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Autophosphorylation of Checkpoint Kinase 2 at Serine 516 Is Required for Radiation-induced Apoptosis

2003; Elsevier BV; Volume: 278; Issue: 38 Linguagem: Inglês

10.1074/jbc.m303795200

ISSN

1083-351X

Autores

Xianglin Wu, Junjie Chen,

Tópico(s)

Microtubule and mitosis dynamics

Resumo

In response to ionizing radiation, checkpoint kinase 2 (Chk2) is activated in an ataxia telangiectasia mutation-dependent manner and induces either cell cycle arrest or apoptosis. Chk2 is also autophosphorylated following DNA damage. It is proposed that autophosphorylation of Chk2 may contribute to Chk2 activation. To fully understand the regulation of Chk2, we mapped an in vitro Chk2 autophosphorylation site at C-terminal serine 516 site (Ser-516). Ser-516 of Chk2 is phosphorylated following radiation in vivo, and this phosphorylation depends on the kinase activity of Chk2. Mutation of this autophosphorylation site (S516A) results in reduced Chk2 kinase activity, suggesting that Chk2 autophosphorylation is required for full kinase activation following DNA damage. Moreover, the S516A mutant of Chk2 is defective in ionizing radiation-induced apoptosis, suggesting that Chk2 autophosphorylation is critical for Chk2 function following DNA damage. In response to ionizing radiation, checkpoint kinase 2 (Chk2) is activated in an ataxia telangiectasia mutation-dependent manner and induces either cell cycle arrest or apoptosis. Chk2 is also autophosphorylated following DNA damage. It is proposed that autophosphorylation of Chk2 may contribute to Chk2 activation. To fully understand the regulation of Chk2, we mapped an in vitro Chk2 autophosphorylation site at C-terminal serine 516 site (Ser-516). Ser-516 of Chk2 is phosphorylated following radiation in vivo, and this phosphorylation depends on the kinase activity of Chk2. Mutation of this autophosphorylation site (S516A) results in reduced Chk2 kinase activity, suggesting that Chk2 autophosphorylation is required for full kinase activation following DNA damage. Moreover, the S516A mutant of Chk2 is defective in ionizing radiation-induced apoptosis, suggesting that Chk2 autophosphorylation is critical for Chk2 function following DNA damage. The maintenance of genome stability relies on the efficient detection of DNA lesions, the rapid transmission of DNA damage signals, and DNA repair. DNA damage signals lead to either cell cycle arrest or apoptosis. Cell cycle arrest in response to DNA damage allows time for DNA repair. On the other hand, if cells fail to repair, they undergo apoptosis. If cells cannot properly respond to DNA damage signals, damaged DNA will be passed onto the daughter cells and the accumulation of mutations will lead to tumorigenesis (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2596) Google Scholar, 2Rouse J. Jackson S.P. Science. 2002; 297: 547-551Crossref PubMed Scopus (555) Google Scholar, 3Meyn M.S. Cancer Res. 1995; 55: 5991-6001PubMed Google Scholar, 4Curman D. Cinel B. Williams D.E. Rundle N. Block W.D. Goodarzi A.A. Hutchins J.R. Clarke P.R. Zhou B.B. Lees-Miller S.P. Andersen R.J. Roberge M. J. Biol. Chem. 2001; 276: 17914-17919Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Thus, the integrity of DNA damage-signaling pathways is critical for tumor prevention. Ataxia telangiectasia mutation (ATM) 1The abbreviations used are: ATM, ataxia telangiectasia mutation; IR, ionizing radiation; Chk2, checkpoint kinase 2; HA, hemagglutinin; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; FHA, forkhead-associated.1The abbreviations used are: ATM, ataxia telangiectasia mutation; IR, ionizing radiation; Chk2, checkpoint kinase 2; HA, hemagglutinin; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; FHA, forkhead-associated. plays a critical role in cellular responses to DNA damage (2Rouse J. Jackson S.P. Science. 