Fig. 6. Ezrin T567A interferes with formation of focal adhesion contacts in Swiss 3T3 cells. Swiss 3T3 cells were microinjected with EFplink derivatives encoding RhoAV14, ROCKΔ3 or Dbl, together with plasmids encoding pBC6-EzrinWT or pBC6-EzrinT567A (50 ng/µl) as indicated. At 4 h post-transfection, cells were fixed, permeabilized and stained for F-actin (green) and paxillin (red). Microinjected cells are indicated by arrowheads.

Fig. 4. (A) Ezrin T567 is required for relocalization induced by ROCK but not PI4P-5K. NIH 3T3 cells were transfected with either 0.5 µg of pEF ROCKΔ3 or PI4P-5K either alone or with 0.3 µg of pBC6-EzrinWT or pBC6-EzrinT567A as indicated, and analysed for ezrin localization and F-actin 18 h later. Transfected cells are indicated by arrowheads. (B) Activated RhoA and its GEFs cannot relocalize ezrin T567A. Cells were transfected with plasmids expressing the indicated proteins and analysed as in (A).

Fig. 2. (A) ROCK activity is required for ezrin relocalization by the oncogenic RhoGEFs Net and Dbl. Cells were transfected with 0.6 µg of pBC6-EzrinWT together with 0.3 µg of EFplink derivatives encoding Net, Dbl or v-Src. Cells were analysed for ezrin and F-actin 18 h later, following a 2 h treatment with Y27632 where indicated, as described in Materials and methods. Panels show a Z stack of confocal images. (B) RhoA.V14-induced ezrin is ROCK dependent. Cells were transfected with 0.6 µg of pBC6-EzrinWT together with pEFplink derivatives encoding either RhoA.V14 (0.3 µg) or the kinase-inactive ROCK mutant ROCKΔ3K105A (0.5 µg), and analysed as above.

Fig. 5. (A) Association of ezrin with the cytoskeleton requires T567. Stable transfectants of Dbl-d- or Net-e-transformed NIH 3T3 cells expressing either VSVG-tagged wild-type ezrin (Dbl Ez-2 and Net Ez-3-7) or ezrin T567A (Dbl EzT567A-10 and Net EzT567A-1) were left untreated or treated for 2 h with Y27632 before fractionation by Triton X-100 extraction. Fractions were analysed for ezrin and actin by immunoblotting. (B) Ezrin is not associated with the cytoskeleton in untransformed or Src-transformed NIH 3T3 cells. Stable transfectants of untransformed Src-transformed NIH 3T3 cell derivatives expressing wild-type VSVG-tagged ezrin (NIH 3T3 Ez-10 and Src-1 Ez-1) were left untreated or treated directly for the indicated times (hours) with Y27632 before fractionation and analysis as above.

Fig. 1. (A) Activated RhoA relocalizes both endogenous and transiently expressed ezrin into actin-containing dorsal protrusions. NIH 3T3 cells were transfected with 0.3 µg of the RhoA.V14 expression plasmid pEF.RhoA.V14 or its parental vector EFplink (top panels), or with these plasmids together with 0.6 µg of the ezrin expression plasmid pBC6-EzrinWT (lower panels). After 18 h, ezrin and F-actin were detected as described in Materials and methods. The nuclear endogenous ezrin staining is non-specific. RhoA expression was confirmed by 9E10 staining (data not shown). Each panel shows a Z stack of confocal images. (B) Relocalization of ezrin by RhoA effector loop mutants. Cells were transfected with 0.6 µg of pBC6-EzrinWT as above together with 0.3 µg each of expression plasmids encoding the indicated RhoA.V14 effector loop mutants. Transfected cells are indicated by arrowheads. Binding affinities of the effector loop mutants for the Rho-binding domains of ROCK, PKN and citron kinases are as follows (Sahai et al., 1998) (+, binding; –, no binding; +/–, intermediate binding): F39A: PKN–, ROCK–, CitronK–; F39V: PKN–, ROCK+, CitronK–; F39L: PKN+, ROCK+/–, CitronK–; E40L: PKN+, ROCK–, CitronK+; E40W: PKN+, ROCK–, CitronK+; E40N: PKN+, ROCK+, CitronK+; E40T: PKN+, ROCK+, CitronK+; Y42C: PKN–, ROCK+, CitronK+. The proportions of cells displaying dorsally relocalized ezrin were as follows: ezrin alone, 25%; ezrin with RhoA.V14WT, 95%; F39A, 19%; F39L, 24%; E40L, 26%; E40W; 37%; F39V, 60%; E40N, 95%; E40T, 82%; and Y42C, 84%.

