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,
Francesc Miralles,1,
Banafshe Larijani,2 and
Richard Treisman1*
Transcription,1 Cell Biophysics Laboratories, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom2
Received 3 September 2007/ Returned for modification 24 October 2007/ Accepted 2 November 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In fibroblasts, the regulation of MAL localization and activity is controlled largely by Rho-dependent changes in the dynamics of actin turnover between its monomeric (G-actin) and filamentous (F-actin) states, and blockade of Rho-induced actin polymerization prevents MAL-mediated activation of SRF target genes (11, 13, 14, 23). MAL constantly circulates between nucleus and cytoplasm in serum-starved cells. Its cytoplasmic steady-state localization is maintained by very efficient CRM1-dependent nuclear export, which also requires its interaction with actin in the nucleus (23). MAL senses the cellular G-actin concentration by direct interaction (Fig. 1A), and reduction of this interaction, whether it results from Rho-induced depletion of the G-actin pool or from direct disruption by actin-binding drugs, such as cytochalasin D (CD), leads to MAL nuclear accumulation (Fig. 1A) (14, 23).
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Here, we investigate the role of the RPEL domain in regulation in detail. We show that the MAL RPEL domain is necessary and sufficient to confer Rho-regulated nuclear accumulation upon a heterologous protein, while that of MC confers constitutive nuclear localization. We show that the RPEL motif defines an actin-binding element. MAL RPEL1 and RPEL2 bind actin relatively strongly, while RPEL3 binds more weakly. In contrast, whereas MC RPEL3 is of an affinity similar to that of MAL, MC RPEL1 and RPEL2 have a much lower affinity for actin. We show that the three RPEL motifs functionally cooperate in MAL to control nucleocytoplasmic shuttling. Using point mutants and protein chimeras, we show that RPEL1 and RPEL2 together control whether the protein is constitutively nuclear or linked to Rho signaling, while RPEL3 is interchangeable between MAL and MC. These results establish a central role for actin binding and the RPEL domain in control of MAL subcellular localization. We propose that MAL regulation is achieved by regulation of actin binding to multiple RPEL motifs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Luciferase reporter assay. Cells in a 24-well dish (30,000 cells/well) were transfected with SRF reporter p3DA.luc (8 ng), reference reporter ptk-RL (20 ng) with or without SRF-VP16 (40 ng), MAL and MAL mutant derivative constructs (10 ng), or C3 transferase (2 ng). Cells were maintained in 0.5% fetal calf serum for 22 h, after which luciferase activities were measured. Firefly luciferase activity was normalized to Renilla luciferase activity. Data from three independent experiments with SEM are expressed relative to reporter activation by coexpressed SRF-VP16. Data with C3 transferase coexpression were normalized to reporter activation by SRF-VP16 and C3 transferase coexpression.
GST pulldown assays. Glutathione-agarose (Sigma) was saturated with glutathione S-transferase (GST) or GST fusion proteins and peptides from Escherichia coli lysates, washed, and used as affinity resin in a binding reaction with total NIH 3T3 cell extract, generated by lysis in binding buffer (50 mM Tris-HCl [pH 8], 100 mM NaCl, 3 mM MgCl2, 0.2 mM EGTA, 0.2 mM ATP, 1 mM dithiothreitol [DTT], and protease inhibitors) through syringing and removal of insoluble material by centrifugation. An equivalent of a confluent 150-mm dish of NIH 3T3 cells was used for two binding reactions. Binding was for 2 h in binding buffer, supplemented with 0.5% TX-100 where indicated. The resin was washed three times in the respective binding buffer without protease inhibitors and subjected to 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with detection of endogenous β-actin (AC-15; Sigma). The blot was Ponceau stained to reveal bait input.
Fluorescence anisotropy.
