Interaction of zyxin with h-warts/LATS1. (A) Schematic diagram of human h-warts/LATS1 and human zyxin showing their domain structures. The hatched bar below the h-warts/LATS1 structure represents the portion used as the bait in the two-hybrid system. Four boldface lines below the human zyxin structure indicate the portions of zyxin encoded by cDNA clones recovered in the two-hybrid screening. (B) Expression of HA-tagged h-warts and FLAG-tagged zyxin in COS7 cells. The lysates (15 μg of protein) expressing both HA-tagged h-warts and FLAG-tagged zyxin were probed with the anti-HA or anti-FLAG antibodies. The lysates of COS7 cells transfected with empty vectors were used as controls. (C) Interaction of zyxin with h-warts/LATS1 in COS7 cells. The same lysates (200 μg of protein) of COS7 cells prepared in (A) were immunoprecipitated with the anti-HA antibody or the anti-FLAG antibody. The immunoprecipitates were probed with anti-HA or anti-zyxin antibodies. (D) Specificity of the h-warts/LATS1 antibody. Lysates of COS7 cells expressing HA-tagged h-warts (lanes 1 and 5), HeLa cell lysate (lane 2), and HeLa cell lysate immunoprecipitated with preimmune IgG (lanes 3 and 7) or with the anti-C1 antibody (lanes 4 and 8) were immunoblotted by preimmune IgG (lanes 1–4) or by the anti-C2 antibody (lanes 5–8). Arrow indicates endogenous h-warts/LATS1. (E) Endogenous h-warts/LATS1 interacts with zyxin. The lysate of COS7 cells expressing HA-tagged zyxin (full-length) was immunoprecipitated with the anti-C1 antibody (lane 3), or with preimmune IgG (lane 2). The immunoprecipitates were probed with the anti-HA antibody. (F) Coimmunoprecipitation of endogenous h-warts/LATS1 and zyxin. The lysate of HeLa cells was immunoprecipitated with preimmune IgG or with the anti-zyxin antibody generated in rabbits as described in Fig. 3. Equal amount of cell lysate was immunoprecipitated with anti–h-warts/LATS1 antibody to quantify the endogenous protein in the aliquot of lysate (input). The immunoprecipitates were probed with the rat anti–h-warts/LATS1(G4) antibody (upper), and the goat anti-zyxin antibody (lower), as indicated.

Interaction of zyxin with h-warts/LATS1. (A) Schematic diagram of human h-warts/LATS1 and human zyxin showing their domain structures. The hatched bar below the h-warts/LATS1 structure represents the portion used as the bait in the two-hybrid system. Four boldface lines below the human zyxin structure indicate the portions of zyxin encoded by cDNA clones recovered in the two-hybrid screening. (B) Expression of HA-tagged h-warts and FLAG-tagged zyxin in COS7 cells. The lysates (15 μg of protein) expressing both HA-tagged h-warts and FLAG-tagged zyxin were probed with the anti-HA or anti-FLAG antibodies. The lysates of COS7 cells transfected with empty vectors were used as controls. (C) Interaction of zyxin with h-warts/LATS1 in COS7 cells. The same lysates (200 μg of protein) of COS7 cells prepared in (A) were immunoprecipitated with the anti-HA antibody or the anti-FLAG antibody. The immunoprecipitates were probed with anti-HA or anti-zyxin antibodies. (D) Specificity of the h-warts/LATS1 antibody. Lysates of COS7 cells expressing HA-tagged h-warts (lanes 1 and 5), HeLa cell lysate (lane 2), and HeLa cell lysate immunoprecipitated with preimmune IgG (lanes 3 and 7) or with the anti-C1 antibody (lanes 4 and 8) were immunoblotted by preimmune IgG (lanes 1–4) or by the anti-C2 antibody (lanes 5–8). Arrow indicates endogenous h-warts/LATS1. (E) Endogenous h-warts/LATS1 interacts with zyxin. The lysate of COS7 cells expressing HA-tagged zyxin (full-length) was immunoprecipitated with the anti-C1 antibody (lane 3), or with preimmune IgG (lane 2). The immunoprecipitates were probed with the anti-HA antibody. (F) Coimmunoprecipitation of endogenous h-warts/LATS1 and zyxin. The lysate of HeLa cells was immunoprecipitated with preimmune IgG or with the anti-zyxin antibody generated in rabbits as described in Fig. 3. Equal amount of cell lysate was immunoprecipitated with anti–h-warts/LATS1 antibody to quantify the endogenous protein in the aliquot of lysate (input). The immunoprecipitates were probed with the rat anti–h-warts/LATS1(G4) antibody (upper), and the goat anti-zyxin antibody (lower), as indicated.

