A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity - PubMed (original) (raw)

A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity

Robert Grosse et al. EMBO J. 2003.

Abstract

Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to define functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay. We identified inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP. These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigella flexneri. VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo. These findings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity.

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Figures

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Fig. 1. VASP domains required for SRF activation and F-actin assembly coincide. (A) SRF activation by VASP mutants. The EVH1 and polyproline-rich regions are shown as filled and open boxes, and the four conserved sequence blocks constituting the EVH2 domain as black squares. (B) Representative SRF activation data. Cells expressed VASP mutants at inputs judged to give comparable protein expression levels (0.05 and 0.15 µg for VASP, VASPΔPP, VASPΔD and YFP–VASP; 0.15 and 0.5 µg for EVH1-PP, EVH2, VASPΔA, VASPΔB, VASPΔC and VASP–DC), together with the SRF reporter gene 3D.Aluc (0.05 µg) and Renilla luciferase transfection control (0.05 µg). Reporter activity is expressed relative to its activation by the constitutively active SRF derivative SRF-VP16. (C) VASP protein expression. Cells expressed VASP mutants as in (B). Cell lysates were analysed by immunoblot using polyclonal VASP antibody (left) or epitope-tag antibody (right). (D) F-actin content. Cells expressed VASP derivatives at levels corresponding to their maximal activity in the SRF reporter gene assay activation. Mean cellular F-actin content was determined relative to that of untransfected cells in the same population using FACS.

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Fig. 2. Intracellular localization of intact VASP, its minimal active derivative EVH2, and the inactive mutants ΔB and DC. NIH 3T3 cells expressing the indicated VASP mutants were maintained in 0.5% FCS for 16 h before fixing for indirect immunofluorescence. Top: Merged image of F-actin (rhodamine–phalloidin; red) and VASP (9E10 epitope-tag; green). Middle: Flag-VASP (9E10 epitope-tag). Bottom: F-actin (rhodamine–phalloidin).

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Fig. 3. The inactive VASP mutants ΔB and DC are interfering mutants. (A) Interference with VASP-induced SRF activation. NIH 3T3 cells transfected with the SRF reporter expressed intact VASP (0.15 µg) or the indicated VASP mutants (0.1, 0.3 and 0.9 µg). The structure of the mutants is shown below. (B) Interference with VASP-induced F-actin accumulation. Cells expressing intact VASP (0.75 µg) and the indicated VASP mutants (4.5 µg) were analysed for mean cellular F-actin content relative to that of untransfected cells in the same population using FACS. (C) The VASP EVH2 block C is sufficient to bind intact VASP. Lysates from cells expressing the indicated VASP mutants were separated by SDS–PAGE (12.5% gel), transferred to a membrane, and probed either with anti-Flag antibodies (upper panel) or with 35S-labelled VASP DC (residues 278–380), produced by in vitro translation (lower panel).

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Fig. 4. Interfering VASP mutants block Vaccinia and Shigella F-actin tail formation. Adherent HeLa cells expressing VASP (0.3 µg) or its derivatives (0.6 µg) were maintained in 10% FCS. Sixteen hours later, the cells were infected for 8 h with Vaccinia (A–D, F) or for 4 h with L.monocytogenes (E) or S.flexneri (F), and processed for immunofluorescence. Cells were stained for F-actin using Alexa 568-phalloidin (red) and for the VASP epitope tag using 9E10 antibody (green). (A–D) VASPΔB blocks Vaccinia actin tail formation. Merged images are shown. Arrows indicate instances of VASP localization to focal adhesions. DAPI staining indicated the presence of viral particles at the tail ends (not shown). Quantitation is shown in (F). (A) Wild-type VASP; similar results were obtained upon infection of cells expressing GFP. (B) VASP EVH2. (C) VASPΔB blocks tail formation. (D) VASP and F-actin localization in the tails of cells expressing intact VASP (A′) or VASP EVH2 (B′). (E and F) Data summaries. The proportion of transfected _Vaccinia_-infected cells with any viral tails in a given field is shown. Data are represented as means ± SEM (n = 3). (E) Vaccinia data. In control infected cells expressing GFP, cells exhibiting tails generally contained 30–60 virus particles with tails. Expression of the interfering mutants reduced the proportion of cells displaying tails, and decreased the number of virus particles with tails to five to 10 per cell. (F) Shigella flexneri data. In control infected cells expressing GFP alone, those cells displaying tails contained only two to eight bacteria with tails. (G) VASPΔB expression allows the formation of L.monocytogenes actin tails. Separate images of VASP and F-actin are shown for infected cells expressing intact VASP (left) or VASPΔB (right). VASP derivatives did not affect the number of bacteria per infected cell. In two independent experiments, 58% and 55% (intact VASP) and 54% and 52% (VASPΔB) of infected cells displayed tails.

