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Results for “Nuclear Localization Signals”

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PubMed2002

Inhibition of nuclear transport of caspase-7 by its prodomain.

Apoptosis is a major form of cell death, characterized by a series of morphological changes induced by cleaving cytoplasmic and nuclear proteins via active caspases. The data presented here show, by fluorescence microscopic and immunoblotting analyses, that a prodomain of caspase-7 inhibits its nuclear translocation and apoptosis-inducing activity. This nuclear localization is dependent on the presence of a basic tetrapeptide that is conserved in mammalian and Xenopus caspase-7 and that is located downstream of a cleavage site between a prodomain and a catalytic protease domain. Furthermore, an attachment of the caspase-7 prodomain (31 amino acids) represses the nuclear transport of a fusion protein of a heterologous protein and the caspase-7 nuclear localization signal (19 amino acids), suggesting that the inhibition of nuclear localization by the prodomain is mediated by the interaction of these short peptides.

Active Transport, Cell Nucleus↗

PubMed2004

Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germline transmission potency.

Embryonic stem (ES) cells are an important tool in developmental biology, genomics, and transgenic methods, as well as in potential clinical applications such as gene therapy or tissue engineering. Electroporation is the standard transfection method for mouse ES (mES) cells because lipofection is quite inefficient. It is also unclear if mES cells treated with cationic lipids maintain pluripotency. We have developed a simple lipofection method for high efficiency transfection and stable transgene expression by employing the nonclassical nuclear localization signal M9 derived from the heterogeneous nuclear ribonucleoprotein A1. In contrast to using 20 microg DNA for 10 x 10(6) cells via electroporation which resulted in 10-20 positive cells/mm2, M9-assisted lipofection of 2 x 10(5) cells with 2 microg DNA resulted in > 150 positive cells/mm2. Electroporation produced only 0.16% EGFP positive cells with fluorescence intensity (FI) > 1000 by FACS assay, while M9-lipofection produced 36-fold more highly EGFP positive cells (5.75%) with FI > 1000. Using 2.5 x 10(6) ES cells and 6 microg linearized DNA followed by selection with G418, electroporation yielded 17 EGFP expressing colonies, while M9-assisted lipofection yielded 72 EGFP expressing colonies. The mES cells that stably expressed EGFP following M9-assisted lipofection yielded > 66% chimeric mice (8 of 12) and contributed efficiently to the germline. In an example of gene targeting, a knock-in mouse was produced from an ES clone screened from 200 G418-resistant colonies generated via M9-assisted lipofection. To our knowledge, this is the first report of generation of transgenic or knock-in mice obtained from lipofected mES cells and this method may facilitate large scale genomic studies of ES developmental biology or large scale generation of mouse models of human disease.

Animals↗

PubMed2004

Correlation between nucleocytoplasmic transport and caspase-3-dependent dismantling of nuclear pores during apoptosis.

During apoptosis (also called programmed cell death), the chromatin condenses and the DNA is cleaved into oligonucleosomal fragments. Caspases are believed to play a major role in nuclear apoptosis. However, the relation between dismantling of nuclear pores, disruption of the nucleocytoplasmic barrier, and nuclear entry of caspases is unclear. We have analyzed nuclear import of the green fluorescent protein fused to a nuclear localization signal (GFP-NLS) in tissue culture cells undergoing apoptosis. Decreased nuclear accumulation of GFP-NLS could be detected at the onset of nuclear apoptosis manifested as dramatic condensation and redistribution of chromatin toward the nuclear periphery. At this step, dismantling of nuclear pores was already evident as indicated by proteolysis of the nuclear pore membrane protein POM121. Thus, disruption of nuclear compartmentalization correlated with early signs of nuclear pore damage. Both these events clearly preceded massive DNA fragmentation, detected by TUNEL assay. Furthermore, we show that in apoptotic cells, POM121 is specifically cleaved at aspartate-531 in its large C-terminal portion by a caspase-3-dependent mechanism. Cleavage of the C-terminal portion of POM121, which is adjoining the nuclear pore complex, is likely to disrupt interactions with other nuclear pore proteins affecting the stability of the pore complex. A temporal correlation of apoptotic events supports a model where caspase-dependent disassembly of nuclear pores and disruption of the nucleocytoplasmic barrier paves the way for nuclear entry of caspases and subsequent activation of CAD-mediated DNA fragmentation.

