Qualitative Highly Divergent Nuclear Export Signals Can Regulate Export by the Competition for Transport Cofactors <i>in Vivo</i>

Heger, Peter; Lohmaier, Jens; Schneider, Grit; Schweimer, Kristian; Stauber, Roland H.

doi:10.1034/j.1600-0854.2001.20804.x

Cited by 26 publications

(55 citation statements)

References 59 publications

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“…However, the fact that both CSR and stability require maintenance of key hydrophobic positions in the NES and show a similar dependence on the identity of the interdigitating hydrophilic residues suggests that they might both depend on the quality of the AID-exportin interaction. Changes in the hydrophilic interdigitating amino acids in an NES have been shown in other systems to affect the quality of exportin-binding and kinetics of intracellular transport (20,21). The dependence of CSR and protein stability on the nature of the NES could therefore simply reflect a requirement for particular kinetics of nucleocytoplasmic shuttling.…”

Section: Discussionmentioning

confidence: 99%

The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence

Geisberger

Rada²,

Neuberger³

2009

Proc. Natl. Acad. Sci. U.S.A.

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The carboxyterminal region of activation-induced deaminase (AID) is required for its function in Ig class switch recombination (CSR) and also contains a nuclear-export sequence (NES). Here, based on an extensive fine-structure mutation analysis of the AID NES, as well as from AID chimeras bearing heterologous NESs, we show that while a functional NES is indeed essential for CSR, it is not sufficient. The precise nature of the NES is critical both for AID stabilization and CSR function: minor changes in the NES can perturb stabilization and CSR without jeopardizing nuclear export. The results indicate that the AID NES fulfills a function beyond simply providing a signal for nuclear export and suggest the possibility that the quality of exportin-binding may be critical to the stabilization of AID and its activity in CSR.activation-induced deaminase ͉ antibody diversification ͉ immunoglobulin class switching ͉ exportin ͉ nuclear transport A ctivation-induced deaminase (AID) plays a central role in antibody gene-diversification, triggering both somatic hypermutation (SHM) and class-switch recombination (CSR) by deaminating deoxycytidine residues within the Ig locus to yield deoxyuridine (1, 2).AID is largely found in the cytoplasm of activated B cells but shuttles between cytoplasm and nucleus (3-6). The C-terminal amino acids of AID comprise a nuclear-export sequence (NES): removal of this NES causes AID to be restricted to the nucleus, whereas its addition to a heterologous nuclear protein drives export of the fusion protein into the cytoplasm (3-5). The sequence of the AID C-terminus conforms well to the consensus established for substrates of the Crm1-dependent export machinery: in keeping with this, export mediated by the AID NES is sensitive to leptomycin B.Analysis of both clinical and contrived mutations in AID have revealed that the AID C-terminal region is also essential for its function in CSR, although not in SHM (7-9). The C-terminal mutations that interfere with AID's ability to potentiate CSR do not compromise its ability to deaminate deoxycytidine in biochemical or bacterial genetic assays. Thus, as previously suggested by others, either the nuclear/cytoplasmic shuttling of AID must be specifically required for CSR or, alternatively, factors specific for CSR must interact with the C-terminal region of AID in an area overlapping the NES (4, 5, 7-9).It was with a view to discriminating these possibilities that we embarked on a refined mutagenic dissection of the AID NES and investigated the ability of heterologous NESs to substitute for the AID NES. Our results suggest that although nuclear export is likely necessary for CSR, this is not the sole function of the AID C-terminal region, with the data raising the possibility that successful CSR depends on the precise quality of AID binding to exportin or a related protein. Results NES Function and CSR Correlate in Alanine-Scanning Mutagenesis.To ascertain whether there was a correlation between export activity and CSR, we performed an alanine-scanning mut...

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Section: Discussionmentioning

confidence: 99%

The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence

Geisberger

Rada²,

Neuberger³

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…18 The NES mediates the RanGTP-dependent Crm1 interaction, is conserved in all mammalian survivin proteins 19 and matches the consensus sequence for leucine-rich NESs. 20 In particular, microinjection of recombinant GST-GFP fusion proteins proved to be a highly reliable and stringent system that allows the quantification of transport in living cells independent of drug treatment or passive diffusion. 11 Microinjection experiments revealed that although the NES proposed by Colnaghi et al 21 ( 96 LTLGEFLKL 104 ) partially overlaps with the NES identified by us, this signal is not sufficient to mediate effective export (manuscript in preparation).…”

