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PubMed2000

Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity.

NFAT transcription factors are highly phosphorylated proteins that are regulated by the calcium-dependent phosphatase calcineurin. We show by mass spectrometry that NFAT1 is phosphorylated on fourteen conserved phosphoserine residues in its regulatory domain, thirteen of which are dephosphorylated upon stimulation. Dephosphorylation of all thirteen residues is required to mask a nuclear export signal (NES), cause full exposure of a nuclear localization signal (NLS), and promote transcriptional activity. An inducible phosphorylation site in the transactivation domain contributes to transcriptional activity. Our data suggest that dephosphorylation promotes NFAT1 activation by increasing the probability of an active conformation, in a manner analogous to that by which depolarization increases the open probability of voltage-gated ion channels. This conformational switch paradigm may explain modification-induced functional changes in other heavily phosphorylated proteins.

Animals↗
PubMed2002

Phosphorylation and dimerization regulate nucleocytoplasmic shuttling of mammalian STE20-like kinase (MST).

Mammalian STE20-like kinase (MST) is a member of the yeast STE20-related kinase family and proteolytically activated by caspase during apoptosis. However, its other cellular functions are not known, including its activation mechanism, substrate(s), and subcellular localization. In this report, using anti-MST monoclonal antibodies, we clearly show that endogenous MST is localized in cytoplasm in a leptomycin B-dependent manner. Analyses with serial deletions and point mutations show that MST has two functional nuclear export signals and, unexpectedly, another localization motif for nuclear import. When cells are treated with leptomycin, monomeric MST is accumulated more rapidly in the nucleus than dimeric MST, indicating that dimerization contributes to the cytoplasmic retention of MST. Okadaic acid, an inhibitor of phosphatase 2A, induces activation of MST and translocation into the nucleus. Using phosphopeptide-specific antibody, we directly show that okadaic acid induces phosphorylation in the activation loop of MST, and, once phosphorylated, MST is rapidly translocated to the nucleus. However, kinase-deficient MST does not enter the nucleus, indicating that phosphorylation and activation is required for okadaic acid-induced nuclear translocation. In apoptotic cells, the activation of MST does not require phosphorylation in the activation loop and occurs through the release of C-terminal regulatory domain by caspase-dependent cleavage. Kinase-deficient MST functions dominant-negatively and represses okadaic acid-induced morphological change indicating that MST plays a role in okadaic acid-induced cellular shrinkage. Our identification of cytoplasmic and nuclear localization motifs and phosphorylation-dependent translocation of MST suggests that regulation of localization is important to the biological function of MST, including its effects on cellular morphology.

3T3 Cells↗
PubMed2002

Identification of a novel transcription factor, ELYS, expressed predominantly in mouse foetal haematopoietic tissues.

BACKGROUND: The precise mechanism governing the generation of haematopoietic stem cells still remains to be understood, partly because the molecules required for early haematopoiesis have not fully been identified. RESULTS: We have identified a novel gene expressed in embryonic haematopoietic tissues, designated ELYS (for embryonic large molecule derived from yolk sac), which has no significant homology with any other known molecules. Based on the cDNA sequence, mouse ELYS protein is composed of 2243 amino acid residues and contains an AT-hook DNA-binding domain, eight nuclear localization signals (NLSs) at the C-terminal region, three nuclear export signals (NESs) and two WD repeats at the N-terminal region. ELYS has a potential to shuttle between the cytoplasm and nucleus. When in the nucleus, ELYS is present in the nuclear matrix. Fusions of the yeast GAL4 DNA-binding domain and various ELYS mutants reveal the presence of transcriptional activation and inhibitory domains. The ELYS gene is predominantly expressed in embryonic haematopoietic tissues, i.e. foetal liver, spleen, and thymus, whereas the expression is down-regulated in the adult. In the aorta-gonad-mesonephros (AGM) region of an 11.5 dpc mouse embryo, ELYS is expressed in the endothelium lining the dorsal aorta. In the adult bone marrow, ELYS is notably expressed in the Lin-/c-kit+/Sca-1+ population. CONCLUSIONS: We have reported the isolation and characterization of a novel molecule, ELYS. ELYS seems to be a nuclear transcription factor associated with both early and mature haematopoietic events.

Amino Acid Sequence↗
PubMed1999

Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region.