Fluorescein isothiocyanate (FITC)-conjugated peptides were synthesized by the Cancer Research UK peptide synthesis facility and quantified using the equation 
492 = 83,000 m–1 cm–1. Actin was purified from rabbit skeletal muscle, rendered nonpolymerizable by incubation in G buffer (2 mM Tris-HCl [pH 8.0], 0.3 mM MgCl2, 0.2 mM EGTA, 0.2 mM ATP, 0.5 mM DTT) with a 10-fold molar excess of LatB (Calbiochem) for 15 h. Uncomplexed actin was polymerized by the addition of 20x initiation buffer (2 M NaCl, 60 mM MgCl2, 10 mM ATP) and removed by ultracentrifugation. Fluorescence anisotropy was measured in a total volume of 50 µl in Mg2+-F buffer (2 mM Tris-HCl, pH 8.0; 100 mM NaCl; 3 mM MgCl2; 0.2 mM EGTA; 0.7 mM ATP; 0.5 mM DTT). FITC-conjugated peptides were used at 0.5 µM while LatB-actin was added from 1 nM up to 59 µM. Plates were read after a minimal coincubation period of 5 h at room temperature to ensure the establishment of binding equilibrium using a Safire2 microplate reader (Tecan) in fluorescence polarization mode (excitation, 470 ± 20 nm; emission, 525 ± 20 nm; 10 reads; 40-µs integration time) and its Magellan software (version 5.03). Anisotropy (A) was calculated by the Magellan software using the formula A = (Iparallel – Iperpendicular)/(Iparallel + 2Iperpendicular), where Iparallel and Iperpendicular denote the fluorescence intensities parallel and perpendicular to the excitation plane, respectively, and a G factor of 1.2041. Nonlinear regression to determine equilibrium dissociation constant (KD) values was done with GraFit version 5.0.11 (Erithacus Software) using the following equation (7):
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Live-cell imaging and photobleaching. Live-cell imaging was performed essentially as described in reference 23. Briefly, for fluorescence loss in photobleaching (FLIP), cells were plated on MatTek dishes (MatTek Corporation), transfected with 50 ng of MAL-green fluorescent protein (GFP) and chimera-GFP plasmids or 20 ng of MAL(1-204)-2GFP or its derivatives, and maintained in phenol red-free medium containing 0.3% serum for 18 h prior to imaging. Cells were stimulated 30 to 60 min prior to imaging, and an area in the cytoplasm was repeatedly bleached for 80 s. For analysis, the background was subtracted, and nuclear fluorescence prior to bleaching was set to 1. At least 10 cells from at least two independent experiments were analyzed per condition. For analysis of nuclear accumulation of MAL(1-204)-2GFP upon stimulation, a stable cell line expressing MAL(1-204)-2GFP was used. The nuclear fluorescence prior to stimulation was set to 0, and the nuclear fluorescence after complete accumulation was set to 1. At least 12 cells from three independent movies were analyzed per condition. For details, see reference 23.
FRET by FLIM. Fluorescence resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy (FLIM) has been described in detail elsewhere (12) and was performed essentially as described in reference 23. NIH 3T3 cells were transfected with 100 ng of MAL-GFP or MC-GFP (donors), with or without 100 ng of MYC-β-actin (acceptor). MYC-β-actin was subsequently detected with Cy3-9E10 monoclonal antibody. The image sequences of the donor were processed by using IP Lab software with automated analysis to provide nonbiased analysis of the data (P. Leboucher and B. Larijani, unpublished data). For details, see reference 23.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since actin binding to the RPEL domain regulates nucleocytoplasmic shuttling of MAL, we next investigated whether differential regulation of MAL and MC result from differences between the proteins’ RPEL domains. Reciprocal exchange of the entire RPEL domain between MAL and MC resulted in exchange of their regulatory properties. MC-N123-MAL, like MC, was predominantly nuclear (Fig. 1C). The two proteins potently activated the SRF reporter to very similar extents, and the activation was not dependent on functional Rho (Fig. 1D). Analysis by FLIP indicated that, like MC itself, MC-N123-MAL was not appreciably exported from the nucleus (Fig. 1E). Conversely, MAL-N123-MC was predominantly cytoplasmic in serum-starved cells (Fig. 1C); it moderately activated the SRF reporter in a Rho-dependent manner, as did MAL (Fig. 1D). Treatment with serum or CD resulted in MAL-N123-MC nuclear accumulation, under which conditions its nuclear export rate was comparable to that of MAL (Fig. 1E).
Together, these results show that the RPEL domains of MAL and MC determine their differential regulation, at the levels of both subcellular localization and activation of SRF-mediated transcription.
The RPEL domain controls dynamic nucleocytoplasmic shuttling of MAL. We next tested whether the RPEL domain is sufficient to mediate Rho-actin-regulated nucleocytoplasmic shuttling in the absence of other MAL or MC sequences. The MAL and MC RPEL domains were fused to PK, a cytoplasmic tetramer of 60-kDa subunits (MAL(2-204)-PK and MC(2-150)-PK) (Fig. 2A) (10). We assessed the behavior of the fusions or point mutant derivatives under a number of conditions previously shown to affect MAL subcellular localization (Fig. 2B) (14, 23).
