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[Cancer Research 65, 10872-10880, December 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

The Syk Tyrosine Kinase Localizes to the Centrosomes and Negatively Affects Mitotic Progression

Déborah Zyss1, Philippe Montcourrier1, Benjamin Vidal1, Christelle Anguille1,2, Fabrice Mérezègue1, Alain Sahuquet1, Paul H. Mangeat1,2 and Peter J. Coopman1

1 Centre National de la Recherche Scientifique UMR5539, Université Montpellier II and 2 Centre de Recherches de Biochimie Macromoléculaire, Centre National de la Recherche Scientifique FRE2593, Montpellier, France

Requests for reprints: Peter J. Coopman, Centre National de la Recherche Scientifique UMR5539, Université Montpellier II, CC107, Place Eugène Bataillon, 34095 Montpellier, France. Phone: 33-467-144731; Fax: 33-467-144286; E-mail: coopman{at}univ-montp2.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We showed previously that the spleen tyrosine kinase Syk is expressed by mammary epithelial cells and that it suppresses malignant growth of breast cancer cells. The exact molecular mechanism of its tumor-suppressive activity remains, however, to be identified. Here, we show that Syk colocalizes and copurifies with the centrosomal component {gamma}-tubulin and exhibits a catalytic activity within the centrosomes. Moreover, its centrosomal localization depends on its intact kinase activity. Centrosomal Syk expression is persistent in interphase but promptly drops during mitosis, obviously resulting from its ubiquitinylation and proteasomal degradation. Conversely, unrestrained exogenous expression of a fluorescently tagged Discosoma sp. red fluorescent protein (DsRed)-Syk chimera engenders abnormal cell division and cell death. Transient DsRed-Syk overexpression triggers an abrupt cell death lacking hallmarks of classic apoptosis but reminiscent of mitotic catastrophe. Surviving stable DsRed-Syk–transfected cells exhibit multipolar mitotic spindles and contain multiple abnormally sized nuclei and supernumerary centrosomes, revealing anomalous cell division. Taken together, these results show that Syk is a novel centrosomal kinase that negatively affects cell division. Its expression is strictly controlled in a spatiotemporal manner, and centrosomal Syk levels need to decline to allow customary progression of mitosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cytoplasmic spleen tyrosine kinase (Syk) was, until recently, uniquely studied in hematopoietic cells. In these cells, Syk is involved in coupling activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation and differentiation (1). We evidenced that Syk is also commonly expressed in mammary epithelial cells and that it acts as a tumor suppressor in breast cancer (2). More precisely, we showed that its expression is significantly decreased or lost in invasive and metastatic breast carcinoma cells. Transfection of Syk into a Syk-negative breast cancer cell line dramatically inhibited its tumorigenic and metastatic capacities in athymic mice. Conversely, overexpression of a kinase-deficient Syk in a Syk-positive breast cancer cell line significantly increased its tumor incidence and growth. Recently, Syk expression was also detected in other cell types (e.g., hepatocytes, fibroblasts, endothelial cells, and neuronal cells), indicating that it is a ubiquitous signaling molecule (3). Its role as a potential tumor suppressor gene has been strengthened in breast cancer (4–6) and evoked in acute lymphoblastic leukemia (7) and gastric carcinoma (8). The loss of Syk expression in a significant fraction of these tumors was associated with a CpG island hypermethylation in the Syk promoter region (8–10). Although Syk was shown to affect cell proliferation, motility, and invasion (2, 11, 12), the exact molecular mechanism of its tumor-suppressive activity remains to be identified.

Our previous observations in athymic mice indicated that suppression of tumor growth by the reintroduction of Syk was the result of aberrant mitosis and cytokinesis (2). The cell cycle is a complex process involving numerous signaling proteins that undergo a strict spatiotemporal regulation, orchestrated by multiple phosphorylation and degradation events. Various protein kinases and their effector proteins play an essential role in controlling cell cycle progression. They are necessary for the completion of mitotic events, such as centrosome separation, bipolar spindle assembly, chromosome segregation, and cytokinesis. Defects in these processes can lead to aneuploidy and genomic instability. Of particular interest for the progression of mitosis are the members of the Aurora, Polo, and NIMA serine/threonine kinase families (13). They exhibit maximal expression and/or catalytic activity during the G2-M phases and transiently relocate to organelles crucial for mitotic progression, such as the microtubule spindle, centrosome, midbody, or kinetochore. Interestingly, these kinases are frequently overexpressed and amplified in many human tumor types (14) and are therefore putative oncogenes and attractive targets for anticancer drug development.

Here, we explored whether the Syk tyrosine kinase, a potential tumor suppressor, is also acting at the mitotic apparatus and affecting mitotic progression. We show that Syk is a new centrosomal kinase that interacts with {gamma}-tubulin, a key centrosome component, and that it is catalytically active within the centrosomes. Although persistent during interphase, centrosomal Syk levels acutely dropped during mitosis. During this phase, Syk is ubiquitinylated and most likely degraded by the proteasome. Sustained expression of a transfected Syk engendered abnormal cell division and nonapoptotic cell death similar to mitotic catastrophe. Together, our observations indicate that Syk is a new spatiotemporal regulated mitotic kinase that negatively affects cell division.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. MCF7, MDA-MB-231, and MDA-MB-435 human breast cancer cell lines and simian COS1 cells were obtained through the American Type Culture Collection (Manassas, VA). The Jurkat lymphoblastic T-cell line was obtained from Dr. Naomi Taylor [Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique (CNRS) UMR5535, Montpellier, France]. Cells were cultured in DMEM containing Glutamax (Life Technologies/Invitrogen Corp., Cergy Pontoise, France) and complemented with 10% fetal bovine serum (FBS) and 30 units/mL penicillin/30 µg/mL streptomycin (complete medium).