The cellular target of leptomycin B (LMB), a nuclear export inhibitor, has been identified as CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins. However, the mechanism by which LMB inhibits CRM1 still remains unclear. CRM1 in a Schizosaccharomyces pombe mutant showing extremely high resistance to LMB had a single amino acid replacement at Cys-529 with Ser. The mutant gene, named crm1-K1, conferred LMB resistance on wild-type S. pombe, and Crm1-K1 no longer bound biotinylated LMB. (1)H NMR analysis showed that LMB bound N-acetyl-L-cysteine methyl ester through a Michael-type addition, consistent with the idea that LMB binds covalently via its alpha, beta-unsaturated delta-lactone to the sulfhydryl group of Cys-529. When HeLa cells were cultured with biotinylated LMB, the only cellular protein bound covalently was CRM1. Inhibition by N-ethylmaleimide (NEM), an alkylating agent, of CRM1-mediated nuclear export probably was caused by covalent binding of the electrophilic structure in NEM to the sulfhydryl group of Cys-529, because the crm1-K1 mutant showed the normal rate for the export of Rev nuclear export signal-bearing proteins in the presence of not only LMB but also NEM. These results show that the single cysteine residue determines LMB sensitivity and is selectively alkylated by LMB, leading to CRM1 inactivation.

Amino Acid Sequence↗
PubMed2002

Nuclear localization of a non-caspase truncation product of atrophin-1, with an expanded polyglutamine repeat, increases cellular toxicity.

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder similar to Huntington's disease, with clinical manifestations including chorea, incoordination, ataxia, and dementia. It is caused by an expansion of a CAG trinucleotide repeat encoding polyglutamine in the atrophin-1 gene. Both patients and DRPLA transgenic mice have nuclear accumulation of atrophin-1, especially an approximately 120-kDa fragment, which appears to represent a cleavage product. We now show that this is an N-terminal fragment that does not correspond to the previously described caspase-3 fragment, or any other known caspase cleavage product. The atrophin-1 sequence contains a putative nuclear localization signal in the N terminus of the protein and a putative nuclear export signal in the C terminus. We have tested the hypothesis that endogenous localization signals are functional in atrophin-1, and that nuclear localization and proteolytic cleavage contribute to atrophin-1 cell toxicity. In transient cell transfection experiments using a neuroblastoma cell line, full-length atrophin-1 with 26 (normal) or 65 (expanded) glutamines localized to both nucleus and cytoplasm, with no significant difference in toxicity between the normal and mutant proteins. A construct with 65 glutamine repeats encoding an N-terminal fragment (which removes an NES) of atrophin-1 similar in size to the truncation product in DRPLA patient tissue, showed increased nuclear labeling, and an increase in cellular toxicity, compared with a similar fragment with 26 glutamines. Full-length atrophin-1 with 65 polyglutamine repeats and mutations inactivating the NES also yielded increased nuclear localization and increased toxicity. These data suggest that truncation enhances cellular toxicity of the mutant protein, and that the NES is a relevant region deleted during truncation. Furthermore, mutating the NLS in the truncated protein shifted atrophin-1 more to the cytoplasm and eliminated the increased toxicity, consistent with the idea that nuclear localization enhances toxicity. In none of the experiments were inclusions visible in the nucleus or cytoplasm suggesting that inclusion formation is unrelated to cell death. These data indicate that truncation of atrophin-1 may alter its ability to shuttle between the nucleus and cytoplasm, leading to abnormal nuclear interactions and cell toxicity.

Animals↗
PubMed2006

Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes.

Influenza virus is a negative strand RNA virus and is one of the rare RNA viruses to replicate in the nucleus. The viral RNA is associated with 4 viral proteins to form ribonucleoprotein particles (RNPs). After cell entry the RNPs are dissociated from the viral matrix protein in the low pH of the endosome and are actively imported into the cell nucleus. After translation of viral mRNAs, the proteins necessary for the assembly of new RNPs (the nucleoprotein and the three subunits of the polymerase complex) are also imported into the nucleus. Apart from these four proteins, part of the newly made matrix protein is also imported and the nuclear export protein (NEP) enters the nucleus probably through diffusion. Finally, NS1 also enters the nucleus in order to regulate a number of nuclear processes. The nuclear localization signals on all these viral proteins and their interaction with the cellular transport system are discussed. In the nucleus, the matrix protein binds to the newly assembled RNPs and NEP then binds to the matrix protein. NEP contains the nuclear export signal necessary for transport of the RNPs to the cytoplasm, necessary for the budding of new virus particles. There appears to be a intricate ballet in exposing and hiding nuclear transport signals which leads to a unidirectional transport of the RNPs to the nucleus at the start of the infection process and an opposite unidirectional export of RNPs at the end of the infection.

