the HSTF association with HSE. In addition, nuclear N-WASP was present in the protein complex that associates with HSE, suggesting that N-WASP participates in suppression of HSP90 transcription. Increased levels of unphosphorylated N-WASP also decreased the activities of Src family kinases in cells but not in experiments in vitro with pure N-WASP and Fyn. Because HSP90 is essential for the activities of Src family kinases, these results suggest that localization of N-WASP modulates Src kinase activity by regulating HSP90 expression.

Active Transport, Cell Nucleus↗
PubMed1994

Competition between nuclear localization and secretory signals determines the subcellular fate of a single CUG-initiated form of FGF3.

The presumed open reading frame for mouse FGF3, starting at the most 5' AUG codon, predicts a hydrophobic N-terminus characteristic of a signal peptide for secretion. However, in reticulocyte lysates and transfected COS-1 cells, the full-length Fgf-3 cDNA is translated almost exclusively from an upstream CUG codon. The resultant products are distributed in both the nucleus and the secretory pathway, implying that the single CUG-initiated form of FGF3 has dual fates. By analysing a series of deletion and replacement mutants and by linking parts of FGF3 to a heterologous protein, we show that secretion is mediated by cleavage adjacent to the previously defined signal peptide, whereas nuclear localization is determined primarily by a classical but relatively weak bipartite motif. In the context of FGF3, nuclear localization also requires the N-terminal sequences which lie upstream of the signal peptide. Thus, the subcellular fate of FGF3 is determined by the competing effects of signals for secretion and nuclear localization within the same protein, rather than by alternative initiation or processing.

Amino Acid Sequence↗
PubMed2004

Identification of a nuclear targeting signal in YopM from Yersinia spp.

YopM is a type III secretion effector from Yersinia which contributes to pathogenicity but whose action still remains unclear. It is an acidic, leucine-rich repeats (LRR) containing protein which migrates to the nucleus of target cells in spite of the fact that it does not contain any classical nuclear localization signal (NLS). Using a yeast approach, we observed that the three first LRRs (LRR1-3) and the 32 C-terminal residues of YopM (YopMC-ter) act as NLSs in yeast. Furthermore, by transfection of HEK293T cells, we observed that YopMC-ter could direct large recombinant EGFP-LexA-AD proteins into the nucleus of mammalian cells confirming that it contains a NLS. Critical residues for nuclear targeting were identified by site-directed mutagenesis in YopMC-ter. In addition, we show that YopMC-ter NLS is crucial for the nuclear targeting of an EGFP-YopM fusion protein.

Active Transport, Cell Nucleus↗
PubMed2000

Ribosomal S6 kinase 2 inhibition by a potent C-terminal repressor domain is relieved by mitogen-activated protein-extracellular signal-regulated kinase kinase-regulated phosphorylation.

Ribosomal S6 kinase 2 (S6K2) is a recently identified serine/threonine protein kinase that phosphorylates the 40 S ribosomal protein S6 in vitro. S6K2 is highly homologous to S6K1 in the core kinase and linker regulatory domains but differs from S6K1 in the N- and C-terminal regions and is differently localized primarily to the nucleus because of a C-terminal nuclear localization signal unique to S6K2. We have recently demonstrated that S6K2 is regulated similarly to S6K1 by the mammalian target of rapamycin pathway and by multiple PI3-K pathway effectors in vivo. However, deletion of the C-terminal domain of S6K2 enhances kinase activity, whereas analogous deletion of S6K1 is inhibitory. Here, we characterize the S6K2 C-terminal motifs that confer this differential regulation. We demonstrate that the inhibitory effects of the S6K2 C-terminal domain are only partly attributable to the nuclear localization signal but that three C-terminal proline-directed potential mitogen-activated protein kinase phosphorylation sites are critical mediators of this inhibitory effect. Site-specific mutation of these sites to alanine completely desensitizes S6K2 to activating inputs, whereas mutation to aspartic acid to mimic phosphorylation results in an activated enzyme which is hypersensitive to activating inputs. Pretreatment of cells with the mitogen-activated protein-extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 inhibited S6K2 activation to a greater extent than S6K1. Furthermore, S6K2 mutants with C-terminal deletion or acidic phosphorylation site mutations displayed greatly reduced U0126 sensitivity. Thus, MEK-dependent inputs to C-terminal phosphorylation sites appear to be essential for relief of S6K2 inhibition but less critical for activation of S6K1. These data suggest a mechanism by which weak PI3-K agonists can regulate S6 phosphorylation and selective translation in the presence of mitogen-activated protein kinase signaling.