Antibodies, cDNA constructs, and drugs. We used mouse monoclonal antibodies (mAb) raised against Syk (clone 4D10, Santa Cruz Biotechnology, Heidelberg, Germany), tyrosine-phosphorylated proteins (clone 4G10, Upstate Biotechnology, Inc./Euromedex, Souffelweyersheim, France), {gamma}-tubulin (clone GTU-88, Sigma, Saint Quentin Fallavier, France), {alpha}-tubulin (clone B-5-1-2, Sigma), FLAG tag (clone M2, Sigma), ubiquitinylated protein (clone FK2, Affinity Research Products, Exeter, United Kingdom), actin (clone mAb350, gift of Dr. Ned Lamb, Institut de Génétique Humaine, CNRS UPR1142, Montpellier, France), and E-cadherin (clone 34, BD Transduction Laboratories, Le Pont de Claix, France). We used rabbit polyclonal antibodies recognizing tyrosine-phosphorylated Syk (Y525/Y526, Y352, and Y323; kindly provided by Dr. Jiong Wu, Cell Signaling Technology, Inc., Beverly, MA), transcription factor IIB (C-18, Santa Cruz Biotechnology), and {gamma}-tubulin (kindly provided by Dr. Michel Bornens, CNRS UMR144, Paris, France). For detecting the centromeres, we used a human autoimmune serum from a calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias (CREST) patient that was kindly provided by Dr. Christian Jaulin [Institut National de la Sante et de la Recherche Medicale (INSERM) EMI0229, CRLC Val d'Aurelle, Montpellier, France].

The following vectors were used for transfection experiments. The human Syk cDNA was isolated from the pCAF1-Syk FLAG-tagged expression vector (2) using BamHI and HindIII and cloned into the Discosoma sp. red fluorescent protein pDsRed-C1 vector (Clontech, Ozyme, Montigny-Le-Bretonneux, France) digested previously with BglII and HindIII. The following vectors were kindly provided: pCAF1-Syk-Y130E (Dr. Susette Mueller, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC), pBOS-H2B-green fluorescent protein (GFP) histone (Dr. Paulo Magalhaes, University of Padua, Padua, Italy), pEGFP-Centrin1 (Dr. Michel Bornens), and pDsRed-clathrin light chain (Dr. Wolfhard Almers, Vollum Institute, Portland, OR).

The 26S proteasome inhibitor MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) and the microtubule-stabilizing (paclitaxel) or microtubule-depolymerizing (nocodazole) drugs were from Sigma. Sodium pervanadate was obtained by mixing 1 mmol/L H2O2 and 1 mmol/L Na3VO4 and incubated on cells for 15 minutes at 37°C.

Cell cycle synchronization and flow cytometry. A G0 arrest was achieved by a 24-hour starvation in serum-free medium (0% FBS). For M-phase block, cells were treated with nocodazole (50 ng/mL) for 16 hours. For a S-phase double-thymidine block, cells were treated with 2 mmol/L thymidine (two 16-hour treatments separated by a 9-hour release). Cells were synchronized by release from these treatments in fresh complete culture medium. Cell cycle distribution was assessed using a standard protocol. After trypsinization, 106 cells were fixed in cold 75% ethanol in PBS and resuspended in sodium citrate buffer (3.8 mmol/L, pH 7) containing 50 µg/mL RNase A and 50 µg/mL propidium iodide or 1 µmol/L To-Pro-3 iodide (Molecular Probes, Cergy Pontoise, France). Cycle analysis was carried out on a FACSCalibur cytometer using the CellQuest software (Becton Dickinson, Le Pont de Claix, France).

Immunoprecipitation and Western blot analysis. Subconfluent cell cultures were lysed in a buffer containing 50 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 0.5% sodium deoxycholate, 1% NP40, 10% glycerol, 1 mmol/L Na3VO4, 50 mmol/L NaF, and an EDTA-free proteinase inhibitor cocktail (Complete, Roche Applied Science, Meylan, France), further called immunoprecipitation buffer. Cleared lysates containing 1 mg protein (as determined with the bicinchoninic acid protein assay; Interchim, Montluçon, France) were used for immunoprecipitation with the relevant antibodies (1 µg) supplemented with 15 µL protein A or G-agarose beads (for 3 hours at 4°C under constant rotation). The washed immunoprecipitates were then subjected to 10% SDS-PAGE. After electroblotting onto polyvinylidene difluoride membranes, the blots were quenched with 5% bovine serum albumin (BSA) in PBS-Tween and incubated with the relevant primary antibody (1 µg/mL) and the corresponding horseradish peroxidase–conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories, Beckman Coulter France, Villepinte, France) in 1% BSA in PBS-Tween. The signal was revealed using enhanced chemiluminescence and detected with Hyperfilm according to the manufacturer's instructions (Amersham Pharmacia Biotech, Orsay, France).

Subcellular fractionations. Cytosolic and membrane fractions from MCF7 cells were prepared according to Jin et al. (15) with minor modifications. Briefly, 107 trypsinized cells were lysed in 1.3 mL hypotonic buffer and subjected to Dounce homogenization (pestle B, 40 strokes). The postnuclear supernatant was centrifuged at 100,000 x g for 30 minutes at 4°C to separate the cytoplasm from the membrane fraction. The membrane pellet was then solubilized in 1.3 mL immunoprecipitation buffer (as detailed above) and used for immunoprecipitation. Cytosolic and nuclear fractions from MCF7 cells were prepared according to Wang et al. (16) with minor modifications. Briefly, 107 trypsinized cells were lysed in 1.3 mL hypotonic buffer complemented with 1.5 mmol/L MgCl2 and subjected to Dounce homogenization (pestle B, 25 strokes). The lysate was centrifuged at 1,000 x g for 10 minutes at 4°C, the supernatant containing the cytosolic fraction. The nuclear fraction (pellet) was then further washed, extracted, and centrifuged as described previously (16). Supernatant and nuclear fractions were finally adjusted to a 150 mmol/L NaCl concentration and used for immunoprecipitation.

Transfections and in vitro kinase assay. Stable and transient transfections were realized using the SuperFect reagent (Qiagen, Courtaboeuf, France) according to the supplier's instructions. For stable transfections, cells were selected in 800 µg/mL G418 (DsRed fusion proteins) or 6 µg/mL blasticidin (H2B-GFP) and resistant clones were pooled and expression evaluated. Transient transfections were analyzed 2 days after transfection.

