Polo-like Kinase 1 and Chk2 Interact and Co-localize to Centrosomes and the Midbody
2003; Elsevier BV; Volume: 278; Issue: 10 Linguagem: Inglês
10.1074/jbc.m211202200
ISSN1083-351X
AutoresLyuben Tsvetkov, Xingzhi Xu, Jia Li, David F. Stern,
Tópico(s)Cancer-related Molecular Pathways
ResumoChk2 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 (1Ward I.M. Wu X. Chen J. J. Biol. Chem. 2001; 276: 47755-47758Google Scholar). 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. 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 (1Ward I.M. Wu X. Chen J. J. Biol. Chem. 2001; 276: 47755-47758Google Scholar). 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. ionizing radiation immunofluorescence phosphatidylinositol (3′) kinase-like kinase ataxia-telangiectasia mutated hydroxyurea SQ/TQ cluster domain Polo-like kinases glutathione S-transferase hemagglutinin immunoblot paraformaldehyde fluorescein isothiocyanate 6′-diamidino-2-phenylindole green fluorescent protein horseradish peroxidase phosphate-buffered saline phospho-Thr phospho-Ser Gray monoclonal antibody 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 (2Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Google Scholar). 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) (3Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Google Scholar). 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) (3Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Google Scholar). Activation of Chk2 by these agents can occur independent of ATM, probably through the related PIKK ATR (3Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Google Scholar, 4Chaturvedi P. Eng W.K. Zhu Y. Mattern M.R. Mishra R. Hurle M.R. Zhang X. Annan R.S. Lu Q. Faucette L.F. Scott G.F. Li X. Carr S.A. Johnson R.K. Winkler J.D. Zhou B.B. Oncogene. 1999; 18: 4047-4054Google Scholar, 5Brown A.L. Lee C.H. Schwarz J.K. Mitiku N. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Google Scholar). 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. 6Bartek J. Falck J. Lukas J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 877-886Google Scholar). Mouse Chk2 −/− ES cells are defective in maintenance of G2 arrest and stabilization of p53 for initiation of G1 arrest (7Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Google Scholar). 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 (8Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Google Scholar,9Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Google Scholar). If DNA damage occurs in S phase, Chk2 phosphorylates Cdc25A and targets it for degradation (10Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Google Scholar). Chk2 and the protein kinase Chk1 regulate the G2/M transition through phosphorylation of their common target Cdc25C at Ser-216 (3Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Google Scholar). Checkpoint-induced phosphorylation of Brca1 at Ser-988 by Chk2 also plays a role in cell survival after DNA damage (11Lee J.S. Collins K.M. Brown A.L. Lee C.H. Chung J.H. Nature. 2000; 404: 201-204Google Scholar). 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 (12Bell D.W. Varley J.M. Szydlo T.E. Kang D.H. Wahrer D.C. Shannon K.E. Lubratovich M. Verselis S.J. Isselbacher K.J. Fraumeni J.F. Birch J.M. Li F.P. Garber J.E. Haber D.A. Science. 1999; 286: 2528-2531Google Scholar). Males and females heterozygous for CHK2*1100delC have an elevated risk of breast cancer (13Meijers-Heijboer H. Van Den Ouweland A. Klijn J. Wasielewski M. De Snoo A. Oldenburg R. Hollestelle A. Houben M. Crepin E. Van Veghel-Plandsoen M. Elstrodt F. Van Duijn C. Bartels C. Meijers C. Schutte M. McGuffog L. Thompson D. Easton D.F. Sodha N. Seal S. Barfoot R. Mangion J. Chang-Claude J. Eccles D. Eeles R. Evans D.G. Houlston R. Murday V. Narod S. Peretz T. Peto J. Phelan C. Zhang H.X. Szabo C. Devilee P. Goldgar D. Futreal P.A. Nathanson K.L. Weber B.L. Rahman N. Stratton M.R. Nat. Genet. 