Active Transport, Cell Nucleus↗
PubMed2006

Oligomerizing potential of a focal adhesion LIM protein Hic-5 organizing a nuclear-cytoplasmic shuttling complex.

Hic-5 is a focal adhesion LIM protein serving as a scaffold in integrin signaling. The protein comprises four LD domains in its N-terminal half and four LIM domains in its C-terminal half with a nuclear export signal in LD3 and is shuttled between the cytoplasmic and nuclear compartments. In this study, immunoprecipitation and in vitro cross-linking experiments showed that Hic-5 homo-oligomerized through its most C-terminal LIM domain, LIM4. Strikingly, paxillin, the protein most homologous to Hic-5, did not show this capability. Gel filtration analysis also revealed that Hic-5 differs from paxillin in that it has multiple forms in the cellular environment, and Hic-5 but not paxillin was capable of hetero-oligomerization with a LIM-only protein, PINCH, another molecular scaffold at focal adhesions. The fourth LIM domain of Hic-5 and the fifth LIM domain region of PINCH constituted the interface for the interaction. The complex included integrin-linked kinase, a binding partner of PINCH, which also interacted with Hic-5 through the region encompassing the pleckstrin homology-like domain and LIM domains of Hic-5. Of note, Hic-5 marginally affected the subcellular distribution of PINCH but directed its shuttling between the cytoplasmic and nuclear compartments in the presence of integrin-linked kinase. Uncoupling of the two signaling platforms of Hic-5 and PINCH through interference with the hetero-oligomerization resulted in impairment of cellular growth. Hic-5 is, thus, a molecular scaffold with the potential to dock with another scaffold through the LIM domain, organizing a mobile supramolecular unit and coordinating the adhesion signal with cellular activities in the two compartments.

Active Transport, Cell Nucleus↗
PubMed2000

Nuclear import and export signals enable rapid nucleocytoplasmic shuttling of the atypical protein kinase C lambda.

The atypical protein kinase C (PKC) isoenzymes, lambda/iota- and zetaPKC, play important roles in cellular signaling pathways regulating proliferation, differentiation, and cell survival. By using green fluorescent protein (GFP) fusion proteins, we found that wild-type lambdaPKC localized predominantly to the cytoplasm, whereas both a kinase-defective mutant and an activation loop mutant accumulated in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal part of the zinc finger domain of lambdaPKC. Leptomycin B treatment induced rapid nuclear accumulation of GFP-lambda as well as endogenous lambdaPKC suggesting the existence of a CRM1-dependent nuclear export signal (NES). Consequently, we identified a functional leucine-rich NES in the linker region between the zinc finger and the catalytic domain of lambdaPKC. The presence of both the NLS and NES enables a continuous shuttling of lambdaPKC between the cytoplasm and nucleus. Our results suggest that the exposure of the NLS in both lambda- and zetaPKC is regulated by intramolecular interactions between the N-terminal part, including the pseudosubstrate sequence, and the catalytic domain. Thus, either deletion of the N-terminal region, including the pseudosubstrate sequence, or a point mutation in this sequence leads to nuclear accumulation of lambdaPKC. The ability of the two atypical PKC isoforms to enter the nucleus in HeLa cells upon leptomycin B treatment differs substantially. Although lambdaPKC is able to enter the nucleus very rapidly, zetaPKC is much less efficiently imported into the nucleus. This difference can be explained by the different relative strengths of the NLS and NES in lambdaPKC compared with zetaPKC.

Amino Acid Sequence↗
PubMed1997

Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.

Animals↗
PubMedDate not supplied

The nuclear function of the nuclear diffusion inhibitory signal of human immunodeficiency virus type 1: critical roles in dominant nuclear localization and intracellular stability.

OBJECTIVES: Human immunodeficiency virus type 1 (HIV-1) Rev is a nucleocytoplasmic shuttling protein with dominant localization in the cell nucleus/nucleolus. To clarify the mechanism(s) that enables such a biased subcellular localization under the co-presence of nuclear/nucleolar targeting (NOS) and nuclear export signals (NES), the properties of another functional domain, a nuclear diffusion inhibitory signal (NIS), was investigated. STUDY DESIGN: The NIS was previously shown to interfere with passive nuclear entry of small proteins. Intracellular distribution and degradation profiles of Rev mutants that harbor mutations in the NIS were analyzed biochemically and cellbiologically. RESULTS: A NIS-deficient Rev mutant, which was no longer functional as Rev, displayed a significantly more rapid degradation profile as a consequence of its dramatic leakage into the cytoplasm. Additionally, disabling the NOS/nuclear localization signal (NLS), as well as the NIS, resulted in further rapid degradation, which also supports the hypothetical role of the nucleolus as a Rev storage site. CONCLUSIONS: It was uncovered that the NIS is playing a key role in HIV-1 Rev function by maintaining the nuclear-dominant localization and the intracellular stability of Rev.