Butadienes↗
PubMed2004

Precisely controlled intracellular delivery of DNA-peptide conjugates.

Precisely controlled intracellular delivery of oligonucleotides was achieved by conjugation with signal peptides and amphiphilic designed peptides. Localization in the cellular nucleus was accelerated by nuclear localizing signals derived from naturally occurring viral proteins and arginine rich designed peptides, and cytoplasmic localization was enhanced by nuclear export signals and membrane fusion peptides.

Amino Acid Sequence↗
PubMed1999

Transcription factors and their genes in higher plants functional domains, evolution and regulation.

A typical plant transcription factor contains, with few exceptions, a DNA-binding region, an oligomerization site, a transcription-regulation domain, and a nuclear localization signal. Most transcription factors exhibit only one type of DNA-binding and oligomerization domain, occasionally in multiple copies, but some contain two distinct types. DNA-binding regions are normally adjacent to or overlap with oligomerization sites, and their combined tertiary structure determines critical aspects of transcription factor activity. Pairs of nuclear localization signals exist in several transcription factors, and basic amino acid residues play essential roles in their function, a property also true for DNA-binding domains. Multigene families encode transcription factors, with members either dispersed in the genome or clustered on the same chromosome. Distribution and sequence analyses suggest that transcription factor families evolved via gene duplication, exon capture, translocation, and mutation. The expression of transcription factor genes in plants is regulated at transcriptional and post-transcriptional levels, while the activity of their protein products is modulated post-translationally. The purpose of this review is to describe the domain structure of plant transcription factors, and to relate this information to processes that control the synthesis and action of these proteins.

Amino Acid Sequence↗
PubMed1998

The Treacher Collins syndrome (TCOF1) gene product, treacle, is targeted to the nucleolus by signals in its C-terminus.

The TCOF1 gene product, treacle, responsible for the craniofacial disorder Treacher Collins syndrome, has been predicted to be a member of a class of nucleolar phosphoproteins based on its primary amino acid sequence. Treacle is a low complexity protein with ten repeating units of acidic and basic residues, each of which contains a large number of putative casein kinase 2 and protein kinase C phosphorylation sites. In addition, the C-terminus of treacle contains multiple putative nuclear localization signals. The overall structure of treacle, as well as sequence similarity to several nucleolar phosphoproteins, predicts that treacle is a member of this class of proteins. Using green fluorescent protein fusion constructs with the full-length and deleted domains of the murine homolog of treacle, we demonstrate that the cellular localization of treacle is nucleolar. This localization is mediated by the last 41 residues of the C-terminus (residues 1262-1302). At least two functional nuclear localization signals have been identified in the protein, one between residues 1176 and 1270 and the second within the last 32 residues of the protein (1271-1302). The nucleolar localization signal is disrupted by two constructs that split the C-terminal region between residues 1270 and 1271. This study provides the first direct analysis of treacle and demonstrates that the protein involved in TCOF1 is a nucleolar protein.

Amino Acid Sequence↗
PubMed2001

Impaired DNA damage response in cells expressing an exon 11-deleted murine Brca1 variant that localizes to nuclear foci.

Both human and mouse cells express an alternatively spliced variant of BRCA1, BRCA1-Delta11, which lacks exon 11 in its entirety, including putative nuclear localization signals. Consistent with this, BRCA1-Delta11 has been reported to reside in the cytoplasm, a localization that would ostensibly preclude it from playing a role in the nuclear processes in which its full-length counterpart has been implicated. Nevertheless, the finding that murine embryos bearing homozygous deletions of exon 11 survive longer than embryos that are homozygous for Brca1 null alleles suggests that exon 11-deleted isoforms may perform at least some of the functions of Brca1. We have analyzed both the full-length and the exon 11-deleted isoforms of the murine Brca1 protein. Our results demonstrate that full-length murine Brca1 is identical to human BRCA1 with respect to its cell cycle regulation, DNA damage-induced phosphorylation, nuclear localization, and association with Rad51. Surprisingly, we show that endogenous Brca1-Delta11 localizes to discrete nuclear foci indistinguishable from those found in wild-type cells, despite the fact that Brca1-Delta11 lacks previously defined nuclear localization signals. However, we further show that DNA damage-induced phosphorylation of Brca1-Delta11 is significantly reduced compared to full-length Brca1, and that gamma irradiation-induced Rad51 focus formation is impaired in cells in which only Brca1-Delta11 is expressed. Our results suggest that the increased viability of embryos bearing homozygous deletions of exon 11 may be due to expression of Brca1-Delta11 and suggest an explanation for the genomic instability that accompanies the loss of full-length Brca1.