To test the catalytic activity of Syk in vitro, simian COS1 cells were transiently transfected with the pCAF1-Syk vector. Syk was immunoprecipitated from the cleared cell lysates and in parallel used for Western blot or autophosphorylation testing. For in vitro kinase assays, the beads were resuspended in 50 µL reaction buffer [10 mmol/L Tris-HCl (pH 8), 10 mmol/L MgCl2, 10 mmol/L MnCl2] together with 10 µCi [{gamma}-32P]ATP (4,500 Ci/mmol, MP Biomedicals, Vannes, France) and agitated for 20 minutes at room temperature. Washed beads were subjected to SDS-PAGE separation, dried, and used for phosphorimage analysis (Molecular Dynamics/Amersham Pharmacia Biotech).

Immunofluorescence microscopy, time-lapse imaging, and karyotype analysis. Cells were grown on 18-mm-diameter glass coverslips in 12-well plates. Cells were either fixed in 3.7% formaldehyde in PBS (20 minutes at room temperature) followed by 0.2% Triton X-100 in TBS (4 minutes) or dehydrated in absolute methanol (10 minutes at –20°C). DNA was visualized by incubation with 0.5 µg/mL cell-permeant Hoechst 33342 dye (Molecular Probes; 10 minutes at room temperature). After incubation with the relevant primary antibodies (10 µg/mL), signal was detected using donkey anti-mouse or anti-rabbit antibodies conjugated with FITC (Jackson ImmunoResearch Laboratories) or goat anti-mouse or anti-rabbit antibodies labeled with tetramethylrhodamine isothiocyanate (TRITC; Sigma; diluted 1:50 in TBS/0.2% gelatin). All antibody incubations were done at room temperature. Coverslips were mounted on glass slides using ProLong antifade medium (Molecular Probes) and observed with a motorized Leica Microsystems (Rueil-Malmaison, France) DMRA2 microscope equipped with an oil immersion x100/1.4 apochromatic objective and a 12-bit Coolsnap FX CCD camera (Princeton Instruments, Roper Scientific, Evry, France), both controlled by the MetaMorph imaging software (Universal Imaging, Roper Scientific).

Live four-dimensional microscopy of transfected cells in glass bottom Petri dishes was carried out with an oil immersion x40/1.25 apochromatic objective on the Leica DMIRE2 microscope. The microscope stage, objective, and culture medium inside the Petri dish were warmed at 37°C by regulated foil heaters (Minco Products, Inc., Minneapolis, MN), whereas the pH of the culture medium containing 8.4 g/L sodium bicarbonate was maintained by bubbling 5% CO2. Sixteen-bit depth images (1 pixel = 0.17 µm) of H2B-GFP histone, DsRed-Syk fluorescence, and phase-contrast microscopy were automatically collected on two Z steps corresponding to the base of cells and 7 to 8 µm above, the best level of focus being selected for the movie. Care was taken to allow low illumination of cells during the experiments to avoid toxicity and bleaching of fluorescence. Sixteen-bit images were processed and rescaled to 8-bit images with MetaMorph and Adobe Photoshop software.

For metaphase spreads, nocodazole-synchronized cells were allowed to swell in 75 mmol/L KCl at 37°C for 15 minutes and fixed by dropwise adding of methanol/acetic acid (3:1, v/v) under gentle vortexing. Cells were washed five times in fixative and dropped onto glass slides. Slides were then air dried, rehydrated in TBS, and used for immunofluorescence.

Apoptosis assays. For the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay, the in situ cell death detection kit (Roche Applied Science) containing fluorescein-labeled nucleotides was used on transfected or treated cells according to the manufacturer's instructions. DNase I treatment (1,000 units for 10 minutes at room temperature) was used as a positive control for DNA fragmentation. The fluorescent pan-caspase inhibitor FITC-Val-Ala-Asp-fluoromethylketone (FITC-VAD-FMK; 5 µmol/L; CaspACE, Promega, Charbonnières, France) was added in the culture medium of transfected or treated cells and incubated for 24 hours at 37°C. Paclitaxel treatment (1 µg/mL, 48 hours at 37°C) was used as a positive control for induction of apoptosis.

Centrosome preparations. Centrosomal fractions from cells were purified using a sucrose gradient according to the original protocol developed by Moudjou and Bornens (17). Briefly, 1 x 107 to 3 x 107 MCF7 or Jurkat cells, treated previously with cytochalasin D and nocodazole, were harvested by trypsinization, washed in half-volume TBS and 10-fold diluted TBS containing 8% sucrose, and then lysed in a 5 mL solution of 1 mmol/L HEPES (pH 7.2), 0.5% NP40, 50 µmol/L MgCl2, 0.1% 2-mercaptoethanol complemented with the Complete proteinase inhibitor cocktail and Na3VO4 (1 mmol/L) and NaF (50 mmol/L) phosphatase inhibitors. HEPES (10 mmol/L final) and DNase I (Boehringer Mannheim, Roche Diagnostics, Meylan, France; 1 µg/mL) were added to the cleared lysate (2,500 x g for 10 minutes) and incubated for 30 minutes on ice. Centrosomes were sedimented into a 0.5 mL cushion of 60% sucrose [in 10 mmol/L PIPES (pH 7.2)/0.1% Triton X-100/0.1% 2-mercaptoethanol] by centrifugation at 10,000 x g for 30 minutes. The interface layer as well as the sucrose cushion were purified further by a discontinuous sucrose gradient consisting of 500 µL of 70%, 300 µL of 50%, and 300 µL of 40% sucrose solutions and centrifuged at 40,000 x g for 1 hour. Fractions were collected from the bottom, 200 µL per fraction, from fractions 1 to 8; the remaining solution was collected as fraction 9. A small aliquot of each fraction was sedimented onto round coverslips in tubes containing a special Plexiglas adapter and then immunodecorated using anti-{gamma}-tubulin antibodies to estimate the centrosome yield in each fraction. Each fraction was then diluted in 1 mL of 10 mmol/L PIPES buffer (pH 7.2) and centrosomes were recovered by centrifugation at 15,000 x g for 10 minutes and boiling in SDS sample buffer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Centrosomal localization of Syk and interaction with {gamma}-tubulin. Unlike its biochemical properties, the subcellular localization and translocation of the nonreceptor tyrosine kinase Syk have not yet been thoroughly explored. Most studies have been done in hematopoietic cells that were transfected with Syk chimeras. Syk was found at membrane patches in activated B lymphocytes (18) as well as in membrane ruffles and focal complexes of Chinese hamster ovary cells (19). Endogenous Syk was observed in the podosomes of actively attaching lymphoblastoid cells (20).