Fig. 3. (A) ROCK can phosphorylate the ezrin C-ERMAD in vitro. NIH 3T3 cells were either mock transfected or transfected with 4 µg of EFplink plasmids encoding 9E10 epitope-tagged ROCKΔ3 or its kinase-inactive derivative ROCKΔ3.K105A. After 18 h, cell extracts were immunoprecipitated with 9E10 antibody and used in immune complex kinases assays with GST or GST–ezrin(280–585) as substrates. Reactions were analysed on a 10% SDS–polyacrylamide gel. Stars indicate presumed autophosphorylation of the kinase. (B) The ROCK effector LIMK-1 and the RhoA effector PKN (PRK1) do not phosphorylate the ezrin C-ERMAD. Cells were transfected with plasmids encoding ROCKΔ3, LIMK-Q1 (the LIMK-1 kinase domain) or intact PKN, and analysed as in (B) with the indicated GST fusion proteins, together with whole calf thymus histones (a kind gift of Dr Robert Nicolas) as substrates. Reactions were analysed on a 15% SDS–polyacrylamide gel. (C) Increased ezrin phosphorylation in transformed cells. Immunoblot analysis of NIH 3T3 cells and derivatives Src-1, Net-e and Dbl-d transformed by the indicated oncogenes using the indicated antibodies. (D) Ezrin phosphorylation is reduced by Y-27632 in RhoGEF-transformed cells. Extracts from derivatives of Src-1-, Net-e- and Dbl-d-transformed cells expressing VSVG-tagged ezrin (Src Ez-1, Net Ez 3-7 and Dbl Ez-2) were analysed using the indicated antibodies after treatment for various times with Y27632.

Fig. 7. (A) Expression of ezrin T567A but not wild-type ezrin restores contact inhibition in Net- and Dbl-transformed NIH 3T3 cells. Derivatives of both untransformed and transformed NIH 3T3 cells expressing ezrin or ezrin T567A were generated and used in secondary focus formation assays. A total of 5000 test cells were seeded among 30 000 untransformed NIH 3T3 cells and cultured in the presence or absence of 10 µM Y27632 for 10 days, when overgrowing colonies were visualized by crystal violet staining. Upper panel: secondary focus formation by NIH 3T3, v-Src-1, Net-e and Dbl-d cells (Sahai et al., 1999). Middle panel: secondary focus formation by derivatives of these cell lines expressing wild-type ezrin (lines NIH 3T3 Ez-10, v-Src-1 Ez-1, Net-e Ez-1 and Dbl-d Ez-2). Similar results were obtained with a further two cell lines in each case. Bottom panel: secondary focus formation by derivatives of the cell lines shown in the upper panel expressing ezrin T567A (lines NIH 3T3 EzT567A-1, v-Src-1 EzT567A-5, Net-e EzT567A-1 and Dbl-d EzT567A-10). Similar results were obtained with a further ezrin T567A transfectant derived from each of the parental transformed cell lines. In the case of Net and Dbl, two further clones derived from independent Net- and Dbl-transformed cell lines (Net-d and Dbl-e; Sahai et al., 1999) were analysed and a similar result obtained. (B) Summary of secondary focus formation assays. Results from two independent experiments using the cell lines as in (A) are shown. (C) Ezrin T567A inhibits primary focus formation by Net and Ras but not by Src. NIH 3T3 cells were transfected with 0.3 µg of EFplink derivatives encoding v-Src, Net or RasR12 either alone or with increasing doses (0.3 or 0.6 µg) of pBC6-EzrinT567A. DNA inputs were kept constant with pBC6 vector DNA. Cells were maintained for 15 days in 5% donor calf serum before staining with crystal violet to visualize foci, which were then counted and compared with the number of foci induced by the respective oncogenes. Under these conditions, v-Src-transfected cells formed ∼175 foci, RasR12 65 foci and Net 125 foci. The diagram shows the average of three independent experiments.