Polo-like Kinase 1 and Chk2 Interact and Co-localize to Centrosomes and the Midbody*

Lyuben Tsvetkov, Xingzhi Xu, Jia LiDagger, and David F. Stern§

From the Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut 06511

Received for publication, November 3, 2002, and in revised form, December 11, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chk2 is a protein kinase intermediary in DNA damage checkpoint pathways. DNA damage induces phosphorylation of Chk2 at multiple sites concomitant with activation. Chk2 phosphorylated at Thr-68 is found in nuclear foci at sites of DNA damage (1). We report here that Chk2 phosphorylated at Thr-68 and Thr-26 or Ser-28 is localized to centrosomes and midbodies in the absence of DNA damage. In a search for interactions between Chk2 and proteins with similar subcellular localization patterns, we found that Chk2 coimmunoprecipitates with Polo-like kinase 1, a regulator of chromosome segregation, mitotic entry, and mitotic exit. Plk1 overexpression enhances phosphorylation of Chk2 at Thr-68. Plk1 phosphorylates recombinant Chk2 in vitro. Indirect immunofluorescence (IF) microscopy revealed the co-localization of Chk2 and Plk1 to centrosomes in early mitosis and to the midbody in late mitosis. These findings suggest lateral communication between the DNA damage and mitotic checkpoints.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Successful completion of the cell division cycle requires that the genome be duplicated accurately, and apportioned equally to daughter cells. Defects in these processes cause genome instability and predispose to cancer. DNA damage is induced by several mechanisms, including exposure to exogenous mutagens and endogenously produced reactive oxygen species. DNA checkpoints actuated by DNA damage or by stalled replication delay cell cycle transitions, providing time for DNA repair, and concomitantly promote DNA repair (2).

The protein kinase Chk2 is an important intermediary of vertebrate DNA damage checkpoint signaling pathways. Chk2 is activated in response to double-strand DNA breaks induced by ionizing radiation (IR)1 through a mechanism primarily involving the phosphatidylinositol (3'-) kinase-like protein kinase (PIKK) ATM (ataxia-telangiectasia-mutated) (3). Chk2 is also activated in response to other DNA-damaging agents, including ultraviolet (UV) light, and by interference with DNA replication, for example by treatment with the ribonucleotide reductase inhibitor hydroxyurea (HU) (3). Activation of Chk2 by these agents can occur independent of ATM, probably through the related PIKK ATR (3-5). The PIKK-dependent phosphorylation of Chk2 in response to DNA damage is required for its activation. Chk2 transmits the checkpoint signal to several downstream signaling pathways leading to an arrest of the cell cycle in G1, S, or G2/M phases (reviewed in Ref. 6). Mouse Chk2 -/- ES cells are defective in maintenance of G2 arrest and stabilization of p53 for initiation of G1 arrest (7). In response to IR, Chk2 phosphorylates p53 at Ser-20 and interferes with p53 binding to MDM2, thereby contributing to p53 stabilization and G1 arrest (8, 9). If DNA damage occurs in S phase, Chk2 phosphorylates Cdc25A and targets it for degradation (10). Chk2 and the protein kinase Chk1 regulate the G2/M transition through phosphorylation of their common target Cdc25C at Ser-216 (3). Checkpoint-induced phosphorylation of Brca1 at Ser-988 by Chk2 also plays a role in cell survival after DNA damage (11).

In addition to its importance in the regulation of tumor suppressor genes TP53 and BRCA1, CHK2 is apparently a tumor suppressor gene itself. CHK2 is defective in a subset of families with variant Li-Fraumeni syndrome with wild type TP53 (12). Males and females heterozygous for CHK2*1100delC have an elevated risk of breast cancer (13). Reports of CHK2 mutations in sporadic and familial human cancers are accumulating (14-20).

Chk2 plays a role in maintenance of G2 arrest after exposure to IR, with Cdc25C being a likely effector in this pathway (3, 7). Since Cdc25C knockout mice have an intact G2 checkpoint arrest (21), it is possible that there are other Chk2 downstream effectors. Genetic studies suggest that the budding yeast Chk2 homolog Rad53 prevents anaphase entry after checkpoint activation through regulation of the Polo kinase Cdc5 (22). DNA damage induces Rad53-dependent phosphorylation of Cdc5 (23). Rad53 and Cdc5 regulate mitotic exit through independent modifications of Bfa1 (24).

There are three Cdc5-homologous Polo-like kinases (PLKs) in mammals: Plk1, Snk, and Fnk/Prk/Plk3. Plk1 functions in mitosis and is most likely to be the mammalian Cdc5 ortholog (reviewed in Ref. 25). Plk1 regulates many aspects of mitosis, including centrosome maturation and orientation, chromosome adhesion, mitotic entry and exit (reviewed in Ref. 26). Also, Plk1 is a target of the DNA damage checkpoint and is inhibited in an ATM-dependent manner after DNA damage (27, 28).

