Correspondence to: Hideyuki Saya, Department of Tumor Genetics and Biology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan. Tel:+81-96-373-5116 Fax:+81-96-373-5120 E-mail:hsaya{at}gpo.kumamoto-u.ac.jp.
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Abstract |
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The mitotic apparatus plays a pivotal role in dividing cells to ensure each daughter cell receives a full set of chromosomes and complement of cytoplasm during mitosis. A human homologue of the Drosophila warts tumor suppressor, h-warts/LATS1, is an evolutionarily conserved serine/threonine kinase and a dynamic component of the mitotic apparatus. We have identified an interaction of h-warts/LATS1 with zyxin, a regulator of actin filament assembly. Zyxin is a component of focal adhesion, however, during mitosis a fraction of cytoplasmic-dispersed zyxin becomes associated with h-warts/LATS1 on the mitotic apparatus. We found that zyxin is phosphorylated specifically during mitosis, most likely by Cdc2 kinase, and that the phosphorylation regulates association with h-warts/LATS1. Furthermore, microinjection of truncated h-warts/LATS1 protein, including the zyxin-binding portion, interfered with localization of zyxin to mitotic apparatus, and the duration of mitosis of these injected cells was significantly longer than that of control cells. These findings suggest that h-warts/LATS1 and zyxin play a crucial role in controlling mitosis progression by forming a regulatory complex on mitotic apparatus.
Key Words: Cdc2, interaction, mitotic spindle, phosphorylation, serine/threonine kinase
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Introduction |
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Mitotic apparatus, which consists of a dynamic array of microtubules and associated proteins, controls mitotic events such as establishment of cell polarity, chromosomal congression and segregation, and finally, cytokinesis. Genetic analyses in yeasts and Drosophila have identified several kinases essential for normal regulation of mitosis (
The warts gene (also known as lats) was identified as a tumor suppressor of Drosophila melanogaster (
A human homologue of the warts/lats gene, termed h-warts/LATS1, has been identified and demonstrated to negatively regulate Cdc2 activity by interacting with Cdc2 in the mitotic phase (
During mitosis, adherent cells change morphology into a spheroid and weakly adherent form. This morphological alteration involves rearrangement of cytoskeletal systems and dissociation of the adhesion apparatus, which are under the control of biochemical status through cell cycle progression (
Zyxin is a component of the focal adhesion complex (
In this study, we have identified the interaction of h-warts/LATS1 with zyxin on the mitotic apparatus during mitosis. The localization of zyxin on the mitotic apparatus appears to be dependent on the presence of h-warts/LATS1 protein. Furthermore, we showed that zyxin is phosphorylated specifically during mitosis, most likely by Cdc2 kinase, and that this phosphorylation controls the association of zyxin with h-warts/LATS1. The interaction between zyxin and h-warts/LATS1 on the mitotic apparatus implicates a significant role for actin regulatory proteins during mitosis.
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Materials and Methods |
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Yeast Two-Hybrid Screening
Yeast strain L40 was used as a host for the two-hybrid screening (
Cell Culture, Synchronization, and Transfections
HeLa, COS7, and U2OS cells were cultured in DME/F12 supplemented with 10% fetal calf serum without antibiotics. HeLa cells were synchronized at the beginning of S phase by double thymidine block and release protocol (first 24 h incubation with 1 mM thymidine, an interval of thymidine-free incubation for 8 h, and second thymidine incubation for 14 h). Mitotic cells were collected by mechanical shake-off from the culture plate 9.5 h after release from S phase. For transient transfection, cells in 6-well plates were transfected using FuGene6 transfection reagent following the manufacturer's instructions (Boehringer Mannheim). To prepare for flow cytometry, cells were trypsinized, fixed with 70% methanol, and DNA were stained with propidium iodide. Cells were subjected to flow cytometry on FACScan® (Becton Dickinson). G1, S, and G2/M populations were calculated with ModFit 2.1 software (Varity).