2002; 297: 547-551Crossref PubMed Scopus (555) Google Scholar). Following DNA damage, ATM is activated and phosphorylates a number of downstream effectors to enforce checkpoint controls (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2596) Google Scholar, 5Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar). One critical substrate is checkpoint kinase 2 (Chk2), a downstream regulator of ATM (6Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1070) Google Scholar). After DNA damage, Chk2 is directly phosphorylated and activated by ATM (5Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar, 6Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1070) Google Scholar, 7Chaturvedi 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: 4047-4054Crossref PubMed Scopus (358) Google Scholar, 8Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (678) Google Scholar). Chk2 may participate in multiple ATM-dependent pathways. For example, Chk2 can phosphorylate p53 at Ser-20 in vitro, and this phosphorylation may be required for the stabilization of p53 and G1 arrest following DNA damage (9Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288PubMed Google Scholar, 10Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300PubMed Google Scholar, 11Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1032) Google Scholar). However, it is also reported that Chk2 is dispensable for p53-mediated G1 arrest (12Jack M.T. Woo R.A. Hirao A. Cheung A. Mak T.W. Lee P.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9825-9829Crossref PubMed Scopus (101) Google Scholar). In addition, Chk2 could regulate the S phase checkpoint by phosphorylating Cdc25A (13Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (860) Google Scholar, 14Falck J. Petrini J.H. Williams B.R. Lukas J. Bartek J. Nat. Genet. 2002; 30: 290-294Crossref PubMed Scopus (314) Google Scholar) or the G2/M transition by regulating Cdc25C (6Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1070) Google Scholar, 15Blasina A. Price B.D. Turenne G.A. McGowan C.H. Curr. Biol. 1999; 9: 1135-1138Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). However, more recent studies using Chk2 knockout mice showed normal S phase and G2/M transition in Chk2-deficient cells (11Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1032) Google Scholar, 16Takai H. Naka K. Okada Y. Watanabe M. Harada N. Saito S. Anderson C.W. Appella E. Nakanishi M. Suzuki H. Nagashima K. Sawa H. Ikeda K. Motoyama N. EMBO J. 2002; 21: 5195-5205Crossref PubMed Scopus (345) Google Scholar). Thus, it remains to be resolved whether Chk2 is involved in certain aspects of the ATM-dependent DNA damage checkpoint controls. The role of Chk2 in the ionizing radiation (IR)-induced apoptosis has been established recently by several groups (12Jack M.T. Woo R.A. Hirao A. Cheung A. Mak T.W. Lee P.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9825-9829Crossref PubMed Scopus (101) Google Scholar, 16Takai H. Naka K. Okada Y. Watanabe M. Harada N. Saito S. Anderson C.W. Appella E. Nakanishi M. Suzuki H. Nagashima K. Sawa H. Ikeda K. Motoyama N. EMBO J. 2002; 21: 5195-5205Crossref PubMed Scopus (345) Google Scholar, 17Xu J. Xin S. Du W. FEBS Lett. 2001; 508: 394-398Crossref PubMed Scopus (65) Google Scholar, 18Peters M. DeLuca C. Hirao A. Stambolic V. Potter J. Zhou L. Liepa J. Snow B. Arya S. Wong J. Bouchard D. Binari R. Manoukian A.S. Mak T.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11305-11310Crossref PubMed Scopus (76) Google Scholar, 19Hirao A. Cheung A. Duncan G. Girard P.M. Elia A.J. Wakeham A. Okada H. Sarkissian T. Wong J.A. Sakai T. De Stanchina E. Bristow R.G. Suda T. Lowe S.W. Jeggo P.A. Elledge S.J. Mak T.W. Mol. Cell. Biol. 2002; 22: 6521-6532Crossref PubMed Scopus (314) Google Scholar, 20Yang S. Kuo C. Bisi J.E. Kim M.K. Nat Cell Biol. 2002; 28: 28Google Scholar). It appears that Chk2 can regulate IR-induced apoptosis through p53-dependent (12Jack M.T. Woo R.A. Hirao A. Cheung A. Mak T.W. Lee P.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9825-9829Crossref PubMed Scopus (101) Google Scholar) or p53-independent (18Peters M. DeLuca C. Hirao A. Stambolic V. Potter J. Zhou L. Liepa J. Snow B. Arya S. Wong J. Bouchard D. Binari R. Manoukian A.S. Mak T.