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Identification of essential sequences for cellular localization in BRMS1 metastasis suppressor.

Rivera J, Megías D, Navas C, Bravo J - PLoS ONE (2009)

Bottom Line: Besides, the combination of a computational searching approach along the utilization of a nuclear export assay, identified a functional motif within the BRMS1 sequence responsible for its nuclear export, that resulted not affected by the highly specific CRM1 inhibitor Leptomycin-B.Interspecies heterokaryon assay demonstrate the capability of BRMS1 to shuttle between the nuclear and cytosolic compartments Our results show for the first time that BRMS1 contains both nuclear import and export signals enabling its nucleo-cytoplasmic shuttling.These findings contributes new data for the understanding of the BRMS1 functions and allow us to speculate that this phenomenon could represent a novel mechanism for regulating the activity of BRMS1 or its associated cytosolic partners.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Group, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain. jrivera@cnio.es

ABSTRACT

Background: Breast cancer metastasis suppressor 1 (BRMS1) reduces the number and the size of secondary tumours in a mouse model without affecting the growth of the primary foci upon its re-expression. Knockdown of BRMS1 expression associates with metastasis. The molecular details on BRMS1 mechanism of action include its ability to function as a transcriptional co-repressor and consistently BRMS1 has been described as a predominantly nuclear protein. Since cellular distribution could represent a potential mechanism of regulation, we wanted to characterize BRMS1 sequence motifs that might regulate its cellular distribution. According to its amino acids sequence, BRMS1 contain two putative nuclear localization signals, however none of them has been proved to work so far.

Methodology/principal findings: By using well known in vivo assays to detect both nuclear import and export signal, we have characterized, in the present study, one functional nuclear localisation signal as necessary and sufficient to promote nuclear transport. Additionally, the outcome of a directed yeast two-hybrid assay identify importin alpha6 as a specific partner of BRMS1 thus speculating that BRMS1 nuclear import could be specifically mediated by the reported nuclear transporter. Besides, the combination of a computational searching approach along the utilization of a nuclear export assay, identified a functional motif within the BRMS1 sequence responsible for its nuclear export, that resulted not affected by the highly specific CRM1 inhibitor Leptomycin-B. Interspecies heterokaryon assay demonstrate the capability of BRMS1 to shuttle between the nuclear and cytosolic compartments

Conclusions/significance: Our results show for the first time that BRMS1 contains both nuclear import and export signals enabling its nucleo-cytoplasmic shuttling. These findings contributes new data for the understanding of the BRMS1 functions and allow us to speculate that this phenomenon could represent a novel mechanism for regulating the activity of BRMS1 or its associated cytosolic partners.

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BRMS1 contains a functional nuclear export motif independent of CRM1.(A) Scheme of the plasmid system used for testing the putative BRMS1 NES motif. Disrupted endogenous NES of Rev protein (Rev1.4) allows the in vivo analysis capability of a BamHI/AgeI inserted sequence fused to eGFP. PCMV: CMV promoter. Representative confocal images of U-2 OS transfected cells with indicated plasmids untreated (−) or treated (+) with ActD (B) or Act-D + LMB (C). Fixed cells were scored for nuclear (N), citosolic (C) or diffuse (N+C) GFP location. Graphics show mean values of percentage of positive cells with S.E.M across three independent experiments.
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pone-0006433-g005: BRMS1 contains a functional nuclear export motif independent of CRM1.(A) Scheme of the plasmid system used for testing the putative BRMS1 NES motif. Disrupted endogenous NES of Rev protein (Rev1.4) allows the in vivo analysis capability of a BamHI/AgeI inserted sequence fused to eGFP. PCMV: CMV promoter. Representative confocal images of U-2 OS transfected cells with indicated plasmids untreated (−) or treated (+) with ActD (B) or Act-D + LMB (C). Fixed cells were scored for nuclear (N), citosolic (C) or diffuse (N+C) GFP location. Graphics show mean values of percentage of positive cells with S.E.M across three independent experiments.

Mentions: To test the export activity of the identified candidate NES in BRMS1, we generated a construct encoding 18 residues (74LKEKLFRERLSQLRLRLE91, hydrophobic residues are underlined) inserted between the export deficient Rev1.4 sequence and eGFP (Figure 5A), rendering the pRev1.4-BRMS1-eGFP plasmid. That insert size has been found to be optimal for export activity [29]. Constructs were transiently over-expressed into different cell types. The use of Act-D has been reported to prevent nucleolar accumulation and nuclear import of Rev protein thereby provoking a cytosolic accumulation of NES-containing protein [33]. Therefore, we treated transfected cells with Act-D to facilitate the detection of weak NES signals [29].