Expression Plasmids
Mammalian expression plasmids were constructed by subcloning the PCR amplified fragment into hemagglutinin 1 (HA)-tagged (pCGN), FLAG-tagged (pBJ-FLAG) vectors. pBJ-FLAG, was constructed by inserting an annealed oligonucleotide between the XhoI and BamHI sites of pBJ-myc. All the PCR products were obtained using PyroBest DNA polymerase (Takara) and we confirmed their sequences. For glutathione-S-transferase (GST) fusion protein expression in bacteria, pGEX2TH-based plasmids were constructed as previously described (
Antibody Preparation
Two polyclonal antibodies against h-warts/LATS1 were generated by injecting rabbits with two synthetic peptides, C1 (PVDPDKLWSDDNEEENVNDTLNG), and C2 (SDEDDQNTGSEIKNRDLVYV), coupled to keyhole limpet-hemocyanin (KLH) via the added NH2-terminal cysteine. GSTh-warts amino acids 136700 and amino acids 136410 were also used for immunizing rats, generating G3 and G4 antisera, respectively. The polyclonal antibody against zyxin was generated in rabbit as previously described (-tubulin (clone B512), vinculin and cyclin B were purchased from Sigma-Aldrich and Transduction Laboratories, respectively.
Immunoprecipitation
Cells were lysed on ice for 30 min with 0.5% NP-40 lysis buffer consisting of 0.5% NP-40, 25 mM Tris-Cl, pH 7.5, 137 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, 2 µg/ml aprotinin, 20 mM ß-glycerophosphate, 1 mM ABSF, 10 µM leupeptin, 1 µM pepstatin, and 1 mM Na3VO4. Lysates were centrifuged at 14,000 g for 20 min. Aliquots of supernatant (200 µg, ~2.0 mg/ml) were incubated for 1 h at 4°C with specific antibodies, and another 1 h incubation after adding 30 µl of protein G/A agarose beads (50% slurry; Calbiochem). After being washed, the bound proteins were analyzed by immunoblotting.
For coimmunoprecipitation of h-warts/LATS1 and zyxin, HeLa cells were lysed on ice for 30 min with five cell volumes of the 0.1% NP-40 lysis buffer (0.1% NP-40, 100 mM NaCl, 25 mM Tris-Cl, pH 8.0, 5 mM EGTA, 1 mM MgCl2 and 5% glycerol supplemented with 1 mM DTT, 2 µg/ml aprotinin, 20 mM ß-glycerophosphate, 1 mM ABSF, 10 µM leupeptin, 1 µM pepstatin, 1 mM Na3VO4, 1 mM benzamidine, and 1 µM microcystin), and centrifuged at 14,000 g for 20 min. Precleared 400 µg quantities of cell lysate (~4 mg/ml) were mixed with 3 µl of anti-zyxin antibody for 3 h on ice, then 15 µl of protein ASepharose beads (Amersham Pharmacia) were added and incubated for another 2 h at 4°C. After washing five times with the 0.1% NP-40 lysis buffer, the lysate was eluted by boiling for 3 min in 62.5 mM Tris-Cl, pH 6.8, containing 2% SDS and 10% glycerol. The elute was removed, made to 10 mM DTT and 0.1% bromophenol blue, boiled again and then subjected to immunoblotting. Quantification analysis was performed by MacBAS v2.5 software.