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11305-11310Crossref PubMed Scopus (76) Google Scholar, 20Yang S. Kuo C. Bisi J.E. Kim M.K. Nat Cell Biol. 2002; 28: 28Google Scholar) pathways. The exact mechanism of the Chk2-dependent apoptosis pathway has not yet been fully understood. Because Chk2 is a DNA damage-activated protein kinase, the function of Chk2 depends on its ability to phosphorylate downstream substrates after DNA damage (21McGowan C.H. Bioessays. 2002; 24: 502-511Crossref PubMed Scopus (85) Google Scholar, 22Dasika G.K. Lin S.C. Zhao S. Sung P. Tomkinson A. Lee E.Y. Oncogene. 1999; 18: 7883-7899Crossref PubMed Scopus (348) Google Scholar). The regulation of Chk2 activation following IR has been well studied. In response to IR, Chk2 is phosphorylated at Thr-68 by ATM (5Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar, 7Chaturvedi 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: 4047-4054Crossref PubMed Scopus (358) Google Scholar, 8Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (678) Google Scholar). The initial phosphorylation of Chk2 at the Thr-68 site can lead to autophosphorylation of Chk2 at Thr-383 and Thr-387, two sites within the activation loop of Chk2 kinase domain (23Lee C.H. Chung J.H. J. Biol. Chem. 2001; 276: 30537-30541Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Mutation of these two residues (Thr to Ala) abolishes Chk2 activation (23Lee C.H. Chung J.H. J. Biol. Chem. 2001; 276: 30537-30541Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), indicating that autophosphorylation of Chk2 may directly affect Chk2 kinase activity. In addition, the phosphorylated Thr-68 site of Chk2 also interacts with the FHA domain of another Chk2 molecule, and thus leads to the formation of Chk2 oligomers (23Lee C.H. Chung J.H. J. Biol. Chem. 2001; 276: 30537-30541Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 24Xu X. Tsvetkov L.M. Stern D.F. Mol. Cell. Biol. 2002; 22: 4419-4432Crossref PubMed Scopus (156) Google Scholar, 25Ahn J.Y. Li X. Davis H.L. Canman C.E. J. Biol. Chem. 2002; 277: 19389-19395Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Chk2 oligomerization may further regulate Chk2 activation, because Chk2 can also autophosphorylate its FHA domain in vitro and these phosphorylation events at the FHA domain lead to dissociation of Chk2 oligomers in vitro (24Xu X. Tsvetkov L.M. Stern D.F. Mol. Cell. Biol. 2002; 22: 4419-4432Crossref PubMed Scopus (156) Google Scholar, 25Ahn J.Y. Li X. Davis H.L. Canman C.E. J. Biol. Chem. 2002; 277: 19389-19395Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). However, the functional significance of these proposed regulations of Chk2 remains to be tested in vivo. To fully understand the complexity of Chk2 regulation, we have mapped an additional Chk2 autophosphorylation site (Ser-516). We have shown that Ser-516 phosphorylation is required for the optimal activation of Chk2 and IR-induced apoptosis in vivo, providing a link between the regulation of Chk2 kinase activity and the function of Chk2 following DNA damage. Constructs—Mammalian expression plasmid encoding HA-tagged Chk2 was described earlier (26Wu X. Webster S.R. Chen J. J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). QuickChange site-directed mutagenesis kit (Strategene) was used to introduce point mutations in the Chk2 coding sequence for the generation of expression constructs encoding Chk2 kinase inactive, S516A, T383A, and T68A mutants. Wild-type or various mutant Chk2 fragments containing Chk2 residues 477–543 were subcloned into the pGEX 5x-3 vector for the expression of these fragments as glutathione S-transferase (GST) fusion proteins in Escherichia coli. These Chk2 fragments were used to map the Chk2 autophosphorylation site at the C terminus. GST-Cdc25C, containing the C-terminal fragment (residues 200–256) of Cdc25C, was used as a Chk2 substrate (27Ward I.M. Wu X. Chen J. J. Biol. Chem. 2001; 276: 47755-47758Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Cell Lines and Culture Conditions—All cell lines were obtained from American Tissue Culture Collection and cultivated