We have examined the localization of phosphorylated and presumably active forms of Chk2. The results suggest the existence of unanticipated links between Chk2 and mitotic checkpoint regulation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Cultures, Transfections, and Treatments-- ATM-deficient (GM5849C) human fibroblasts were obtained from Coriell Institute for Medical Research, Camden, NJ. Other cell lines were obtained from American Type Culture Collection. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in the presence of antibiotics in a humidified incubator at 37 °C. Cells were treated with nocodazole (Sigma) (250 ng/ml) for 16 h and adriamycin (Sigma) (0.5 µM) for 1 h. Cells were irradiated in a Mark I 137Cs irradiator (Shepard). Cells were transfected by calcium phosphate precipitation with 5 µg of plasmid DNA and were analyzed after 40 h.

Antibodies-- Rabbit polyclonal anti-phospho-Thr-68-Chk2 previously described (29) was affinity-purified by phosphopeptide affinity chromatography. Rabbit polyclonal anti-Chk2 antibody was produced by immunization with recombinant GST-Chk2 produced in Escherichia coli. Mouse anti-Plk1 mAb mixture was obtained from Zymed Laboratories; mouse anti-Chk2 mAb (clone 2CHK01) from Neomarkers; and affinity-purified rabbit polyclonal anti-phospho-Thr-26/PSer-28 antibody was a generous gift of Yi Tan (Cell Signaling Technology). Mouse anti-gamma -tubulin mAb (clone GTU-88) and mouse anti-FLAG affinity agarose gel were obtained from Sigma; mouse anti-hemagglutinin (HA) mAb (16B12) from BabCo/Covance; horseradish peroxidase (HRP)-conjugated rat anti-HA mAb (3F10) and rat anti-HA mAb (3F10) from Roche Molecular Biochemicals; and HRP-conjugated secondary antibodies were obtained from Pierce.

Plasmids and Recombinant Proteins-- pcDNA-HAChk2 and pcDNA-HAChk2T68A were previously described (29). Plk1 coding sequences from an expressed sequence tag (EST) clone (GenBankTM accession no. BE 900300, obtained from Research Genetics) were amplified by PCR and cloned into pcDNA.3-3xFLAG vector, resulting in pcDNA.3-FLAGPlk1. Plk1 deletion mutants were generated by cloning of PCR-amplified DNA fragments into the pcDNA.3-3xFLAG vector. Plk1T82A was derived from pcDNA.3-FLAGPlk1 by PCR-based site-directed mutagenesis. pGEX2TK-GSTChk2, pGEX2TK-GSTChk2 (D368A), pGEX2TK-Chk2-(1-221), and His-FLAG-Chk2(D347A) previously described (29) were used for protein expression in E. coli.

Immunoprecipitation and Immunoblotting-- HEK 293-T cells were washed in PBS and solubilized in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5% Nonidet P-40, 120 mM NaCl) containing a protease inhibitor mixture (Roche Molecular Biochemicals). Cells were collected and centrifuged at 13 000 × g for 10 min at 4 °C. Immunoprecipitation was carried out by incubating 500 µg of protein lysate with 5 µg of anti-Chk2, 1.5 µg of anti-Plk1, 3 µg of anti-HA, and 10 µl of anti-FLAG affinity agarose gel (Sigma) at 4 °C overnight. For immunoblot (IB) analysis, nitrocellulose membranes were blocked in 3% nonfat dry milk in TBST (0.5% Tween-20, 120 mM NaCl, 50 mM Tris-HCl, pH 7.5) for 1 h at room temperature, and incubated with either anti-phospho-Thr-68-Chk2 (1:4000), anti-phospho-Thr-26/Ser-28-Chk2 (1:2000), anti-Plk1 antibody (1:500), mouse anti-Chk2 antibody (1:200), anti-HA-HRP antibody (1:1000), or anti-FLAG-HRP antibody (1: 2000) overnight at 4 °C. HRP-conjugated rabbit anti-mouse and goat anti-rabbit IgG (Pierce)(1:10,000) were used as secondary antibodies. Immunoblotted proteins were detected by ECLTM chemiluminescence reagents (Amersham Biosciences).

Immunofluorescence Microscopy-- Cells grown on poly-D-lysine coated culture slides (BD PharMingen) were washed in PBS, fixed for 15 min in PBS containing 4 or 0.5% paraformalaldehyde (PFA), and permeabilized in Triton buffer (0.5% Triton X-100 in PBS). Fixed cells were blocked in blocking solution (2% bovine serum albumin, 0.1% Tween, PBS) for 30 min at 37 °C in a humidified chamber. Immunostaining was performed using anti-PThr-68-Chk2 antibody (0.5 µg/ml), anti-Plk1 antibody (1: 100), anti-gamma -tubulin antibody (1:1000), anti-PThr-26/PSer-28-Chk2 antibody (1:300), mouse anti-Chk2 antibody (1:50), mouse anti-HA mAb (16B12) (1:300), or rat anti-HA mAb (3F10) (1:300) for 30 min at 37 °C in a humidified chamber, followed by three washes in blocking buffer. Cells were incubated with anti-mouse Rhodamine (Rd) (1:1000), anti-mouse fluorescein isothiocyanate (FITC) (1:100), anti-rabbit-FITC (1:100) secondary antibodies. DNA was stained with 6'-diamidino-2-phenylindole (DAPI) in mounting solution (Vector Laboratories). IF microscopy was performed using a Nikon Microphot-FX microscope using ×40 and ×60 Plan Apo objectives. Images were captured with a Spot digital camera (Diagnostic Instruments Inc.) and processed using Adobe Photoshop.

