Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL
2004; Springer Nature; Volume: 23; Issue: 20 Linguagem: Inglês
10.1038/sj.emboj.7600404
ISSN1460-2075
AutoresGuido Posern, Francesc Miralles, Sebastian Guettler, Richard Treisman,
Tópico(s)Cell death mechanisms and regulation
ResumoArticle23 September 2004free access Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL Guido Posern Guido Posern Transcription Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London, UKPresent address: Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Search for more papers by this author Francesc Miralles Francesc Miralles Search for more papers by this author Sebastian Guettler Sebastian Guettler Search for more papers by this author Richard Treisman Corresponding Author Richard Treisman Search for more papers by this author Guido Posern Guido Posern Transcription Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London, UKPresent address: Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Search for more papers by this author Francesc Miralles Francesc Miralles Search for more papers by this author Sebastian Guettler Sebastian Guettler Search for more papers by this author Richard Treisman Corresponding Author Richard Treisman Search for more papers by this author Author Information Guido Posern1, Francesc Miralles, Sebastian Guettler and Richard Treisman 1Transcription Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London, UK *Corresponding author. Transcription Laboratory, Room 401, Cancer Research UK, PO Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. Tel.: +44 207 269 3271; Fax: +44 207 269 3093; E-mail: [email protected] The EMBO Journal (2004)23:3973-3983https://doi.org/10.1038/sj.emboj.7600404 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Nuclear accumulation of the serum response factor coactivator MAL/MKL1 is controlled by its interaction with G-actin, which results in its retention in the cytoplasm in cells with low Rho activity. We previously identified actin mutants whose expression promotes MAL nuclear accumulation via an unknown mechanism. Here, we show that actin interacts directly with MAL in vitro with high affinity. We identify a further activating mutation, G15S, which stabilises F-actin, as do the activating actins S14C and V159N. The three mutants share several biochemical properties, but can be distinguished by their ability to bind cofilin, ATP and MAL. MAL interaction with actin S14C is essentially undetectable, and that with actin V159N is weakened. In contrast, actin G15S interacts more strongly with MAL than the wild-type protein. Strikingly, the nuclear accumulation of MAL induced by overexpression of actin S14C is substantially dependent on Rho activity and actin treadmilling, while that induced by actin G15S expression is not. We propose a model in which actin G15S acts directly to promote MAL nuclear entry. Introduction Actin has a long history of proposed connections with nuclear events as well as its classical role as a cytoskeletal component. Recent studies suggest potential roles for actin and its relatives in chromatin remodelling, transcription and RNA export (reviewed by Olave et al, 2002; Bettinger et al, 2004). Among these roles is that played by actin to control its own expression both transcriptionally and at the level of mRNA translation, localisation and stability (for references see Lyubimova et al, 1999; Sotiropoulos et al, 1999). Autoregulation of actin transcription occurs via a mechanism in which G-actin binds to and controls the activity of MAL/MKL1, a coactivator of the serum response factor (SRF) transcription factor (Miralles et al, 2003). SRF, a MADS-box protein, controls a large number of growth factor-inducible and muscle-specific genes through the mutually exclusive association of different SRF cofactors (Murai and Treisman, 2002; Miralles et al, 2003; Wang et al, 2004), and the expression of many of these genes is thus influenced by G-actin level. In fibroblasts, MAL and its relative MAL16/MKL2 are predominantly cytoplasmic in the absence of Rho signalling, but accumulate in the nucleus upon Rho activation (Cen et al, 2003; Miralles et al, 2003; Du et al, 2004; C Perez-Sanchez, unpublished