Interaction of zyxin with h-warts/LATS1. (A) Schematic diagram of human h-warts/LATS1 and human zyxin showing their domain structures. The hatched bar below the h-warts/LATS1 structure represents the portion used as the bait in the two-hybrid system. Four boldface lines below the human zyxin structure indicate the portions of zyxin encoded by cDNA clones recovered in the two-hybrid screening. (B) Expression of HA-tagged h-warts and FLAG-tagged zyxin in COS7 cells. The lysates (15 μg of protein) expressing both HA-tagged h-warts and FLAG-tagged zyxin were probed with the anti-HA or anti-FLAG antibodies. The lysates of COS7 cells transfected with empty vectors were used as controls. (C) Interaction of zyxin with h-warts/LATS1 in COS7 cells. The same lysates (200 μg of protein) of COS7 cells prepared in (A) were immunoprecipitated with the anti-HA antibody or the anti-FLAG antibody. The immunoprecipitates were probed with anti-HA or anti-zyxin antibodies. (D) Specificity of the h-warts/LATS1 antibody. Lysates of COS7 cells expressing HA-tagged h-warts (lanes 1 and 5), HeLa cell lysate (lane 2), and HeLa cell lysate immunoprecipitated with preimmune IgG (lanes 3 and 7) or with the anti-C1 antibody (lanes 4 and 8) were immunoblotted by preimmune IgG (lanes 1–4) or by the anti-C2 antibody (lanes 5–8). Arrow indicates endogenous h-warts/LATS1. (E) Endogenous h-warts/LATS1 interacts with zyxin. The lysate of COS7 cells expressing HA-tagged zyxin (full-length) was immunoprecipitated with the anti-C1 antibody (lane 3), or with preimmune IgG (lane 2). The immunoprecipitates were probed with the anti-HA antibody. (F) Coimmunoprecipitation of endogenous h-warts/LATS1 and zyxin. The lysate of HeLa cells was immunoprecipitated with preimmune IgG or with the anti-zyxin antibody generated in rabbits as described in Fig. 3. Equal amount of cell lysate was immunoprecipitated with anti–h-warts/LATS1 antibody to quantify the endogenous protein in the aliquot of lysate (input). The immunoprecipitates were probed with the rat anti–h-warts/LATS1(G4) antibody (upper), and the goat anti-zyxin antibody (lower), as indicated.

In vitro interaction of h-warts/LATS1 and zyxin. (A) Schematic diagram of deletion mutant zyxin showing their domain structures. The NH₂-terminal two-thirds mutant (Δ1) contains four copies of a proline rich cluster and the nuclear exporting signal (NES), and the COOH-terminal third mutant (Δ2) contains three copies of the LIM domain. LIM domains were constructed tandemly or independently as indicated (Δ3–Δ7). The HA-tag epitope or the GST region of the fusion protein is not shown. (B) The lysates of COS7 cells expressing the indicated HA-tagged zyxin full-length and mutants (lanes 1–3) were subjected to incubation with either GST alone (lanes 4, 6, and 8) or GST–h-warts/LATS1 (amino acids 136–700) resin (lanes 5, 7, and 9). The bound fraction was analyzed by SDS-PAGE, followed by immunoblotting with anti-HA antibody. (C) In vitro binding assay between recombinant h-warts/LATS1 and zyxin. 10 μg of the purified GST-zyxin deletion mutants, as shown in (A), were immobilized to resins, and incubated with purified His-tagged h-warts/LATS1 (1.0 μg of protein) generated in baculovirus-infected Sf9 cells. The bound fraction was analyzed by immunoblotting with the anti-C2 antibody.