For phosphatase treatment, cells were permeabilized with 0.1% Triton X-100 in PBS for 30 s at room temperature, followed by three washes with PBS, and one with phosphatase buffer, and then incubated with 4 units/µl lambda -phosphatase (New England Biolabs) for 20 min at 30 °C. After treatment, cells were washed in PBS and fixed as in the basic protocol.

In Vitro Kinase Assays-- Anti-Plk1 antibody (1:500) was incubated at 4 °C overnight with 500 µg of protein lysate. Plk1 immunoprecipitates were incubated with 10 µg of alpha -casein at 30 °C for 5 min in 20 µl of kinase buffer (20 mM HEPES pH 7.4, 50 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, and 1 µM ATP) supplemented with 5 µCi of [gamma -32P]ATP (>5000 Ci/mmol, AA0018, Amersham Biosciences). The reactions were stopped with the addition of 20 µl of 2× Laemmli gel electrophoresis sample buffer. The samples were separated by SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue, dried, and visualized by autoradiography.

For detection of the kinase activity of exogenous FLAG-Plk1 protein, with phosphospecific antibodies, 300 µg of protein lysate was incubated with 10 µl of anti-FLAG affinity agarose gel. Anti-FLAG immunoprecipitates were incubated with 2 µg of recombinant GST-Chk2-(1-221) or His-FLAG-Chk2D347A at 30 °C for 10 min in 20 µl of kinase buffer with 25 µM ATP. The samples were analyzed by IB with anti-phospho-Thr-68-Chk2 antibody (1:4000).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of PThr-68-Chk2 Antibody-- Although Chk2 is activated by DNA damage, little is known about regulation of Chk2 during the normal cell cycle. The yeast homolog of Chk2, Rad53, is required for normal delay of late-firing replication origins, and timely induction of ribonucleotide reductase for the synthesis of nucleotide precursors (30, 31). This suggests that mammalian Chk2 may have important functions in normal cell cycle progression.

Damage-dependent activation of Chk2 is accompanied by the phosphorylation of a cluster of SQ/TQ sites near the N terminus of Chk2 termed the SCD (32-34). These sites are consensus targets for phosphorylation by PIKKs, including the Chk2 regulators Atm and Atr. Phosphorylation of Chk2 at one or more of the SCD sites is required for Chk2 activation after DNA damage (32-35).

Atm and Atr have different preferences for phosphorylation within the SCD in vitro (34). The predominant site phosphorylated in response to double-stranded breaks is Thr-68, which is required for intact responses to DNA damage (33). ATM is the major Chk2 regulator in this response, and preferentially phosphorylates Thr-68 in vitro. In response to replication block or UV exposure, this site is phosphorylated independently of ATM, probably by ATR (34).

In order to localize forms of Chk2 phosphorylated at this site, we produced and affinity-purified a phosphospecific antibody, alpha -PThr-68-Chk2, raised against a phosphopeptide containing the region surrounding phospho-Thr-68. In lysates from irradiated 293T cells, but not-mock-irradiated cells, alpha -PThr-68-Chk2 recognized endogenous Chk2 (lower band, Fig. 1A, lanes 2 and 4) and transiently expressed HA-Chk2 (upper band, Fig. 1A, lane 2). However, this antibody did not recognize transiently expressed HA-Chk2T68A, in which the target phosphorylation site has been replaced with A (Fig. 1A, lane 4). In addition, the alpha -PThr-68-Chk2 antibody recognized endogenous Chk2 as a single band that increased in intensity with escalating radiation dose, while alpha -Chk2 antibody detected approximately equal amounts of Chk2 protein in all samples (Fig. 1B). Recombinant Chk2 undergoes autophosphorylation at multiple sites when expressed in bacteria (29). alpha -PThr-68-Chk2 recognized GST-Chk2-WT, but not GST-Chk2D368A, which has a mutation that eliminates kinase catalytic activity (Fig. 1C).


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Fig. 1.   Characterization of alpha -PThr-68-Chk2 antibody. A, extracts from HEK 293T cells transiently expressing HA-Chk2-wild type (lanes 1 and 2) or HA-Chk2T68A (lanes 3 and 4) were mock-irradiated (lanes 1 and 3) or irradiated with 5 Gy (lanes 2 and 4) and immunoblotted with alpha -PThr-68-Chk2, alpha Chk2, or alpha HA antibodies. B, IB of extracts from HEK 293T cells exposed to indicated doses of IR analyzed by immunoblotting with alpha -PThr-68-Chk2, or alpha -Chk2. C, IB of purified recombinant GST-Chk2-WT and kinase-defective GST-Chk2 D368A fusion proteins, produced in bacteria, and immunoblotted with alpha -PThr-68-Chk2 or alpha -Chk2. D, IF analysis of non-irradiated (0 Gy) and irradiated (4 Gy) HEK-293, HT-1080, and GM5849C cells with alpha -PThr-68-Chk2 antibody. Bar is 10 µm long.