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A mutations) resulted in constitutive nuclear localization of MAL(2-204)-PK. Nuclear accumulation, whether induced by serum stimulation or mutation of the RPEL motifs, was dependent on the RPEL domain B2 region (Fig. 2B, compare left and right bars) (14, 23). This result supports the notion that the B2 region is a nuclear localization signal that is regulated by actin binding (23). Like MC, MC(2-150)-PK was nuclear in unstimulated cells (Fig. 2B). Serum stimulation and LatB treatment did not affect nuclear localization of either MC or MC(2-150)-PK, although the coexpression of β-actin R62D (19) resulted in a moderate relocalization of both MC and MC(2-150)-PK to the cytoplasm, suggesting that the MC RPEL domain may, in principle, bind to actin. Deletion of the B2 region in MC and MC(2-150)-PK resulted in their substantial or complete cytoplasmic localization, respectively (data not shown). Taken together, these observations show that the RPEL domain is sufficient to respond to Rho-actin signaling and that the different behaviors of MAL and MC reflect different properties of their RPEL domains. To assess the dynamics of RPEL fusion proteins, we linked the MAL and MC RPEL domains to a double-GFP tag [MAL(1-204)-2GFP and MC(1-150)-2GFP] (Fig. 3A). In serum-starved cells, MAL(1-204)-2GFP was cytoplasmic; its basal rate of nuclear import, revealed by the rate of its nuclear accumulation upon LMB treatment, was comparable to that following serum or CD stimulation, as previously observed for MAL-GFP (Fig. 3B) (23). To monitor the nuclear export of MAL(2-204)-2GFP, we again performed FLIP experiments. Under the experimental conditions, the nuclear fluorescence of GFP rapidly decayed during cytoplasmic bleaching (Fig. 3C). The nuclear fluorescence of MC(1-150)-2GFP, like that of MAL-GFP in the presence of LMB, was largely insensitive to cytoplasmic bleaching. Thus, the RPEL domain of MC cannot facilitate nuclear export. Bleaching of nuclear MAL(1-204)-2GFP fluorescence following CD treatment or serum stimulation was somewhat more efficient and was comparable to the that of the actin-binding-deficient xxx derivative of MAL(1-204)-2GFP. Similar results were previously observed with MAL-GFP (23). In summary, the MAL and MC RPEL domains suffice for conferring actin-controlled and CRM1-dependent nucleocytoplasmic shuttling or constitutively nuclear localization on MRTFs.
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The isolated RPEL motif defines an actin-binding element. To investigate the basis for the differential actin-binding properties of the MAL and MC RPEL domains, we analyzed the properties of the individual RPEL motifs. Phylogenetic analysis shows that each of the three MAL RPEL motifs is more closely related to the corresponding motifs in the other family members than to the other motifs in MAL. RPEL1 and RPEL2 are most divergent between MAL and MC, while RPEL3 is relatively conserved (Fig. 4C).
We first performed GST pulldown assays using individual RPEL motifs as baits. GST fusions of MAL RPEL1 or RPEL2 efficiently recruited β-actin from cell lysates, while MAL RPEL3 recovered β-actin inefficiently (Fig. 4D, left). In this assay, the MAL RPEL1 RA substitution greatly reduced but did not abolish interaction with actin, whereas the analogous substitutions in RPEL2 and RPEL3 had a much greater effect. A more severe, charge-reversal mutation in MAL RPEL1, RR81/82DD, blocked its interaction with actin in this assay. In contrast to MAL, MC RPEL1 and RPEL2 did not recover detectable amounts of β-actin in this assay, although MC RPEL3 appeared to bind actin similarly to MAL RPEL3 (Fig. 4D, right).