We studied the subcellular localization of endogenous Syk in MCF7 cells using immunocytochemistry. In formaldehyde-fixed and Triton X-100–permeabilized cells, Syk was found in the cytoplasm as well as in plasma membrane extensions (Fig. 1A, top). This localization is consistent with the observation that cytoplasmic Syk can be recruited at the cell membrane and activated by transmembrane receptors, such as integrins (19). In agreement with previous studies, we also observed a faint nuclear signal (16, 18). Most surprisingly, in methanol-fixed cells, Syk was also prominently present in paired dot-like structures evoking centrosomes. This assumption was undeniably confirmed by the colocalization of Syk with {gamma}-tubulin, a crucial component of the pericentriolar material responsible for nucleation of microtubules (Fig. 1A, middle and insets). In parallel, Syk-deficient MDA-MB-231 human breast cancer cells were transiently transfected with a DsRed-tagged Syk cDNA construct. Besides a diffuse cytoplasmic staining, Syk was also strongly present at membrane ruffles and microvilli (Fig. 1A, bottom). The presence of Syk in centrosomes was confirmed by its matching localization with enhanced GFP (EGFP)–tagged Centrin1, a centriolar component (Fig. 1A, bottom and insets). Time-lapse videomicroscopic analysis of the transfected DsRed-Syk showed the dynamic nature of both membrane extensions and centrosomes in which Syk localizes (Video 1).



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  		Figure 1. Centrosomal localization of Syk and interaction with {gamma}-tubulin. A, MCF7 cells grown on coverslips were fixed with formaldehyde (form.), permeabilized with Triton X-100 (TX), and stained for Syk with the 4D10 antibody and a FITC-labeled secondary antibody (top left). DNA was visualized with the cell-permeant Hoechst 33342 dye (top right). Double immunostaining on methanol-fixed (MeOH) MCF7 cells with the monoclonal anti-Syk antibody (FITC; middle left) and a polyclonal anti-{gamma}-tubulin antibody (TRITC; middle right). Syk-deficient MDA-MB-231 cells were transiently cotransfected with the pDsRed-Syk and pEGFP-Centrin1 expression vectors (bottom). Insets, enlargements of the boxed centrosomal regions; arrows, Syk localized at plasma membrane extensions. Bar, 10 µm. B, centrosomes were isolated and enriched from MCF7 and Jurkat cells using a discontinuous sucrose gradient (see Materials and Methods). mAbs against Syk (4D10) and {gamma}-tubulin (GTU-88; {gamma}-tub) were used for Western blot (WB) analysis. C, cleared lysates from MCF7 cells (2 mg protein) were used for immunoprecipitation (IP) with the Syk or {gamma}-tubulin antibodies and protein G-agarose beads and separated on SDS-PAGE gels under reducing (+DTT) and nonreducing conditions (-DTT).

 
The centrosome colocalization experiments were further substantiated with biochemical approaches. Centrosomes from exponentially growing unsynchronized MCF7 cells were isolated and enriched using a discontinuous sucrose gradient. Western blotting detection of Syk and {gamma}-tubulin revealed their presence in the same centrosome-enriched fractions (Fig. 1B, top). This observation was also corroborated in centrosome preparations from Jurkat T lymphoblastic cells, thereby evidencing that the presence of Syk in centrosomes is not restricted to nonhematopoietic cells (Fig. 1B, bottom). Several centrosome components physically interact, albeit perhaps indirectly, with {gamma}-tubulin and coimmunoprecipitate (e.g., BRCA1; ref. 21). Correspondingly, Syk and {gamma}-tubulin mutually coimmunoprecipitated in lysates from unsynchronized MCF7 cells (Fig. 1C). Immunoprecipitated complexes were analyzed on SDS-PAGE gels under reducing (+DTT) and nonreducing (–DTT) conditions to unambiguously distinguish {gamma}-tubulin from the immunoglobulin heavy chains: both conditions generated comparable results. These experiments indicate that Syk physically localizes to the centrosomes and interacts with {gamma}-tubulin.

Importance of the Syk catalytic activity for its centrosomal localization. We next investigated the importance of the Syk catalytic activity for its centrosomal localization. Hence, two mutant Syk forms were generated: (a) a kinase-negative mutant in which the ATP-binding domain was inactivated (K402R; ref. 2) and (b) a mutant possessing both a greatly reduced receptor interaction and an increased basal intrinsic kinase activity (Y130E; ref. 22). The altered catalytic activities of these mutants were verified by evaluating their autophosphorylation capacity ([{gamma}-32P]ATP incorporation; Fig. 2A) and their ability to coimmunoprecipitate other tyrosine-phosphorylated proteins (anti-phosphotyrosine blotting; Fig. 2B) after transfection in COS1 cells. As expected, the K402R mutant lacked any detectable kinase activity, whereas the Y130E mutant showed an increased capacity to phosphorylate itself and other proteins. DsRed-labeled fusion proteins of wild-type and mutant Syk forms were then transiently expressed in Syk-negative MDA-MB-231 cells and their subcellular localization was studied by fluorescence microscopy. As observed previously, wild-type Syk was present at both plasma membrane extensions and centrosomes (Fig. 2C, top). Intriguingly, the kinase-negative mutant exclusively localized at membrane extensions but was no longer present in the centrosomes. Conversely, the constitutively active mutant localized predominantly at the centrosomes in which it generated prominent phosphotyrosine-containing epitopes (Fig. 2C, bottom). Centrosomes also exhibited an intense phosphotyrosine signal in cells transfected with wild-type but not kinase-negative mutant Syk (data not shown). These observations denote that the Syk kinase activity is crucial for its centrosomal localization and indicate that Syk exhibits a catalytic activity at the centrosomal level. Because the Y130 residue lies near a coiled coil of {alpha}-helices located in between the two SH2 domains (22), its phosphorylation or replacement by a negatively charged amino acid might cause conformational changes of the Syk protein and affect its centrosomal targeting.



