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Results for “Nuclear Localization Signals”

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PubMed2003

Defective importin beta recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations.

The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding/bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein in vitro and in vivo, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMPbeta. The SRY-R133W mutant shows reduced IMPbeta binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMPbeta binding, whereas SRY-R75N and SRY-R76P show normal IMPbeta binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.

Active Transport, Cell Nucleus↗

PubMed2002

A short peptide at the C terminus is responsible for the nuclear localization of RAG2.

The RAG1 and RAG2 proteins are the lymphoid-specific factors essential for V(D)J recombination, the process that leads to the diversification of antigen receptors on B and T lymphocytes. Nucleolar/nuclear localization of RAG1 is mediated by four basic domains, which are the binding sites for the nuclear transport proteins SRP1 and RCH1, and by a nuclear localization signal (NLS) in the fifth basic domain. The C-terminal region of RAG2 from amino acids (aa) 417 to 484 shows a homology with the PHD domain of other proteins involved in chromatin-mediated gene regulation by protein-protein interactions. Mutations in this domain were shown to be responsible for several diseases and in some case lead to altered subcellular localization of proteins. We found that the C-terminal PHD domain of RAG2 is not responsible for the nuclear localization of the protein. We report here the characterization of a region (aa 491-527) in the C-terminal domain of RAG2, downstream of the putative PHD domain, which directs the nuclear localization of the protein.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Characterization of nuclear localization of a hepatitis B virus precore protein derivative P22.

Both of hepatitis B virus core protein and a precore protein derivative, named P22, have been shown to localize in the nucleus. Although P22 has ten additional amino acid residues at its amino-terminus, both proteins contain the same nuclear localization signal. In order to understand the mechanism that regulates the activity of this signal, we have studied the nuclear localization of P22 and compared it with that of core protein. It was found that both cytosolic and nuclear fractions of P22 were phosphorylated but to a lesser extent when compared with cytosolic core protein. This distinction was likely attributed to different conformations between these two proteins since the density gradient analysis revealed a different particle formation for P22 in the cytosol. When expressed in Vero cells synchronized by serum deprivation, P22 remained in the cytosol during G0 and G1 phases, accumulated gradually in the nucleus during S phase, and largely localized in the nucleus when cells were confluent. On the other hand, the core protein was transported into the nucleus during mid-G1 phase, shuttled back to the cytosol in S phase and again accumulated in the nucleus when cells were confluent. Interestingly, when aphidicolin was used to arrest the cells in late G1 phase, both proteins were found to accumulate in the nuclei. These results indicated that although both P22 and core proteins possessed the same nuclear localization signal, the cellular regulation of their nuclear transport was not identical and might involve different molecular mechanisms.

Animals↗

PubMed2002

Cytoplasmic localization of Wis1 MAPKK by nuclear export signal is important for nuclear targeting of Spc1/Sty1 MAPK in fission yeast.

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1(+) does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.

Active Transport, Cell Nucleus↗

PubMed2003

DYRK1A accumulates in splicing speckles through a novel targeting signal and induces speckle disassembly.

The protein kinase DYRK1A is distributed throughout the nucleoplasm, accumulating in speckle-like regions. We have found that this punctuated nuclear distribution is determined by the contribution of several elements. Although the nuclear import is mediated by two distinct nuclear localization signals, one at the N-terminus and the other located in the linker region, between subdomains X and XI of the catalytic domain, the accumulation in speckles that are SC35 positive depends on a sequence motif that is located C-terminal to the kinase domain and comprises a histidine tail. A similar sequence is also responsible for the targeting of cyclin T1. Therefore the histidine-rich region represents a novel splicing speckle targeting signal. Moreover, overexpression of DYRK1A induces speckle disassembly. Such disassembly is DYRK1A activity specific, since the overexpression of a DYRK1A kinase inactive mutant, the paralogous DYRK1B or a chimeric protein DYRK1B that has been directed to the speckles via the DYRK1A targeting signal, leaves the SC35 speckle pattern untouched. Thus DYRK1A protein kinase may play a role in regulating the biogenesis of the splicing speckle compartment.

Animals↗

PubMed2003

Domains of Gln3p interacting with karyopherins, Ure2p, and the target of rapamycin protein.