2002; 31: 55-59Google Scholar). Reports of CHK2 mutations in sporadic and familial human cancers are accumulating (14Matsuoka S. Nakagawa T. Masuda A. Haruki N. Elledge S.J. Takahashi T. Cancer Res. 2001; 61: 5362-5365Google Scholar, 15Hofmann W.K. Miller C.W. Tsukasaki K. Tavor S. Ikezoe T. Hoelzer D. Takeuchi S. Koeffler H.P. Leuk. Res. 2001; 25: 333-338Google Scholar, 16Ingvarsson S. Sigbjornsdottir B.I. Huiping C. Hafsteinsdottir S.H. Ragnarsson G. Barkardottir R.B. Arason A. Egilsson V. Bergthorsson J.T. Breast Cancer Res. 2002; 4: R4Google Scholar, 17Miller C.W. Ikezoe T. Krug U. Hofmann W.K. Tavor S. Vegesna V. Tsukasaki K. Takeuchi S. Koeffler H.P. Genes Chromosomes Cancer. 2002; 33: 17-21Google Scholar, 18Aktas D. Arno M. Rassool F. Mufti G. Leuk. Res. 2002; 26: 985Google Scholar, 19Allinen M. Huusko P. Mantyniemi S. Launonen V. Winqvist R. Br. J. Cancer. 2001; 85: 209-212Google Scholar, 20Hangaishi A. Ogawa S. Qiao Y. Wang L. Hosoya N. Yuji K. Imai Y. Takeuchi K. Miyawaki S. Hirai H. Blood. 2002; 99: 3075-3077Google Scholar). Chk2 plays a role in maintenance of G2 arrest after exposure to IR, with Cdc25C being a likely effector in this pathway (3Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Google Scholar,7Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Google Scholar). Since Cdc25C knockout mice have an intact G2 checkpoint arrest (21Chen M.S. Hurov J. White L.S. Woodford-Thomas T. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 3853-3861Google Scholar), 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 (22Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Google Scholar). DNA damage induces Rad53-dependent phosphorylation of Cdc5 (23Cheng L. Hunke L. Hardy C.F. Mol. Cell. Biol. 1998; 18: 7360-7370Google Scholar). Rad53 and Cdc5 regulate mitotic exit through independent modifications of Bfa1 (24Hu F. Wang Y. Liu D. Li Y. Qin J. Elledge S.J. Cell. 2001; 107: 655-665Google Scholar). 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. 25Nigg E.A. Curr. Opin. Cell Biol. 1998; 10: 776-783Google Scholar). Plk1 regulates many aspects of mitosis, including centrosome maturation and orientation, chromosome adhesion, mitotic entry and exit (reviewed in Ref. 26Donaldson M.M. Tavares A.A. Hagan I.M. Nigg E.A. Glover D.M. J. Cell Sci. 2001; 114: 2357-2358Google Scholar). Also, Plk1 is a target of the DNA damage checkpoint and is inhibited in an ATM-dependent manner after DNA damage (27Smits V.A. Klompmaker R. Arnaud L. Rijksen G. Nigg E.A. Medema R.H. Nat. Cell Biol. 2000; 2: 672-676Google Scholar,28van Vugt M.A. Smits V.A. Klompmaker R. Medema R.H. J. Biol. Chem. 2001; 276: 41656-41660Google Scholar). 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. 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. Rabbit polyclonal anti-phospho-Thr-68-Chk2 previously described (29Xu X. Tsvetkov L.M. Stern D.F. Mol. Cell. Biol. 2002; 22: 4419-4432Google Scholar) was affinity-purified by phosphopeptide affinity chromatography. Rabbit polyclonal anti-Chk2 antibody was produced by immunization with recombinant GST-Chk2 produced inEscherichia 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-γ-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. pcDNA-HAChk2 and pcDNA-HAChk2T68A were previously described (29Xu X. Tsvetkov L.M. Stern D.F. Mol. Cell. Biol. 2002; 22: 4419-4432Google Scholar). 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 (29Xu X. Tsvetkov L.M. Stern D.F. Mol. Cell. Biol. 2002; 22: 4419-4432Google Scholar) were used for protein expression in E. coli. HEK 293-T cells were washed in PBS and solubilized in lysis buffer (50 mmTris-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 ECLTMchemiluminescence reagents (Amersham Biosciences). 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-γ-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 λ-phosphatase (New England Biolabs) for 20 min at 30 °C. After treatment, cells were washed in PBS and fixed as in the basic protocol. 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 α-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 [γ-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). 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 (30Navas T.A. Sanchez Y. Elledge S.J. Genes Dev. 1996; 10: 2632-2643Google Scholar, 31Santocanale C. Diffley J.F. Nature. 1998; 395: 615-618Google Scholar). 