Specificity of the anti-zyxin antibody. (A) Total lysates of HeLa cells (lanes 1 and 3), and U2OS cells (lanes 2 and 4) were resolved in 12% SDS-PAGE, followed by immunoblotting with either the preimmune IgG (lanes 1 and 2) or the anti-zyxin antibody (lanes 3 and 4). (B) Subcellular localization of endogenous zyxin. Subconfluently grown U2OS cells were fixed with 4% paraformaldehyde and processed for indirect immunofluorescent staining with the anti-zyxin antibody labeled by FITC. DNA was visualized by propidium iodide (a), and F-actin by rhodamine-phalloidin (b). (C) Distribution of zyxin during the cell cycle. U2OS cells were prepared as in (B, a). Representative cells in prophase (a), metaphase (b), anaphase (c), and telophase (d) are shown. (D) Detection of zyxin on the mitotic apparatus. U2OS cells were fixed with acetone/methanol solution and processed for immunostaining with the anti-zyxin antibody labeled with FITC (a and d) and anti-tubulin antibody labeled with Cy3 (b and e). The merged picture of the left two panels are shown in the right panel (c and f). Representative cells in metaphase (a–c) and telophase (d–f) are shown. Bars, 10 μm.

Colocalization of zyxin and h-warts/LATS1 to the mitotic apparatus. (A) Localization of h-warts/LATS1 at the different phases of the cell cycle. U2OS cells were preextracted with MSB (microtubule stabilizing buffer, including 0.5% Triton X-100) and fixed with cold methanol, followed by incubation with rat anti–h-warts antibody and mouse anti–α-tubulin antibody. Immunofluorescence staining was performed with FITC-conjugated anti–mouse IgG antibody (a, d, g, j and m) and Cy3-conjugated anti–rat IgG antibody (b, e, h, k, and n). The merged pictures of the upper two panels are shown in the lower panel (c, f, i, l, and o). Representative cells in interphase (a–c), metaphase (d–f), anaphase (g–i), telophase (j–l), and later telophase (m–o) are shown. (B) Distribution of zyxin during mitosis. U2OS cells were preextracted with 7.5 μg/ml digitonin/KHM, and fixed with cold methanol, after incubation with rabbit anti-zyxin antibody and mouse anti–α-tubulin antibody. Immunofluorescence staining was performed with Texas red–conjugated anti–mouse IgG antibody (a, d, g, j, and m) and FITC-conjugated anti–rabbit IgG antibody (b, e, h, k, and n). The merged pictures of the upper two panels are shown in the lower panel (c, f, i, l, and o). Representative cells in prometaphase (a–c), metaphase (d–f), anaphase (g–i), telophase (j–l), and later telophase (m–o) are shown. (C) Identical distribution of h-warts/LATS1 and zyxin to the mitotic apparatus. U2OS cells were prepared and fixed as in (A) and processed for incubation with rabbit anti-zyxin antibody (a and d) and rat anti–h-warts antibody (b and e). Immunofluorescence staining was performed with FITC-conjugated anti–rabbit antibody and Cy3-conjugated anti–rat IgG antibody. The merged pictures of the left two panels are shown in the right panel (c and f). Bars, 10 μm.