DNA damage induces phosphorylation of Chk2 at Thr-68 in nuclear foci at the sites of DNA damage (1). To determine if alpha -PThr-68-Chk2 can detect these foci, we immunostained irradiated 293T, HT-1080 and GM5849C cells. Immunoreactive foci were formed following exposure to IR in 293T and HT-1080 cells, but not in GM5849C cells, which lack functional ATM (Fig. 1D).

Phosphorylated Chk2 Is Localized to Centrosomes-- IF of untreated 293 cells with alpha -PThr-68-Chk2 antibody produced a staining pattern characteristic of proteins associated with centrosomes (Fig. 2A). Signals generally consisted of single or paired nuclear dots in interphase cells, separated dots flanking condensed chromatin in metaphase, and single dots adjacent to chromatin in telophase (Fig. 2A). Similar staining patterns with alpha -PThr-68-Chk2 were observed with three additional cell lines: WI38, HT-1080, U2OS, and GM5849C (AT cells) (data not shown). The signal is ATM-independent since it was similar in ATM-deficient GM5849C cells.


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Fig. 2.   Centrosomal localization of phosphorylated Chk2. A, IF analysis of HEK-293 cells with alpha -PThr-68-Chk2 antibody detected with FITC. DAPI staining marks nuclei. B, IF analysis of U2-OS cells with alpha -PThr-68-Chk2 (FITC) and alpha gamma -tubulin (rhodamine). C, centrosome and midbody localization of phosphorylated Chk2. IF of U2-OS cells with alpha -PThr-68-Chk2 (red) and alpha -PThr-26/PSer-28-Chk2 (green) antibodies, DAPI (blue). D, phosphopeptide immunogen used to elicit alpha -PThr-68-Chk2 antibody was used for competition with the antigen in IF analysis of U2-OS cells with alpha -PThr-68-Chk2 and alpha gamma -tubulin antibodies. E, double IF of U2-OS cells with alpha -PThr-68-Chk2 and alpha gamma -tubulin antibodies after in situ permeabilization and treatment with 4 units/µl lambda -phosphatase or buffer for 30 min at 25 °C. Bars are 10 µm long.

Additional controls were performed to verify specificity of the antibody. IF background was low with control nonspecific IgG as primary antibody (data not shown). Competition with the oligophosphopeptide used as antigen for production of alpha -PThr-68-Chk2 eliminated the IF signal (Fig. 2D). gamma -tubulin nucleates microtubule assembly and is concentrated at centrosomes (36). To confirm the localization of Chk2 to the centrosome, we performed double IF using alpha -PThr-68-Chk2 and alpha gamma -tubulin antibodies in U2-OS cells (Fig. 2B). The PThr-68-Chk2 and gamma -tubulin fluorescence signals overlapped at the strong, centrosome-like foci seen with alpha -PThr-68-Chk2, providing direct evidence for centrosomal localization of phosphorylated Chk2 (Fig. 2, B and D). However, in contrast to PThr-68-Chk2 signals, the gamma -tubulin signal was not competed with the PThr-68-containing phosphopeptide (Fig. 2D). In order to verify that the alpha -PThr-68-Chk2 signal is a result of Chk2 phosphorylation, rather than high local concentration of Chk2 or other artifact, we treated permeabilized U2-OS cells with lambda -phosphatase. Phosphatase treatment eliminated the PThr-68-Chk2 signal, but the gamma -tubulin signal remained although slightly reduced (Fig. 2E). Incubation with buffer did not change either gamma -tubulin or PThr-68-Chk2 signals (data not shown).

Two additional Chk2-reactive antibodies, alpha Chk2, which should recognize all forms of Chk2, and alpha -PThr-26/PSer-28-Chk2, were tested. Anti-PThr-26/PSer-28-Chk2 should recognize additional Chk2 regulatory sites in the SCD (34). Although S28 does not seem to be a good substrate for either ATM or ATR, Thr-26 is evidently a good ATR substrate in vitro (34). Treatment of 293 and U2OS cells with IR, UV, or hydroxyurea (HU)-induced reactivity of exogenously expressed HA-Chk2 with alpha -PThr-26/PSer-28-Chk2 antibody analyzed by IB (data not shown). The antibody detects endogenous Chk2 after exposure to IR and doxorubicin, but not in untreated cells (data not shown). Finally, alpha -PThr-26/PSer-28-Chk2 antibody recognizes wild-type HA-Chk2 but not HA-Chk2T26A/S28A with amino acid substitutions at the immunogenic sites (data not shown). IF with alpha -PThr-26/PSer-28-Chk2 in U2OS cells yielded a centrosome-like pattern (Fig. 2C). In contrast, alpha Chk2 detected a diffuse nuclear and cytoplasmic localization that overlapped with focal alpha -PThr-68-Chk2 fluorescence at centrosomes (Fig. 3A).


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Fig. 3.   Localization of Chk2 to the centrosome. A, dual IF of U2-OS cells with alpha -PThr-68-Chk2 and alpha Chk2 antibodies. Arrowheads mark coincident staining between alpha -PThr-68-Chk2 and a subset the alpha Chk2 signal. B, dual IF of 293T cells transiently expressing HA-Chk2 with alpha gamma -tubulin (red) and alpha HA (green) antibodies after mild fixation (0.5% PFA) and Triton extraction. DAPI (blue) staining marks nuclei. C, co-IF of 293T cells transiently expressing HA-Chk2 with alpha -PThr-68-Chk2 (green) and alpha HA (red) antibodies after mild fixation (0.5% PFA) and Triton extraction. Bars are 10 µm long.