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  		Figure 2. Importance of the Syk catalytic activity for its centrosomal localization. A, FLAG-tagged wild-type (WT) and mutant (K402R and Y130E) Syk forms were transiently expressed in COS1 cells. After 48 hours, immunoprecipitates obtained with the anti-FLAG (M2) mAb were incubated with [{gamma}-32P]ATP, separated by SDS-PAGE, and analyzed by autoradiography or Western blotting with the anti-FLAG antibody (loading control). B, wild-type and Y130E mutated FLAG-Syk forms were immunoprecipitated from transiently transfected COS1 cells using the anti-FLAG antibody. After SDS-PAGE, immunoprecipitates were analyzed by Western blotting using the mAb against tyrosine-phosphorylated proteins (4G10; P-Tyr). C, wild-type and mutated DsRed-Syk forms were transiently expressed in MDA-MB-231 cells for 48 hours (left). Centrosomes ({gamma}-tubulin, GTU-88) or tyrosine-phosphorylated proteins (4G10) were detected by immunofluorescence using a FITC-labeled secondary antibody (right). Insets, enlargements of the boxed centrosomal regions; arrowheads, Syk localized at plasma membrane extensions. Bar, 10 µm.

 
We also analyzed the phosphorylation status of endogenous Syk in subcellular fractions (membrane, cytosol, and nucleus) and centrosome preparations of MCF7 cells. Syk was evidently much less abundant in the membrane and nuclear fractions compared with the cytosol (Fig. 3A and B). Because antibodies recognizing the phosphorylated Y130 residue are currently nonexisting, we blotted immunoprecipitated Syk with a mixture of anti-phosphotyrosine Syk antibodies recognizing the activation loop (Y525/Y526) essential for its function (23) and tyrosine residues crucial for its interaction with phospholipase C{gamma}1 (Y352; ref. 24) or Cbl (Y323; ref. 25). Unless overexposition of the Western blots, no tyrosine-phosphorylated Syk was detected neither in the subcellular fractions (Fig. 3A and B) nor in the purified centrosomes (data not shown). This confirms the low steady-state phosphorylation status and minimal basal intrinsic activity of Syk in unstimulated cells. As the natural extracellular signals and receptors activating Syk in breast cancer cells are still unidentified, we tested the effect of a pervanadate treatment, known to activate Syk (26), on its phosphorylation status. Consequently, Syk in all subcellular fractions (Fig. 3A and B) and in purified centrosomes (Fig. 3C) was phosphorylated on tyrosine residues and the phosphorylation signal intensity matched the Syk protein levels. This indicates that all these cellular compartments can contain phosphorylated Syk and that endogenous centrosomal Syk can be phosphorylated also on residues other than Y130.



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  		Figure 3. Presence of phosphorylated Syk in different subcellular compartments. A, cytosolic (Cyt) and membrane (Mem) fractions were prepared from MCF7 cells that were either untreated (-PV) or treated with sodium pervanadate (+PV). Endogenous Syk was immunoprecipitated, separated by SDS-PAGE, and analyzed by Western blotting using an anti-Syk (4D10) mAb or a mixture of polyclonal anti-phosphotyrosine-Syk antibodies (P-Syk; Y525/Y526, Y352, and Y323). Fractionation efficacy was confirmed by Western blotting of total cell lysates with an anti-E-cadherin (clone 34) mAb. B, cytosolic and nuclear fractions were prepared from untreated or pervanadate-treated MCF7 cells, and immunoprecipitated Syk was analyzed by Western blotting as detailed above. Fractionation efficacy was confirmed by Western blotting of total cell lysates with a monoclonal anti-actin (mAb350) antibody or a polyclonal antibody recognizing the transcription factor IIB. C, centrosomes were isolated and enriched from pervanadate-treated MCF7 cells using a discontinuous sucrose gradient and analyzed by Western blotting as detailed above or with a polyclonal antibody against {gamma}-tubulin.

 
Variations in centrosomal Syk levels during mitosis. Kinases involved in the progression of mitosis generally exhibit variations in expression levels or activity throughout the cell cycle, typically peaking during particular phases of mitosis. They associate transiently with mitotic structures, such as centrosome, kinetochore, microtubule spindle, or midbody (13). We therefore analyzed the expression levels of Syk from MCF7 cells synchronized in G1 phase (serum withdrawal), S phase (double-thymidine block), or M phase (nocodazole treatment) by Western blotting on Syk immunoprecipitates and total cell lysates. Synchronization efficiency was confirmed by flow cytometric analysis of the corresponding cell cycle distribution. Syk levels were comparable between nonsynchronized cells and cells synchronized in G1 and S phases (Fig. 4A). However, following release from nocodazole block in early mitosis and subsequent progression through mitosis, Syk levels decreased slightly but significantly compared with the steady-state {alpha}-tubulin levels (20-80 minutes after withdrawal).



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  		Figure 4. Variations in centrosomal Syk levels during mitosis. A, MCF7 cells were either nonsynchronized (NS) or synchronized at various stages of the cell cycle using serum starvation (G0-G1 phase), double-thymidine block (S phase), or nocodazole treatment (M phase). Percentages of cells at the various stages of the cell cycle were quantified by flow cytometry using propidium iodide. Cell lysates (50 µg protein) were either directly used for electrophoresis or immunoprecipitated (1 mg protein) with monoclonal Syk antibody (4D10) and protein G-agarose. After SDS-PAGE, protein levels were analyzed by Western blotting using Syk (4D10) and {alpha}-tubulin (B-5-1-2) mAbs (loading control). B and C, Syk expression was detected using the mAb (4D10) and a FITC-labeled (green) secondary antibody on nonsynchronized MCF7 cells grown on coverslips and fixed with methanol (B) or formaldehyde and Triton X-100 (C). The different phases of mitosis were identified using Hoechst 33342 dye (blue) to monitor the DNA condensation status and polyclonal anti-{gamma}-tubulin (TRITC; red) to locate the centrosomes. Arrowheads, centrosomes in interphase cells; full arrows and dotted arrows, evidenced and presumed centrosome positions in mitotic cells that are based on the {gamma}-tubulin immunolocalization, respectively. D, lysates of nonsynchronized, MG132 proteasome inhibitor–treated (5 or 16 hours), or nocodazole-synchronized (16 hours treated and 90 minutes released) MCF7 cells were immunoprecipitated with the mAb recognizing ubiquitinylated proteins (FK2; Ubiq-prot) and Syk was detected using Western blotting with the mAb (4D10). Ubiq-Syk, high-molecular-weight ubiquitinylated Syk species.