data). MAL associates with G-actin in vivo, and depletion of the G-actin pool upon activation of Rho releases MAL for nuclear import (Sotiropoulos et al, 1999; Posern et al, 2002; Miralles et al, 2003). Actin mutants have given useful insight into the mechanism by which Rho controls SRF activity. In support of the notion that G-actin is the regulator, the overexpression of nonpolymerising β-actin mutants such as actins R62D, G13R and actin-VP16 inhibits SRF activation and MAL nuclear accumulation (Posern et al, 2002). Consistent with this, MAL can be recovered in immunoprecipitates of these mutants and wild-type actin, and their overexpression prevents MAL nuclear accumulation (Miralles et al, 2003). It has remained unclear whether MAL interacts directly with G-actin. We also identified two actin mutants, actins S14C and V159N, which stabilise F-actin and whose expression strongly activates SRF (Posern et al, 2002). Curiously, however, although cells expressing these mutants accumulate MAL in the nucleus and exhibit an increase in the F-/G-actin ratio, their absolute level of G-actin is if anything slightly increased relative to that of untransfected cells (Posern et al, 2002; Miralles et al, 2003). We suggested two mechanisms to explain these observations: either the mutants were incapable of interacting with the SRF coactivator (i.e. MAL), allowing its release to the nucleus, or they acted independently of the actin treadmilling cycle to induce its activation (Posern et al, 2002). In this paper, we study the connection between activating actin mutants and SRF activation in the light of our identification of MAL as an actin-regulated SRF coactivator. We show that wild-type actin binds the N-terminal RPEL domain of MAL directly in vitro, and identify a new activating actin mutant, actin G15S, in a two-hybrid screen with this domain. All three activating actin mutants, actins G15S, S14C and V159N, share the ability to stabilise F-actin. They all exhibit a decreased ability to bind the C-terminal half of gelsolin, and an enhanced affinity for profilin, suggesting that their structure mimics that of ATP-actin. However, they can also be distinguished biochemically by their ability to interact with nucleotide, cofilin and MAL. Although actin G15S binds MAL more strongly than wild-type actin, the S14C mutation greatly reduces the actin–MAL interaction. Strikingly, the ability of actin S14C to induce MAL nuclear accumulation is strongly dependent on the actin treadmilling cycle and basal Rho activity, while G15S- and V159N-induced MAL translocation is not. We propose that whereas actin S14C releases MAL from the inhibitory effect of wild-type G-actin by diluting the endogenous G-actin pool, actin G15S (and probably V159N) may act directly to induce MAL nuclear translocation. Results Actin interacts directly with the MAL RPEL motifs in vitro We previously showed that MAL can be recovered in immunoprecipitates of cytoplasmic actin. The interaction was dependent on the integrity of the MAL N-terminal RPEL motifs (Miralles et al, 2003). It remained unclear, however, whether this complex involves a direct interaction between MAL and actin or requires additional proteins. To test whether the actin-MAL complex can form in vitro, we passed whole-cell extracts over affinity beads comprising MAL RPEL motifs 2 and 3 fused to GST. As controls we used mutant RPEL derivatives containing P → A or R → D mutations in each RPEL motif, which were previously shown to be defective for actin binding in vivo (Miralles et al, 2003). After extensive washing, bound proteins were eluted using the G-actin-binding drug swinholide A, which competes with MAL for actin binding (Miralles et al, 2003). A 42 kDa protein was prominent in the eluate from the wild-type but not mutant GST fusion protein; no other proteins in the 10–300 kDa Mr range bound specifically (Figure 1A). Similar results were obtained when proteins were eluted from the affinity beads by boiling in SDS–PAGE loading buffer (data not shown). An immunoblot experiment confirmed that the 42 kDa protein was indeed β-actin (Figure 1B). Purified β-actin also bound the wild-type but not the mutant MAL affinity beads, and this interaction was resistant