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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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Importin β activity is required for MRTF-A nuclear accumulation. (A) Impβ was silenced in R332 cells (NIH 3T3 cells stably expressing MRTF-A–GFP) using RNA interference, and the localisation of the protein was analysed under different conditions. FCS, fetal calf serum; CD, Cytochalasin D (2 μM); LMB, leptomycin B. (⩽100 cells per point, n=3 independent experiments, error bars indicate s.e.m. values). Right: western blotting showing Impβ depletion (B) Similar as in panel A, serum stimulation. The Impβ knockdown was rescued by transient transfection of an siRNA-resistant form of Impβ–mCherry. mCherry empty plasmid was used as control. (C) Inhibition of SRF reporter 3D.A-Luc activation after Impβ depletion. Three independent experiments were performed; error bars represent s.e.m. values. (D) The localisation of transiently transfected MRTF-A–GFP in resting MDA-MB-231 cells. Phalloidin staining in red. Right: quantification. (E) Impβ–MRTF-A interaction detected by proximity ligation assay, using anti-impβ and anti-MRTF-A antibodies, is reduced in cells transfected with a β-actin expression plasmid, identified by coexpressed GFP marker. PLA was scored as cytoplasmic foci per cell (error bar indicates s.e.m. values; n=55; ***P<0.001, unpaired Mann–Whitney test).
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f3: Importin β activity is required for MRTF-A nuclear accumulation. (A) Impβ was silenced in R332 cells (NIH 3T3 cells stably expressing MRTF-A–GFP) using RNA interference, and the localisation of the protein was analysed under different conditions. FCS, fetal calf serum; CD, Cytochalasin D (2 μM); LMB, leptomycin B. (⩽100 cells per point, n=3 independent experiments, error bars indicate s.e.m. values). Right: western blotting showing Impβ depletion (B) Similar as in panel A, serum stimulation. The Impβ knockdown was rescued by transient transfection of an siRNA-resistant form of Impβ–mCherry. mCherry empty plasmid was used as control. (C) Inhibition of SRF reporter 3D.A-Luc activation after Impβ depletion. Three independent experiments were performed; error bars represent s.e.m. values. (D) The localisation of transiently transfected MRTF-A–GFP in resting MDA-MB-231 cells. Phalloidin staining in red. Right: quantification. (E) Impβ–MRTF-A interaction detected by proximity ligation assay, using anti-impβ and anti-MRTF-A antibodies, is reduced in cells transfected with a β-actin expression plasmid, identified by coexpressed GFP marker. PLA was scored as cytoplasmic foci per cell (error bar indicates s.e.m. values; n=55; ***P<0.001, unpaired Mann–Whitney test).

Mentions: We next sought to identify the import machinery involved in MRTF-A nuclear import. As Impβ has been shown to function as a nuclear import receptor for proteins containing a conventional bipartite NLS, we tested its involvement in MRTF-A nuclear import by use of siRNA to silence its expression. Knockdown of Impβ in NIH 3T3 cells stably expressing MRTF-A–GFP resulted in the substantial inhibition of LMB -induced MRTF-A nuclear accumulation (Figure 3A). Similar results were obtained when MRTF-A nuclear accumulation was induced by cytochalasin D, which interferes with actin–MRTF-A interaction, and thereby blocks actin-mediated MRTF-A export (Vartiainen et al, 2007). The effect of Impβ depletion on serum-stimulated nuclear accumulation was more marked (Figure 3A). Probably, this reflects the fact that serum stimulation, unlike CD or LMB treatment, does not completely block nuclear export (Vartiainen et al, 2007), although we cannot exclude the possibility that basal import involves additional Impβ-independent import signal(s) active only in unstimulated conditions. Expression of an siRNA-resistant Impβ derivative restored nuclear accumulation of MRTF-A in response to serum stimulation, indicating that the effect was specific (Figure 3B). Depletion of Impβ also impaired serum- or cytochalasin D-induced activation of an SRF reporter gene (Figure 3C). In MDA-MB-231 cells, in which MRTF-A is nuclear even under resting conditions (Medjkane et al, 2009), Impβ depletion restored its cytoplasmic localisation (Figure 3D).