in RPMI 1640 supplemented with 10% fetal bovine serum. To establish stable cell lines expressing HA-tagged wild-type or mutant Chk2, HCT15 cells were transfected with plasmids encoding the indicated HA-tagged wild-type or mutant Chk2. G418-resistant clones were isolated, and exogenous Chk2 expression was confirmed by Western blotting using anti-HA or anti-Chk2 antibody. Clones that have similar expression levels of Chk2 were used in this study. The stable cell lines are cultivated in RPMI 1640 supplemented with 10% fetal bovine serum plus 200 μg/ml G418. Sf9 insect cells were cultivated in Grace's insect media supplemented with 10% fetal bovine serum at 28 °C. Antibodies—The generation of anti-Chk2 (number 7) monoclonal antibody and anti-Chk2pT68 antibody were reported earlier (27Ward I.M. Wu X. Chen J. J. Biol. Chem. 2001; 276: 47755-47758Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). To generate phospho-specific antibodies against Chk2 Ser-516 site, Ser(P)-516 (pS516) peptide (CVLAQP(p)STSRKR) was coupled to keyhole limpet hemocyanin (Pierce) and used as antigen to immunize rabbits. Anti-Ser(P)-516 antibody was affinity-purified as described previously (27Ward I.M. Wu X. Chen J. J. Biol. Chem. 2001; 276: 47755-47758Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Anti-Chk2 pT383pT387 antibody is a gift from Dr. Jay H. Chung. In Vitro Chk2 Kinase Assay—Active GST-Chk2 isolated from SF9 cells was used as kinase and incubated with substrates for 30 min at 30 °C in 30 μl of kinase buffer (50 mm Tris, pH 7.5, 10 mm MgCl2, with 10 μm ATP and 10 μCi of [γ-32P]ATP). The reactions were stopped by the addition of 30 μl of 2× Laemmli's SDS sample buffer. Proteins were separated by 12.5% SDS-PAGE, transferred onto polyvinylidene difluoride membrane. 32P incorporation in substrates was visualized by autoradiography. Mapping Chk2 Autophosphorylation Site—HA-tagged Chk2 was immunoprecipitated from extracts of HCT15 cells stably expressing HA-tagged wild-type Chk2 or S516A mutant. In vitro autophosphorylation reactions were performed to label Chk2 with 32P radioisotope. Proteins were separated on 7.5% SDS-PAGE, and gel slices corresponding to Chk2 were excised. In-gel digestion with trypsin, peptide extraction, and HPLC separation were performed by the Mayo Clinic protein core facility. Phospho-peptide spotted on the paper was visualized by autoradiography and corresponding peptides were sequenced by Edman degradation. Dephosphorylation of Chk2—293T cells were radiated. Cells were harvested 1 h after IR.Chk2 was precipitated with anti-Chk2A antibody. Precipitated Chk2 was treated with Lambda phophatase (BioLab), mixture of Lambda phosphatase inhibitor (1 mm NaF, 1 mm sodium vanadate, 25 mm β-glycerophophate, and 20 nm microcystin) and Lambda phosphatase, respectively. Western blots were done with anti-Chk2 and anti-pS516. Radiation-induced Apoptosis—To assay radiation-induced apoptosis, HCT116, HCT116 with Chk2-/- (28Jallepalli P.V. Lengauer C. Vogelstein B. Bunz F. J. Biol. Chem. 2003; 278: 20475-20479Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), HCT15, and HCT15 cell line expressing wild-type or S516A mutant of Chk2 were plated in 6-cm culture plates. 28 or 36 h after 10 or 30 Gy of radiation, cells were trypsinized, washed once with 1× phosphate-buffered saline with 1% bovine serum albumin, and fixed in methanol/acetic acid solution (3:1) for 1 h. Fixed cells were dropped onto the glass slides, and the slides were dried. Cells were stained with Hoechst solution, and the numbers of cells with fragmented nuclei were counted under the microscope. For TUNEL assay, cells were fixed in 1% formaldehyde in phosphatebuffered saline on ice for 15 min, washed once with phosphate-buffered saline, and resuspended in 50 μl of reaction buffer (10 μl of 5× buffer, 0.04 μm of BrdUrdUTP, 0.5 μl of TdT). The reactions were carried out at 37 °C for 40 min. After that, cells were washed with rinsing buffer and then incubated with fluorescein isothiocyanate-conjugated anti-BrdUrd mAb for 30 min at 22 °C. Fluorescein isothiocyanate-positive