Limits...
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.

Show MeSH

Related in: MedlinePlus

NLS1 is necessary and sufficient for nuclear targeting of BRMS1.(A) Schemes of full length (FL) BRMS1, deletion mutants and their controls (pHM830 and pHM840). Plasmids express a triple GFP/insert/β-Gal fusion protein. (B) Confocal images of U-2 OS transfected cells expressing GFP-β-Gal NLS constructs. (C) A construct encompassing the residues KRKK from NLS1, resemble the location pattern of the FL. KRKS, represents a point mutant where the last positively charged residue shifted to uncharged. (D) Total cell lysates from U-2 OS untransfected (mock) or transfected cells with the indicated constructs were analysed by Western Blotting using an anti-GFP antibody. Molecular markers are shown in kDa.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2713406&req=5

pone-0006433-g001: NLS1 is necessary and sufficient for nuclear targeting of BRMS1.(A) Schemes of full length (FL) BRMS1, deletion mutants and their controls (pHM830 and pHM840). Plasmids express a triple GFP/insert/β-Gal fusion protein. (B) Confocal images of U-2 OS transfected cells expressing GFP-β-Gal NLS constructs. (C) A construct encompassing the residues KRKK from NLS1, resemble the location pattern of the FL. KRKS, represents a point mutant where the last positively charged residue shifted to uncharged. (D) Total cell lysates from U-2 OS untransfected (mock) or transfected cells with the indicated constructs were analysed by Western Blotting using an anti-GFP antibody. Molecular markers are shown in kDa.

Mentions: To check whether NLS1 and NLS2 are required for BRMS1 nuclear import, we engineered truncated forms of BRMS1 (Figure 1A) and inserted them into the NLS mapping vector pHM830 [20]. The resulting fusion protein is large enough to prevent its passive diffusion to the nucleus, since its size is far above the diffusion limit for the mammalian nuclear pore complex [21]. This system has extensively been used for the unambiguous identification of the NLS activity of both conventional [20] and non conventional import signals [22].


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

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

NLS1 is necessary and sufficient for nuclear targeting of BRMS1.(A) Schemes of full length (FL) BRMS1, deletion mutants and their controls (pHM830 and pHM840). Plasmids express a triple GFP/insert/β-Gal fusion protein. (B) Confocal images of U-2 OS transfected cells expressing GFP-β-Gal NLS constructs. (C) A construct encompassing the residues KRKK from NLS1, resemble the location pattern of the FL. KRKS, represents a point mutant where the last positively charged residue shifted to uncharged. (D) Total cell lysates from U-2 OS untransfected (mock) or transfected cells with the indicated constructs were analysed by Western Blotting using an anti-GFP antibody. Molecular markers are shown in kDa.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006433-g001: NLS1 is necessary and sufficient for nuclear targeting of BRMS1.(A) Schemes of full length (FL) BRMS1, deletion mutants and their controls (pHM830 and pHM840). Plasmids express a triple GFP/insert/β-Gal fusion protein. (B) Confocal images of U-2 OS transfected cells expressing GFP-β-Gal NLS constructs. (C) A construct encompassing the residues KRKK from NLS1, resemble the location pattern of the FL. KRKS, represents a point mutant where the last positively charged residue shifted to uncharged. (D) Total cell lysates from U-2 OS untransfected (mock) or transfected cells with the indicated constructs were analysed by Western Blotting using an anti-GFP antibody. Molecular markers are shown in kDa.
Mentions: To check whether NLS1 and NLS2 are required for BRMS1 nuclear import, we engineered truncated forms of BRMS1 (Figure 1A) and inserted them into the NLS mapping vector pHM830 [20]. The resulting fusion protein is large enough to prevent its passive diffusion to the nucleus, since its size is far above the diffusion limit for the mammalian nuclear pore complex [21]. This system has extensively been used for the unambiguous identification of the NLS activity of both conventional [20] and non conventional import signals [22].

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