Colocalization of zyxin and h-warts/LATS1 to the mitotic apparatus. (A) Localization of h-warts/LATS1 at the different phases of the cell cycle. U2OS cells were preextracted with MSB (microtubule stabilizing buffer, including 0.5% Triton X-100) and fixed with cold methanol, followed by incubation with rat anti–h-warts antibody and mouse anti–α-tubulin antibody. Immunofluorescence staining was performed with FITC-conjugated anti–mouse IgG antibody (a, d, g, j and m) and Cy3-conjugated anti–rat IgG antibody (b, e, h, k, and n). The merged pictures of the upper two panels are shown in the lower panel (c, f, i, l, and o). Representative cells in interphase (a–c), metaphase (d–f), anaphase (g–i), telophase (j–l), and later telophase (m–o) are shown. (B) Distribution of zyxin during mitosis. U2OS cells were preextracted with 7.5 μg/ml digitonin/KHM, and fixed with cold methanol, after incubation with rabbit anti-zyxin antibody and mouse anti–α-tubulin antibody. Immunofluorescence staining was performed with Texas red–conjugated anti–mouse IgG antibody (a, d, g, j, and m) and FITC-conjugated anti–rabbit IgG antibody (b, e, h, k, and n). The merged pictures of the upper two panels are shown in the lower panel (c, f, i, l, and o). Representative cells in prometaphase (a–c), metaphase (d–f), anaphase (g–i), telophase (j–l), and later telophase (m–o) are shown. (C) Identical distribution of h-warts/LATS1 and zyxin to the mitotic apparatus. U2OS cells were prepared and fixed as in (A) and processed for incubation with rabbit anti-zyxin antibody (a and d) and rat anti–h-warts antibody (b and e). Immunofluorescence staining was performed with FITC-conjugated anti–rabbit antibody and Cy3-conjugated anti–rat IgG antibody. The merged pictures of the left two panels are shown in the right panel (c and f). Bars, 10 μm.

Colocalization of zyxin and h-warts/LATS1 to the mitotic apparatus. (A) Localization of h-warts/LATS1 at the different phases of the cell cycle. U2OS cells were preextracted with MSB (microtubule stabilizing buffer, including 0.5% Triton X-100) and fixed with cold methanol, followed by incubation with rat anti–h-warts antibody and mouse anti–α-tubulin antibody. Immunofluorescence staining was performed with FITC-conjugated anti–mouse IgG antibody (a, d, g, j and m) and Cy3-conjugated anti–rat IgG antibody (b, e, h, k, and n). The merged pictures of the upper two panels are shown in the lower panel (c, f, i, l, and o). Representative cells in interphase (a–c), metaphase (d–f), anaphase (g–i), telophase (j–l), and later telophase (m–o) are shown. (B) Distribution of zyxin during mitosis. U2OS cells were preextracted with 7.5 μg/ml digitonin/KHM, and fixed with cold methanol, after incubation with rabbit anti-zyxin antibody and mouse anti–α-tubulin antibody. Immunofluorescence staining was performed with Texas red–conjugated anti–mouse IgG antibody (a, d, g, j, and m) and FITC-conjugated anti–rabbit IgG antibody (b, e, h, k, and n). The merged pictures of the upper two panels are shown in the lower panel (c, f, i, l, and o). Representative cells in prometaphase (a–c), metaphase (d–f), anaphase (g–i), telophase (j–l), and later telophase (m–o) are shown. (C) Identical distribution of h-warts/LATS1 and zyxin to the mitotic apparatus. U2OS cells were prepared and fixed as in (A) and processed for incubation with rabbit anti-zyxin antibody (a and d) and rat anti–h-warts antibody (b and e). Immunofluorescence staining was performed with FITC-conjugated anti–rabbit antibody and Cy3-conjugated anti–rat IgG antibody. The merged pictures of the left two panels are shown in the right panel (c and f). Bars, 10 μm.