Gln3p is a GATA-type transcription factor responsive to the quality of nitrogen and carbon. In preferred nitrogen such as glutamine, Gln3p is phosphorylated and sequestered in the cytoplasm in a manner that is dependent on the target of rapamycin (TOR) protein and Ure2p. In nonpreferred nitrogen or nitrogen starvation, Gln3p is dephosphorylated and imported into the nucleus via karyopherin alpha/Srp1p. Upon reintroduction of preferred nitrogen, Gln3p is exported from the nucleus by Crm1p/Xpo1p. Although recent work has provided insights into Gln3p, a more detailed understanding is needed to elucidate the mechanism of its localization and function. In this study, we show that Gln3p contains canonical nuclear localization signal and nuclear export signal sequences necessary for its localization and interaction with its relevant karyopherins. In addition, we identify an N-terminal domain of Gln3p interacting with Ure2p and a C-terminal region for binding to TOR. Finally, we find a lysine/arginine-rich domain essential for the rapamycin-sensitive function, but dispensable for its localization. Our results reveal key domains of Gln3p important for its function and regulation.

Binding Sites↗

PubMed2004

Phosphorylation of RCC1 in mitosis is essential for producing a high RanGTP concentration on chromosomes and for spindle assembly in mammalian cells.

Spindle assembly is subject to the regulatory controls of both the cell-cycle machinery and the Ran-signaling pathway. An important question is how the two regulatory pathways communicate with each other to achieve coordinated regulation in mitosis. We show here that Cdc2 kinase phosphorylates the serines located in or near the nuclear localization signal (NLS) of human RCC1, the nucleotide exchange factor for Ran. This phosphorylation is necessary for RCC1 to generate RanGTP on mitotic chromosomes in mammalian cells, which in turn is required for spindle assembly and chromosome segregation. Moreover, phosphorylation of the NLS of RCC1 is required to prevent the binding of importin alpha and beta to RCC1, thereby allowing RCC1 to couple RanGTP production to chromosome binding. These findings reveal that the cell-cycle machinery directly regulates the Ran-signaling pathway by placing a high RanGTP concentration on the mitotic chromosome in mammalian cells.

Animals↗

PubMed2000

Identification of the isoforms of Ca(2+)/Calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization.

Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT-PCR followed by the "nested" PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was delta2. Immunostaining suggested that the delta2 isoform was localized predominantly at the Golgi apparatus. The localization of the delta2 isoform at the Golgi apparatus was also observed in NG108-15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108-15 cells. In contrast to the alphaB and delta3 isoforms that entered the nucleus, as reported, the gammaA isoform was excluded from the nucleus in the transfected NG108-15 cells. These results suggest that the 15-amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the gammaA isoform.

Amino Acid Sequence↗

PubMed1998

Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells depends critically on import of the viral preintegration complex into the nucleus. Recent evidence suggests that viral protein R (Vpr) plays a key regulatory role in this process by binding to karyopherin alpha, a cellular receptor for nuclear localization signals, and increasing its affinity for the nuclear localization signals. An in vitro binding assay was used to investigate the role of Vpr in docking of the HIV-1 preintegration complex (PIC) to the nuclear pore complex. Mutant HIV-1 PICs that lack Vpr were impaired in the ability to dock to isolated nuclei and recombinant nucleoporins. Although Vpr by itself associated with nucleoporins, the docking of Vpr+ PICs was dependent on karyopherin beta and was blocked by antibodies to beta. Vpr stabilized docking by preventing nucleoporin-stimulated dissociation of the import complex. These results suggest a biochemical mechanism for Vpr function in transport of the HIV-1 genome across the nuclear pore complex.

Cell Line↗

PubMed2000

Self-association of coilin reveals a common theme in nuclear body localization.