Identification of essential sequences for cellular localization in BRMS1 metastasis suppressor.

Rivera J, Megías D, Navas C, Bravo J - PLoS ONE (2009)

BRMS1 contains a functional nuclear export motif independent of CRM1.(A) Scheme of the plasmid system used for testing the putative BRMS1 NES motif. Disrupted endogenous NES of Rev protein (Rev1.4) allows the in vivo analysis capability of a BamHI/AgeI inserted sequence fused to eGFP. PCMV: CMV promoter. Representative confocal images of U-2 OS transfected cells with indicated plasmids untreated (−) or treated (+) with ActD (B) or Act-D + LMB (C). Fixed cells were scored for nuclear (N), citosolic (C) or diffuse (N+C) GFP location. Graphics show mean values of percentage of positive cells with S.E.M across three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713406&req=5

pone-0006433-g005: BRMS1 contains a functional nuclear export motif independent of CRM1.(A) Scheme of the plasmid system used for testing the putative BRMS1 NES motif. Disrupted endogenous NES of Rev protein (Rev1.4) allows the in vivo analysis capability of a BamHI/AgeI inserted sequence fused to eGFP. PCMV: CMV promoter. Representative confocal images of U-2 OS transfected cells with indicated plasmids untreated (−) or treated (+) with ActD (B) or Act-D + LMB (C). Fixed cells were scored for nuclear (N), citosolic (C) or diffuse (N+C) GFP location. Graphics show mean values of percentage of positive cells with S.E.M across three independent experiments.
Mentions: To test the export activity of the identified candidate NES in BRMS1, we generated a construct encoding 18 residues (74LKEKLFRERLSQLRLRLE91, hydrophobic residues are underlined) inserted between the export deficient Rev1.4 sequence and eGFP (Figure 5A), rendering the pRev1.4-BRMS1-eGFP plasmid. That insert size has been found to be optimal for export activity [29]. Constructs were transiently over-expressed into different cell types. The use of Act-D has been reported to prevent nucleolar accumulation and nuclear import of Rev protein thereby provoking a cytosolic accumulation of NES-containing protein [33]. Therefore, we treated transfected cells with Act-D to facilitate the detection of weak NES signals [29].

Bottom Line: Besides, the combination of a computational searching approach along the utilization of a nuclear export assay, identified a functional motif within the BRMS1 sequence responsible for its nuclear export, that resulted not affected by the highly specific CRM1 inhibitor Leptomycin-B.Interspecies heterokaryon assay demonstrate the capability of BRMS1 to shuttle between the nuclear and cytosolic compartments Our results show for the first time that BRMS1 contains both nuclear import and export signals enabling its nucleo-cytoplasmic shuttling.These findings contributes new data for the understanding of the BRMS1 functions and allow us to speculate that this phenomenon could represent a novel mechanism for regulating the activity of BRMS1 or its associated cytosolic partners.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Group, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain. jrivera@cnio.es

ABSTRACT

Background: Breast cancer metastasis suppressor 1 (BRMS1) reduces the number and the size of secondary tumours in a mouse model without affecting the growth of the primary foci upon its re-expression. Knockdown of BRMS1 expression associates with metastasis. The molecular details on BRMS1 mechanism of action include its ability to function as a transcriptional co-repressor and consistently BRMS1 has been described as a predominantly nuclear protein. Since cellular distribution could represent a potential mechanism of regulation, we wanted to characterize BRMS1 sequence motifs that might regulate its cellular distribution. According to its amino acids sequence, BRMS1 contain two putative nuclear localization signals, however none of them has been proved to work so far.

Methodology/principal findings: By using well known in vivo assays to detect both nuclear import and export signal, we have characterized, in the present study, one functional nuclear localisation signal as necessary and sufficient to promote nuclear transport. Additionally, the outcome of a directed yeast two-hybrid assay identify importin alpha6 as a specific partner of BRMS1 thus speculating that BRMS1 nuclear import could be specifically mediated by the reported nuclear transporter. Besides, the combination of a computational searching approach along the utilization of a nuclear export assay, identified a functional motif within the BRMS1 sequence responsible for its nuclear export, that resulted not affected by the highly specific CRM1 inhibitor Leptomycin-B. Interspecies heterokaryon assay demonstrate the capability of BRMS1 to shuttle between the nuclear and cytosolic compartments

Conclusions/significance: Our results show for the first time that BRMS1 contains both nuclear import and export signals enabling its nucleo-cytoplasmic shuttling. These findings contributes new data for the understanding of the BRMS1 functions and allow us to speculate that this phenomenon could represent a novel mechanism for regulating the activity of BRMS1 or its associated cytosolic partners.

Show MeSH
Related in: MedlinePlus