 
We subsequently assessed by immunocytochemistry whether any variation in Syk subcellular localization or expression level was noticeable during the cell cycle and particularly throughout mitosis. The different phases of mitosis were identified using Hoechst 33342 dye to monitor the DNA condensation status and anti-{gamma}-tubulin antibodies to locate the centrosomes. Although Syk appeared intensely centrosomal during interphase (Figs. 1A, middle, and 2C, top) and prophase (Fig. 4B, top), its centrosomal levels abruptly dropped or vanished during metaphase (Fig. 4B, bottom) and remained alike for the rest of mitosis (data not shown). {gamma}-Tubulin levels in the same metaphase cell and Syk levels in neighboring interphase cells were used as internal standards. Loss of centrosomal Syk was not due to a general lack of cellular Syk because overall cytoplasmic and submembranous Syk levels did not noticeably change during mitosis (Fig. 4C). This explains why only slight variations in total Syk protein levels were observed in MCF7 cells withdrawn from nocodazole block (Fig. 4A). These observations show that a fraction of the total cellular Syk specifically localizes to the centrosomes in which Syk levels are spatiotemporally regulated.

The abrupt decline of proteins involved in control of the cell cycle is generally the result of their ubiquitinylation and subsequent proteasomal degradation. Hence, we tested whether Syk is ubiquitinylated in asynchronous and mitotic MCF7 cells using Western blotting of Syk after immunoprecipitation of ubiquitinylated proteins. As expected, high molecular weight ubiquitinylated Syk species were evidenced in cells treated with the MG132 proteasome inhibitor, which, in addition, arrests cells in the G2-M phase. Interestingly, ubiquitinylated Syk was also observed in mitotic cells released from nocodazole arrest but not in asynchronous cells (Fig. 4D). These observations indicate that the rapid disappearance of Syk at the centrosomes during mitosis is the result of its ubiquitinylation and subsequent proteasomal degradation.

Cell death induced by transient Syk overexpression. To assess the significance of the centrosomal Syk loss during mitosis, we tested the consequences of its temporary sustained overexpression. Syk-negative MDA-MB-231 cells, stably expressing histone H2B-GFP as a chromatin marker, were transiently transfected with the DsRed-Syk vector. Startlingly, numerous Syk-overexpressing cells displayed an abnormal, unhealthy morphology. Time-lapse videomicroscopy revealed that cells apparently entering cell division never completed mitosis. After a healthy period, cells rounded up, exhibited an atypical DNA condensation, and finally died in an explosive manner (Fig. 5A; Video 2). Conversely, cells transfected with the empty DsRed vector (not shown) or a mock DsRed-clathrin cDNA entered and accomplished mitosis without difficulty (Fig. 5B; Video 3), thereby excluding a toxic effect of the DsRed protein. Although the phenotype of sustained Syk overexpression evokes programmed cell death, DsRed-Syk overexpression did not engender phenomena classically associated with apoptosis. Suffering cells did not exhibit DNA fragmentation as evaluated with the TUNEL assay (Fig. 5C, top). In addition, the fluorescent FITC-VAD-FMK pan-caspase inhibitor neither accumulated in the DsRed-Syk-transfected cells nor prevented cell death (Fig. 5C, bottom), thus excluding the involvement of any caspase activity. Correspondingly, we evidenced previously that apoptosis did not account for the tumor growth inhibition in athymic mice injected with Syk-transfected MDA-MB-435 cells (2). Different forms of cell death lacking biochemical hallmarks of classic apoptosis have been reported and can occur during mitosis (27, 28). The nearly absence of mitoses, the rounding up of cells, and the chromatin condensation preceding cell death suggest that sustained Syk overexpression induces a cell death reminiscent of mitotic catastrophe.



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  		Figure 5. Cell death induced by transient Syk overexpression. A, MDA-MB-231 Syk-deficient cells stably expressing H2B-GFP histone were transiently transfected with the pDsRed-Syk vector and after 24 hours continuously followed by time-lapse videomicroscopy for an additional 15 hours. Left and right, fluorescent images of DsRed-Syk and H2B-GFP proteins, respectively; middle, phase-contrast (Phase) images of the transfected cells. After a healthy 24-hour period following transfection (0 hours; thin arrows, centrosomal Syk), transfected cells began to round up (9 hours), condensed their chromatin (closed arrowheads), and rapidly died in an explosive manner (15 hours; thick arrows). B, MDA-MB-231/H2B-GFP cells expressing DsRed-clathrin maintained a healthy morphology and displayed normal mitosis (open arrowheads). C, MDA-MB-231 cells transiently transfected with the DsRed-Syk vector were subjected to a TUNEL assay after fixation or incubated with the fluorescent FITC-VAD-FMK pan-caspase inhibitor before fixation. DNase and paclitaxel treatments were used as positive controls, respectively, for the induction of DNA fragmentation and caspase activation (FITC; green). DNA was visualized with the cell-permeant Hoechst 33342 dye (blue). Bar, 10 µm.