We have found that coilin, the marker protein for Cajal bodies (coiled bodies, CBs), is a self-interacting protein, and we have mapped the domain responsible for this activity to the amino-terminus. Together with a nuclear localization signal, the self-interaction domain is necessary and sufficient for localization to CBs. Overexpression of various wild-type and mutant coilin constructs in HeLa cells results in disruption of both CBs and survival motor neurons (SMN) gems. Additionally, we have identified a cryptic nucleolar localization signal (NoLS), within the coilin protein, which may be exposed in specific coilin phospho-isoforms. The implications of these findings are discussed in light of the fact that other proteins known to localize within nuclear bodies (e. g., PML, SMN and Sam68) can also self-associate. Thus protein self-interaction appears to be a general feature of nuclear body marker proteins.

Cell Nucleolus↗

PubMed1998

Functional conservation of the transportin nuclear import pathway in divergent organisms.

Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding proteins (heterogeneous nuclear ribonucleoproteins [hnRNP]) represented by hnRNP A1, which shuttle continuously between the nucleus and the cytoplasm. hTRN1 interacts with the M9 region of hnRNP A1, a 38-amino-acid domain rich in Gly, Ser, and Asn, and mediates the nuclear import of M9-bearing proteins in vitro. Saccharomyces cerevisiae transportin (yTRN; also known as YBR017c or Kap104p) has been identified and cloned. To understanding the nuclear import mediated by yTRN, we searched with a yeast two-hybrid system for proteins that interact with it. In an exhaustive screen of the S. cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p. We delineated a ca.-50-amino-acid region in Nab2p, termed NAB35, which specifically binds yTRN and is similar to the M9 motif. NAB35 also interacts with hTRN1 and functions as a nuclear localization signal in mammalian cells. Interestingly, yTRN can also mediate the import of NAB35-bearing proteins into mammalian nuclei in vitro. We also report on additional substrates for TRN as well as sequences of Drosophila melanogaster, Xenopus laevis, and Schizosaccharomyces pombe TRNs. Together, these findings demonstrate that both the M9 signal and the nuclear import machinery utilized by the transportin pathway are conserved in evolution.

Amino Acid Sequence↗

PubMed1999

The FAR1 locus encodes a novel nuclear protein specific to phytochrome A signaling.

The phytochrome family of photoreceptors has a well-defined role in regulating gene expression in response to informational light signals. Little is known, however, of the early steps of phytochrome signal transduction. Here we describe a new Arabidopsis mutant, far1 (far-red-impaired response), which has reduced responsiveness to continuous far-red light, but responds normally to other light wavelengths. This phenotype implies a specific requirement for FAR1 in phyA signal transduction. The far1 locus maps to the south arm of chromosome 4, and is not allelic to photomorphogenic loci identified previously. All five far1 alleles isolated have single nucleotide substitutions that introduce stop codons in a single ORF. The FAR1 gene encodes a protein with no significant sequence similarity to any proteins of known function. The FAR1 protein contains a predicted nuclear localization signal and is targeted to the nucleus in transient transfection assays. This result supports an emerging view that early steps in phytochrome signaling may be centered in the nucleus. The FAR1 gene defines a new multigene family, which consists of at least four genes in Arabidopsis. This observation raises the possibility of redundancy in the phyA-signaling pathway, which could account for the incomplete block of phyA signaling observed in the far1 mutant.

Alleles↗

PubMed1994

Molecular mechanisms of androgen action.