to latrunculin B but sensitive to swinholide A (Figure 1C). These results complement previous experiments in which co-immunoprecipitation of MAL and actin was disrupted by cytochalasin D or swinholide A, which also promoted MAL nuclear accumulation, but not by latrunculin B, which did not (Miralles et al, 2003). Figure 1.MAL binds directly to actin. (A) Actin does not associate with other cellular proteins on the RPEL motif. NIH3T3 cells were lysed by syringing in detergent-free buffer and the high-speed G-actin supernatant was affinity-precipitated using GST-MAL(met)(1–171) or its derivatives PP34/78AA (PP) or RR33/77DD (RR), which carry point mutations in each of the two RPEL motifs. Bound proteins were eluted with 500 nM swinholide A, separated by 6–16% gradient PAGE and detected by silver staining. The diagonal arrows indicate contaminating GST fusion proteins and asterisks mark nonspecifically retained polypeptides. (B) Precipitated proteins from a GST-MAL(met)(1–171) affinity precipitation experiment of the type shown in (A) were analysed by immunoblotting with β-actin antibody. (C) Affinity precipitation of purified biotinylated nonmuscle actin using GST-MAL(met)(1–171) or its derivatives. Where indicated, 1 μM latrunculin B or 100 nM swinholide A was included in the binding reaction. Bound actin was separated by 12% SDS–PAGE and detected by overlay with peroxidase-conjugated streptavidin. (D) Native 6% polyacrylamide gel electrophoresis assay of complex formation between GST-MAL(met)(1–171) derivatives and ADP- or AMP-PNP-loaded nonmuscle actin (left and right panels, respectively), with detection by Coomassie blue (CB) staining or anti-β-actin immunoblot. (E) Inhibition of skeletal muscle α-actin polymerisation by MAL(fl)2–261 (left), which contains all three RPEL motifs, and gelsolin(S4–6) (right). Data are mean of two independent experiments. Download figure Download PowerPoint Some actin-binding proteins strongly discriminate between different nucleotide-bound forms of actin. We therefore tested the nucleotide-binding requirements of actin–MAL interaction using a native gel electrophoresis assay. Wild-type or mutant GST-MAL fusion proteins were mixed with ADP- or AMP-PNP-bound actin, separated on a native gel and actin detected by immunoblotting. Actin–MAL complex formation was detected regardless of the bound nucleotide (Figure 1D). These results show that additional accessory proteins are not required for binding of actin to the MAL RPEL motifs. Finally, we determined the apparent affinity of the intact RPEL domain, by exploiting its ability to inhibit actin polymerisation (Hertzog et al, 2002). We used skeletal muscle α-actin since it is readily purified and recovers MAL as efficiently as β-actin in co-immunoprecipitation experiments (see Supplementary Figure S1). As a positive control, we evaluated gelsolin segments 4–6 (gelsolin(S4–6)). Increasing amounts of the MAL RPEL domain or gelsolin(S4–6) led to complete inhibition of F-actin assembly at comparable concentrations (Figure 1E). The data are consistent with an apparent Kd of 24 nM for MAL–actin, and an apparent Kd of 76 nM for gelsolin(S4–6), in good agreement with published data (Way et al, 1989). Profilin overexpression induces MAL nuclear accumulation and SRF activation (Sotiropoulos et al, 1999; Miralles et al, 2003), and consistent with this, MAL is not recovered in profilin immunoprecipitates (Supplementary Figure S1). Thus, high-affinity MAL–actin interaction allows it to compete effectively with profilin for free G-actin. Identification of actin G15S, a novel MAL-activating protein To identify cDNAs encoding proteins that interact with the MAL N-terminal region in an unbiased way, we set up a yeast two-hybrid assay. A GAL4 fusion protein containing MAL residues 1–631 was used to screen an NIH3T3 cDNA library, and all cDNAs recovered were counterscreened against a mutant MAL containing RPEL motif point mutations. From 600 000 clones screened, we recovered a mouse γ-actin cDNA containing the mutation G15S and two other cDNAs whose characterisation will be described in full elsewhere. Colony growth and liquid β-galactosidase assays showed that binding of γ-actin G15S to MAL was dependent