cells are counted as apoptotic cells. Chk2 Oligomerization Assay—GST-Chk2 FHA fragment (containing residues 61–226) generated in E. coli cells were used to pull down Chk2 from whole cell extract as indicated. Proteins bound to GST-Chk2 FHA domain were eluted and separated on SDS-PAGE. Western blot was performed using anti-HA or anti-Chk2 antibodies to determine the oligomerization of Chk2. Autophosphorylation Site at the Chk2 C Terminus—Autophosphorylation of Chk2 increases following DNA damage (see Ref. 6Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1070) Google Scholar and Fig. 1A). To determine Chk2 autophosphorylation sites, we generated stable cell lines expressing HA-tagged Chk2 in Chk2-deficient HCT15 cells. Chk2 was immunoprecipitated from control and IR-treated HCT15 cells expressing wild-type or kinase-dead Chk2. As shown in Fig. 1A, the kinase activity of Chk2 increased following DNA damage. The phosphorylation of Chk2 in vitro depends on the kinase activity of Chk2, suggesting that this phosphorylation is due to autophosphorylation of Chk2, rather than a contaminating kinase(s). To determine the autophosphorylation sites of Chk2, the 32P-labeled Chk2 bands were excised, and in-gel trypsinization, extraction, and HPLC separation were performed. The HPLC fractions were spotted on a polyvinylidene difluoride membrane, and radiolabeled phospho-peptide was visualized by autoradiography. Only one major spot was found in samples derived from wild-type Chk2 (Fig. 1B). The radio-labeled peptide was sequenced by Edman degradation (Fig. 1B), and was found to correspond to the C terminus of Chk2 (495KFQDLLSEE NESTALPQVLAQPSTSR519) Chk2 Is Autophosphorylated in Vitro at Ser-516 —Due to the limited amount of Chk2 we were able to purify from the cell, we could not determine the phosphorylation sites within this peptide by phosphopeptide sequencing. There are 6 potential phosphorylation sites (serine or threonine) within the peptide we sequenced. To further map the autophosphorylation site(s) within this peptide, we generated GST fusion proteins containing the residues 477–543 of Chk2 as wild-type or mutant (Ser/Thr mutated to Ala) proteins (Fig. 1C). Only mutant 3 was not phosphorylated by Chk2 in vitro (Fig. 1D), suggesting that one or more of the sites mutated in mutant 3 is the autophosphorylation site. Further studies using single mutants of each potential phosphorylation site within mutant 3 demonstrated that Ser-516 is the only autophosphorylation site within this peptide (Fig. 1E). Ser-516 of Chk2 Is Phosphorylated in Vivo Following DNA Damage—To investigate whether Ser-516 is indeed phosphorylated in vivo, we generated a Ser-516 phospho-specific antibody. The phospho-specific antibody (pS516) recognized wildtype Chk2 only after DNA damage (Fig. 2A), and phosphatase-treatment abolished the ability of this antibody to recognize Chk2 (Fig. 2A). In addition, this phospho-specific antibody failed to recognize the Ser-516 to Ala mutant of Chk2 after IR (Fig. 3B), confirming the specificity of this pS516 phosphspecific antibody. Taken together, these data indicate that Chk2 is phosphorylated at Ser-516 in intact cells following DNA damage.Fig. 3Phosphorylation of Ser-516 site occurs late in the activation of Chk2 following DNA damage. A, the expression levels of wild-type or mutant Chk2 in HCT15 stable cell lines are similar to that of endogenous Chk2 in A549 cells. B, Thr-68 phosphorylation of Chk2 is required for phosphorylation of Chk2 at the Ser-516 site. HCT15 cells stably expressing wild-type (WT), Thr-68 to Ala (T68A), or Ser-516 to Ala (S516A) mutants of Chk2 were mock-treated or treated with 10 Gy of IR. Western blots were performed using anti-Chk2, anti-pT68, or pS516 phospho-specific antibodies. C, Chk2 Thr-383/387 sites are phosphorylated before the phosphorylation of Ser-516 site. Indicated cells are mocktreated or irradiated (10 Gy) and collected one hour later. Chk2 was immunoprecipitated and blotted with anti-Chk2, anti-pT383pT387, or anti-S516P antibodies. D, Chk2 S516A mutant does not affect Chk2 