Amino Acid Sequence↗
PubMed2006

Separating the anti-apoptotic and mitotic roles of survivin.

Survivin is a bifunctional protein that acts as a suppressor of apoptosis and has an essential role in mitosis. To date whether these two functions can be divorced has not been addressed. Here we show that the linker region between the BIR (baculovirus inhibitor of apoptosis repeat) domain of survivin and COOH-terminal alpha helix may be the key to separating its roles. When overexpressed survivin is present in interphase cells and shuttles between the cytoplasm and nucleus. Here we identify a rev-like nuclear exportation signal (NES) in the central domain of survivin and demonstrate that point mutations within this region cause accumulation of survivin in the nucleus. Interestingly cells expressing NES mutants exhibit reduced survival after X-irradiation. Moreover, cells expressing survivin(L98A)-green fluorescent protein (GFP) showed increased poly(ADP-ribose) polymerase-cleavage and caspase-3 activity after tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment compared with cells expressing full-length survivin-green fluorescent protein. These data suggest a direct link between the interphase localization of survivin and cellular responsiveness to apoptotic stimuli. Using a cell proliferation assay, we also found that ectopic expression of NES mutants can complement for depletion of endogenous survivin, indicating that they can execute the mitotic duties of survivin. Thus we demonstrate for the first time that 1) survivin has a functional NES; 2) nuclear accumulation of overexpressed survivin correlates with increased sensitivity of cells to ionising radiation; and 3) the anti-apoptotic and mitotic roles of survivin can be separated through mutation of its NES. Separating these two functions of survivin could open up new possibilities for therapeutic strategies aimed at eliminating cancer cells yet preserving normal cell viability.

Apoptosis↗
PubMed2005

Overexpression of Ets-1 in human hematopoietic progenitor cells blocks erythroid and promotes megakaryocytic differentiation.

Ets-1 is a widely expressed transcription factor implicated in development, tumorigenesis and hematopoiesis. We analyzed Ets-1 gene expression during human erythroid and megakaryocytic (MK) differentiation in unilineage cultures of CD34+ progenitor cells. During erythroid maturation, Ets-1 is downmodulated and exported from the nucleus into the cytoplasm through an active mechanism mediated by a leucine-rich nuclear export signal. In contrast, during megakaryocytopoiesis Ets-1 increases and remains localized in the nucleus up to terminal maturation. Overexpression of Ets-1 in erythroid cells blocks maturation at the polychromatophilic stage, increases GATA-2 and decreases both GATA-1 and erythropoietin receptor expression. Conversely, Ets-1 overexpressing megakaryocytes are characterized by enhanced differentiation and maturation, coupled with upmodulation of GATA-2 and megakaryocyte-specific genes. We show that Ets-1 binds to and activates the GATA-2 promoter, in vitro and in vivo, indicating that one of the pathways through which Ets-1 blocks erythroid and promotes MK differentiation is via upmodulation of GATA-2 expression.

Active Transport, Cell Nucleus↗
PubMed2005

Nuclear targeting and cell cycle regulatory function of human BARD1.

The BARD1 gene is mutated in a subset of breast and ovarian cancers, implicating BARD1 as a potential tumor suppressor. BARD1 gains a ubiquitin E3 ligase activity when heterodimerized with BRCA1, but the only known BRCA1-independent BARD1 function is a p53-dependent proapoptotic activity stimulated by nuclear export to the cytoplasm. We described previously the nuclear-cytoplasmic shuttling of BARD1, and in this study, we identify the transport sequences that target BARD1 to the nucleus and show that they are essential for BARD1 regulation of the cell cycle. We used deletion mapping and mutagenesis to define two active nuclear localization signals (NLSs) present in human BARD1 that are not conserved in rodent BARD1. Site-directed mutagenesis of the primary bipartite NLS abolished BARD1 nuclear import and caused its cytoplasmic accumulation. Using flow cytometry and 5-bromo-2-deoxyuridine incorporation assays, we discovered that transiently expressed BARD1 can elicit a p53-independent cell cycle arrest in G1 phase, and that this was abrogated by mutation of the BARD1 NLS but not by mutation of the nuclear export signal. Thus, BARD1 regulation of the cell cycle is a nuclear event and may be linked to its induced expression during mitosis and its possible involvement in the DNA damage checkpoint.