Alternative Splicing↗
PubMed2004

Importin 7 and importin alpha/importin beta are nuclear import receptors for the glucocorticoid receptor.

The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin alpha selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin alpha-importin beta heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin alpha bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding.

Active Transport, Cell Nucleus↗
PubMed2003

Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3.

Numerous cellular proteins are able to localize to the nucleus due to the fact that they possess a nuclear localization signal (NLS) in their amino acid sequence. Nuclear localization sequences recognized by the importin alpha/beta heterodimer are found in cellular proteins capable of performing many diverse functions, ranging from chromatin remodeling to cell cycle regulation. Evidence has been presented that suggests individual importin alpha homologues are present at varying levels in different adult tissues. Other data have shown that specific subsets of NLSs found in different cellular proteins are recognized by individual importin alpha homologues with varying affinities. This evidence led us to hypothesize that due to the specific cargoes they carry, the mammalian embryo has different developmental requirements for individual importin alpha homologues. The results of the studies presented here indicate that importin alpha/beta-mediated import occurs throughout early cleavage in the porcine embryo, as determined by a reporter protein microinjection assay, and that multiple importin alpha homologues are present throughout early cleavage, as determined by immunocytochemical analysis. An RNA interference approach was used in an attempt to determine the developmental requirements for specific importin alpha homologues during early cleavage in the porcine embryo. Results from this study showed that fertilized porcine embryos injected with double stranded RNA (dsRNA) corresponding to the importin alpha homologue karyopherin alpha3 had significantly fewer nuclei following four days of culture than did embryos injected with dsRNA for another importin alpha homologue, karyopherin alpha2, or two control groups. This is the first report indicating that mammalian embryos may have differential developmental requirements for specific nuclear trafficking pathways.

Animals↗
PubMed1992

Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is a widespread receptor-coupled signalling system at the plasma membrane of most eukaryotic cells. The existence of an entirely separate nuclear phosphoinositide signalling system is suggested from evidence that purified nuclei synthesize PtdInsP2 and phosphatidylinositol 4-phosphate (PtdInsP) in vitro and that a transient decrease in the mass of these lipids occurs when Swiss 3T3 cells are cultured in the presence of insulin-like growth factor-1 (IGF-1). These IGF-1-dependent changes in inositol lipids coincide with an increase in nuclear diacyglycerol and precede translocation to the nucleus and activation of protein kinase C (refs 5, 6). Circumstantial evidence that links these changes with mitosis comes from the isolation of a 3T3 clone that expresses the type-1 IGF receptor and binds IGF-1 peptide but does not respond mitogenically or show transient mass changes in nuclear inositol lipids. A key question is how IGF-1 initiates the rapid breakdown of PtdInsP and PtdInsP2 in the nucleus. Here we present evidence that nuclei of 3T3 cells contain the beta-isozyme of phosphoinositidase C, whereas the gamma-isozyme is confined to the cytoplasm and that IGF-1 treatment stimulates exclusively the activity of nuclear phosphoinositidase C.

3T3 Cells↗
PubMed2001

TaCRK3 encodes a novel Theileria annulata protein kinase with motifs characteristic of the family of eukaryotic cyclin dependent kinases: a comparative analysis of its expression with TaCRK2 during the parasite life cycle.

The TaCRK3 gene from the bovine apicomplexan parasite Theileria annulata, encodes a 46 kDa polypeptide with strong homology to the eukaryotic family of cyclin-dependent kinases. TaCRK3 does not show significant alignment with any particular CDK group, other than the Pfmrk kinases from the related apicomplexans Plasmodium falciparum and Plasmodium yoelii. It has a putative bipartite nuclear localization signal and is located to parasite nuclei by IFAT. Protein levels are constitutive throughout differentiation of the intra-lymphocytic macroschizont. This contrasts with the expression pattern of TaCRK2 (Kinnaird et al., 1996, Mol. Microbiol., 22, 293-302) which is closely related to the eukaryotic CDK1 /2 families involved in regulation of cell cycle progression. TaCRK2 is also located to the parasite nuclei but has no nuclear localization signal and exhibits transient up-regulation in protein levels during mid-merogony. However compared to TaCRK3, it shows down-regulation near the end of merogony. We predict that TaCRK3 may have a role in regulation of gene transcription while TaCRK2 is more likely to be involved in control of parasite nuclear division.

Amino Acid Motifs↗
PubMed2001

Karyopherins and nuclear import.