In Vitro Pull-Down Assay and Solution-Binding Assay
For pull-down assay, 30 µg of GST fusion protein was immobilized on glutathione-agarose, and equilibrated with 0.5% NP-40 lysis buffer. Aliquots of cell lysate (200 µg, ~2.0 mg/ml) were incubated with the glutathione-agarose for 1 h. The bound proteins were analyzed by immunoblotting. In vitro solution binding assay was performed as described (
In Vitro Kinase Assay
Synchronized HeLa cells were washed with ice-cold PBS and lysed on the plate with RIPA buffer as previously described (-[32P]ATP (3,000 Ci/mmol; Amersham Pharmacia), 1 µM microcystin, 8 µg of cell lysates, and 10 µg of GST-zyxin. Each reaction mixture was then chilled and mixed with 30 µl of glutathione-agarose beads (50% slurry) and 0.5 ml of ice-cold TNE buffer, followed by rocking for 30 min at 4°C. The glutathione-agarose beads were washed and boiled in 30 µl of Laemmli sample buffer to elute GST fusion proteins. The samples were resolved by 8% SDS-PAGE and visualized by autoradiography. The result of gel analyses were quantified by MacBAS (Fujifilm).
Depletion of Cdc2
Aliquots of 150 µl of mitotic cell lysate (1.0 mg/ml) were incubated with 50 µl of p13-suc1 agarose beads (50% slurry; Upstate Biotechnology) at 4°C for 45 min. After centrifugation, 50 µl of fresh p13 suc1-beads was added to the supernatant and incubated for an additional 30 min. The Cdc2 kinase assay was performed by the SignaTECT assay system (Promega) in which biotinylated peptide derived from histone H1 was used as a substrate and radiolabeled phosphorylated substrate was recovered with streptoavidin matrix. The purified active Cdc2 kinase was prepared as previously described (
Immunofluorescence Microscopy
U2OS cells were grown on a 35-mm petri dish to ~70% confluence, fixed with 4% paraformaldehyde/PBS, pH 7.4, for 15 min at room temperature, followed by permeabilization with 0.2% Triton X-100/PBS, otherwise fixed with a methanol/acetone solution for 10 min on ice. A preextraction protocol was performed either with 7.5 µg/ml digitonin in KHM buffer (25 mM Hepes-KOH, pH 7.2, 125 mM potassium acetate, 2.5 mM magnesium acetate) or with microtubule stabilizing buffer (MSB; 80 mM Pipes-KOH, pH 6.8, 5 mM EGTA, 1 mM MgCl2 containing 0.5% Triton X-100) for 5 min at room temperature before fixing with chilled absolute methanol for 10 min at -20°C as described (-tubulin antibody (B512). This was followed by incubation with FITC-conjugated antirabbit/mouse IgG antibody (Amersham Pharmacia), Cy3-conjugated anti-rat IgG antibody (Amersham Pharmacia), and Texas redconjugated anti-mouse IgG antibody (Molecular Probes). The stained cells were mounted with 1,4-diazabicyclo-[2,2,2]-octane/glycerol, and observed with confocal microscopy (Fluoview; Olympus). Images were obtained separately by independent excitation at 488/568 nm to minimize overlapping signals, and were processed using Photoshop software (Adobe).
Microinjection
U2OS cells were grown on 35-mm petri dishes to 75% confluency and microinjected using semi-automatic micro-manipulator/injector (Eppendorf 5171/5246). Cells in prometaphase were selected for injection on the basis of morphology by phase contrast images, and injected with a 1.0 mg/ml solution of purified GST fusion proteins and rhodamine-tubulin (Cytoskeleton) in PBS, pH 6.9, supplemented with a final concentration of 0.5 mM GTP. The microscopical stage was maintained at 37°C and the procedure was completed within 20 min to minimize pH changes. After injection, cells were incubated at 37°C for 20 min for rhodamine-tubulin to be distributed to the mitotic spindle, followed by detergent preextraction (7.5 µg/ml digitonin in KHM buffer) and a methanol fixation protocol as described above.
Synchronized HeLa cells were injected into the cytoplasm with a combination of 1.0 mg/ml solution of purified GST fusion proteins together with 1.0 mg/ml ß-galactosidase (Sigma-Aldrich). Cells were fixed with 0.5% glutaraldehyde/PBS, pH 7.2, at subsequent time points, followed by incubation with 1 mg/ml of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) in 150 mM NaCl, 0.01% sodium deoxycholate, 0.02% NP-40, 2 mM MgCl2, 5 mM K3Fe(CN)6, and 5 mM K4Fe(CN)6 for 12 h at 37°C. After removing the X-gal solution, cells were overlaid with acreto-orcein (Merck) in 60% acetic acid to visualize chromatin.