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)

Importin β activity is required for MRTF-A nuclear accumulation. (A) Impβ was silenced in R332 cells (NIH 3T3 cells stably expressing MRTF-A–GFP) using RNA interference, and the localisation of the protein was analysed under different conditions. FCS, fetal calf serum; CD, Cytochalasin D (2 μM); LMB, leptomycin B. (⩽100 cells per point, n=3 independent experiments, error bars indicate s.e.m. values). Right: western blotting showing Impβ depletion (B) Similar as in panel A, serum stimulation. The Impβ knockdown was rescued by transient transfection of an siRNA-resistant form of Impβ–mCherry. mCherry empty plasmid was used as control. (C) Inhibition of SRF reporter 3D.A-Luc activation after Impβ depletion. Three independent experiments were performed; error bars represent s.e.m. values. (D) The localisation of transiently transfected MRTF-A–GFP in resting MDA-MB-231 cells. Phalloidin staining in red. Right: quantification. (E) Impβ–MRTF-A interaction detected by proximity ligation assay, using anti-impβ and anti-MRTF-A antibodies, is reduced in cells transfected with a β-actin expression plasmid, identified by coexpressed GFP marker. PLA was scored as cytoplasmic foci per cell (error bar indicates s.e.m. values; n=55; ***P<0.001, unpaired Mann–Whitney test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Importin β activity is required for MRTF-A nuclear accumulation. (A) Impβ was silenced in R332 cells (NIH 3T3 cells stably expressing MRTF-A–GFP) using RNA interference, and the localisation of the protein was analysed under different conditions. FCS, fetal calf serum; CD, Cytochalasin D (2 μM); LMB, leptomycin B. (⩽100 cells per point, n=3 independent experiments, error bars indicate s.e.m. values). Right: western blotting showing Impβ depletion (B) Similar as in panel A, serum stimulation. The Impβ knockdown was rescued by transient transfection of an siRNA-resistant form of Impβ–mCherry. mCherry empty plasmid was used as control. (C) Inhibition of SRF reporter 3D.A-Luc activation after Impβ depletion. Three independent experiments were performed; error bars represent s.e.m. values. (D) The localisation of transiently transfected MRTF-A–GFP in resting MDA-MB-231 cells. Phalloidin staining in red. Right: quantification. (E) Impβ–MRTF-A interaction detected by proximity ligation assay, using anti-impβ and anti-MRTF-A antibodies, is reduced in cells transfected with a β-actin expression plasmid, identified by coexpressed GFP marker. PLA was scored as cytoplasmic foci per cell (error bar indicates s.e.m. values; n=55; ***P<0.001, unpaired Mann–Whitney test).
Mentions: We next sought to identify the import machinery involved in MRTF-A nuclear import. As Impβ has been shown to function as a nuclear import receptor for proteins containing a conventional bipartite NLS, we tested its involvement in MRTF-A nuclear import by use of siRNA to silence its expression. Knockdown of Impβ in NIH 3T3 cells stably expressing MRTF-A–GFP resulted in the substantial inhibition of LMB -induced MRTF-A nuclear accumulation (Figure 3A). Similar results were obtained when MRTF-A nuclear accumulation was induced by cytochalasin D, which interferes with actin–MRTF-A interaction, and thereby blocks actin-mediated MRTF-A export (Vartiainen et al, 2007). The effect of Impβ depletion on serum-stimulated nuclear accumulation was more marked (Figure 3A). Probably, this reflects the fact that serum stimulation, unlike CD or LMB treatment, does not completely block nuclear export (Vartiainen et al, 2007), although we cannot exclude the possibility that basal import involves additional Impβ-independent import signal(s) active only in unstimulated conditions. Expression of an siRNA-resistant Impβ derivative restored nuclear accumulation of MRTF-A in response to serum stimulation, indicating that the effect was specific (Figure 3B). Depletion of Impβ also impaired serum- or cytochalasin D-induced activation of an SRF reporter gene (Figure 3C). In MDA-MB-231 cells, in which MRTF-A is nuclear even under resting conditions (Medjkane et al, 2009), Impβ depletion restored its cytoplasmic localisation (Figure 3D).

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