Proteins of the karyopherin alpha and karyopherin beta families play a central role in nucleocytoplasmic transport. Recently, crystal structures of karyopherin alpha and its complexes with nuclear localization signal peptides, a karyopherin beta2-Ran complex and complexes of full-length and fragments of karyopherin beta1 with import substrates, Ran and nucleoporins have been solved. These karyopherin structures provide valuable insights into understanding the molecular mechanism of nuclear import, especially substrate recognition, substrate release by GTPase and interactions with the nuclear pore complex.

Active Transport, Cell Nucleus↗
PubMed1997

A novel class of RanGTP binding proteins.

The importin-alpha/beta complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of approximately 20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-beta. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-beta, it prevents the activation of Ran's GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-beta, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.

Amino Acid Sequence↗
PubMed1997

Import of human and Thermoplasma 20S proteasomes into nuclei of HeLa cells requires functional NLS sequences.

Proteasomes are present both in the nucleus and cytoplasm of eukaryotic cells. Their localization is regulated and changes during the cell cycle. Nuclear localization signal (NLS) type sequences were identified in proteasomes from various organisms. In addition, acidic complementary sequences were identified (cNLS) which could interact with the positively charged NLS, masking or unmasking them and thereby modulating nuclear import. In this paper we show that fluorescently labeled human erythrocyte 20S proteasomes accumulate in the nucleus of digitonin-permeabilized cells. This translocation is ATP-dependent and occurs through the nuclear pore complex as is shown by blocking of the nuclear pores with wheat germ agglutinin. In addition, we used 20S proteasomes from Thermoplasma acidophilum as a model system. Recombinant 20S proteasomes from the archaebacterium Thermoplasma acidophilum are imported into nuclei of HeLa and 3T3 cells similar to their eukaryotic counterpart. We constructed mutants in the putative NLS and cNLS region to study their effect on import. The NLS mutant was not imported into nuclei and showed cytoplasmic staining only. This indicates that this sequence is indeed responsible for nuclear targeting. Mutational studies of the cNLS do not support the involvement of this sequence in regulation of nuclear transport.

3T3 Cells↗
PubMed2000

Acetylation by PCAF enhances CIITA nuclear accumulation and transactivation of major histocompatibility complex class II genes.

The class II transactivator (CIITA), the master regulator of the tissue-specific and interferon gamma-inducible expression of major histocompatibility complex class II genes, synergizes with the histone acetylase coactivator CBP to activate gene transcription. Here we demonstrate that in addition to CBP, PCAF binds to CIITA both in vivo and in vitro and enhances CIITA-dependent transcriptional activation of class II promoters. Accordingly, E1A mutants defective for PCAF or CBP interaction show reduced ability in suppressing CIITA activity. Interestingly, CBP and PCAF acetylate CIITA at lysine residues within a nuclear localization signal. We show that CIITA is shuttling between the nucleus and cytoplasm. The shuttling behavior and activity of the protein are regulated by acetylation: overexpression of PCAF or inhibition of cellular deacetylases by trichostatin A increases the nuclear accumulation of CIITA in a manner determined by the presence of the acetylation target lysines. Furthermore, mutagenesis of the acetylated residues reduces the transactivation ability of CIITA. These results support a novel function for acetylation, i.e., to regulate gene expression by stimulating the nuclear accumulation of an activator.

Acetylation↗
PubMed2002

The NH2-terminal domain of Golgin-160 contains both Golgi and nuclear targeting information.

Golgin-160 is a member of the golgin family of Golgi-localized membrane proteins. The COOH-terminal two-thirds of golgin-160 is predicted to form a coiled-coil, with an NH(2)-terminal "head" domain. To identify the Golgi targeting information in golgin-160, full-length and deletion constructs tagged with green fluorescent protein were generated. The head domain alone was targeted to the Golgi complex in the absence of assembly with endogenous golgin-160. Further truncations from both ends of the head domain narrowed the Golgi targeting information to 85 amino acids between residues 172 and 257. Surprisingly, certain truncations of the head domain also specifically accumulated in the nucleus. Both a nuclear localization signal (masked in the full-length protein) and information for nuclear retention contributed to the nuclear localization of these truncations. Because the golgin-160 head is cleaved by caspases during apoptosis, we examined the localization of epitope-tagged proteins corresponding to all potential caspase cleavage fragments. Our data suggest that three of six fragments could be targeted to the nucleus, provided that they are released from Golgi membranes after cleavage. The finding that both Golgi and nuclear targeting information is present in the same region of golgin-160 suggests that this protein may have more than one function.

Active Transport, Cell Nucleus↗