Active Transport, Cell Nucleus↗

PubMed1993

Nuclear import of the human androgen receptor.

Nuclear import of the human androgen receptor was investigated by immunocytochemical analysis of androgen receptor deletion and substitution mutants, which were transiently expressed in COS-1 cells. The signal responsible for nuclear import is encoded by amino-acid residues 608-625 and is functionally similar to the bipartite nucleoplasmin nuclear-localization signal. Although the subcellular distribution of androgen receptors mutated in the DNA-binding domain was unchanged compared with the wild-type androgen receptor, in the presence of ligand these mutations resulted in part of the receptor population forming clusters. Depending on the presence or absence of the bipartite nuclear localization signal, clusters were formed in the nucleus or in the cytoplasm, respectively. Expression of the wild-type androgen receptor in different cell lines revealed a cell-line-specific subcellular distribution of the unliganded receptor. The androgen receptor was predominantly nuclear when expressed in HeLa cells, whereas mainly cytoplasmic staining was observed when it was expressed in COS-1 cells. In the presence of hormone, the androgen receptor was located in the nucleus, independent of the cell line that was expressing the receptor. Anti-androgens and various steroid hormones induced the nuclear localization of the wild-type androgen receptor in a dose-dependent way, without activating transcription of an androgen-regulated reporter gene. This indicates that the inability of the tested compounds to activate transcription is not due to inhibited nuclear import.

Amino Acid Sequence↗

PubMed2003

Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination.

As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N- and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.

Active Transport, Cell Nucleus↗

PubMed1996

Nuclear multicatalytic proteinase subunit RRC3 is important for growth regulation in hepatocytes.

Multicatalytic proteinases (MCPs) are macromolecular structures involved in the intracellular degradation of many types of proteins. MCPs are composed of a 20S "core" of both structural (alpha) and presumed catalytic (beta) subunits, in association with regulatory proteins. They are characteristically found in both the nucleus and cytoplasm of cells, although mechanisms governing the subcellular distribution of MCPs are not known. RRC3, an alpha subunit of rat MCPs, contains both a putative nuclear localization signal (NLS) and a potential tyrosine phosphorylation site which could play a role in nuclear import, and the nuclear form of RRC3 appears to be involved in the regulation of cell growth. Here we have generated a variety of RRC3 expression constructs to study features of RRC3 important in nuclear localization and cell growth. PCR was utilized to develop constructs containing point mutations in either the putative NLS (K51 mutated to A) or at a potential tyrosine phosphorylation site (Y121 mutated to F), and an epitope from influenza hemagglutinin (HA) was added in triplicate to the C-terminus of the constructs as a means of identification. RRC3 constructs were then made in which the nucleotide sequence near the translation initiation site of RRC3 was modified in such a way that the amino acid sequence of the protein translated from the constructs is unchanged from that of normal RRC3, thus allowing differential modulation of endogenous RRC3 with antisense oligonucleotide treatment. These N-terminally modified constructs are designated mC3, mC3NLS, and mC3y. In vitro transcription/translation reactions with these constructs produced the expected products, which were immunoprecipitated with a mouse monoclonal anti-HA antibody. Immunohistochemical studies with hepatocyte cell lines transiently transfected with either mC3NLS or mC3y showed only cytoplasmic staining, whereas cells transfected with mC3 had a staining pattern typical of endogenous RRC3 (both cytoplasmic and nuclear) with strong staining of the nuclear perimeter. Immunoblot analyses of subcellular fractions from stably transfected CWSV1 cells showed mC3 product in both the cytosol and nucleus of cells, whereas mC3NLS or mC3y products were restricted to the cytosol. CWSV1 cells stably transfected with the pTet-Splice vector containing no insert (as a control) were markedly inhibited (80%) in cell growth and showed altered morphology when treated with antisense oligonucleotides targeted to endogenous RRC3, reproducing previous studies. Similarly, CWSV1 cells stably transfected with either mC3NLS or mC3y constructs showed analogous growth inhibition and morphologic alteration upon antisense treatment. In contrast, CWSV1 cells stably transfected with the mC3 construct showed normal growth and morphology following antisense oligonucleotide treatment, demonstrating that replenishment of nuclear RRC3 was necessary and sufficient to relieve growth inhibition. In 32P-metabolic labeling studies, mC3 was tyrosine-phosphorylated in cytosol as the full-length protein (M(r) 36000). mC3NLS was also phosphorylated in cytosol, whereas mC3y was not. Nuclear mC3 showed phosphorylation of a M(r) 27000 processed form while neither mC3NLS nor mC3y showed any phosphorylated nuclear products. Our results show that nuclear RRC3 is important in control of cell growth and that both the NLS and Y121 are important in nuclear localization of RRC3. Control of nuclear import by tyrosine phosphorylation may represent a novel regulatory mechanism, and our results further suggest that RRC3 may travel as a maverick subunit.