Mitosis-specific phosphorylation of zyxin. (A) Posttranslational modification of zyxin during the cell cycle. HeLa cells were synchronized at the beginning of S phase by the double thymidine block method. After release from S phase, cells were harvested at the indicated time points (lanes 1–7). To block cells at mitosis, nocodazole was added into the medium 6 h after release from S phase and incubated for another 6 h (N, lane 8). After removal of nocodazole, cells were incubated in nocodazole-free media for another 3 h to obtain interphase cells (NR, lane 9). Samples were analyzed for DNA content by flow cytometry. The percentages of the cells in G1, S, and G2/M are shown in the upper panel. Each sample was subjected to immunoblotting with anti-zyxin antibody, anti-vinculin antibody, and anti-cyclin B antibody. (B) Phosphatase treatment of zyxin. Endogenous zyxin was immunoprecipitated from interphase (lanes 1 and 2), mitotic (lanes 3 and 4) and nocodazole-treated (lanes 5 and 6) lysate and incubated with 100 units of calf intestine alkaline phosphatase (CIAP) (lanes 2, 4, and 6) or without CIAP (lanes 1, 3, and 5) at 37°C for 30 min. Equal amount of precipitants were immunoblotted with the anti-zyxin antibody. Arrows indicate the endogenous zyxin. (C) Detection of zyxin-kinase activity in mitotic cell lysate. GST-zyxin (full-length) was incubated in the presence of γ-[³²P]ATP with either buffer alone (lane 1), interphase cell lysate (lane 2), or mitotic cell lysate (lane 3) for 30 min at 25°C. GST-zyxin (full-length) was analyzed by SDS-PAGE and autoradiography. (D) Phosphorylation region of zyxin. Purified GST-zyxin, full-length and truncated mutants, as indicated (left; Coomassie blue staining), were subjected to in vitro kinase assay with interphase/mitotic cell lysate as described in (C) (right, autoradiography). Note that a fraction of protein was converted into the slow-mobility form after incubation with mitotic lysate in GST-zyxin (full-length) and zyxinΔ1.

Characterization of zyxin-kinase. (A) Effect of protein kinase inhibitors on the zyxin-kinase activity in mitotic cell lysate. GST-zyxin (full-length) was incubated for 30 min at 25°C in the presence of γ-[³²P]ATP and mitotic cell lysate with the indicated protein kinase inhibitors as follows: 100 nM staurosporine (broad serine/threonine kinase inhibitor), 100 μM olomoucine (Cdc2 inhibitor), 100 μM U0126 (MEK1 inhibitor), 1 μM PKI (protein kinase A inhibitor), 100 nM calphostin C (protein kinase C inhibitor), 100 μM SB203580 (p38 kinase inhibitor), or without kinase inhibitor (mitotic cell lysate alone). Results are presented as the percentage of the levels of GST-zyxin phosphorylation, where 100% represents the phosphorylation level after incubation with mitotic cell lysate alone. (B) Depletion of Cdc2 from mitotic cell lysate. Mitotic cell lysate (lane 1) was incubated with p13-suc1–conjugated agarose beads to deplete Cdc2 (lanes 2 and 4). Mock depletion was performed by incubating the mitotic cell lysate with the same volume of unconjugated agarose beads (lanes 3 and 5). Unbound cytosolic proteins were immunoblotted with anti-Cdc2 (top) or anti–β-tubulin antibodies (bottom). (C) Histone H1 kinase activity of mitotic cell lysates. The peptide derived from histone H1 was incubated in the presence of γ-[³²P]ATP with mitotic cell lysate, mock-depleted lysate, or Cdc2-depleted lysate. Results are presented as percentage of the phosphorylation levels of the peptide, where 100% represents the phosphorylation level after incubation with the mitotic cell lysate. (D) Effect of Cdc2 depletion on the zyxin-kinase activity in mitotic cell lysate. GST-zyxin (full-length) was incubated in the presence of γ-[³²P]ATP with either buffer alone (lane 1), mock-depleted mitotic cell lysate (lane 2), or Cdc2-depleted mitotic cell lysate (lane 3), or Cdc2-depleted mitotic cell lysate supplemented with purified active Cdc2 complex (lane 4) for 30 min at 25°C. GST-zyxin (full-length) was analyzed by SDS-PAGE and autoradiography. (E) Direct phosphorylation of zyxin by Cdc2. GST-zyxin (full-length) was incubated in the presence of γ-[³²P]ATP with (lane 1) or without active Cdc2 complex (lane 2).