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Results |
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Identification of h-warts/LATS1 Interacting Protein
To identify proteins that physically interact with h-warts/LATS1, we conducted a HeLa cDNA library screen by the yeast two-hybrid method. We used a region of amino acids 394675, which does not contain the kinase domain of h-warts/LATS1, as the bait for the screen (Fig 1 A). From 2.3 x 106 initial transformants, 53 clones were found to confer both the His+ and LacZ+ phenotypes on L40 containing pBTM116HA/h-warts. These positive clones were subjected to secondary screening to eliminate false positives, and confirmed the specific interaction between h-warts/LATS1 and protein encoded by a library cDNA within the yeast cells. Among the positive clones, we isolated four independent cDNA clones encoding partial protein fragments that derived from zyxin, a regulator of actin assembly (Fig 1 A). The other h-warts/LATS1-interacting proteins identified in this screen will be described elsewhere.
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Interaction of Zyxin with h-warts/LATS1
To examine whether zyxin interacts with h-warts/LATS1 in intact cells, we coexpressed zyxin with h-warts/LATS1 in COS7 cells (Fig 1 B). Full-length zyxin and full-length h-warts/LATS1 were tagged with FLAG and HA epitopes, respectively, at their NH2 termini. When the lysate coexpressing FLAG-zyxin with HA-h-warts/LATS1 was immunoprecipitated with the anti-FLAG antibody, HA-h-warts/LATS1 was detected in the FLAG-zyxin immune complex (Fig 1 C, lane 1). Conversely, FLAG-zyxin was detected in the HA-h-warts/LATS1 immune complex (Fig 1 C, lane 4). Neither FLAG-zyxin nor HA-h-warts/LATS1 was detected in the unrelated control IgG immunoprecipitates from lysates expressing both proteins (data not shown).
An additional experiment to confirm the interaction of zyxin with h-warts/LATS1 was performed. Two polyclonal antibodies were raised against h-warts/LATS1 by injecting two KLH-conjugated synthetic polypeptides, C1 (amino acids 1,0411,063) and C2 (amino acids 1,1111,130), into rabbits. Endogenous h-warts/LATS1 was immunoprecipitated by the anti-C1 antibody and was detected at immunoblotting by the anti-C2 antibody (Fig 1 D, lane 8). When the COS7 cell lysate expressing HA-tagged zyxin (full-length) was immunoprecipitated with the anti-C1 antibody, HA-zyxin (full-length) was coprecipitated with endogenous h-warts/LATS1 (Fig 1 E, lane 3).
Furthermore, we tested whether endogenous zyxin and h-warts/LATS1 interact in vivo. Using anti-zyxin antibody generated in rabbits, zyxin was immunoprecipitated from HeLa cell lysate. The endogenous h-warts/LATS1 was detected in the immunoprecipitate (Fig 1 F). Although comparison of the band profiles revealed that the small fraction of h-warts/LATS1 (~9%) interacts with zyxin, these results indicate the association of h-warts/LATS1 and zyxin in intact cells.
Since zyxin harbors three copies of LIM domain in the COOH-terminal third of the molecule (Fig 2 A), known as a proteinprotein interacting structure, we first examined whether the LIM domain of zyxin serves as a binding interface for h-warts/LATS1 interaction. COS7 cell lysates expressing HA-tagged zyxin either full-length, NH2-terminal two-thirds (1) or COOH-terminal third (
2), which contains three LIM domains, were incubated with GSTh-warts/LATS1 (amino acids 136700) fusion protein bound to glutathione-agarose beads (Fig 2 B, lanes 13). The pull-down assay revealed that HA-zyxin
2 coprecipitated with GSTh-warts/LATS1 but HA-zyxin
1 did not (Fig 2 B, lanes 7 and 9). Unexpectedly, the full-length HA-zyxin was hardly detectable in the GSTh-warts precipitate (Fig 2 B, lane 5).