Animals↗

PubMed2002

Functional domains and DNA-binding sequences of RFLAT-1/KLF13, a Krüppel-like transcription factor of activated T lymphocytes.

RFLAT-1/KLF13, a member of the Krüppel-like family of transcription factors, was identified as a transcription factor expressed 3-5 days after T lymphocyte activation. It binds to the promoter of the chemokine gene RANTES (regulated on activation normal T cell expressed and secreted) and regulates its "late" expression in activated T-cells. In this study, a series of experiments to define the functional domains of RFLAT-1/KLF13 were undertaken to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this factor. Using the GAL4 fusion system, distinct transcriptional activation and repression domains were identified. The RFLAT-1 minimum activation domain is localized to amino acids 1-35, whereas the repression domain resides in amino acids 67-168. Deletion analysis on the RFLAT-1 protein further supports these domain functions. The RFLAT-1 activation domain is similar to that of its closest family member, basic transcription element-binding protein 1. This domain is highly hydrophobic, and site-directed mutagenesis demonstrated that both negatively charged and hydrophobic residues are important for transactivation. The nuclear localization signal of RFLAT-1 was also identified using the RFLAT-1/green fluorescence protein fusion approach. RFLAT-1 contains two potent, independent nuclear localization signals; one is immediately upstream of the zinc finger DNA-binding domain, and the other is within the zinc fingers. Using mutational analysis, we also determined that the critical binding sequence of RFLAT-1 is CTCCC. The intact CTCCC box on the RANTES promoter is necessary for RFLAT-1-mediated RANTES transcription and is also required for the synergy between RFLAT-1 and NF-kappaB proteins.

3T3 Cells↗

PubMed1999

Characterization of the MEN1 gene product, menin, by site-specific polyclonal antibodies.

The gene associated with multiple endocrine neoplasia type 1 (MEN 1), designated MEN1, has recently been identified. This gene shows no homology to other known genes, and its expression is not restricted to endocrine organs as estimated by northern blotting. Expression of the MEN1 gene product, menin, has been studied only in a few tissues. In this report, expression of menin in various cells and mouse tissues was studied using two polyclonal antibodies against menin. Expression of menin as a 76 kDa single protein was observed in all cell lines examined, regardless of origin. Two nuclear localization signals of the menin have been reported, but through the study of mutant menin in lymphocytes from subjects with MEN 1, impaired nuclear localization of the mutant menin was observed even though the mutant retained one of the two nuclear localization signals (NLSs). Menin was stable in vitro with a half-life of over 24 h at 37 degrees C. In the cell, the half-life of wild-type menin was about 10 h, while that of the mutant was about 2 h. The mutant rapidly disappeared from the nucleus.

Animals↗

PubMed2000

Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus.

Smad4 plays a pivotal role in all transforming growth factor beta (TGF-beta) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-beta signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-beta signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-beta signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm.

Active Transport, Cell Nucleus↗

PubMed2003

Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates.

60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other. We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran.GTP. Export of 60S subunits is coupled with that of the nucleo- cytoplasmic shuttling protein NMD3. Human NMD3 (hNMD3) contains a CRM-1-dependent leucine-rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation. When present in Xenopus oocytes, both wild-type and export-defective mutant hNMD3 proteins bind to newly made nuclear 60S pre-export particles at a late step of subunit maturation. The export-defective hNMD3, but not the wild-type protein, inhibits export of 60S subunits from oocyte nuclei. These results indicate that the NES mutant protein competes with endogenous wild-type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export. We propose that NMD3 acts as an adaptor for CRM1-Ran.GTP-mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast.