on the RPEL motifs: it was severely reduced by R → D or P → A mutations at single RPEL motifs, and undetectable upon mutation of both motifs (Figure 2A). Intrigued by this observation, and our previous finding that mutations at the β-actin nucleotide-binding pocket can substantially affect its activity in the SRF activation assay (Posern et al, 2002), we tested whether the G15S mutation affects the ability of actin to repress SRF activity. Expression of γ-actin G15S strongly potentiated activity of the SRF reporter gene 3D.A-Luc in serum-starved cells; in contrast, expression of wild-type γ-actin, as with wild-type β-actin, suppressed activation of the reporter following serum stimulation (Figure 2B). Figure 2.Identification of actin G15S, an activator of MAL and SRF. (A) Yeast two-hybrid interactions between γ-actin G15S and MAL(met)(1–631) or mutant derivatives containing point changes in either or both RPEL motifs. Upper panels: trans-illuminated images of colony growth on selective (−His) or nonselective (control) medium. Lower histogram: interaction quantification by liquid-culture Gal4-lacZ reporter gene assay (WT γ-actin=100; error bars: s.e.m.; n=3) (B) γ-Actin G15S expression activates SRF. Cells were transfected with SRF reporter 3D.A-Luc (40 ng), together with wild-type γ-actin (100, 250 or 500 ng) or γ-actin G15S (100 or 250 ng), maintained in 0.3% FCS for 40 h and then serum-stimulated where indicated. Data are means of three independent experiments; error bars: s.e.m. (C) Reporter activation by β-actin G15S and other β-actin mutants. Cells were transfected with reporter and the indicated β-actin mutants and treated as in (B). (D) β-Actin G15S expression induces MAL nuclear accumulation. Cells expressing MAL(fl)-HA (50 ng) and β-actin G15S (500 ng) were processed for immunofluorescence 24 h after transfection. Confocal sections of 0.3 μm thickness show actin G15S (anti-Flag; red) and nuclear accumulation of MAL(fl) (anti-HA; green). The merged picture shows the stack of both actin and MAL sections. Download figure Download PowerPoint We next introduced the G15S mutation into β-actin. Expression of β-actin G15S also substantially activated expression of the SRF reporter in serum-starved cells, to levels comparable to those achieved by expression of the activating mutants actins S14C and V159N (Figure 2C; Posern et al, 2002). Consistent with its ability to activate SRF, β-actin G15S expression induced nuclear accumulation of the SRF coactivator MAL in NIH3T3 cells, but did not itself accumulate in the nucleus (Figure 2D). To allow comparison with our other 'activating' actin mutants, all further studies were performed with β-actin G15S. Actin G15S stabilises F-actin Upon transient overexpression in fibroblasts, actins S14C and V159N exhibit properties suggesting that they stabilise F-actin (Posern et al, 2002), consistent with structural studies of yeast actin V159N (Belmont and Drubin, 1998; Belmont et al, 1999a). We therefore measured intracellular levels of F-actin and G-actin in cells expressing the different actins. Cells were stained with phalloidin, which specifically binds F-actin, or DNase I, which specifically binds G-actin, and staining was quantified in transfected cells relative to untransfected cells in the same population using the FACS (Geneste et al, 2002; Posern et al, 2002). Cells expressing wild-type actin exhibit an increase of approximately 40% in both F- and G-actin levels. In this assay, overexpression of actin G15S altered the balance between F- and G-actin in favour of F-actin, as observed previously for actins S14C and V159N (Figure 3A). To corroborate this result, we used detergent extraction and centrifugation to produce F-actin- and G-actin-enriched supernatant and pellet fractions from cells expressing Flag-tagged actin. In this assay, serum stimulation led to increased recovery of wild-type actin in the F-actin fraction (Figure 3B). In contrast, depolymerisation of the cytoskeleton by the actin-binding drugs swinholide A or latrunculin B, or coexpression of C3 transferase or the G-actin-binding protein profilin, caused Flag-actin to accumulate in the G-actin fraction (Figure 3B). Actin G15S was recovered in increased