 
Abnormal mitosis and aberrant cell division induced by stable Syk transfection. Although cell death is generally the end point of anomalous mitosis, selected cells can survive and exhibit an abnormal ploidy. Flow cytometric cell cycle analysis of stable DsRed-Syk-transfected MDA-MB-435 cells showed the presence of important fractions of hypodiploid cells (<2n), indicative of cell death (19%), as well as hypertetraploid cells (>4n), evocating abnormal cell division (19%; Fig. 6A, left). These phenomena coincide with a considerable reduction (2.5-fold) of cells in G1 phase without significantly affecting the relative number of cells in S and G2-M phases. These anomalies were not observed in untransfected cells (Fig. 6A) or in control cells transfected with DsRed-clathrin (Fig. 6A, right) or DsRed alone (data not shown). To elucidate whether the hypertetraploid cells are the consequence of unequal chromosome segregation or the result of chromosome rearrangements, we enumerated the total chromosome numbers and the amount of dicentric chromosomes on metaphase spreads. As most established cancer cell lines, MDA-MB-435 cells displayed a high degree of karyotypic complexity with a considerable variation in chromosome numbers (29). However, chromosome counts revealed a significant higher variation in chromosome number in DsRed-Syk-transfected cells (Fig. 6B, table), suggesting an uneven chromosome distribution during cell division. Cancer cells frequently show chromosomal instability resulting in gaps, breaks, dicentric and ring chromosomes, chromatid exchanges, deletions, duplications, and translocations. Dicentric chromosomes are caused by Robertsonian translocations that combine the long arms of two acrocentric chromosomes. To evaluate the presence of new chromosomal rearrangements in Syk-transfected cells, we counted the number of dicentric chromosomes using a centromere staining on 22 metaphase spreads using a human autoimmune CREST serum. Both DsRed-Syk-transfected and untransfected MDA-MB-435 cells consistently contained one dicentric chromosome, indicating the absence of this common type of constitutional chromosome rearrangement (Fig. 6B, right). We therefore conclude that the hypertetraploid DNA content is most likely caused by the presence of extra copies of whole chromosomes resulting from a disproportionate chromosome distribution. This substantiates our former observations on tumors formed by Syk-transfected MDA-MB-435 cells. Using fluorescence in situ hybridization analysis with centromeric probes for chromosomes 17 and X, we observed that these cells contained supernumerary copies of these chromosomes (2). Immunocytochemical observations on DsRed-Syk-transfected MDA-MB-435 cells also strengthen this concept. These cells frequently contained supernumerary centrosomes and multiple abnormally sized nuclei and exhibited atypical multipolar metaphase spindles (Fig. 6C). Likewise, aberrant mitoses were also observed in tumors formed by Syk-transfected MDA-MB-435 cells (2) and Syk-transfected BT549 cells (5). Finally, asymmetrical cell division producing a mother cell and a daughter cell with a significantly different size (nucleus and cytosol) was observed in DsRed-Syk-transfected cells (Fig. 6C). Taken together, these results show that stable Syk transfection either engenders immediate cell death or triggers abnormal mitosis, thereby generating endopolyploid cells that might ultimately undergo cell death.



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  		Figure 6. Abnormal mitosis and aberrant cell division induced by stable Syk transfection. A, the cell cycle distribution of MDA-MB-435 cells stably expressing DsRed-Syk or DsRed-clathrin cDNA was analyzed by flow cytometry using To-Pro-3 to detect DNA and compared with untransfected cells. Note that the DsRed-Syk-expressing subpopulation, unlike the untransfected cells, exhibited the presence of important fractions of hypodiploid cells (<2n) in addition to cells containing an abnormal hypertetraploid DNA content (>4n; left). Transfection of DsRed-clathrin did not affect the cell cycle compared with untransfected cells (right). B, MDA-MB-435 cells stably transfected with DsRed-Syk and untransfected cells were synchronized by overnight nocodazole treatment and sorted by fluorescence-activated cell sorting. Metaphase spreads were stained with Hoechst 33342 (blue) and CREST-SH serum detecting the centromeres followed by a FITC-labeled secondary antibody (green). Total chromosome numbers and the amount of dicentric chromosomes (inset) per cell were counted microscopically on 22 metaphases. Bar, 10 µm. C, MDA-MB-435 Syk-deficient cells stably transfected with pDsRed-Syk were stained with anti-{gamma}-tubulin (GTU-88) or anti-{alpha}-tubulin (B-5-1-2) mAbs followed by a FITC-labeled secondary antibody (green). DNA was visualized with the cell-permeant Hoechst 33342 dye (blue). Insets, enlarged regions containing supernumerary centrosomes; arrows, atypical multipolar mitotic spindles; closed arrowhead, midbody of an asymmetrically dividing DsRed-Syk positive cell; open arrowhead, a normally dividing untransfected cell. Bar, 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The cytoplasmic tyrosine kinase Syk was believed to be exclusively involved in a variety of hematopoietic cell responses, particularly immunoreceptor-mediated signaling events (1). In the last years, Syk expression has been evidenced in a wide variety of nonhematopoietic tissues, suggesting it to be a ubiquitous signaling molecule with a wide-ranging physiologic function (3). Furthermore, we and others showed that Syk expression is significantly decreased or lost in tumors progressing toward a malignant phenotype, indicating that Syk is a potential tumor and metastasis suppressor (2, 4–6). The exact molecular mechanisms by which Syk affects cell division and exerts its tumor-suppressive activity remain, however, to be identified.

It is well established that Syk is a cytoplasmic kinase that can be recruited at the cell membrane forming a complex with activated transmembrane receptors. Here, we show that Syk also localizes to the centrosomes, the major organizing center of interphase and mitotic spindle microtubules. Syk not only colocalizes but also physically interacts and copurifies with the intrinsic centrosome component {gamma}-tubulin. Unlike {gamma}-tubulin, Syk is not an exclusive centrosome component but is also clearly present at cell membrane extensions in which active actin remodeling occurs. Such a dual subcellular localization is not conflicting and has also been observed with other proteins, such as reggie-1/flotillin-2 (30). Our observations with the kinase-negative and constitutively active Syk mutants show that its kinase activity is essential for its centrosomal localization and that Syk exerts a catalytic activity within the centrosomes. In line with our observations, phosphotyrosine epitopes have been detected at the centrosomes and were suggested to be dependent on Syk expression (31). The presence of a protein tyrosine kinase at the centrosomes is unique and unexpected because centrosomal kinases involved in control of mitosis, such as members of the Polo and Aurora families, phosphorylate their substrates on serine/threonine residues (13). Furthermore, unlike Syk, theses kinases exhibit an oncogenic activity and positively affect cell division (32).