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Fig. 5. Serum-induced SRF activation requires functional VASP. (A) Interference with serum-induced SRF activation. Cells transfected with the SRF reporter and plasmids expressing VASP mutants (0.1, 0.3 and 0.9 µg) were analysed for reporter activity before and after serum stimulation. Inset: VASP immunoblot. (B) Interference by the VASP mutants is signal pathway-specific. Cells transfected with NLex.ElkC and LexOP.Luc (left; ERK pathway) or CAGA12.Luc (right; SMAD pathway) together with control plasmid RLTK and expressed VASP mutants (0.9 µg). Luciferase activity was determined following stimulation with 15% FCS (left) or 2 ng/ml TGFβ (right). (C) Functional VASP is required for maximal serum-induced SRF activation. MVD7 cells, transfected with SRF reporter 3D.Aluc and RLTK control plasmid, expressed intact VASP or YFP–VASP, together with VASPΔB (0.9 µg). Reporter activity was determined before and after serum stimulation.

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Fig. 6. VASP functions in the Rho–mDia–actin pathway to SRF activation. (A) Functional Rho is required for efficient VASP-induced SRF activation. Cells transfected with SRF reporter expressed intact VASP (0.02, 0.05, 0.15 µg) or VASP mutants (ΔPP: 0.02, 0.05, 0.15 µg; EVH2: 0.05, 0.2, 0.6 µg), together with C3 transferase (0.1 µg) as indicated. (Inset) Immunoblot analysis of VASP protein levels. (B) Functional Rho and mDia1 are required for VASP-induced SRF activation and F-actin accumulation. Left: SRF activation. Cells transfected with SRF reporter and expressing C3 transferase (0.1 µg) or interfering mDia1 mutant F1F2Δ1 (Copeland and Treisman, 2002) (0.9 µg) were processed as in (A). Inset: VASP immunoblot of cell lysates. Right: F-actin accumulation. Cells expressed intact VASP (0.75 µg) with either C3 transferase (0.5 µg) or mDia1 mutant F1F2Δ1 (4.5 µg). Mean cellular F-actin content of the transfected cells was determined relative to that of untransfected cells in the same population using FACS. (C) Functional VASP is required for efficient mDia1- induced SRF activation. Left: cells transfected with SRF reporter expressed activated mDia1 mutants FH1FH2 (0.03 µg) or FH2 (0.3 µg) (Copeland and Treisman, 2002), and VASPΔB (0.1, 0.3 and 0.9 µg). Right: mDia derivatives. Ellipse, Rho-binding domain; D, DAD domain. Immunoblot for mDia1 indicates that VASPΔB expression does not affect mDia1 protein levels.

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Fig. 7. Physical interaction between VASP and mDia1. (A) Association of endogenous VASP and mDia1. NIH 3T3 cell extracts or buffer were immunoprecipitated either with crosslinked anti-mDia1 or control anti-GFP beads (left panels), or with crosslinked anti-VASP or control anti-GFP beads (right panels). The precipitates were analysed by immunoblot, with protein detection by anti-mDia1 (upper panels) or anti-VASP (lower panels). Protein input in the lysate lanes (left) was 1/30 that used for the IP. Removal of irrelevant lanes from the scanned image of a single gel is indicated by gaps. (B) Two regions of mDia1 mediate VASP interaction. Lysates were prepared from cells expressing Flag-VASP and the indicated HA-mDia1 mutants, immunoprecipitated using Flag–agarose, and analysed by immunoblotting with HRP-coupled anti-HA. Bottom: mDia1 expression levels in the lysate. (C) Two regions of VASP mediate interactions with mDia1. Lysates were prepared from expressing HA-VASP derivatives and either Flag-mDia1 ΔRBD (left) or 9E10-tagged mDia1 F1F2 or F2 (right). Immunoprecipitates prepared using Flag–agarose (left) or myc–agarose (right) were analysed by immunoblotting with HRP-coupled anti-HA, or with 9E10 and secondary anti-mouse HRP-coupled antibodies as indicated. (Bottom) VASP expression levels in the lysate.

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Fig. 8. Function of VASP in SRF activation. (A) Functional domains of VASP. The EVH1, polyproline-rich and EVH2 domains are shown as in Figure 1, with the three PKA phosphorylation sites indicated as circles. Functional roles of the EVH2 domain are indicated at the top and biochemical roles of the EVH2 subdomains at the bottom. (B) Proposed role for VASP in Rho-controlled F-actin assembly. Two Rho effector pathways control F-actin accumulation through its stabilization (ROCK → LIMK → cofilin pathway) or its assembly (mDia/VASP pathway). The dotted line indicates that the precise role of VASP in the mDia pathway remains unclear. SRF activity reflects depletion of the cellular G-actin pool, which may derepress an SRF coactivator.

References

    1. Arsenian S., Weinhold,B., Oelgeschlager,M., Ruther,U. and Nordheim,A. (1998) Serum response factor is essential for mesoderm formation during mouse embryogenesis. EMBO J., 17, 6289–6299. - PMC - PubMed
    1. Bachmann C., Fischer,L., Walter,U. and Reinhard,M. (1999) The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding and actin bundle formation. J. Biol. Chem., 274, 23549–23557. - PubMed
    1. Bear J.E., Loureiro,J.J., Libova,I., Fassler,R., Wehland,J. and Gertler,F.B. (2000) Negative regulation of fibroblast motility by Ena/VASP proteins. Cell, 101, 717–728. - PubMed
    1. Bear J.E., Krause,M. and Gertler,F.B. (2001) Regulating cellular actin assembly. Curr. Opin. Cell Biol., 13, 158–166. - PubMed
    1. Bear J.E. et al. (2002) Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell, 109, 509–521. - PubMed

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