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An actin-regulated importin α/β-dependent extended bipartite NLS directs nuclear import of MRTF-A.

Pawłowski R, Rajakylä EK, Vartiainen MK, Treisman R - EMBO J. (2010)

Bottom Line: We show that MRTF-A contains an unusually long bipartite nuclear localisation signal (NLS), comprising two basic elements separated by 30 residues, embedded within the RPEL domain.Binding of the Impα-Impβ heterodimer to the intact MRTF-A RPEL domain occurs competitively with G-actin.Thus, MRTF-A contains an actin-sensitive nuclear import signal.

View Article: PubMed Central - PubMed

Affiliation: Transcription Laboratory, Cancer Research UK, London Research Institute, London, UK.

ABSTRACT
Myocardin-related transcription factors (MRTFs) are actin-regulated transcriptional coactivators, which bind G-actin through their N-terminal RPEL domains. In response to signal-induced actin polymerisation and concomitant G-actin depletion, MRTFs accumulate in the nucleus and activate target gene transcription through their partner protein SRF. Nuclear accumulation of MRTFs in response to signal is inhibited by increased G-actin level. Here, we study the mechanism by which MRTF-A enters the nucleus. We show that MRTF-A contains an unusually long bipartite nuclear localisation signal (NLS), comprising two basic elements separated by 30 residues, embedded within the RPEL domain. Using siRNA-mediated protein depletion in vivo, and nuclear import assays in vitro, we show that the MRTF-A extended bipartite NLS uses the importin (Imp)α/β-dependent import pathway, and that import is inhibited by G-actin. Interaction of the NLS with the Impα-Impβ heterodimer requires both NLS basic elements, and is dependent on the Impα major and minor binding pockets. Binding of the Impα-Impβ heterodimer to the intact MRTF-A RPEL domain occurs competitively with G-actin. Thus, MRTF-A contains an actin-sensitive nuclear import signal.

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The B2 and B3 elements constitute an extended bipartite NLS sufficient for nuclear import of a heterologous protein. NIH 3T3 cells were transfected with the PK fusion proteins and analysed by immunofluorescence microscopy 24 h later with or without a 30-min treatment with 30 nM leptomycin B (LMB). (A) Schematic representation of pyruvate kinase (PK) fusion proteins. (B) The B2 and B3 elements constitute a bipartite NLS, and the B2 element possesses weak autonomous NLS activity. (C) Nuclear accumulation of MRTF-A(111–166)–PK requires intact B2 and B3 elements.
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f2: The B2 and B3 elements constitute an extended bipartite NLS sufficient for nuclear import of a heterologous protein. NIH 3T3 cells were transfected with the PK fusion proteins and analysed by immunofluorescence microscopy 24 h later with or without a 30-min treatment with 30 nM leptomycin B (LMB). (A) Schematic representation of pyruvate kinase (PK) fusion proteins. (B) The B2 and B3 elements constitute a bipartite NLS, and the B2 element possesses weak autonomous NLS activity. (C) Nuclear accumulation of MRTF-A(111–166)–PK requires intact B2 and B3 elements.

Mentions: As independent mutation of either the B2 or B3 sequences effectively abolished MRTF-A nuclear import, we speculated that together the two elements might constitute a bipartite nuclear import signal. To test whether this is the case, we assessed the subcellular localisation of fusion proteins in which peptide sequences encompassing B2, B3 or both elements were joined to the normally cytoplasmic pyruvate kinase protein (Figure 2A). Fusion proteins containing either B2 or B3 alone were predominantly cytoplasmic (Figure 2B). In contrast, the fusion protein containing the entire putative NLS sequence (111/166–PK) localised exclusively to the nucleus, and this was dependent on the integrity of the two basic elements, as seen with MRTF-A itself (Figure 2C). Actin overexpression did not cause relocalisation of 111/166–PK to the cytoplasm (data not shown), consistent with the observation that the entire RPEL domain is required for correct MRTF-A regulation (Miralles et al, 2003; Vartiainen et al, 2007; Guettler et al, 2008).