oligomerization. Extracts from indicated cell lines were prepared before and after radiation. Oligomerization of Chk2 was examined by GST-Chk2 FHA domain pull-down. The left panel indicates the equal expression of wild-type or mutant Chk2 in these HCT15 derivative cell lines.View Large Image Figure ViewerDownload (PPT) Ser-516 Is an in Vivo Autophosphorylation Site—To confirm that Ser-516 is an autophosphorylation site in vivo, we used Chk2-deficient HCT15 cells stably expressing wild-type or kinase-inactive Chk2. As shown in Fig. 2B, Ser-516 site was only phosphorylated in cells expressing wild-type Chk2 after DNA damage, but not in cells expressing a kinase-inactive form of Chk2, suggesting that indeed Ser-516 is an autophosphorylation site in vivo. Autophosphorylation of Chk2 at Ser-516 Correlates with Thr-68 Phosphorylation of Chk2 Following DNA Damage—It is not clear whether autophosphorylation of Chk2 at Ser-516 contribute to the regulation of Chk2 following DNA damage. We first examined whether there is any difference between Chk2 autophosphorylation and phosphorylation of Chk2 at Thr-68, an ATM-dependent phosphorylation site required for Chk2 activation. As shown in Fig. 2C, Ser-516 phosphorylation correlates with phosphorylation of Chk2 at Thr-68 following various doses of radiation. Time course studies indicates that the maximal autophosphorylation of Ser-516 occurs slightly later than Thr-68 phosphorylation (Fig. 2D), suggesting that Chk2 may be phosphorylated at Thr-68 before it can be autophosphorylated at Ser-516. Ser-516 Phosphorylation Occurs Late in the Activation of Chk2 Following DNA Damage—To examine the sequential regulation of various phosphorylation events on Chk2, we established HCT15 cell lines expressing various phosphorylation mutants of Chk2 (Fig. 3A and data not shown). The expression levels of these exogenous Chk2 in HCT15 cells are similar to that of endogenous Chk2 in A549 cells or other cell lines (Fig. 3A and data not shown). As shown in Fig. 3B, although we failed to detect Ser-516 phosphorylation in the Chk2T68A mutant, we readily detected Thr-68 phosphorylation in the Chk2 S516A mutant following DNA damage. Thus, it is likely that phosphorylation of Chk2 at Ser-516 requires initial Thr-68 phosphorylation in vivo. Besides Thr-68, Thr-383 and Thr-387 have recently been identified as Chk2 autophosphorylation sites (23Lee C.H. Chung J.H. J. Biol. Chem. 2001; 276: 30537-30541Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). These phosphorylation events occur at the Chk2 activation loop and are required for the activation of Chk2 (23Lee C.H. Chung J.H. J. Biol. Chem. 2001; 276: 30537-30541Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). As shown in Fig. 3C, we observed that the Thr-383/T387 sites were phosphorylated following DNA damage in S516A mutant, suggesting that Ser-516 phosphorylation is not required for Chk2 phosphorylation at the activation loop. In agreement with that Ser-516 phosphorylation requires Chk2 kinase activity (Fig. 2B), we failed to detect Ser-516 phosphorylation in Chk2T383A mutant (Fig. 3C), because this mutant abolished Chk2 kinase activity in vivo (data not shown). These data suggest that Thr-383/387 phosphorylation occurs before Ser-516 phosphorylation following DNA damage. Chk2 oligomerizaton has recently been proposed to be important for Chk2 activation following DNA damage. The oligomerization of Chk2 is mediated through the interaction between Chk2 FHA domain and the Thr-68 phosphorylation site of Chk2. Both S516A and T383A mutants can still be phosphorylated by ATM at the Thr-68 site (Fig. 3B and data not shown). As shown in Fig. 3D, both mutants still retain the ability to form oligomers following DNA damage. Taken together, these data suggest the following steps in Chk2 activation following DNA damage. First, Chk2 is phosphorylated by ATM at the Thr-68 site. This initial phosphorylation event leads to the oligomerization of Chk2 and the phosphorylation of Chk2 at the activation loop (Thr-383/387). Thr-383/387 phosphorylation increases Chk2 kinase