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 (32Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Google Scholar, 33Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Google Scholar, 34Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Google Scholar). 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 (32Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Google Scholar, 33Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Google Scholar, 34Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Google Scholar, 35Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936Google Scholar). Atm and Atr have different preferences for phosphorylation within the SCD in vitro (34Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Google Scholar). The predominant site phosphorylated in response to double-stranded breaks is Thr-68, which is required for intact responses to DNA damage (33Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Google Scholar). 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 (34Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Google Scholar). In order to localize forms of Chk2 phosphorylated at this site, we produced and affinity-purified a phosphospecific antibody, α-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, α-PThr-68-Chk2 recognized endogenous Chk2 (lower band, Fig.1 A, lanes 2 and4) and transiently expressed HA-Chk2 (upper band, Fig. 1 A, lane 2). However, this antibody did not recognize transiently expressed HA-Chk2T68A, in which the target phosphorylation site has been replaced with A (Fig. 1 A,lane 4). In addition, the α-PThr-68-Chk2 antibody recognized endogenous Chk2 as a single band that increased in intensity with escalating radiation dose, while α-Chk2 antibody detected approximately equal amounts of Chk2 protein in all samples (Fig.1 B). Recombinant Chk2 undergoes autophosphorylation at multiple sites when expressed in bacteria (29Xu X. Tsvetkov L.M. Stern D.F. Mol. Cell. Biol. 2002; 22: 4419-4432Google Scholar). α-PThr-68-Chk2 recognized GST-Chk2-WT, but not GST-Chk2D368A, which has a mutation that eliminates kinase catalytic activity (Fig. 1 C). DNA damage induces phosphorylation of Chk2 at Thr-68 in nuclear foci at the sites of DNA damage (1Ward I.M. Wu X. Chen J. J. Biol. Chem. 2001; 276: 47755-47758Google Scholar). To determine if α-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. 1 D). IF of untreated 293 cells with α-PThr-68-Chk2 antibody produced a staining pattern characteristic of proteins associated with centrosomes (Fig.2 A). 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. 2 A). Similar staining patterns with α-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. 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 α-PThr-68-Chk2 eliminated the IF signal (Fig. 2 D). γ-tubulin nucleates microtubule assembly and is concentrated at centrosomes (36Schiebel E. Curr. Opin. Cell Biol. 2000; 12: 113-118Google Scholar). To confirm the localization of Chk2 to the centrosome, we performed double IF using α-PThr-68-Chk2 and αγ-tubulin antibodies in U2-OS cells (Fig. 2 B). The PThr-68-Chk2 and γ-tubulin fluorescence signals overlapped at the strong, centrosome-like foci seen with α-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 γ-tubulin signal was not competed with the PThr-68-containing phosphopeptide (Fig. 2 D). In order to verify that the α-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 λ-phosphatase. Phosphatase treatment eliminated the PThr-68-Chk2 signal, but the γ-tubulin signal remained although slightly reduced (Fig.2 E). Incubation with buffer did not change either γ-tubulin or PThr-68-Chk2 signals (data not shown). Two additional Chk2-reactive antibodies, αChk2, which should recognize all forms of Chk2, and α-PThr-26/PSer-28-Chk2, were tested. Anti-PThr-26/PSer-28-Chk2 should recognize additional Chk2 regulatory sites in the SCD (34Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Google Scholar). 