To measure RPEL-actin binding affinities quantitatively under conditions of solution-binding equilibrium, we employed fluorescence anisotropy assays. Increasing amounts of nonpolymerizable LatB-actin were titrated into binding reactions containing a constant amount of 32-residue RPEL peptides, N-terminally modified with fluorescein. The fluorescence anisotropy at 525 nm was measured, and the equilibrium dissociation constants were derived as described in Materials and Methods. MAL RPEL1 and RPEL2 bound actin with comparable affinities of 5.4 ± 0.5 µM and 2.3 ± 0.2 µM, respectively (Fig. 4E). MAL RPEL3 bound three to seven times weaker, with a KD of only 18.8 ± 1.0 µM. The RA mutation decreased the affinities of MAL RPEL1 and RPEL2 for actin to 16.4 ± 2.1 µM and 15.0 ± 3.3 µM, respectively, and reduced the affinity of MAL RPEL3 to below the detection limit, as did the RPEL1 charge reversal mutation. Actin bound to MC RPEL1 with a KD of 15.4 ± 0.8 µM, threefold lower than to MAL RPEL1, but strikingly, binding of actin to MC RPEL2 was undetectable under our assay conditions. The affinity of MC RPEL3 was very similar to that of MAL RPEL3, with a KD of 16.6 ± 0.2 µM. Taken together, these data demonstrate that the RPEL motif defines an actin-binding element with a wide range of affinities for actin and suggest that the different properties of the MAL and MC RPEL domains may reflect differences in the affinities of their RPEL motifs for actin.
The three MAL RPEL motifs functionally cooperate in regulation.
We next used the insights from the actin-binding analysis to address the functional significance of RPEL motifs for MAL regulation. To this end, we introduced the RA mutation into each of the RPEL motifs, generating single (x23; 1x3; 12x), double (1xx; x2x; xx3), and triple (xxx) MAL RPEL domain mutants (Fig. 5A). The proteins were expressed in NIH 3T3 cells and analyzed for subcellular localization and their potential to activate an SRF reporter.
Under serum-starved conditions, wild-type MAL was found detectably nuclear (nuclear or with a comparable intensity in the nucleus and cytoplasm) in less than 10% of the cells (Fig. 5B) (14). An alanine substitution at RPEL1, RPEL2, and RPEL3 increased this to approximately 25%, 40%, and 70% of the cells, respectively. Compared to single RPEL mutants, all double RPEL mutants displayed a much stronger tendency to accumulate in the nucleus, and MAL 1xx and xxx were virtually indistinguishable, with 90% of cells displaying predominantly nuclear localization (Fig. 5B). Following serum stimulation, all proteins were predominantly nuclear in >80% of the cells (data not shown). The ability of these mutants to activate the SRF reporter correlated well with the proportion of cells displaying appreciable nuclear accumulation (Fig. 5C). Single-alanine substitutions reduced reporter dependence on Rho, with mutation of RPEL3 showing the greatest effect, while double substitutions further decreased Rho dependence (Fig. 5C). The integrity of all three RPEL motifs is thus required for MAL regulation.
The alanine-substituted MAL RPEL1 and RPEL2 peptides both retain an affinity for actin similar to that of MAL RPEL3 (Fig. 4E). To analyze the role of actin binding to RPEL1 more thoroughly, we introduced the RPEL1 charge reversal mutation (RR81/82DD), which effectively abolishes actin binding in vitro, into MAL (MAL-1DD) (Fig. 5A). MAL-1DD was more severely deregulated than MAL x23: it was cytoplasmic in only 30% of the cells, pancellular in about 60%, and nuclear in approximately 10% (Fig. 5D), and it activated the SRF reporter more strongly (Fig. 5E). We used a similar approach with MAL RPEL2, in this case precisely exchanging it for MC RPEL2, which does not bind actin detectably in vitro (MAL-2MC) (Fig. 5A). This mutant also exhibited striking deregulation, with nuclear localization in 20% of the cells and with most of the remainder displaying pancellular localization (Fig. 5D); the expression of MAL-2MC strongly activated the SRF reporter substantially independently of Rho (Fig. 5E).
These results demonstrate that the three MAL RPEL motifs functionally cooperate, with even the weakest actin-binding motif, RPEL3, playing an important role in MAL regulation. Mutations in any single MAL RPEL motif are sufficient to cause deregulation of MAL subcellular localization, provided such mutations effectively abolish RPEL-actin interaction in vitro. Taken together, the results suggest that MAL regulation involves assembly of a higher-order actin-MAL complex(es) (see Discussion).
The RPEL1-RPEL2 unit specifies differential regulation of MAL and MC. The different actin-binding affinities of MAL and MC RPEL1 and RPEL2 suggest that the reason for the differential regulation of MAL and MC might reside in the unit containing these two RPEL motifs. Indeed, the observation above that substitution of MAL RPEL2 by that of MC results in substantial deregulation of MAL subcellular localization and activity is consistent with this idea. To address the role of the RPEL motifs in MC localization more systematically, we generated a series of MAL-MC chimeras, in which increasing extents of N-terminal sequences were reciprocally exchanged, with crossover points in the center of the RPEL-RPEL linker sequence (see Fig. 6A for nomenclature). The chimeras were analyzed for their subcellular localization and their ability to activate an SRF reporter in serum-starved cells, and their nuclear export rates were analyzed using the FLIP assay.