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We next sought to delineate in detail the region(s) of zyxin important for its association with h-warts/LATS1 using GST fusion proteins containing a full-length or various truncated zyxin (Fig 2 A). Same amounts of GST-zyxin fusion proteins bound to glutathione-agarose beads were incubated with baculoviral expressed His-tagged h-warts (full-length), and the retained proteins were analyzed by immunoblotting with anti-C2 antibody. While GST-zyxin-LIM1/2 (3) and GST-zyxin-LIM1/2/3 (
2) were found to bind with purified h-warts protein, the other GST-zyxin mutants did not (Fig 2 C). This in vitro binding experiment demonstrated that zyxin directly interacts with h-warts/LATS1 and that the region containing both LIM1 and LIM2 domains of zyxin is essential for their binding.
Interestingly, consistent with the findings shown in Fig 2 A, GST-zyxin (full-length) did not interact with His-tagged h-warts/LATS1. However, their association was detected in in vivo binding assays (Fig 1C, Fig E, and Fig f). Based on these findings, we speculated that the LIM1/2 domains are masked in full-length zyxin and that an intramolecular and/or intermolecular modification may regulate the interaction between zyxin and h-warts/LATS1.
Localization of h-warts/LATS1Zyxin Complex to Mitotic Apparatus
Next, we compared the subcellular localization of h-warts/LATS1 and zyxin by immunocytochemical analysis. We raised antibody against zyxin (amino acids 2435) in rabbits by the procedure described by
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To examine localization in detail, cells were processed in a detergent-preextraction protocol which washes out the free cytoplasmic proteins. As previously reported, h-warts/LATS1 locates to centrosomes during interphase (Fig 4 A, ac). When cells enter prometaphase/metaphase, h-warts/LATS1 translocates toward the mitotic spindle array as well as the spindle poles (Fig 4 A, df). As the cell commits to anaphase (Fig 4 A, gi) and later to telophase (Fig 4 A, jl), h-warts/LATS1 is found progressively at the bundle of microtubules at the midzone, called the central spindle, that connects daughter cells (Fig 4 A, mo). The detergent-preextraction immunostaining procedure showed that a fraction of zyxin was also found to associate with mitotic apparatus during mitosis. As cells entered mitosis, when assembly of the mitotic spindle begins from spindle poles, zyxin initially located to the mitotic spindle (Fig 4 B, ac), and this association with the mitotic spindle became prominent by metaphase (Fig 4 B, df). As cells proceeded into anaphase, zyxin was detected on the spindle between segregated sister chromatids, which are composed of bidirectional overlapping polar spindle (Fig 4 B, gi). In late anaphase, zyxin became detectable as a distinct wide band extending across the midzone of the central spindle (Fig 4 B, jl). This central spindle staining persisted into telophase when the spindle compacts into the midbody (Fig 4 B, mo). These specific staining patterns on the mitotic apparatus were neither detected with the preimmune IgG nor with anti-vinculin antibody (data not shown). When cells were double-stained for h-warts/LATS1 and zyxin, the yellow color produced by superimposing green and red demonstrated that h-warts/LATS1 and zyxin colocalize on the mitotic spindle, spindle poles and midbody of the dividing cells (Fig 4 C). The dynamic changes in zyxin and h-warts/LATS1 localization on the mitotic apparatus is identical, suggesting that zyxin interacts with h-warts/LATS1 on the mitotic apparatus, including mitotic spindle and central spindle at midzone of the dividing cells.