amounts in the F-actin fraction compared to wild-type actin, as were actins S14C and V159N (Figure 3C; Posern et al, 2002). Moreover, coexpression of actin G15S and wild-type actin led to an increased recovery of wild-type actin in the F-actin fraction (Figure 3D). These results show that like actins S14C and V159N, actin G15S can copolymerise with wild-type actin to generate F-actin of increased stability. Figure 3.Actin G15S stabilises filament formation. (A) Transfected cells expressing the indicated Flag-tagged actins (1 μg) were stained for the Flag epitope and either TRITC-phalloidin (for F-actin) or FITC-DNase I (for G-actin). The FACS was used to quantify mean levels of F- or G-actin in the transfected population relative to those of the untransfected population (error bars: s.e.m.; n=3). (B) Actin fractionation lysates were prepared from cells expressing wild-type Flag-actin (1 μg) with C3 transferase (50 ng) or profilin (500 ng) coexpression, or treatment with latrunculin B (0.3 μM, 1 h), swinholide A (100 nM, 1 h) or FCS (15%, 10 min) as indicated. Flag-actin in each supernatant (S) and pellet (P) fraction was detected by immunoblotting using anti-Flag antibodies. (C) Actin fractionation lysates were prepared from cells expressing the indicated Flag-tagged actin mutants (1 μg) and analysed by anti-Flag immunoblotting as in (B). (D) Activating actin mutants copolymerise with and stabilise wild-type F-actin. Actin fractionation lysates were prepared from cells expressing the indicated Flag-tagged actins (1 μg) together with HA-tagged wild-type actin (500 ng), and analysed by anti-HA immunoblot. Download figure Download PowerPoint Activating actins share altered gelsolin- and profilin-binding properties To gain insight into the changed conformation of the activating actins, we studied their interaction with different actin-binding proteins, initially focusing on proteins that discriminate between different nucleotide-bound states of the molecule. The C-terminal half of the F-actin severing protein gelsolin, comprising segments 4–6, specifically binds wild-type ADP-bound G-actin (Laham et al, 1993). We used the GST-gelsolin(S4–6) fusion protein in pulldown assays with high-speed supernatant extracts of transiently transfected NIH3T3 cells expressing Flag-tagged wild-type or mutant actins. All the activating mutants were recovered inefficiently in comparison to wild-type actin and the nonpolymerisable mutant R62D (Figure 4A, upper); in a control experiment, purified wild-type ADP-actin, but not AMP-PNP-actin, effectively bound GST-gelsolin(S4–6) (Figure 4A, lower). The decreased affinity of the activating actins for GST-gelsolin(S4–6) suggests that their structure does not readily adopt a conformation characteristic of ADP-actin. Figure 4.Activating actin mutants have both shared and distinct properties. (A) Activating actins exhibit reduced affinity for the C-terminal half of gelsolin. Upper panel: G-actin supernatants from cells expressing the indicated actins (2 μg) were affinity-precipitated using GST-gelsolin(S4–6), and bound proteins were detected by immunoblotting with anti-Flag antibodies. Lower panels: native gel electrophoresis assay of complex formation between GST-gelsolin(S4–6) and ADP- or AMP-PNP-loaded nonmuscle actin, with detection by Coomassie blue or anti-β-actin immunoblot. (B) Interaction of actins with endogenous profilin. Upper panels: Extracts as in (A) were immunoprecipitated with anti-Flag and analysed for profilin (anti-profilin; upper) and actin (anti-Flag; lower). Cells were in 0.5% serum except where stimulated with 15% serum for the indicated times (min). Lower panel: Control immunoblot for profilin in the lysate. (C) Interaction of actins with endogenous cofilin. Extracts were prepared and analysed as in (B). (D) Nucleotide binding by actin mutants. Actin immunoprecipitates were prepared as in (B), followed by nucleotide determination using the luciferase-based ATP assay. Immunoblot: recovered actin in each sample. Data are mean±s.e.m. (n=4). (E) Nucleotide binding by actin mutants. Actins were prepared as in (D) and nucleotide was identified by Mono-Q anion exchange chromatography. UV absorbance at 254 nm from a representative