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To determine which parts of the RPEL domain specify MAL-like regulation, we replaced MC RPEL domain sequences with the corresponding sequences from MAL (Fig. 6A). Replacement of the MC N extension with that of MAL had little effect on MC subcellular localization or its ability to activate the SRF reporter. Substitution of the MC N extension and RPEL1 sequences with those of MAL had a greater effect: most cells expressed MAL-N1-MC throughout the nucleus and cytoplasm, and as with MAL, SRF reporter activation was now substantially Rho dependent (Fig. 6B and C). Replacement of the MC N extension, RPEL1, and RPEL2 by the equivalent sequences from MAL conferred authentic MAL-like regulation on MC, at the levels of both steady-state subcellular localization and of SRF reporter activation (Fig. 6B and C). MAL-N12-MC accumulated in the nucleus upon LMB treatment, indicating that it shuttles continuously through the nucleus, and analysis of its nuclear export rates by FLIP indicated that it behaves very similarly to intact MAL or MAL-N123-MC upon serum or CD stimulation (Fig. 6D; compare Fig. 1E). Finally, we tested whether substitution of MC RPEL2 by that of MAL is sufficient to confer MAL-like regulation on MC. The resulting mutant, MC-2MAL, behaved effectively identically to MC itself (Fig. 6E and F). These results show that the regulatory properties of MAL require the integrity of its RPEL1-RPEL2 unit, consistent with the analysis presented in Fig. 5.
Taken together, the behaviors of MAL-MC chimeras demonstrate that the different regulatory behaviors of MAL and MC are both specified by the identity of their RPEL1-RPEL2 unit.
The functional significance of RPEL3 depends on its context.
The results presented above show that the different regulatory behaviors of MAL and MC are specified by the identity of their RPEL1-RPEL2 unit. The data also suggest that although both MAL RPEL3 and MC RPEL3 bind actin with a similarly low affinity, actin binding by RPEL3 is functionally relevant only in the context of MAL. To test this idea, we examined the functional significance of RPEL3 RA mutations, which abolish actin binding in vitro (Fig. 4). The RA mutation of MAL RPEL3 induced strong deregulation of MAL at the levels of both localization and reporter activation, as described above, while the analogous mutation of MC RPEL3 affected neither MC nuclear localization nor its ability to activate the SRF reporter (Fig. 6G and H). In contrast, the MC RPEL3 mutation caused pronounced deregulation in the context of MAL-N12-MC (MAL-N12-MC 3x): while the intact protein behaved very similarly to MAL, its RPEL3 RA derivative was nuclear in about 40% of the cells, pancellular in approximately 50%, and cytoplasmic in about only 10% and strongly activated the SRF reporter. These results show that RPEL3 is functionally interchangeable between MAL and MC and suggest that its ability to confer regulation depends on the identity of the RPEL1-RPEL2 unit N-terminal to it. Mutation of RPEL motifs 1 and 2 (MAL xx3) (Fig. 5A) results in a degree of deregulation comparable to that of MAL 12x which suggests that it is the ability of the RPEL1-RPEL2 unit to bind actin that determines whether actin binding to RPEL3 is significant for MAL regulation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A working model of MC family RPEL domain function is shown in Fig. 7. We propose that changes in actin-MAL interaction control the relative abundance of complexes which differ in their competence for nuclear export and import. Our fluorescence anisotropy studies show that two MAL RPEL motifs represent high-affinity actin-binding sites, while the third is weak; nevertheless, all three sites in MAL must be competent to bind actin for proper regulation to occur, indicating that they functionally cooperate in vivo. The higher affinity of RPEL1 and RPEL2 requires that more severe mutations must be introduced into these sites to significantly affect their activity. The B2 sequence within the RPEL domain is required for MAL nuclear import, which can be inhibited by actin overexpression; actin binding is also required for CRM1-dependent nuclear export (14, 23). One simple interpretation of our data is thus that the B2 sequence is a target for as-yet-unidentified nuclear import factors and that it is occluded by actin interacting with the neighboring RPEL motifs, particularly the adjacent RPEL3 (Fig. 7, top). According to this view, MAL does not necessarily need to be devoid of actin to enter the nucleus, but resolution of this issue awaits further studies. Conversely, export factors would bind only to the actin-bound RPEL domain. While Fig. 7 shows interaction predominantly with actin bound to all three RPEL motifs, our data do not rule out other modes of interaction.