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Specific Phosphorylation of Zyxin during Mitosis
Since h-warts/LATS1 was reported to be posttranslationally modified in a cell cyclespecific manner and to play a critical role in cell cycle regulation (
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For an additional approach to demonstrate the specific phosphorylation of zyxin during mitosis, in vitro kinase assay using cell lysates was employed. We used cell lysates from various phases in the cell cycle as sources of the enzyme and GST-zyxin as the substrate. GST-zyxin was phosphorylated significantly with mitotic cell lysate (Fig 5 C, lane 3). To determine the phosphorylation region(s) in the molecule GST-zyxin1 and GST-zyxin
2 were processed for the in vitro kinase assay. GST-zyxin
1, which corresponds to the NH2-terminal two thirds of zyxin, was significantly phosphorylated with mitotic cell lysate whereas no phosphorylation was detected in GST-zyxin
2 (Fig 5 D).
Characterization of Zyxin-Kinase during Mitosis
To address the class to which zyxin-kinase belongs, we used various specific inhibitors that have been developed for kinases. The kinase assay was performed with mitotic cell lysates which were preincubated with various kinase inhibitors (Fig 6 A). GST-zyxin phosphorylation activity in the cell lysate was specifically inhibited not only when the mitotic cell lysate was preincubated with the broad serine/threonine kinase inhibitor staurosporine (100 nM), but also after preincubation with olomoucine, which is a specific inhibitor of Cdc2 (Fig 6 A). Therefore, to examine whether Cdc2 kinase is the responsible kinase for zyxin phosphorylation in the mitotic cell lysate, we prepared mitotic cell lysate depleted of Cdc2, which should contain the full complement of mitotically active kinases except for Cdc2, including kinases activated downstream of Cdc2 (Fig 6 B). By monitoring the total protein concentration and the Cdc2 kinase activity of the depleted mitotic cell lysate (Fig 6 C), we found that phosphorylation of GST-zyxin was significantly inhibited by depleting Cdc2 from the mitotic cell lysate (Fig 6 D, lane 3). Addition of active Cdc2 complex to the Cdc2-depleted mitotic cell lysate restored the phosphorylation activity (Fig 6 D, lane 4), indicating that loss of zyxin phosphorylating activity was due to removal of Cdc2 but not to other components in the lysate. Furthermore, the purified Cdc2 kinase complex was sufficient for zyxin phosphorylation without requiring any other components (Fig 6 E). These data demonstrate that Cdc2 is a kinase responsible for the mitosis-specific phosphorylation of zyxin in vitro, even though other active kinases are present in the mitotic lysate.
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Phosphorylation of Zyxin by Cdc2 Regulates Binding to h-warts/LATS1
Three lines of evidence suggest that zyxin interacts with h-warts/LATS1 during mitosis and that the interaction is regulated by the phosphorylation of zyxin. First, zyxin undergoes posttranslational modification during mitosis, which was shown to be phosphorylation. Second, h-warts/LATS1 binds to the region containing LIM1 and LIM2 domains of zyxin and an intra/inter-molecular modification of zyxin may be required for h-warts/LATS1 to access to the binding domains. Third, a fraction of zyxin is distributed to the mitotic apparatus, where it colocalizes with h-warts/LATS1 protein. Since our results demonstrated that Cdc2 is the kinase responsible for mitotic phosphorylation of zyxin, we postulated that the Cdc2-mediated phosphorylation of zyxin promotes interaction between zyxin and h-warts/LATS1. To test this possibility, GST-zyxin (full-length) was phosphorylated by active Cdc2 complex, followed by incubation with recombinant His-tagged h-warts/LATS1. His-tagged h-warts/LATS1 precipitated with phosphorylated GST-zyxin (Fig 7 A, lane 5) while the control unphosphorylated GST-zyxin did not (Fig 7 A, lanes 2 and 4).
