Abstract
BackgroundBreast 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 FindingsBy 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 α6 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 compartmentsConclusions/SignificanceOur 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
Highlights
Breast cancer metastasis suppressor 1 (BRMS1), one of the members of a recently described family of proteins known as metastasis suppressors, inhibit the development of secondary foci without affecting the growth of the primary tumour
Analysis and prediction of cellular localization signals based on BRMS1 amino-acid sequence by the PSORT II server [18] reveals the presence of two putative nuclear localization signals (NLSs) at residues 198–205 (PPSKRKKA) and 238–245 (PQKRKSD) fulfilling the loose consensus of the classical monopartite NLS which requires a lysine in the P1 position followed by basic residues in positions P2 and P4 to yield a consensus sequence of K K/R X K/R [19]
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]
Summary
Breast cancer metastasis suppressor 1 (BRMS1), one of the members of a recently described family of proteins known as metastasis suppressors, inhibit the development of secondary foci without affecting the growth of the primary tumour. These results were demonstrated by ectopic expression into highly metastatic cells in an experimental in vivo assay (reviewed in [1]). 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. According to its amino acids sequence, BRMS1 contain two putative nuclear localization signals, none of them has been proved to work so far
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