experiment is shown, with elution volumes for ADP and ATP indicated. The asterisk indicates a nonspecifically recovered component also present in precipitates from mock-transfected cells. Download figure Download PowerPoint We next used the co-immunoprecipitation assay to examine the ability of the mutants to associate with profilin, which favours nucleotide exchange on actin and preferentially binds ATP-actin (see Perelroizen et al, 1995; Pollard and Borisy, 2003). Extracts were prepared from cells expressing the Flag-tagged actins, immunoprecipitated using anti-Flag antibodies and recovery of cellular profilin was monitored by immunoblotting. Profilin was recovered in immunoprecipitates of wild-type actin, but not the nonpolymerisable mutant R62D (Figure 4B). In this assay, profilin was more effectively recovered by the activating actin mutants than by the wild-type protein (Figure 4B). Serum stimulation led to a rapid decrease in the recovery of profilin associated with actin (Figure 4B, right). Taken together, these results suggest that the activating actins cannot enter a conformation readily adopted by ADP-bound wild-type G-actin, and an increased tendency to enter a conformation preferred by ATP-bound wild-type G-actin. In contrast, the nonpolymerising mutant R62D, which retains the ability to inhibit MAL nuclear accumulation, appears to adopt a conformation more characteristic of ADP-bound G-actin. The activating mutants interact differentially with cofilin and nucleotide Cofilin binding discriminates in favour of ADP-bound actin (Carlier et al, 1997). We therefore also evaluated the ability of the mutant actins to associate with cellular cofilin using the co-immunoprecipitation assay. In contrast to their similar interactions with gelsolin(S4–6) and profilin, however, the activating actins exhibited very different behaviour in this assay: cofilin was efficiently recovered in all the actin immunoprecipitates save that of the activating mutant G15S (Figure 4C). Given that the behaviour of the activating mutants in the gelsolin(S4–6)- and profilin-binding assays suggested that they might adopt an 'ATP-like' conformation more readily than wild-type actin, we next sought to confirm the identity of the nucleotide associated with the different mutants. Each of the actins was recovered from transfected cells by anti-Flag immunoprecipitation (IP) and bound ATP was quantified using a luciferase enzymatic assay (Chen et al, 1995; Chen and Rubenstein, 1995; Schuler et al, 1999). In this assay, less ATP was recovered with the nonpolymerisable R62D and the activating V159N actin mutants than with wild-type actin, while more ATP was recovered with the activating mutant G15S; in contrast, activating actin S14C was recovered virtually free of ATP (Figure 4D). The relatively high amounts of ADP recovered in these assays may reflect hydrolysis during actin isolation. Essentially identical results were obtained when nucleotide association was evaluated by HPLC assay (Figure 4E; Rosenblatt et al, 1995). Taken together with the results in the preceding section, these data show that although all three activating mutants share a number of properties, they nevertheless can be distinguished biochemically according to their ability to bind nucleotide (S14C defective) and cofilin (G15S defective). Activating actins also exhibit distinct MAL-binding properties We next examined the interaction between the activating actins and MAL. Actins were immunoprecipitated from extracts of cells transfected with Flag-tagged actins and recovery of endogenous MAL was assessed by immunoblotting. In this assay, the different activating actins again behaved differently. MAL binding to actin S14C was essentially undetectable; in contrast, actin G15S bound MAL more effectively than wild-type actin (Figure 5A). To increase the sensitivity of the assay, we repeated it, this time overexpressing MAL as well as the actins. Again, MAL binding to actin S14C was undetectable, while actin G15S bound more efficiently than wild-type actin, and V159N less effectively (Figure 5B). Neither wild-type nor mutant actins associated with the MAL mutant PP34/78AA, which carries P → A changes in its RPEL motifs (Figure 