activity and further phosphorylates Chk2 at Ser-516 site. Autophosphorylation at Ser-516 Is Required for Optimal Activation of Chk2 Following IR—To investigate the role of Ser-516 autophosphorylation in Chk2 activation, we used HCT15 cells expressing wild-type or the Ser-516 Ala mutant (S516A) of Chk2. Ectopically expressed wild-type and mutant Chk2 were immunoprecipitated before and after DNA damage. In vitro Chk2 kinase assays were performed to compare the activity of S516A mutant with that of wild-type Chk2. As shown in Fig. 4, the kinase activity of S516A mutant, as determined by its ability to phosphorylate Cdc25C, was decreased compared with that of wild-type Chk2. Densitometry analysis indicated that the kinase activity of Chk2 S516A mutant was decreased by about 50%. Interestingly, the basal activity of Chk2 S516A mutant was also reduced (Fig. 4). There was also reduced, but not absent, Chk2 autophosphorylation in the S516A mutant, suggesting the existence of additional Chk2 autophosphorylation sites (for example Thr-383/387 sites) as suggested by early studies (23Lee C.H. Chung J.H. J. Biol. Chem. 2001; 276: 30537-30541Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Autophosphorylation at Ser-516 Is Required for IR-induced Apoptosis—Recent studies suggest that Chk2 regulates ionizing radiation (IR)-induced apoptosis (12Jack M.T. Woo R.A. Hirao A. Cheung A. Mak T.W. Lee P.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9825-9829Crossref PubMed Scopus (101) Google Scholar, 17Xu J. Xin S. Du W. FEBS Lett. 2001; 508: 394-398Crossref PubMed Scopus (65) Google Scholar, 18Peters M. DeLuca C. Hirao A. Stambolic V. Potter J. Zhou L. Liepa J. Snow B. Arya S. Wong J. Bouchard D. Binari R. Manoukian A.S. Mak T.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11305-11310Crossref PubMed Scopus (76) Google Scholar, 19Hirao A. Cheung A. Duncan G. Girard P.M. Elia A.J. Wakeham A. Okada H. Sarkissian T. Wong J.A. Sakai T. De Stanchina E. Bristow R.G. Suda T. Lowe S.W. Jeggo P.A. Elledge S.J. Mak T.W. Mol. Cell. Biol. 2002; 22: 6521-6532Crossref PubMed Scopus (314) Google Scholar). Indeed, we found increased apoptosis in HCT116 cells that contain wildtype Chk2, but not in HCT116 Chk2-/- cells (Fig. 5A) (28Jallepalli P.V. Lengauer C. Vogelstein B. Bunz F. J. Biol. Chem. 2003; 278: 20475-20479Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). This suggests that Chk2 is involved in IR-induced apoptosis in human cells. To test whether Ser-516 phosphorylation is required for Chk2 function in IR-induced apoptosis, we again used HCT15 cell lines stably expressing wild-type or mutant Chk2. As shown in Fig. 5B, increased apoptosis following IR was observed in cells expressing wild-type Chk2 but not in parental HCT15 cells, confirming that Chk2 is required for IR-induced apoptosis. Mutation of S516A in Chk2 greatly reduced apoptosis following IR (Fig. 5, B and C). TUNEL assays using three independent S516A mutant cell lines were performed to verify that the S516A mutant is defective in IR-induced apoptosis (Fig. 5C). Taken together, these data suggest that autophosphorylation of Chk2 at Ser-516 is critical for its function following IR. In this study, we have identified Ser-516 as a new Chk2 autophosphorylation site. Ser-516 is phosphorylated in vivo after the initial activation of Chk2 by Thr-68 and Thr-383/387 phosphorylation. Mutation of Chk2 at Ser-516 reduces Chk2 kinase activity and abolishes the IR-induced apoptosis, suggesting that autophosphorylation of Chk2 at Ser-516 is required for full activation of Chk2 and Chk2 function following DNA damage. Using in-gel digestion and HPLC purification, we have only mapped one of the autophosphorylation sites (Ser-516) on Chk2. There are apparently additional Chk2 autophosphorylation sites (see Fig. 4). Two of them (Thr-383 and Thr-387), reside at the activation loop of Chk2 kinase domain, have been reported earlier (23Lee C.H. Chung J.H. J. Biol. Chem. 2001; 276: 30537-30541Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Mutations of Thr-383 and Thr-387 to Ala abolished Chk2 