Androgens directly regulate a vast number of physiological events. These direct androgen effects are mediated by a nuclear receptor that exhibits four major functions or activities: steroid binding, DNA binding, transactivation, and nuclear localization. The SBD consists of a hydrophobic pocket of amino acids that exhibits high-affinity, androgen-specific binding. Based on studies of mutant AR, it appears that a number of different amino acids contribute to the steroid binding characteristics of the AR. The DNA binding domain confers sequence-specific binding to structures called androgen-responsive elements. The specificity of steroid binding and DNA binding provides a crucial basis for androgen-specific regulation of target genes. The nuclear localization signal shares homology with known nuclear localization signals and, coupled with the presence of androgens, is responsible for localizing the AR to the nucleus. The transactivation functions reside mostly in the NH2 terminus but the responsible domains are as yet poorly defined. Though the different domains can act as independent moieties, one domain can clearly alter the behavior of another domain. For instance, the SBD appears to inhibit the transactivating functions until steroid is bound and the amino terminus prevents DNA binding activity until steroid is bound. The relative ease of introducing mutations with polymerase chain reaction technology will facilitate further delineation of critical amino acids and domains responsible for the various activities of the AR. The recent cloning and characterization of AR promoters revealed that the AR genes are driven by a TATA-less promoter characteristics of housekeeping genes. Analysis of transcription rates, mRNA levels, and protein levels indicates that androgens and pkA and pkC pathways modulate expression of AR mRNA and protein. This indicates that the same signal pathways that interact to regulate androgen target genes also regulate the levels of AR in the target tissues. Surprisingly few androgen-regulated genes have been well characterized for the mechanisms by which androgen regulates the gene. The C(3), Slp, probasin, PSA, and hKLK2 genes have provided examples where androgens regulate transcription. Posttranscriptional regulation by androgens has been demonstrated for the SVP1, 2, 3, and 4 and AR genes. The mechanisms underlying posttranscriptional regulation are poorly defined but substantial progress has been made in defining the critical elements that mediate transcriptional effects of androgens. Transcriptional effects are mediated through binding of androgen-AR complexes to specific DNA sequences called AREs. Simple AREs such as those found in C(3) and kallikrein genes tend to be permissive in that GR and PR can also act through the same element.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgens↗

PubMed2002

Localization of the secretory granule marker protein chromogranin B in the nucleus. Potential role in transcription control.

Chromogranins A (CGA) and B (CGB) are two major Ca(2+) storage proteins of the secretory granules of neuroendocrine cells. Nevertheless, we found in the present study that CGB was also localized in the nucleus. In immunogold electron microscopy using bovine adrenal medullary chromaffin cells, it was found that the number of CGB-labeled gold particles localized per microm(2) of the nucleus was equivalent to 20% that of CGB-labeled gold particles localized per microm(2) of the secretory granules. Considering that CGB is estimated to exist in the 0.1-0.2-mm range in the secretory granules of bovine chromaffin cells, 20% of these amounts to 20-40 microm. In addition, transfection of CGA and CGB into nonneuroendocrine COS-7 and NIH3T3 cells repeatedly indicated the nuclear localization of CGB in addition to its usual localization in the cytoplasm. Moreover, immunoblot and immunogold electron microscopy analyses of neuroendocrine PC12 cells also showed the existence of endogenous CGB in both the cytosol and the nucleus. Nuclear routing of CGB did not appear to depend entirely upon the nuclear localization signal as some of the nuclear localization signal mutant CGB were still targeted to the nucleus. In gene array assay, CGB was shown to either induce or suppress transcription of many genes including those of transcription factors. Of these we have analyzed eight genes, four induced (zinc finger protein, MEF2C, hCRP2, abLIM) and four suppressed (hcKrox, T3-receptor, troponin C, integrin) using the quantitative reverse transcription-PCR method and spectrophotometry to determine the transcription levels of each mRNA. CGB was shown to increase the transcription of zinc finger protein, MEF2C, hCRP2, and abLIM by 2.5-5-fold while suppressing that of hcKrox, T3-receptor, troponin C, and integrin by 60-75%. Given that MEF2C and hcKrox genes are transcription factors, these results pointed to the transcription control role of CGB in the nucleus.

3T3 Cells↗

PubMed2001

Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits.

The Ku antigen is a heteromeric (Ku70/Ku80), mostly nuclear protein. Ku participates in multiple nuclear processes from DNA repair to V(D)J recombination to telomere maintenance to transcriptional regulation and serves as a DNA binding subunit and allosteric regulator of DNA-dependent protein kinase. While some evidence suggests that subcellular localization of Ku may be subject to regulation, how Ku gains access to the nucleus is poorly understood. In this work, using a combination of indirect immunofluorescence and direct fluorescence, we have demonstrated that transfer of the Ku heterodimer to the nucleus is determined by basic nuclear localization signals in each of the Ku subunits that function independently. A bipartite basic nuclear localization signal between amino acids 539-556 of Ku70 was observed to be required for nuclear import of full-length Ku70 monomer, while a short Ku80 motif of four amino acids from 565-568 containing three lysines was required for the nuclear import of full-length Ku80. Ku heterodimers containing only one nuclear localization signal accumulated in the nucleus as efficiently as wild-type Ku, while site directed mutagenesis inactivating the basic motifs in each subunit, resulted in a Ku heterodimer that was completely localized to the cytoplasm. Lastly, our results indicate that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization, markedly reduced the accumulation of Ku70 without affecting heterodimer formation in mammalian cells.