In addition to centrosome-associated staining, we detected phosphorylated Chk2 with alpha -PThr-68-Chk2 and alpha -PThr-26/PSer-28-Chk2 antibodies at the midbody, the central part of the cytokinetic bridge, in telophase (Fig. 2C).

HA-Chk2 Localizes to Centrosomes-- Under basal conditions, the bulk of Chk2 is not phosphorylated, and Chk2 is characterized by a diffuse nuclear IF pattern. We hypothesized that a minor subset of Chk2 is phosphorylated without induced DNA damage, and it is this subset that is associated with discrete basal localization. Fixation at reduced PFA concentrations (1 and 0.5%) and pre-extraction of cells with 0.1% Triton X-100 permitted visualization of a subpopulation of overexpressed HA-Chk2 with centrosomal staining pattern and which colocalizes with gamma -tubulin (Fig. 3B). Under these conditions, alpha HA and alpha -PThr-68-Chk2 signals overlapped, including at the centrosomes (Fig. 3C). Staining for HA-Chk2 with two different alpha HA antibodies yielded the same localization pattern (data not shown).

Chk2 Interacts with Plk1-- On the basis of localization of Chk2 and Plk1 and suggested communication between their signaling pathways (22, 24, 37, 38), we determined whether Chk2 and Plk1 physically interact. HA-tagged Chk2 and FLAG-tagged Plk1 coimmunoprecipitated when transiently expressed in 293T cells (Fig. 4A, lanes 3 and 6), and endogenous Chk2 and Plk1 co-immunoprecipitated from 293T cells (Fig. 4B, lanes 3 and 4). There was more Plk1 protein in immunoprecipitates from nocodazole-treated cells, but this may simply reflect the higher expression of Plk1 in mitotic cells (Fig. 4B, lanes 9 and 10) (reviewed in Ref. 25). The Chk2/Plk1 interaction was not affected by Adriamycin (doxorubicin)-induced DNA damage (Fig. 4B, lane 4). However, Adriamycin induced the Chk2 mobility shift associated with phosphorylation (Fig. 4B, lanes 2 and 4), and inhibited in vitro Plk1 kinase activity toward alpha -casein (Fig. 4B, line 10).


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Fig. 4.   Chk2 interacts with Plk1. A, co-immunoprecipitation of HA-Chk2 and FLAG-Plk1. Whole cell extracts from HEK 293T cells transiently expressing HA-Chk2 (lanes 1, 2, 3, 5, and 6) and/or FLAG-Plk1 (lanes 2-6) were immunoprecipitated with mouse IgG (lanes 2 and 5), alpha FLAG antibody (lanes 1 and 3), or alpha HA antibody (lanes 4 and 6). Western blot analysis of immunoprecipitates was performed with alpha FLAG (lower panel) and alpha -HA antibodies (upper panel). B, co-IP of endogenous Chk2 and Plk1. HEK 293-T cells were treated with 0 (lanes 1, 2, 7, and 8) or 250 ng/ml nocadazole (lanes 3, 4, 5, 6, 9, and 10) for 16 h and with 0 (lanes 1, 3, 5, 6, 7, and 9) or 0.5 µM (lanes 2, 4, 8, and 10) Adriamycin for 1 h. Extracts from the cells were IP with preimmune serum (lane 5), mouse IgG (lane 6), alpha Chk2 antibody (lanes 1-4), and alpha Plk1 antibody (lanes 7-10). Western blot analyses were performed to detect Chk2 (lower left panel) and Plk1 (upper left and upper right panels) in the immunoprecipitates. Portions of Plk1 immunoprecipitates were used for in vitro kinase assays. Kinase activity of Plk1 was detected by incorporation of [gamma -32P]ATP into alpha -casein, used as a substrate (middle right). Amounts of Plk1 used for in vitro kinase assay were monitored by IB analysis of Plk1 immunoprecipitates (upper right). alpha -Casein was detected by Coomassie Brilliant Blue staining (lower right).