activity (23Lee C.H. Chung J.H. J. Biol. Chem. 2001; 276: 30537-30541Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), suggesting that these two autophosphorylation sites are crucial for Chk2 activation. Thus, it appears that different autophosphorylation sites on Chk2 regulate distinct steps in Chk2 activation. Indeed, a complex picture of Chk2 activation following DNA damage is emerging. After DNA damage, phosphorylation of Chk2 at Thr-68 by ATM leads to the initial oligomerization of Chk2, mediated by the interaction between the Chk2 Thr68 phosphorylation site and the Chk2 FHA domain (24Xu X. Tsvetkov L.M. Stern D.F. Mol. Cell. Biol. 2002; 22: 4419-4432Crossref PubMed Scopus (156) Google Scholar, 25Ahn J.Y. Li X. Davis H.L. Canman C.E. J. Biol. Chem. 2002; 277: 19389-19395Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 29Ahn J. Prives C. J. Biol. Chem. 2002; 16: 16Google Scholar). It is possible, although not yet proven, that the oligomerization of Chk2 may lead to autophosphorylation and full activation of Chk2. Our results presented here agree with this complex model of Chk2 activation following DNA damage. Despite intensive studies in the last few years, the exact function of Chk2 in DNA damage responses is not fully understood. What is the function of Chk2? Recent studies suggest that Chk2 may regulate IR-induced apoptosis (12Jack M.T. Woo R.A. Hirao A. Cheung A. Mak T.W. Lee P.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9825-9829Crossref PubMed Scopus (101) Google Scholar, 16Takai H. Naka K. Okada Y. Watanabe M. Harada N. Saito S. Anderson C.W. Appella E. Nakanishi M. Suzuki H. Nagashima K. Sawa H. Ikeda K. Motoyama N. EMBO J. 2002; 21: 5195-5205Crossref PubMed Scopus (345) Google Scholar, 17Xu J. Xin S. Du W. FEBS Lett. 2001; 508: 394-398Crossref PubMed Scopus (65) Google Scholar, 18Peters M. DeLuca C. Hirao A. Stambolic V. Potter J. Zhou L. Liepa J. Snow B. Arya S. Wong J. Bouchard D. Binari R. Manoukian A.S. Mak T.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11305-11310Crossref PubMed Scopus (76) Google Scholar, 19Hirao A. Cheung A. Duncan G. Girard P.M. Elia A.J. Wakeham A. Okada H. Sarkissian T. Wong J.A. Sakai T. De Stanchina E. Bristow R.G. Suda T. Lowe S.W. Jeggo P.A. Elledge S.J. Mak T.W. Mol. Cell. Biol. 2002; 22: 6521-6532Crossref PubMed Scopus (314) Google Scholar, 20Yang S. Kuo C. Bisi J.E. Kim M.K. Nat Cell Biol. 2002; 28: 28Google Scholar). Indeed, we observed IR-induced apoptosis in HCT116 cells containing wild-type Chk2, but not in HCT116-derived Chk2-/- cells. We also detected a 3-fold increase in apoptosis in wildtype mouse embryonic fibroblast but not in Chk2-deficient mouse embryonic fibroblast cells following radiation (data not shown). In addition, Chk2 is required for IR-induced apoptosis in HCT15-Chk2 wild-type cells but not in HCT15-Chk2 T68A cells, which do not have Chk2 kinase activity (30Lou Z. Minter-Dykhouse K. Wu X. Chen J. Nature. 2003; 421: 957-961Crossref PubMed Scopus (287) Google Scholar). All these studies suggest that Chk2 and Chk2 kinase activity are required for IR-induced apoptosis. Using HCT15 cells, we have shown that indeed autophosphorylation of Chk2 at Ser-516 is required for IR-induced apoptosis (Fig. 5, B and C). The IR-induced apoptosis observed in this study is mediated through a p53-independent pathway, because only mutant p53 exists in HCT15 cells (31Bell 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: 2528-2531Crossref PubMed Scopus (746) Google Scholar). Future studies will reveal, in detail, the regulation of Chk2-dependent apoptosis following DNA damage. We thank Drs. Larry Karnitz and Jann Sarkaria for stimulating conversation and members in Dr. Junjie Chen's laboratory for helpful discussions, and we also thank the protein facility core of the Mayo Clinic for sequencing phospho-peptides of Chk2. We also thank Drs. Bert Vogelstein and Fred Bunz for providing HCT116 and HCT116 Chk2-/- cell lines and Dr. Jay H. Chung for providing anti-pT383/pT387 phospho-specific antibody.

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