Amino Acid Sequence↗
PubMed2006

Cytoplasmic Clb2 is required for timely inactivation of the mitotic inhibitor Swe1 and normal bud morphogenesis in Saccharomyces cerevisiae.

Subcellular localization is an important determinant of substrate and functional specificity for cyclin-cyclin dependent kinase (CDK) complexes. This work addresses the cytoplasmic function of the budding yeast mitotic cyclin Clb2, which is mostly nuclear but is also present in the bulk cytoplasm and at the mother-bud neck. Clb2 contains two leucine-rich nuclear export signals (NESs)--one of which we newly describe here--that maintain its presence in the cytoplasm. Yeast strains bearing mutations in one or both of these NESs have elongated buds, indicative of a G2/M cell cycle delay. A small number of these cells exhibit a filamentous-like morphology under conditions that do not normally induce filamentous growth. These phenotypes are enhanced by deletion of the other three mitotic cyclins (CLB1,3,4) and are dependent on expression of Swe1, the yeast Cdk1 inhibitory kinase. Deltaclb1,3,4 Deltabud3 cells, which fail to localize Clb2 to the bud neck, also exhibit a Swe1-dependent elongated bud phenotype. Our results support a model in which cytoplasmic Clb2-Cdk1 is required for timely inactivation of Swe1 at the G2/M transition and bud neck targeting of Clb2 contributes to the efficiency of this process. Cytoplasmic Clb2 may also be important for repression of filamentous growth.

Binding Sites↗
PubMed1997

The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export.

BACKGROUND: The human immunodeficiency virus (HIV-1) uses the viral protein Rev to regulate gene expression by promoting the export of unspliced and partially spliced viral transcripts. Rev has been shown to function in a variety of organisms, including Saccharomyces cerevisiae. The export activity of Rev depends on a nuclear export signal (NES), which is believed to interact either directly or indirectly with the nuclear pore complex to carry out its export function. Crm1p is a member of the importin-beta protein family, other members of which are known to be directly involved in nuclear import. Crm1p has recently been shown to contribute to nuclear export in vertebrate systems. Here, we have studied this mechanism of nuclear to cytoplasmic transport. RESULTS: Viable mis-sense mutations in the CRM1 gene substantially reduced or eliminated the biological activity of Rev in S. cerevisiae, providing strong evidence that Crm1p also contributes to transport of Rev NES-containing proteins and ribonucleoproteins in this organism. Crm1p interacted with FG-repeat-containing nuclear pore proteins as well as Rev, and we have demonstrated that the previously described two-hybrid interaction between Rev and the yeast nuclear pore protein Rip1p is dependent on wild-type Crm1p. CONCLUSIONS: We conclude that Crm1p interacts with the Rev NES and nuclear pore proteins during delivery of cargo to the nuclear pore complex. Our findings also agree well with current experiments on Crm1p orthologs in Schizosaccharomyces pombe and in vertebrate systems.

Biological Transport↗
PubMed1997

Mediators of nuclear protein import target karyophilic proteins to pore complexes of cytoplasmic annulate lamellae.

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes of Xenopus laevis. In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin beta and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central "transporter" and closely resemble those of "transport intermediates" found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.

Animals↗
PubMed2006

Structural phylogenetic analysis of activation-induced deaminase function.

In mammals, activation-induced deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. SHM and CSR activities require separate regions within AID. A chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES) at the AID C terminus is necessary for CSR, and has been suggested to associate with CSR-specific cofactors. CSR appeared late in AID evolution, during the emergence of land vertebrates from bony fish, which only display SHM. Here, we show that AID from African clawed frog (Xenopus laevis), but not pufferfish (Takifugu rubripes), can induce CSR in AID-deficient mouse B cells, although both are catalytically active in bacteria and mammalian cell systems, albeit at decreased level. Like mammalian AID, Takifugu AID is actively exported from the cell nucleus by CRM1, and the Takifugu NES can substitute for the equivalent region in human AID, indicating that all the CSR-essential NES motif functions evolutionarily predated CSR activity. We also show that fusion of the Takifugu AID catalytic domain to the entire human noncatalytic domain restores activity in mammalian cells, suggesting that AID features mapping within the noncatalytic domain, but outside the NES, influence its function.

Amino Acid Sequence↗
PubMed1998

SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex.

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

Amino Acid Sequence↗