Distinct nuclear import and export pathways mediated by members of the karyopherin β family

Moroianu, Junona

doi:10.1002/(sici)1097-4644(19980801)70:2<231::aid-jcb9>3.0.co;2-p

Cited by 63 publications

(17 citation statements)

References 86 publications

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“…These identified proteins were subdivided into eight groups according to their previously published functions (Matunis et al . 1996, Moroianu 1998, Stopka et al . 2000, Goldberg et al .…”

Section: Resultsmentioning

confidence: 99%

Identification of cell-specific targets of sumoylation during mouse spermatogenesis

Xiao¹,

Pollack²,

Andrusier³

et al. 2016

Reproduction

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Recent findings suggest diverse and potentially multiple roles of SUMO in testicular function and spermatogenesis. However, SUMO targets remain uncharacterized in the testis due to the complex multicellular nature of testicular tissue, the inability to maintain and manipulate spermatogenesis in vitro, and the technical challenges involved in identifying low-abundance endogenous SUMO targets. In this study, we performed cell-specific identification of sumoylated proteins using concentrated cell lysates prepared with de-sumoylation inhibitors from freshly purified spermatocytes and spermatids. One-hundred and twenty proteins were uniquely identified in the spermatocyte and/or spermatid fractions. The identified proteins are involved in the regulation of transcription, stress response, microRNA biogenesis, regulation of major enzymatic pathways, nuclear-cytoplasmic transport, cell cycle control, acrosome biogenesis, and other processes. Several proteins with important roles during spermatogenesis were chosen for further characterization by co-immunoprecipitation, co-localization and in-vitro sumoylation studies. GPS-SUMO software was used to identify consensus and non-consensus sumoylation sites within the amino acid sequences of the proteins. The analyses confirmed the cell-specific sumoylation and/or SUMO interaction of several novel, previously uncharacterized SUMO targets such as CDK1, RNAP II, CDC5, MILI, DDX4, TDP-43 and STK31. Furthermore, several proteins that were previously identified as SUMO targets in somatic cells (e.g., KAP1, MDC1) were identified as SUMO targets in germ cells. Many of these proteins have a unique role in spermatogenesis and during meiotic progression. This research opens a novel avenue for further studies of SUMO at the level of individual targets.

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“…These identified proteins were subdivided into eight groups according to their previously published functions (Matunis et al . 1996, Moroianu 1998, Stopka et al . 2000, Goldberg et al .…”

Section: Resultsmentioning

confidence: 99%

Identification of cell-specific targets of sumoylation during mouse spermatogenesis

Xiao¹,

Pollack²,

Andrusier³

et al. 2016

Reproduction

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“…Thus, both the ESE-1 NES1 and NES2 sequences are sufficient to mediate nuclear export. Because NES motifs conforming to the (L/I)X2-4(L/I)X1-4(L/I)X(L/I) consensus sequence activate nuclear export by binding directly to the CRM1 nuclear exporter protein [24], we next tested the role of CRM1 in the nuclear export mediated by each ESE-1 NES motifs. MCF-12A cells transfected with the GFP-NES1-SAR or GFP-NES2-SAR constructs were treated with the CRM1-specific inhibitor leptomycin B [25], which resulted in the redistribution of GFP-NES1-SAR (Figure 2C, panels 3 & 4) and GFP-NES2-SAR (Figure 2C, panels 9 & 10), respectively, from the cytoplasm both to the nuclear and cytoplasmic compartments.…”

Section: Resultsmentioning

confidence: 99%

Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism

et al. 2011

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BackgroundThe ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR) subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells.ResultsTo map the minimal functional nuclear localization (NLS) and nuclear export (NES) signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242HGKRRR247) in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102LCNCALEELRL112) and another to the DNA binding domain (DBD), (NES2: 275LWEFIRDILI284). Moreover, analysis of a putative NLS located in the DBD (316GQKKKNSN323) by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions.ConclusionsThese data highlight that ESE-1 contains NLS and NES signals that play a critical role in regulating its subcellular localization and function, and that an intact SAR domain mediates MEC transformation exclusively in the cytoplasm, via a novel nontranscriptional mechanism, whereby the SAR motif is accessible for ligand and/or protein interactions. These findings are significant, since they provide novel molecular insights into the functions of ETS transcription factors in mammary cell transformation.

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“…In eukaryotic cells, the presence of the nuclear envelope provides a layer of separation between the cytoplasm, where transcription factors and chromatin remodeling enzymes are synthesized, and the nucleus and the chromatin contained therein. Several intracellular trafficking systems have been described [4], [5]; each uses a distinct mechanism to interact with nuclear cargoes (for export to the cytoplasm) and cytoplasmic cargoes (for import to the nucleus). These trafficking systems transport their intracellular cargoes through the nuclear pore complexes on the nuclear envelope and position the cargoes in the proper cellular compartment at the correct time during the cell cycle and development.…”

Section: Introductionmentioning

confidence: 99%

Identification of Karyopherin α1 and α7 Interacting Proteins in Porcine Tissue

et al. 2012

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Specialized trafficking systems in eukaryotic cells serve a critical role in partitioning intracellular proteins between the nucleus and cytoplasm. Cytoplasmic proteins (including chromatin remodeling enzymes and transcription factors) must gain access to the nucleus to exert their functions to properly program fundamental cellular events ranging from cell cycle progression to gene transcription. Knowing that nuclear import mediated by members of the karyopherin α family of transport receptors plays a critical role in regulating development and differentiation, we wanted to determine the identity of proteins that are trafficked by this karyopherin α pathway. To this end, we performed a GST pull-down assay using porcine orthologs of karyopherin α1 (KPNA1) and karyopherin α7 (KPNA7) and prey protein derived from porcine fibroblast cells and used a liquid chromatography and tandem mass spectrometry (LC-MS/MS) approach to determine the identity of KPNA1 and KPNA7 interacting proteins. Our screen revealed that the proteins that interact with KPNA1 and KPNA7 are generally nuclear proteins that possess nuclear localization signals. We further validated two candidate proteins from this screen and showed that they are able to be imported into the nucleus in vivo and also interact with members of the karyopherin α family of proteins in vitro. Our results also reveal the utility of using a GST pull-down approach coupled with LC-MS/MS to screen for protein interaction partners in a non-traditional model system.

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Distinct nuclear import and export pathways mediated by members of the karyopherin β family

Cited by 63 publications

References 86 publications

Identification of cell-specific targets of sumoylation during mouse spermatogenesis

Identification of cell-specific targets of sumoylation during mouse spermatogenesis

Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism

Identification of Karyopherin α1 and α7 Interacting Proteins in Porcine Tissue

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