An actin-regulated importin α/β-dependent extended bipartite NLS directs nuclear import of MRTF-A.

Pawłowski R, Rajakylä EK, Vartiainen MK, Treisman R - EMBO J. (2010)

The B2 and B3 elements constitute an extended bipartite NLS sufficient for nuclear import of a heterologous protein. NIH 3T3 cells were transfected with the PK fusion proteins and analysed by immunofluorescence microscopy 24 h later with or without a 30-min treatment with 30 nM leptomycin B (LMB). (A) Schematic representation of pyruvate kinase (PK) fusion proteins. (B) The B2 and B3 elements constitute a bipartite NLS, and the B2 element possesses weak autonomous NLS activity. (C) Nuclear accumulation of MRTF-A(111–166)–PK requires intact B2 and B3 elements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2964165&req=5

f2: The B2 and B3 elements constitute an extended bipartite NLS sufficient for nuclear import of a heterologous protein. NIH 3T3 cells were transfected with the PK fusion proteins and analysed by immunofluorescence microscopy 24 h later with or without a 30-min treatment with 30 nM leptomycin B (LMB). (A) Schematic representation of pyruvate kinase (PK) fusion proteins. (B) The B2 and B3 elements constitute a bipartite NLS, and the B2 element possesses weak autonomous NLS activity. (C) Nuclear accumulation of MRTF-A(111–166)–PK requires intact B2 and B3 elements.
Mentions: As independent mutation of either the B2 or B3 sequences effectively abolished MRTF-A nuclear import, we speculated that together the two elements might constitute a bipartite nuclear import signal. To test whether this is the case, we assessed the subcellular localisation of fusion proteins in which peptide sequences encompassing B2, B3 or both elements were joined to the normally cytoplasmic pyruvate kinase protein (Figure 2A). Fusion proteins containing either B2 or B3 alone were predominantly cytoplasmic (Figure 2B). In contrast, the fusion protein containing the entire putative NLS sequence (111/166–PK) localised exclusively to the nucleus, and this was dependent on the integrity of the two basic elements, as seen with MRTF-A itself (Figure 2C). Actin overexpression did not cause relocalisation of 111/166–PK to the cytoplasm (data not shown), consistent with the observation that the entire RPEL domain is required for correct MRTF-A regulation (Miralles et al, 2003; Vartiainen et al, 2007; Guettler et al, 2008).

Bottom Line: We show that MRTF-A contains an unusually long bipartite nuclear localisation signal (NLS), comprising two basic elements separated by 30 residues, embedded within the RPEL domain.Binding of the Impα-Impβ heterodimer to the intact MRTF-A RPEL domain occurs competitively with G-actin.Thus, MRTF-A contains an actin-sensitive nuclear import signal.

View Article: PubMed Central - PubMed

Affiliation: Transcription Laboratory, Cancer Research UK, London Research Institute, London, UK.

ABSTRACT
Myocardin-related transcription factors (MRTFs) are actin-regulated transcriptional coactivators, which bind G-actin through their N-terminal RPEL domains. In response to signal-induced actin polymerisation and concomitant G-actin depletion, MRTFs accumulate in the nucleus and activate target gene transcription through their partner protein SRF. Nuclear accumulation of MRTFs in response to signal is inhibited by increased G-actin level. Here, we study the mechanism by which MRTF-A enters the nucleus. We show that MRTF-A contains an unusually long bipartite nuclear localisation signal (NLS), comprising two basic elements separated by 30 residues, embedded within the RPEL domain. Using siRNA-mediated protein depletion in vivo, and nuclear import assays in vitro, we show that the MRTF-A extended bipartite NLS uses the importin (Imp)α/β-dependent import pathway, and that import is inhibited by G-actin. Interaction of the NLS with the Impα-Impβ heterodimer requires both NLS basic elements, and is dependent on the Impα major and minor binding pockets. Binding of the Impα-Impβ heterodimer to the intact MRTF-A RPEL domain occurs competitively with G-actin. Thus, MRTF-A contains an actin-sensitive nuclear import signal.

Show MeSH
Related in: MedlinePlus