Plk1 Overexpression Induces Chk2 Phosphorylation-- To understand further the functional interactions between Chk2 and Plk1, we co-expressed HA-Chk2 and FLAG-Plk1 in 293T cells and analyzed the cell lysates by IB with alpha -PThr-68-Chk2 and alpha -PThr-26/PSer-28-Chk2 (Fig. 5B). Co-expression of HA-Chk2 and FLAG-Plk1 promoted phosphorylation of least at 2 sites (Thr-26 and/or Ser-28 and Thr-68) in the SCD of HA-Chk2 (Fig. 5B). We next expressed full-length and truncated forms of FLAG-Plk1 in 293-T cells. FLAG-Plk1-(1-330), FLAG-Plk1-(1-408), and FLAG-Plk1-(1-480) have deletions in the C terminus, that enhance their kinase activity (39-42) (Fig. 5A, lanes 4-6). Two other proteins, FLAG-Plk1(K82A), a kinase defective (KD) mutant and FLAG-Plk1(330-CT), with a deleted kinase domain, have no apparent kinase activity (Fig. 5A, lanes 3 and 7) (39-42). Fluorescence-activated sorter analysis of 293T cells co-expressing green fluorescence protein (GFP) and FLAG-Plk1 wild type, FLAG-Plk1-KD and Plk1-(330-CT), showed accumulation of 34, 37, and 56% of transfected cells in G2/M in comparison to 19% of cells expressing GFP only (data not shown). However, expression of FLAG-Plk1-(1-330), FLAG-Plk1-(1-408), and FLAG-Plk1-(1-480) had no effect on cell cycle distribution of transfected cells, consistent with previous reports (39-42). Expressed FLAG-Plk1 wild type, FLAG-Plk1-(1-330), FLAG-Plk1-(1-408), and FLAG-Plk1-(1-480), which have normal or enhanced kinase activity, increased phosphorylation of endogenous Chk2 at Thr-68 (Fig. 5A, lines 2, 4, 5, and 6). In contrast, phosphorylation of Chk2 Thr-68 was unchanged in the cells with expressed FLAG-Plk1-KD or FLAG-Plk1-(330-CT), which have no associated kinase activity (Fig. 5A, lanes 3 and 7). The correlation of Thr-68 phosphorylation with the kinase activity of FLAG-Plk1 mutants, but not with G2/M cell arrest, indicates that Thr-68 phosphorylation is dependent upon FLAG-Plk1 kinase activity.


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Fig. 5.   Plk1 overexpression induces Chk2 phosphorylation. A, lysates from 293T vector-transfected cells (lane 1) or cells transiently expressing wild type FLAG-Plk1 (lane 2), FLAG-Plk1-KD (lane 3), FLAG-Plk1-(1-330) (lane 4), FLAG-Plk1-(1-480) (lane 5), FLAG-Plk1-(1-408) (lane 6), and FLAG-Plk1 (330-CT) were analyzed by IB with alpha -PThr-68-Chk2, alpha Chk2, and alpha FLAG antibodies. The kinase activity of the exogenous Plk1 was measured by in vitro kinase assay with alpha FLAG immunoprecipitates from the same lysates and 10 µg of alpha -casein substrate in presence of [gamma -32P]ATP. alpha -Casein was detected by Coomassie Brilliant Blue staining. B, lysates from 293T cells transiently expressing HA-Chk2 only or HA-Chk2 and FLAG-Plk1 were analyzed by IB with alpha -PThr-68-Chk2, alpha -PThr-26/PSer-28-Chk2, alpha HA, and alpha FLAG antibodies.

Plk1 Phosphorylates Chk2 in Vitro-- Immune complexes containing FLAG-Plk1 transiently expressed in 293T cells phosphorylated recombinant His-FLAG-Chk2-KD and GST-Chk2-(1-221) in vitro (Fig. 6). This was due to Plk1 activity in the immune complexes, since FLAG-Plk1-KD was not associated with kinase activity toward recombinant Chk2 (Fig. 6).


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Fig. 6.   Plk1 phosphorylates Chk2 in vitro. A, alpha FLAG immunoprecipitates from lysates from 293T vector-transfected cells or cells transiently expressing FLAG-Plk1 or FLAG-Plk1-KD 293T cells were incubated with 2 µg of recombinant GST-Chk2-(1-221) (A) or His-FLAG-Chk2D347A (B) under kinase assay conditions with ATP. Proteins from the reactions were separated by SDS-PAGE, and immunoblotted with alpha -PThr-68-Chk2 antibody for detection of phosphorylation at Thr-68 Chk2, alpha FLAG antibody for FLAG-Plk1 and His-FLAG-Chk2D347A, and alpha Chk2 antibody for GST-Chk2-(1-221).

Chk2 and Plk1 Co-localize to Centrosomes and the Midbody-- Plk1 is associated with the kinetochores of condensed chromosomes, the centrosome in prophase and metaphase, and the midbody, later in mitosis (43, 44). Early in mitosis (prophase and metaphase) Plk1 and PThr-68-Chk2 were apparently associated with the centrosomes (Fig. 7, A and B). Later in mitosis, PThr-68-Chk2 was detected at the centrosomes and the midbody, whereas the Plk1 signal was localized only to the midbody (Fig. 7C). This suggests that Plk1 is not required to maintain centrosomal localization of Chk2.


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Fig. 7.   Co-localization of Chk2 and Plk1 to the centrosome and the midbody. Co-immunostaining of U2-OS cells with alpha -PThr-68-Chk2 (green) and alpha Plk1 (red) antibodies. Signals of alpha -PThr-68-Chk2 and alpha Plk1 antibodies overlap at the centrosome in prophase (A) and metaphase (B) cells and at the midbody later in mitosis (C). DAPI staining marks the location of DNA. Bars are 10 µm long.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We found hat subpopulations of Chk2, phosphorylated at sites required for DNA damage responses, co-localize with centrosomes and midbodies. The results are surprising, because numerous studies have implicated Chk2 and its yeast homologs in DNA damage responses and DNA replication checkpoints, but Chk2 has not been directly linked to activity of checkpoints that detect assembly of the mitotic spindle. Potential activities of Chk2 at these sites include normal or checkpoint regulation of the centrosome cycle and/or cytokinesis. Alternatively, phospho-Chk2 could provide a lateral connection between these processes and DNA damage and replication inhibition responses.