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Furthermore, to examine whether zyxin associates with h-warts/LATS1 specifically during mitosis in the intact cells, endogenous zyxin was immunoprecipitated from either interphase or mitotic cell lysate and probed with antih-warts/LATS1 antibody. The endogenous h-warts/LATS1 coprecipitated with zyxin from mitotic lysate was found to be more abundant than that from interphase cell lysate (Fig 7 B, lane 4). These results indicates that zyxin interacts with h-warts/LATS1 during mitosis, and that the interaction is regulated by phosphorylation of zyxin. This mitosis-specific interaction explains why the proportion of the zyxin-binding h-warts/LATS1 to its entire pool was less than 10% in asynchronized cells (Fig 1 F). It is also interesting to note that the zyxin-binding h-warts/LATS1 migrated slower than its entire pool (Fig 1 F), suggesting that mitotic phosphorylated form of h-warts/LATS1 (
Zyxin Is Targeted to the Mitotic Apparatus by Interacting with h-warts/LATS1
To test whether dynamic changes in zyxin localization on the mitotic apparatus is dependent on h-warts/LATS1 distribution, we attempted to disrupt endogenous zyxin localization by injecting excessive amounts of an h-warts/LATS1 fragment (amino acids 136700) that is shown to bind preferentially to zyxin (Fig 2 B). Prophase/prometaphase U2OS cells were microinjected with purified GSTh-warts/LATS1(136700) fusion protein, or control GST protein. Rhodamine-labeled tubulin was injected together with the GST fusion proteins not only to distinguish injected cells but to monitor mitotic spindle organization as the cell cycle proceeds. After injection, cells were subjected to the detergent-preextraction immunostaining with anti-zyxin antibody. Signal of endogenous zyxin on the mitotic apparatus was diminished or disappeared in 64% of the GSTh-warts/LATS1(136700)injected cells (n = 11), whereas it was decreased in only 18% of the GST-injected control cells (n = 12; Fig 8). These observations indicate that association of zyxin with h-warts/LATS1 is essential for targeting as well as dynamic localization of zyxin on the mitotic apparatus during mitosis.
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Biological Significance of h-warts/LATS1-Zyxin Complex on the Mitotic Apparatus
Since h-warts/LATS1 is speculated to be involved in mitotic control, the h-warts/LATS1-zyxin complex on the mitotic apparatus may play a role in proper execution of the mitotic process. To test this possibility, we examined whether h-warts/LATS1(136700) microinjected cells can proceed through mitosis normally. HeLa cells, synchronized at S phase, were coinjected with GSTh-warts/LATS1 (136700) or GST-mock and ß-galactosidase to detect injected cells. Cells were fixed at various time points after release from an S phase block, and the frequency of mitotic cells (mitotic index) in cells having ß-galactosidase activity was determined (Fig 9 A). While the control experiments exhibited a peak mitotic index at 10.5 h after release from S phase and a decrease after 12 h, h-warts/LATS1(136700)injected cells revealed a high mitotic index after 10.5 h and continued to increase until 12 h after the release (Fig 9 B). Apparently, cells injected with h-warts/LATS1(136700) had a prolonged mitotic phase. These observations suggest that disruption of h-warts/LATS1-zyxin complex leads to impairment of normal mitotic progression.
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Discussion |
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In searching for cellular targets of the h-warts/LATS1 tumor suppressor protein, we have identified zyxin which plays a central role in actin assembly and organization. During interphase, zyxin is found at cellular locations that are enriched in actin filaments, including the leading edge and focal adhesion plaques, whereas h-warts/LATS1 is localized to centrosome. However, during mitosis, when zyxin is dissociated from focal adhesions, a fraction of the free cytoplasmic zyxin becomes associated with mitotic spindles. We have shown that zyxin is phosphorylated during mitosis and that the mitotic phosphorylation is required for association with h-warts/LATS1 on the mitotic apparatus. These findings implicate dual functions for zyxin: not only does it play a key role in cell adhesion and cytoskeletal organization in interphase cells, but it also acts as a participant in mitotic control by forming a complex with h-warts/LATS1 on the mitotic apparatus.