The expression and activity of mitotic kinases that localize at the centrosomes generally peak during mitosis (e.g., Aurora-related protein kinase 3; ref. 33). On the contrary, centrosomal Syk levels significantly dropped or disappeared during mitosis. Most interestingly, the checkpoint kinase Chk1 was shown recently to localize also in interphase but not mitotic centrosomes (34). The observation that the overall cellular Syk expression decreased only slightly during mitosis compared with centrosomal Syk denotes its restricted spatiotemporal degradation at the centrosomal level. As a matter of fact, we evidenced that Syk is ubiquitinylated during mitosis. Interestingly, the active 26S proteasome (35) and certain E3-ubiquitin ligases (36) were reported to localize to the centrosome. Accordingly, Syk might be both ubiquitinylated and locally degraded at the centrosomal level explaining its restricted loss during mitosis.

The observation that centrosomal Syk levels significantly dropped during mitosis strongly suggests that Syk affects mitosis in a negative, brake-like manner, the loss of centrosomal Syk allowing cell division to occur accurately. Supporting this idea, transient and stable Syk transfections generated cells with abnormal phenotypes associated with aberrant mitosis. A strong and sustained exogenous Syk expression in transiently transfected cells induced an abrupt nonapoptotic cell death. The roundup morphology and chromatin condensation in Syk-overexpressing cells with a disastrous phenotype strongly suggest the occurrence of a mitosis-associated cell death, reminiscent of mitotic catastrophe (27, 28). Besides, stable Syk-transfected cells contained multiple, abnormally sized nuclei as well as supernumerary centrosomes and displayed multipolar mitotic spindles. We hypothesize that the sustained overexpression of transfected Syk throughout the cell cycle may prevail over its proteasomal degradation, thereby perturbing the mitotic progression and ultimately leading to mitotic catastrophe. Depending on the exogenous Syk expression levels or the repair mechanisms, cells may escape from this arrest and undergo abnormal mitosis or cytokinesis resulting in endopolyploid cells, finally leading to cell death and/or aneuploidy (37). Unlike Syk, expression of the Polo-like kinase Plk2 (38) and the checkpoint kinase Chk2 (39) needed to be repressed to generate mitotic catastrophe. This consolidates their apparent opposing roles in the control of mitosis.

Identification of the Syk activating signals/receptors and its substrates/effectors in breast epithelial cells is crucial to unveil its action mechanism because these cells do not express immunoreceptors. Integrin receptors that are activated by extracellular matrix molecules and that mediate interactions with the actin cytoskeleton are preferential candidates. Experiments with hematopoietic cells show the tyrosine phosphorylation of Syk following integrin contact with ligand and show that Syk activity regulates integrin-mediated attachment (20). Most interestingly, recent evidence was found for ß1 integrin–dependent Syk activation in the H2B mammary epithelial cell line and the MCF7 breast carcinoma cell line (6). Intriguingly, {alpha}-tubulin, a key component of microtubules and centrosomes, was shown to be phosphorylated by Syk in vitro and in vivo (2, 31). Tyrosine-phosphorylated tubulin can be assembled into microtubules, and preassembled microtubules can be phosphorylated by Syk in vitro (31). In conjunction with our results, these observations indicate that Syk can phosphorylate components of both microtubules and centrosomes and suggest that it might directly operate at the level of the mitotic machinery. The centrosomal localization of Syk will undeniably contribute to the unraveling of its tumor suppressor mechanism. We showed previously that Syk-negative cells exhibit an increased tumorigenic and metastatic capacity and accumulate biochemical features evocative of malignant progression (2). Here, we show that centrosomal Syk negatively affects progression of mitosis. Consequently, its absence in centrosomes of tumor cells could allow uncontrolled mitosis and cause genetic instability and malignant progression. Interestingly, well-known tumor suppressors have recently been found at centrosomes (e.g., BRCA1 and p53) and affect centrosome function and cell cycle progression (21, 40, 41). As for these proteins, it is unlikely that the tumor suppressor activity of Syk could be attributed uniquely to its centrosomal component without taking into account its presence in other subcellular compartments.

In conclusion, our results show that, besides serine/threonine protein kinases, tyrosine kinases can also be present and spatiotemporally regulated at the centrosomes. Moreover, they show that mitotic progression can also be negatively regulated by phosphorylation events, backing the fact that Syk acts as a tumor suppressor. The disappearance of endogenous centrosomal Syk during mitosis and the aberrant mitotic phenotype after sustained exogenous Syk expression point toward a role for Syk as a kinase controlling cell division. Our results support and extend the concept that centrosomes are more than solely microtubule-organizing centers but catalytic platforms assembling positive and negative signaling molecules controlling cell cycle checkpoints and stress responses (42, 43).


   Acknowledgments
 
Grant support: Association pour la Recherche sur le Cancer 3111 and 4311, Ligue Nationale contre le Cancer (Comité de l'Hérault), Fondation pour la Recherche Médicale, Groupement des Entreprises Françaises dans la Lutte contre le Cancer Languedoc-Roussillon, and CNRS and Languedoc-Roussillon Region BDI Ph.D. fellowship (D. Zyss).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Wolfhard Almers, Michel Bornens, Christian Jaulin, Ned Lamb, Paulo Magalhaes, Susette Mueller, Naomi Taylor, and Jiong Wu for the generous gifts of reagents; Jean-Philippe Chambon and Stephen Baghdiguian (TUNEL), Fabrice Raynaud, Christian Jaulin, Charles Theillet, and Béatrice Orsetti (karyotype/centromere analysis), Claude Celati (centrosome preparations), and Christian Roy (cell fractionation) for technical advice; and Anne Morel for technical help. Some experiments have been done with the assistance of the Montpellier RIO Imaging (CNRS FRE2593) and Flow Cytometry (INSERM U475) facilities.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 4/13/05. Revised 8/24/05. Accepted 9/23/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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