Active Transport, Cell Nucleus↗

PubMed1992

Nuclear localization of Agrobacterium VirE2 protein in plant cells.

The Agrobacterium single-stranded DNA (ssDNA) intermediate T-strand is likely transferred to the plant cell nucleus as a complex with a single VirD2 molecule at its 5' end and multiple VirE2 molecules along its length. VirD2 contains a nuclear localization signal (NLS); however, because the T-strand is principally coated with VirE2 molecules, VirE2 also might assist in nuclear uptake. Indeed, VirE2 fused to a reporter protein localizes to plant cell nuclei, a process mediated by two amino acid sequences with homology to the bipartite NLS of Xenopus nucleoplasmin. Moreover, tumorigenicity of an avirulent virE2 mutant is restored when inoculated on transgenic plants expressing VirE2, supporting in planta function of VirE2.

Amino Acid Sequence↗

PubMed1990

Gene structure of nuclear lamin LIII of Xenopus laevis; a model for the evolution of IF proteins from a lamin-like ancestor.

The lamin LIII gene of Xenopus laevis has been characterized. The gene is duplicated in the Xenopus genome. The transcribed region spreads over 22 kb of genomic DNA encoding 12 exons. Two alternatively spliced mRNAs are observed which encode LIII isoforms that differ only by the 12 C-terminal amino acids which, however, both contain the CaaX motif known to be the target of post-translational modifications. The intron pattern of the lamin LIII gene is strikingly similar to that of an invertebrate intermediate filament (IF) gene over the entire protein coding sequence. The similarity in gene structure is restricted to the rod domain when compared with vertebrate types I-III IF genes. Our data suggest a model of how IF proteins evolved from a lamin-like ancestor by deletion of two signal sequences; the nuclear localization signal and the C-terminal ras-related CaaX motif. The data rule out the previously proposed hypothesis that IF proteins evolved from an intronless ancestor with an early divergence of neuronal and non-neuronal IF proteins. Together with the data presented in the accompanying paper by Dodemond et al. it can be concluded that the tail domains of lamins and invertebrate IF proteins, but not those of vertebrate IF proteins, are homologous. Thus, the different vertebrate IF proteins probably evolved by combination of the central rod domain with different tail domains by exon shuffling.

Amino Acid Sequence↗

PubMed1991

Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2.

Polymerase basic protein 2 (PB2), a component of the influenza virus polymerase complex, when expressed alone from cloned cDNA in the absence of other influenza virus proteins, is transported into the nucleus. In this study, we have examined the nuclear translocation signal of PB2 by making deletions and mutations in the PB2 sequence. Our studies showed that two distant regions in the polypeptide sequence were involved in the nuclear translocation of PB2. In one region, four basic residues (K-736 R K R) played a critical role in the nuclear translocation of PB2, since the deletion or mutation of these residues rendered the protein totally cytoplasmic. However, seven residues (M K R K R N S) of this region, including the four basic residues, failed to translocate a cytoplasmic reporter protein into the nucleus, suggesting that these sequences were necessary but not sufficient for nuclear translocation. Deletion of another region (amino acids 449 to 495) resulted in a mutant protein which was cytoplasmic with a perinuclear distribution. This novel phenotype suggests that a perinuclear binding step was involved prior to translocation of PB2 across the nuclear pore and that a signal might be involved in perinuclear binding. Possible involvement of these two signal sequences in the nuclear localization of PB2 is discussed.

Amino Acid Sequence↗

PubMed2004

Nuclear import of the BTB/POZ transcriptional regulator Kaiso.

Kaiso is a BTB/POZ transcription factor that functions in vitro as a transcriptional repressor of the matrix metalloproteinase gene matrilysin and the non-canonical Wnt signaling gene Wnt-11, and as an activator of the acetylcholine-receptor-clustering gene rapsyn. Similar to other BTB/POZ proteins (e.g. Bcl-6, PLZF, HIC-1), endogenous Kaiso localizes predominantly to the nuclei of mammalian cells. To date, however, the mechanism of nuclear import for most POZ transcription factors, including Kaiso, remain unknown. Here, we report the identification and characterization of a highly basic nuclear localization signal (NLS) in Kaiso. The functionality of this NLS was verified by its ability to target a heterologous beta-galactosidase/green-fluorescent-protein fusion protein to nuclei. The mutation of one positively charged lysine to alanine in the NLS of full-length Kaiso significantly inhibited its nuclear localization in various cell types. In addition, wild-type Kaiso, but not NLS-defective Kaiso, interacted directly with the nuclear import receptor Importin-alpha2 both in vitro and in vivo. Finally, minimal promoter assays using a sequence-specific Kaiso-binding-site fusion with luciferase as reporter demonstrated that the identified NLS was crucial for Kaiso-mediated transcriptional repression. The identification of a Kaiso NLS thus clarifies the mechanism by which Kaiso translocates to the nucleus to regulate transcription of genes with diverse roles in cell growth and development.