Regulation of the Interaction between h-warts/LATS1 and Zyxin
We have shown a physical association of zyxin with h-warts/LATS1 and that a region containing the first and second LIM domains (LIM1/2) of zyxin is responsible for the interaction. The LIM domain is a protein binding motif which is found in a wide variety of proteins involved in transcription, cell adhesion, and cytoskeletal organization (
Although zyxin was first described as a phosphoprotein (
As predicted by our hypothesis, the Cdc2-dependent phosphorylation has been shown to allow the full-length zyxin to interact with h-warts/LATS1 (Fig 7 A). In fact, zyxin associates with h-warts/LATS1 preferentially during mitosis (Fig 7 B), and this interaction might be significant for the subsequent cellular events in cell division. However, the binding of phosphorylated full-length zyxin to h-warts/LATS1 was not as efficient as that of the 2 protein (Fig 7 A), so the possibility can not be excluded that the interaction is regulated by not only phosphorylation but other unknown modifications of full-length zyxin.
Spatial Control of Zyxin Localization during Mitosis
Zyxin has been shown to dissociate from focal adhesion plaques and to distribute diffusely into the cytoplasm coincidentally with the mitotic disappearance of focal adhesions. Subsequently, a fraction of this free cytoplasmic zyxin becomes colocalized with h-warts/LATS1 on the mitotic apparatus (Fig 3 and Fig 4). Since zyxin plays a critical role in the actin filament assembly at focal adhesions (
Dynamic Interaction of h-warts/LATS1Zyxin Protein Complex on the Mitotic Apparatus
The interaction between zyxin and h-warts/LATS1 on the mitotic apparatus implicates a significant role of actin regulatory proteins during mitosis. Zyxin serves as a scaffold for gathering actin regulatory proteins, such as Ena/VASPprofilactin complex or vav-small GTPase complex, at focal adhesion plaques (
The duration of mitosis in cells injected with h-warts/LATS1(136700) fragment, which significantly perturbed zyxin specifying to the mitotic apparatus, was significantly longer than that of control cells, mainly due to delay in exit from mitosis (Fig 9). An intriguing explanation for the observation is that the mislocalization of zyxin, as well as zyxin-binding partners for actin polymerization, induces discoordination of contractile ring in dividing cells, and thereby delays their exit from mitosis. Alternatively, h-warts/LATS1 enzymatic activity may be modulated by zyxin, so that disruption of their interaction results in inactivation of h-warts/LATS1 itself. It can not be excluded, however, that another, as yet unknown, protein(s) is also functionally impaired by h-warts/LATS1(136700) fragments to produce these results.
To maintain genomic stability and proper ploidy, it is crucial that cell division occurs at the end of anaphase after chromosome segregation. The molecular mechanism through which h-warts/LATS1 serves as a tumor suppressor is unknown, but one possible explanation is that h-warts/LATS1 may play a critical role in cell division processes by forming a complex with zyxin on the mitotic apparatus. Abrogation of this interaction may involve failure in normal mitotic progression, leading to chromosomal instability, which is a hallmark of malignant tumors.
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Footnotes |
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1 Abbreviations used in this paper: DMPK, myotonic dystrophy protein kinase; GST, glutathione-S-transferase; HA, hemagglutinin 1; KLH, keyhole limpet-hemocyanin; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside.
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Acknowledgements |
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We thank Drs. Q. Hu, H. Maruta, and A. Kikuchi for providing the pCGN, pGEX2TH, and pBJ-Myc plasmids, respectively; Dr. K. Tanabe for critically reading the manuscript; Dr. J. Moon for editorial assistance; Drs. Y. Arima, S. Honda, M. Nitta, and H. Nakamura for valuable suggestions; Mr. K. Ida (Olympus) and Mr. K. Ueda (Uniscience) for technical assistance; and T. Arino for secretarial assistance.
This work was supported by a grant for Cancer Research from the Ministry of Education, Science and Culture of Japan (H. Saya).
Submitted: 16 November 1999
Revised: 14 April 2000
Accepted: 19 April 2000
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References |
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