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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 comprise a putative NES motif that compare to leucine-rich NES.(A) Schematic representation of the BRMS1 structure showing the putative NES sequence boundaries identified by NetNES 1.1 server. (B) Alignment of conserved hydrophobic residues (in boxes) from different BRMS1 orthologs. Numbers refer to amino acid residues. Human putative NES-BRMS1 sequence is compared with known NES, ranging from highest (MAPKK; PKI and Rev) to the weakest (p53) activity. A generally accepted loose consensus sequence, where X represents any amino acid and Φ any large hydrophobic residue (L, I, V, F, M) is shown.
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pone-0006433-g004: BRMS1 comprise a putative NES motif that compare to leucine-rich NES.(A) Schematic representation of the BRMS1 structure showing the putative NES sequence boundaries identified by NetNES 1.1 server. (B) Alignment of conserved hydrophobic residues (in boxes) from different BRMS1 orthologs. Numbers refer to amino acid residues. Human putative NES-BRMS1 sequence is compared with known NES, ranging from highest (MAPKK; PKI and Rev) to the weakest (p53) activity. A generally accepted loose consensus sequence, where X represents any amino acid and Φ any large hydrophobic residue (L, I, V, F, M) is shown.

Mentions: We inspected its entire amino-acid sequence looking for a leucine, or any other large hydrophobic rich region similar to those previously identified as the transport signals that can mediate nuclear export [30], [31]. We noticed a large cluster of hydrophobic residues closely spaced in the amino terminal half of the protein that would fulfil the loose consensus of a leucine-rich NES. This result was confirmed after submission of BRMS1 sequence to the NetNES 1.1 database server [16]. The server assigned NES score values over the threshold for residues L83 to L88, which in addition to an over-representation of D, E and S residues suggest that this region conforms to the established criteria for a NES [30]. As shown in Figure 4 the alignment of all the BRMS1 orthologs deposited in the databases, identified by blastp search [32] demonstrates the high conservation of this candidate NES sequence among the different species, including the regularly conserved spacing between the critical residues for export function.


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

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

BRMS1 comprise a putative NES motif that compare to leucine-rich NES.(A) Schematic representation of the BRMS1 structure showing the putative NES sequence boundaries identified by NetNES 1.1 server. (B) Alignment of conserved hydrophobic residues (in boxes) from different BRMS1 orthologs. Numbers refer to amino acid residues. Human putative NES-BRMS1 sequence is compared with known NES, ranging from highest (MAPKK; PKI and Rev) to the weakest (p53) activity. A generally accepted loose consensus sequence, where X represents any amino acid and Φ any large hydrophobic residue (L, I, V, F, M) is shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006433-g004: BRMS1 comprise a putative NES motif that compare to leucine-rich NES.(A) Schematic representation of the BRMS1 structure showing the putative NES sequence boundaries identified by NetNES 1.1 server. (B) Alignment of conserved hydrophobic residues (in boxes) from different BRMS1 orthologs. Numbers refer to amino acid residues. Human putative NES-BRMS1 sequence is compared with known NES, ranging from highest (MAPKK; PKI and Rev) to the weakest (p53) activity. A generally accepted loose consensus sequence, where X represents any amino acid and Φ any large hydrophobic residue (L, I, V, F, M) is shown.
Mentions: We inspected its entire amino-acid sequence looking for a leucine, or any other large hydrophobic rich region similar to those previously identified as the transport signals that can mediate nuclear export [30], [31]. We noticed a large cluster of hydrophobic residues closely spaced in the amino terminal half of the protein that would fulfil the loose consensus of a leucine-rich NES. This result was confirmed after submission of BRMS1 sequence to the NetNES 1.1 database server [16]. The server assigned NES score values over the threshold for residues L83 to L88, which in addition to an over-representation of D, E and S residues suggest that this region conforms to the established criteria for a NES [30]. As shown in Figure 4 the alignment of all the BRMS1 orthologs deposited in the databases, identified by blastp search [32] demonstrates the high conservation of this candidate NES sequence among the different species, including the regularly conserved spacing between the critical residues for export function.

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