Active Transport, Cell Nucleus↗

PubMed2005

Contribution of nuclear and extranuclear polyQ to neurological phenotypes in mouse models of Huntington's disease.

In postmortem Huntington's disease brains, mutant htt is present in both nuclear and cytoplasmic compartments. To dissect the impact of nuclear and extranuclear mutant htt on the initiation and progression of disease, we generated a series of transgenic mouse lines in which nuclear localization or nuclear export signal sequences have been placed N-terminal to the htt exon 1 protein carrying 144 glutamines. Our data indicate that the exon 1 mutant protein is present in the nucleus as part of an oligomeric or aggregation complex. Increasing the concentration of the mutant transprotein in the nucleus is sufficient for and dramatically accelerates the onset and progression of behavioral phenotypes. Furthermore, nuclear exon 1 mutant protein is sufficient to induce cytoplasmic neurodegeneration and transcriptional dysregulation. However, our data suggest that cytoplasmic mutant exon 1 htt, if present, contributes to disease progression.

Animals↗

PubMed2005

Analysis of nuclear transport signals in the human apurinic/apyrimidinic endonuclease (APE1/Ref1).

The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1's nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1's N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8-13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1's nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export.

Acetylation↗

PubMed2000

A method for the large-scale cloning of nuclear proteins and nuclear targeting sequences on a functional basis.

We describe here a selection strategy allowing the cloning of sequences that contain a functional nuclear targeting signal. Our method relies on the use of green fluorescent protein fusion proteins to identify nuclear targeting sequences. Transfected cells expressing nuclear protein fusions were isolated on the basis of their nuclear fluorescence using flow cytometry and the transfected DNAs were recovered after bacterial transformation with total DNA from pools of sorted cells. Starting from a cDNA expression library, in which only 1% of the expressed proteins were nuclear, we obtained a 70-fold enrichment in nuclear protein-encoding clones after a single round of selection. Among the 63 clones that have been partially sequenced to date, 25 (40%) corresponded to known nuclear proteins and 13 (20%) to previously uncharacterized sequences. Despite their ability to target the green fluorescent protein marker to the cell nucleus, about half of the cloned sequences did not encode canonical basic or bipartite nuclear localization signals. The method can thus be applied to the large-scale cloning of functional nuclear targeting sequences, which opens the way to a wide investigation of nuclear import mechanisms and to the identification of previously unknown nuclear proteins.

Animals↗

PubMed2004

14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1).

p27(Kip1) (p27), a CDK inhibitor, migrates into the nucleus, where it controls cyclin-CDK complex activity for proper cell cycle progression. We report here that the classical bipartite-type basic amino-acid cluster and the two downstream amino acids of the C-terminal region of p27 function as a nuclear localization signal (NLS) for its full nuclear import activity. Importin alpha3 and alpha5, but not alpha1, transported p27 into the nucleus in conjunction with importin beta, as evidenced by an in vitro transport assay. It is known that Akt phosphorylates Thr 157 of p27 and this reduces the nuclear import activity of p27. Using a pull-down experiment, 14-3-3 was identified as the Thr157-phosphorylated p27NLS-binding protein. Although importin alpha5 bound to Thr157-phosphorylated p27NLS, 14-3-3 competed with importin alpha5 for binding to it. Thus, 14-3-3 sequestered phosphorylated p27NLS from importin alpha binding, resulting in cytoplasmic localization of NLS-phosphorylated p27. These findings indicate that 14-3-3 suppresses importin alpha/beta-dependent nuclear localization of Thr157-phosphorylated p27, suggesting implications for cell cycle disorder in Akt-activated cancer cells.

14-3-3 Proteins↗