In the absence of DNA damage, most Chk2 molecules are not phosphorylated. Activation of Chk2 by DNA damage or inhibition of replication coincides with Chk2 phosphorylation and requires Chk2 kinases Atm or Atr. Mutation of the phosphorylation site at Thr-68 impairs the regulation of Chk2 by DNA damage. The correlation of Chk2 phosphorylation at sites within its SCD and its activation suggests that the Chk2 phosphospecific antibodies used here mark functionally active forms of Chk2.

ATM and ATR phosphorylate different sites in Chk2 SCD with overlapping but distinct substrate specificity in vitro (34). The presence of phosphorylated Chk2 at the mitotic structures that we observed in AT cells indicates that there might be other kinases involved, presumably including ATR. Centrosomes and the midbody are sites of localization of non-PIKK protein kinases that might phosphorylate Chk2. Although the Chk2 SQ/TQ sites monitored in these experiments are sites for PIKK-dependent phosphorylation in the DNA damage response, these same sites may also be targets for PIKK-independent phosphorylation. Chk2 expressed in E. coli can transphosphorylate kinase-defective Chk2 at Thr-68 (29) and Thr-26 or Ser-28 (data not shown). The finding that at least one conventional (non-PIKK) Ser/Thr kinase, Chk2, can phosphorylate this site suggests that, in centrosomes and other loci with phospho-Chk2, other protein kinases may be responsible for Chk2 phosphorylation. Recently it was reported that another non-PIKK, Plk3 can phosphorylate the SCD of Chk2 in vitro and can contribute to its full activation (45). In yeast and mammals, high local concentrations of Chk2 may enable Chk2-Chk2 transphosphorylation, which may, in turn, be sufficient to activate Chk2. In the DNA damage response, localization of Chk2 to sites of DNA damage may be mediated by the FHA domain which itself recognizes phosphopeptides. Recruitment of Chk2 to centrosomes could be mediated through binding of the FHA domain of Chk2 to phosphoproteins located there. This may deliver Chk2 in close proximity to activating kinases at these sites. Non-PIKK Ser/Thr kinases localized to centrosomes include Aurora family kinases (reviewed in Ref. 46), and at some phases of the cell cycle, spindle checkpoint kinases Mps1, Bub1, and BubR1 (reviewed in Ref. 47). Cell cycle-dependent localization of PLKs to the centrosomes, kinetochores, and midbody has been reported (43, 44).

Proper regulation of centrosome duplication and maturation is vital for maintenance of a bipolar mitotic spindle. Aberrant centrosome and spindle numbers are common hallmarks of aggressive cancers. Mutations in genes associated with DNA damage responses, including p53, have been implicated in aberrant partitioning of centrosomes after cell division, and genomic instability. Cells lacking p53 or with mutated p53 have abnormal numbers of centrosomes (48). Centrosome amplification in tumor and cell lines is linked to mutations in p53 (48), abnormal expression of MDM2 (49), and p21CIP1 (50). The mechanisms for these changes are not completely understood, and may reflect direct regulation of centrosome maturation and stability through these pathways, or indirect effects on survival of changes resulting in multinucleation and polyploidy (51).

A second Chk2 substrate, Brca1, partly co-localizes with Chk2 in untreated cells (11), and displays centrosomal localization (52). Brca1 associates with the centrosome in mitotic cells and interacts with gamma -tubulin. It is interesting that the Brca1 fraction interacting with gamma -tubulin is hypophosphorylated (52). Brca1 disassociation from centrosomes could be phosphorylation dependent and mediated by Chk2 phosphorylation. Mutations in the DNA repair protein Brca2 (53) or overexpression of the Chk2 kinase ATR (54) also affect centrosome number.

The localization of Chk2 to components of the mitotic apparatus and its physical association with Plk1 suggest that Chk2 functions in the regulation of mitosis. In support of this, we observed the Plk1-dependent phosphorylation of several sites within the SCD of Chk2. Plk1 is expressed in S, G2, and M phases of cell cycle and undergoes phosphorylation-dependent activation in G2 and M, with a peak in its activity in mitosis (39). We speculate that Chk2 phosphorylation occurs locally at the sites of the interaction between Plk1 and Chk2. These sites, according to our results from colocalization experiments, could be the centrosomes and midbody, although the possibility of Chk2-Plk1 interaction elsewhere cannot be ruled out. Another Polo-like kinase Plk3, phosphorylates Chk2 after IR (45). The effect of IR on Plk1 and Plk3 activities is diametrically opposite; Plk1 is inhibited while Plk3 is activated. Chk2 regulation by Plk1 is probably a part of a different mechanism because we observed that Plk1 phosphorylated Chk2 without exposure of the cells to DNA damaging agents. Also, the interaction between Plk1 and Chk2 is not regulated by exposure of the cells to Adriamycin, a DNA damaging agent. Overexpression of wild type Plk1, kinase-defective Plk1, and an N-terminal-deleted Plk1-(330-CT), caused the accumulation of transfected cells in G2/M and later a mitotic catastrophe and apoptosis, which could activate DNA damage checkpoint as a secondary event (36, 38, 39). This is unlikely to be related to Chk2 regulation since we found that Chk2 phosphorylation correlates with the overexpression of Plk1 mutant protein with normal or elevated kinase activity but not with the overexpression of Plk1 proteins causing G2/M arrest. Definition of the circumstances in which Plk1 phosphorylates Chk2 may reveal new Chk2 functions in the normal cell cycle, or in cross-talk between mitotic regulation and DNA damage checkpoints.