Active Transport, Cell Nucleus↗

PubMed2005

Functional analysis of natural mutations in two TWIST protein motifs.

The basic helix-loop-helix protein Twist, a transcriptional repressor, is essential for embryogenesis in both invertebrates and vertebrates. Haploinsufficiency of the human TWIST1 gene, which causes the craniosynostosis disorder Saethre-Chotzen syndrome (SCS), is related to failure to repress transcription of CDKN1A (which encodes p21/WAF1/CIP1), promoting osteoblast differentiation. We have examined the functional significance of natural TWIST1 variants present in craniosynostosis patients and in their healthy relatives. Both deletion and duplication variants of the glycine-rich tract Gly5AlaGly5 inhibited E2A (E12/E47)-dependent transcription of CDKN1A to a similar degree as wild-type protein, indicating that the length of this glycine tract is not critical for efficient transcriptional repression. We also evaluated a newly identified heterozygous TWIST1 variant (c.115C>G, encoding p.Arg39Gly), located within a putative nuclear localization signal (NLS), that was present in a child with mild SCS and her clinically unaffected father and grandmother. Unlike wild-type protein, this mutant required cotransfected E12 to localize to the nucleus, indicating that the NLS, including amino acid 39, is essential for nuclear localization; inhibition of E2A-dependent transcription of CDKN1A occurred normally. This analysis further dissects the structure-function relationships of TWIST and corroborates with phenotypic observations of disease expressivity.

Amino Acid Motifs↗

PubMed1997

The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein.

Protein import into the nucleus and export from the nucleus are signal-mediated processes that require energy. The nuclear transport process about which the most information is currently available is classical nuclear localization signal (NLS)-mediated nuclear import. However, details concerning the signal-mediated export of proteins and RNAs as well as alternative nuclear import pathways are beginning to emerge. An example of this is the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein which, by virtue of its M9 domain, is actively exported from the nucleus and imported into the nucleus via a novel pathway mediated by the recently characterized transportin protein. Here we report that the shuttling hnRNP K protein contains a novel shuttling domain (termed KNS) which has many of the characteristics of M9, in that it confers bi-directional transport across the nuclear envelope. KNS-mediated nuclear import is dependent on RNA polymerase II transcription, and we show that a classical NLS can override this effect. Furthermore, KNS accesses a separate import pathway from either classical NLSs or M9. This demonstrates the existence of a third protein import pathway into the nucleus and thereby defines a new type of nuclear import/export signal.

Amino Acid Sequence↗

PubMed1998

Identification of three major sentrinization sites in PML.

Acute promyelocytic leukemia arises following a reciprocal chromosome translocation t(15;17), which generates PML-retinoic acid receptor alpha fusion proteins (PML-RARalpha). We have shown previously that wild type PML, but not PML-RARalpha, is covalently modified by the sentrin family of ubiquitin-like proteins (Kamitani, T., Nguyen, H. P., Kito, K., Fukuda-Kamitani, T., and Yeh, E. T. H. (1998) J. Biol. Chem. 273, 3117-3120). To understand the mechanisms underlying the differential sentrinization of PML versus PML-RARalpha, extensive mutational analysis was carried out to determine which Lys residues are sentrinized. We show that Lys65 in the RING finger domain, Lys160 in the B1 Box, and Lys490 in the nuclear localization signal contributes three major sentrinization sites. The PML mutant with Lys to Arg substitutions in all three sites is expressed normally, but cannot be sentrinized. Furthermore, the triple substitution mutant is localized predominantly to the nucleoplasm, in contrast to wild type PML, which is localized to the nuclear bodies. Thus, sentrinization of PML, in the context of the RING finger and the B1 box, regulates nuclear body formation. Furthermore, we showed that sentrinization of PML-RARalpha could be restored by overexpression of sentrin, but not by retinoic acid treatment. These studies provide novel insight into the pathobiochemistry of acute promyelocytic leukemia and the sentrinization pathway.

Animals↗