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 (34Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Google Scholar). Treatment of 293 and U2OS cells with IR, UV, or hydroxyurea (HU)-induced reactivity of exogenously expressed HA-Chk2 with α-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, α-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 α-PThr-26/PSer-28-Chk2 in U2OS cells yielded a centrosome-like pattern (Fig. 2 C). In contrast, αChk2 detected a diffuse nuclear and cytoplasmic localization that overlapped with focal α-PThr-68-Chk2 fluorescence at centrosomes (Fig.3 A). In addition to centrosome-associated staining, we detected phosphorylated Chk2 with α-PThr-68-Chk2 and α-PThr-26/PSer-28-Chk2 antibodies at the midbody, the central part of the cytokinetic bridge, in telophase (Fig. 2 C). 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 γ-tubulin (Fig. 3 B). Under these conditions, αHA and α-PThr-68-Chk2 signals overlapped, including at the centrosomes (Fig. 3 C). Staining for HA-Chk2 with two different αHA antibodies yielded the same localization pattern (data not shown). On the basis of localization of Chk2 and Plk1 and suggested communication between their signaling pathways (22Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Google Scholar, 24Hu F. Wang Y. Liu D. Li Y. Qin J. Elledge S.J. Cell. 2001; 107: 655-665Google Scholar, 37Toczyski D.P. Galgoczy D.J. Hartwell L.H. Cell. 1997; 90: 1097-1106Google Scholar, 38Pellicioli A. Lee S.E. Lucca C. Foiani M. Haber J.E. Mol. Cell. 2001; 7: 293-300Google Scholar), we determined whether Chk2 and Plk1 physically interact. HA-tagged Chk2 and FLAG-tagged Plk1 coimmunoprecipitated when transiently expressed in 293T cells (Fig.4 A, lanes 3 and6), and endogenous Chk2 and Plk1 co-immunoprecipitated from 293T cells (Fig. 4 B, 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. 4 B, lanes 9 and 10) (reviewed in Ref.25Nigg E.A. Curr. Opin. Cell Biol. 1998; 10: 776-783Google Scholar). The Chk2/Plk1 interaction was not affected by Adriamycin (doxorubicin)-induced DNA damage (Fig. 4 B, lane 4). However, Adriamycin induced the Chk2 mobility shift associated with phosphorylation (Fig. 4 B, lanes 2 and 4), and inhibited in vitro Plk1 kinase activity toward α-casein (Fig. 4 B, line 10). 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 α-PThr-68-Chk2 and α-PThr-26/PSer-28-Chk2 (Fig. 5 B). 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.5 B). 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 (39Mundt K.E. Golsteyn R.M. Lane H.A. Nigg E.A. Biochem. Biophys. Res. Commun. 1997; 239: 377-385Google Scholar, 40Jang Y.J. Ma S. Terada Y. Erikson R.L. J. Biol. Chem. 2002; Google Scholar, 41Jang Y.J. Lin C.Y. Ma S. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1984-1989Google Scholar, 42Seong Y.S. Kamijo K. Lee J.S. Fernandez E. Kuriyama R. Miki T. Lee K.S. J. Biol. Chem. 2002; 28: 28Google Scholar) (Fig. 5 A, 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. 5 A, lanes 3 and7) (39Mundt K.E. Golsteyn R.M. Lane H.A. Nigg E.A. Biochem. Biophys. Res. Commun. 1997; 239: 377-385Google Scholar, 40Jang Y.J. Ma S. Terada Y. Erikson R.L. J. Biol. Chem. 2002; Google Scholar, 41Jang Y.J. Lin C.Y. Ma S. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1984-1989Google Scholar, 42Seong Y.S. Kamijo K. Lee J.S. Fernandez E. Kuriyama R. Miki T. Lee K.S. J. Biol. Chem. 2002; 28: 28Google Scholar). 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 (39Mundt K.E. Golsteyn R.M. Lane H.A. Nigg E.A. Biochem. Biophys. Res. Commun. 1997; 239: 377-385Google Scholar, 40Jang Y.J. Ma S. Terada Y. Erikson R.L. J. Biol. Chem. 2002; Google Scholar, 41Jang Y.J. Lin C.Y. Ma S. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1984-1989Google Scholar, 42Seong Y.S. Kamijo K. Lee J.S. Fernandez E. Kuriyama R. Miki T. Lee K.S. J. Biol. Chem. 2002; 28: 28Google Scholar). 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. 5 A, 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. 5 A, lanes 3 and7). 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. 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). Plk1 is associated with the kinetochores of condensed chromosomes, the centrosome in prophase and metaphase, and the midbody, later in mitosis (43Arnaud L. Pines J. Nigg E.A. Chromosoma. 1998; 107: 424-429Google Scholar, 44Golsteyn R.M. Mundt K.E. Fry A.M. Ni
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