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Even though MC and MAL RPEL domain mutants may have residual affinity for actin, our data show that this must be below the threshold for effective interaction with actin at the G-actin concentrations prevailing in vivo. These proteins are therefore refractory to actin-mediated export, although actin overexpression does cause some redistribution of MC to the cytoplasm. The conservation of RPEL3 in MC is puzzling, since MC does not appear to be regulated by actin, at least in our system. It remains possible that other unknown MC family regulatory factors interact with RPEL3, and we are currently seeking to identify such proteins.
It is likely that actin recruitment to MAL RPEL motifs involves positive cooperativity, either through direct actin-actin interactions or allosteric mechanisms. We speculate that such cooperativity might confer resistance of the Rho-actin-MAL-SRF system to subtle fluctuations in G-actin concentration while allowing "switch-like" activation of MAL once the G-actin concentration drops below a specific threshold. Cooperativity in actin binding may explain the discrepancy between our fluorescence anisotropy results, which show that individual RPEL motifs recruit actin with micromolar affinity and the nanomolar affinity of the intact MAL RPEL domain previously estimated from actin polymerization inhibition studies (18). Although functional cooperativity between multiple actin-binding motifs or domains has previously been observed for many F-actin regulatory proteins (see, e.g., references 4, 20, and 26), the actin-MAL interaction appears unique in that functional cooperation occurs between G-actin binding sites, and the actin-binding partner is the target, not the regulator.
While this work was in progress, a study of MC subcellular localization in 10T1/2 cells was presented by others (9). That study proposed that in addition to MAL RPEL2, the basic region B2, together with RPEL3 and/or basic region B1, determine MAL cytoplasmic localization. Our data demonstrate that the RPEL domain is necessary and sufficient to mediate MC- or MAL-like subcellular localization properties, and therefore do not support this interpretation. We also note that chimeras used for the study by Hinson et al. used the MAL(met) isoform of MAL, which does not contain MAL RPEL1 (14). Nevertheless, the possibility that the discrepancy reflects cell line-specific effects cannot be excluded.
The cytoplasmic steady-state localization of MAL(2-204)-PK and MAL(1-204)-2GFP is more sensitive to their expression level than that of intact MAL (S.G. and M.K.V., unpublished observations). It is possible that this reflects a lack of transcription-mediated cytoskeletal homeostasis upon the expression of RPEL domain fusions, although we cannot rule out the possibility that it is a consequence of the fusion.
What is the functional advantage of uncoupling SRF activation from Rho-actin signaling in muscle cells via MC? The specialized role of actin in differentiated muscle cells might impinge on the actin polymerization cycle in a way that might interfere with growth factor signaling via the actin cytoskeleton. Indeed, actin filaments are stabilized in muscle cells, as documented by their relative resistance to CD or LatB (for references, see reference 25), which rely on actin filament turnover. Filament stabilization in muscle cells is, for example, conferred by the actin-filament binder tropomyosin, which inhibits ADF/cofilin activity (15). Maintenance of smooth and cardiac muscle identity may require MC as an actin-independent SRF coactivator.
In conclusion, our data show that regulation of MAL does not merely require its interaction with actin per se but is dependent on specific signal-mediated changes in actin's interaction with the different RPEL motifs, and possibly other sequences, within the RPEL domain. The elucidation of the molecular mechanism by which binding of multiple actin molecules controls MAL activity will form an interesting subject for future work.
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ACKNOWLEDGMENTS |
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This work was funded by Cancer Research UK. M.K.V. is supported by an EMBO long-term fellowship, and S.G., a fellow of the Studienstiftung des deutschen Volkes, by a Boehringer Ingelheim Fonds predoctoral scholarship.
The authors have no conflicting financial interests.
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FOOTNOTES |
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Published ahead of print on 19 November 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: Institute of Biotechnology, University of Helsinki, Viikinkaari 9, Helsinki 00014, Finland.
Present address: Basic Medical Sciences Division, St. George's Hospital, University of London Medical School, Cranmer Terrace, London SW17 0RE, United Kingdom.
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