Vertebrate and budding yeast Chk2 are important in the DNA damage-dependent arrest of mitotic entry and exit. However, Chk2 and Rad53 are not, apparently, regulated through or required for the mitotic spindle checkpoint (redundant functions have not been entirely ruled out). Moreover, unlike Plk1, it is not clear that Chk2 functions are required in a normal cell cycle, rather than being called into play only after genotoxic damage. In human testes, Chk2 protein is more abundant in spermatogonia (mitotic division), than in spermatocytes (meiotic division), indicating a possible mitotic role for Chk2 (55).

Plk-1 is a multifunctional mitotic protein kinase involved in many mitotic events, including centrosome maturation and formation of the mitotic spindle (56). Plk1 regulates mitotic entry by promoting translocation of cyclin B1 to the nucleus (57) and by inhibiting export of Cdc25C from the nucleus (58). Plx may also directly enhance Cdc25 catalytic activity (59, 60). In contrast to Plk1, Chk2 is a negative regulator of Cdc25C. DNA damage-activated Chk2 inhibits Cdc25C activity through phosphorylation at a 14-3-3 binding site (3) that leads to preferential accumulation of Cdc25C in the cytoplasm (61-63). Close proximity of Chk2 and Plk1 suggests a possibility for mutual regulation and regulation of other Cdc2 regulators, such as Myt1, Mik1, and Wee1. At anaphase, phosphorylation of yeast Cdc5 and other PLKs are involved in regulation of chromosome cohesion, and both entry and exit from mitosis. In Xenopus, Plx1 promotes anaphase progression through phosphorylation of APC/C and cohesin (64, 65).

CDC5 inhibits Rad53 in the process of adaptation to unrepairable DNA damage (37, 38), and was implicated as a possible Rad53 effector (22). Recent work suggests that Cdc5 and Rad53 antagonistically regulate mitotic exit pathways through the common regulatory target Bfa1 (24). It is noteworthy that Bfa1 is located at spindle pole bodies, where it regulates activity of TEM1, the limiting regulator of CDK inactivation required for mitotic egress. Perhaps localization of Chk2 to centrosomes and kinetochores (data not shown) reflects interaction with a similar mitotic exit complex.

Chk2 is an intermediary in pathways regulating DNA damage responses and checkpoint arrest. The unexpected localization of phosphorylated forms of Chk2 to centrosomes and midbodies; physical interaction and colocalization with Plk1; and phosphorylation by Plk1 suggest hitherto unexplored connections of these DNA damage checkpoint pathways with chromosome mechanics. Since centrosome and spindle aberrations are common in tumor cells with dysfunctional checkpoint pathways, these findings shed new light on the basis of genomic instability.

    ACKNOWLEDGEMENTS

We thank Marc Schwartz, Jonathan McMenamin-Balano, Soo-Jung Lee, and other members of the Stern laboratory for helpful comments. We are grateful to J. Rose for providing the IF microscope.

    FOOTNOTES

* This work was supported by United States Army Medical Research and Materiel Command (USAMRMC) DAMD 17-98-1-8272, U. S. Public Health Service (USPHS) R01CA82257, Anna Fuller fellowship in molecular oncology (to L. T.), Susan G. Komen Breast Cancer Foundation Fellowship PDF2000 719 (to L. T.), and USAMRMC DAMD 17-01-1-0464 (to L. T.), USAMRMC DAMD 17-01-1-0465 (to X. X.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by USAMRMC Predoctoral Training Program in Breast Cancer Research DAMD 17-99-1-9461.

§ To whom correspondence should be addressed: Dept. of Pathology, School of Medicine, Yale University, 310 Cedar St., BML 342, New Haven, CT 06511. Tel.: 203-785-4832; Fax: 203-785-7467; E-mail: Df.stern@yale.edu.

Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M211202200

    ABBREVIATIONS

The abbreviations used are: IR, ionizing radiation; IF, immunofluorescence; PIKK, phosphatidylinositol (3') kinase-like kinase; ATM, ataxia-telangiectasia mutated; HU, hydroxyurea; SCD, SQ/TQ cluster domain; PLKs, Polo-like kinases; GST, glutathione S-transferase; HA, hemagglutinin; IB, immunoblot; PFA, paraformaldehyde; FITC, fluorescein isothiocyanate; DAPI, 6'-diamidino-2-phenylindole; GFP, green fluorescent protein; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; PThr, phospho-Thr; PS, phospho-Ser; Gy, Gray; mAb, monoclonal antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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