5B; Miralles et al, 2003). MAL nuclear accumulation in response to serum stimulation is dependent on two basic sequence elements (Miralles et al, 2003). MALΔB1ΔB2, which lacks these basic sequences, remained cytoplasmic upon expression of actins S14C, G15S and V159N, indicating that the basic sequences are also required for MAL nuclear accumulation induced by the activating actins (Figure 5C). A control experiment confirmed that MALΔB1ΔB2 nevertheless remains competent to bind wild-type actin as well as the R62D and G15S mutants (Figure 5C). Figure 5.Interaction of MAL with wild-type and mutant actins. (A) Interaction of actins with endogenous MAL from NIH3T3 cells. Upper panels: extracts from cells expressing the indicated Flag-tagged actin mutants (1 μg) were immunoprecipitated with anti-Flag or control antibodies, and analysed for MAL (anti-MAL; upper) and actin (anti-Flag; lower). Lower panels: control immunoblots for MAL (anti-MAL; upper) and actin expression (anti-Flag) in the lysate. ctrl. Trf.: no actin; ctrl. Ab: wild-type actin, anti-HA IP. (B) Interaction of actins with overexpressed HA-tagged MAL(met) and HA-MAL(met) PP34/78AA (containing mutated RPEL motifs) (1 μg). Cells were transfected and processed as in (A). Data in lanes 1–3 are from Miralles et al (2003). (C) Nuclear accumulation induced by the activating actins requires MAL basic box regions. Upper panels: cells in 0.5% FCS expressing MALΔB1B2-HA (0.1 μg), which lacks the basic regions, and activating actins (1 μg) were fixed and stained for MAL (anti-HA; green), actins (anti-Flag; red) and DNA (Hoechst 33258; blue). Numbers show proportion of cells with predominantly nuclear MAL. Lower panels: control co-immunoprecipitation experiments performed as in (B). (D) Overexpression of nonpolymerisable actin R62D, but not the F-actin-stabilising mutants, promotes relocalisation of MAL(met)1–471 to the cytoplasm. Cells expressing HA-tagged MAL(met)1–471 with vector (mock) or Flag-tagged actins were stained and scored as in (C). Download figure Download PowerPoint We previously showed that MAL(1–471), a MAL mutant lacking its C-terminal sequences, which include the dimerisation interface, accumulates in the nucleus under conditions of basal Rho signalling (Miralles et al, 2003). Overexpression of nonpolymerisable actin R62D, which binds MAL, is sufficient to relocalise MAL(1–471) to the cytoplasm under these conditions (Figure 5D, top rows). In contrast, none of the activating actins, even actins G15S and V159N, which can interact with MAL, relocalised MAL(1–471) to the cytoplasm (Figure 5D, lower rows). Binding of these actins to MAL is thus incapable of retaining it in the cytoplasm. MAL activation by actins exhibits differential dependence on actin treadmilling We previously proposed that activating actin mutants might potentiate SRF activation either because of their inability to bind a putative coactivator (MAL) or because their binding activates it (Posern et al, 2002). The different MAL-binding properties of the mutants described above are consistent with the notion that the mutants use different mechanisms to induce MAL nuclear accumulation. To investigate this issue, we tested the dependence of SRF reporter gene activation on basal Rho activity, using C3 transferase coexpression to inactivate endogenous Rho (Hill et al, 1995). As previously observed, expression of C3 transferase completely abolished serum-induced activation of the SRF reporter gene, but did not affect its activation by coexpression of an activated mDia mutant (Hill et al, 1995; Copeland and Treisman, 2002). Expression of C3 transferase substantially inhibited reporter activation induced by actin S14C expression but had no significant effect upon activation by actin G15S (Figure 6A). In these experiments, the effect of C3 transferase upon activation by actin V159N was of borderline statistical significance, however. Figure 6.Activating actins exhibit different requirements for actin treadmilling in SRF activation. (A) SRF reporter activation by activating actins is differentially sensitive to Rho inactivation. Cells transfected with SRF reporter expressed activating actins (500 ng
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