Regulation of h-warts/LATS1-zyxin association. (A) Association of h-warts/LATS1 with Cdc2-mediated phosphorylation of zyxin. 10 μg of GST-zyxin (full-length) was incubated with active Cdc2 complex with or without ATP for 30 min at 25°C as described in Fig. 6 E, followed by in vitro binding assay as in Fig. 2 C. After kinase reaction in the absence of ATP (lane 4), or in the presence of ATP (lane 5), GST-zyxin (full-length) were incubated with 1 μg of His-tagged h-warts/LATS1 (lane 1). Control experiments were performed without kinase reaction using GST-zyxin (full-length) (lane 2) or GST-zxyinΔ2 (lane 3). Bound fraction was analyzed by SDS-PAGE, followed by immunoblotting with the anti-C2 antibody. (B) Mitosis-specific interaction of h-warts/LATS1 and zyxin. The lysate of interphase (lanes 1 and 2) or mitotic (lanes 3 and 4) HeLa cells was immunoprecipitated with preimmune IgG (lanes 1 and 3) or with the anti-zyxin antibody (lanes 2 and 4). The immunoprecipitates were probed with anti–h-warts/LATS1 antibody (G4) or with anti-zyxin antibody as in Fig. 1 F.

Effect of h-warts/LATS1(136–700) induction on mitosis progression. (A) HeLa cells synchronized at the beginning of S phase were coinjected with GST–h-warts/LATS1(136–700) or GST and β-galactosidase. After release from S phase, cells were fixed and analyzed for β-galactosidase activity. Chromatin is visualized by staining cells with aceto-orcein, with which mitotic condensed chromatin can be easily identified by its strong staining. Representative microscopic pictures of uninjected cells (a) from 10.5 h after release from S phase, GST-injected cells (b) and GST–h-warts/LATS1(136–700) (c) from 12 h after the release. (B) Mitotic indices in cells injected with GST–h-warts/LATS1(136–700), GST-mock, and β-galactosidase alone. Mitotic index was determined by counting the percentage of cells with condensed chromatin and rounded-up morphology among β-galactosidase-positive cells. Approximately 150 cells were injected and scored for each experiment. Data are mean ± SD of the six different experiments.

Effect of h-warts/LATS1(136–700) induction on mitosis progression. (A) HeLa cells synchronized at the beginning of S phase were coinjected with GST–h-warts/LATS1(136–700) or GST and β-galactosidase. After release from S phase, cells were fixed and analyzed for β-galactosidase activity. Chromatin is visualized by staining cells with aceto-orcein, with which mitotic condensed chromatin can be easily identified by its strong staining. Representative microscopic pictures of uninjected cells (a) from 10.5 h after release from S phase, GST-injected cells (b) and GST–h-warts/LATS1(136–700) (c) from 12 h after the release. (B) Mitotic indices in cells injected with GST–h-warts/LATS1(136–700), GST-mock, and β-galactosidase alone. Mitotic index was determined by counting the percentage of cells with condensed chromatin and rounded-up morphology among β-galactosidase-positive cells. Approximately 150 cells were injected and scored for each experiment. Data are mean ± SD of the six different experiments.

Disruption of endogenous zyxin localization on the mitotic apparatus by introducing h-warts/LATS1 protein fragments. U2OS cells were microinjected with 1.0 mg/ml of GST–h-warts/LATS1 (amino acids 135–700) or GST alone, supplemented with rhodamine-labeled tubulin. After preextraction and fixation, cells were incubated with rabbit anti-zyxin antibody and labeled with FITC-conjugated anti–rabbit IgG antibody. Cells with rhodamine-labeled mitotic spindles (a, d, g, and j) were evaluated for zyxin staining (b, e, h, and k). Representative pictures injected with GST alone (a–f), or GST–h-warts (amino acids 135–700; g–l) in metaphase and telophase are shown. The bottom panels (c, f, i, and l) show the phase-contrast images of the corresponding cells. Bars, 10 μm.