Section: Introductionmentioning

confidence: 99%

Survivin’s Dual Role: An Export’s View

Knauer¹,

Mann²,

Stauber³

2007

Cell Cycle

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“…The putative NES (aa 95À103 ) loosely fits a consensus sequence proposed by, Heger et al 16 L-x(2,3)-(FILVMP)-x(2)-(LI)-x-(LIV). Furthermore, COILS algorithms indicate the typical COOHterminal coiled-coil conformation (aa 127À147 ), usually present in survivin but missing in other IAP family members.…”

Section: Resultsmentioning

confidence: 99%

“…This is consistent with previous studies in which chromosome region maintenance 1-mediated, NES-dependent nuclear export of human survivin was blocked by LMB. 10,16 Survivin is a subunit of the chromosomal passenger complex (CPC), consisting of Aurora-B, Borealin and INCENP, and is essential for proper Conserved dual role of survivin B Luthringer et al chromosome segregation and cytokinesis. As Crm1 is involved in tethering the CPC to the centromere by interacting with survivin's NES, 10 SDSURVL might affect various stages of cell proliferation, from proper kinetochore attachment to spindle formation.…”

Section: Discussionmentioning

confidence: 99%

“…16 In short, 48 h after transfection cells were treated with LMB (6 nM, 30 min), washed in phosphate-buffered saline (PBS), and fixed in 4% (w/v) paraformaldehyde (PFA; 5 min, RT). After additional washing of cells with PBS and subsequent counterstaining of nuclei with DRAQ5 (5 mM; Biostatus Ltd., Shepshed, UK), images were acquired on a Zeiss 710 confocal LSM (Zeiss, Göttingen, Germany).…”

Section: Methodsmentioning

confidence: 99%

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Poriferan survivin exhibits a conserved regulatory role in the interconnected pathways of cell cycle and apoptosis

Luthringer¹,

Isbert²,

Müller³

et al. 2010

Cell Death Differ

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Survivin orchestrates intracellular pathways during cell division and apoptosis. Its central function as mitotic regulator and inhibitor of cell death has major implications for tumor cell proliferation. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution a complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulator, a survivin-like protein (SURVL) of one of the earliest-branching metazoan taxa was identified and functionally characterized. SURVL of the sponge Suberites domuncula shares considerable similarities with its metazoan homologs, ranging from conserved exon/intron structure to presence of protein-interaction domains. Whereas sponge tissue shows a low steady-state level, SURVL expression was significantly upregulated in rapidly proliferating primmorph cells. In addition, challenge of tissue and primmorphs with heavy metal or lipopeptide stimulated SURVL expression, concurrent with the expression of a newly discovered caspase. Complementary functional analyses in transfected HEK-293 cells revealed that heterologous expression of a SURVL-EFGP fusion not only promotes proliferation but also enhances resistance to cadmiuminduced cell death. Taken together, these results suggest both a deep evolutionary conserved dual role of survivin and an equally conserved central position in the interconnected pathways of cell cycle and apoptosis. Apoptosis has a crucial role not only in countless physiological processes (e.g., elimination of redundant cells during vertebrate embryonic development) but also during pathogenesis. As apoptosis relies on the clear-cut function of numerous proand antiapoptotic factors, any malfunction on their part or disruption of their respective interplay inevitably leads to dysfunctional cellular metabolism. The ensuing delayed or premature cell death often manifests in severe pathologies, including carcinogenesis and neurodegenerative diseases.The groundbreaking discovery of the Caenorhabditis elegans proteins cell death abnormality (Ced)-9, homolog of human antiapoptotic Bcl-2, and Ced-3, homolog of human caspase-1, has shown the molecular conservation of the apoptotic cell death program along a broad range of Metazoa, from nematodes to vertebrates.1 Since then, the fundamental role of apoptosis has been increasingly recognized in various physiological processes. In addition, several invertebrate model organisms revealed a significant potential for analyses of apoptotic mechanisms, not only under an evolutionary point of view. Thus, the identification of the human crumbs homolog 1 and its causative role in the manifestation of retinitis pigmentosa was based on the discovery of mutational variants of the Drosophila melanogaster protein crumbs.2 In addition, loss-of-function mutations of the C. elegans dystrophin-1 gene confirmed the role of human dystrophin in the manifestation...

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Qualitative Highly Divergent Nuclear Export Signals Can Regulate Export by the Competition for Transport Cofactors in Vivo

Cited by 26 publications

References 59 publications

The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence

The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence

Survivin’s Dual Role: An Export’s View

Poriferan survivin exhibits a conserved regulatory role in the interconnected pathways of cell cycle and apoptosis

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