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• W2070632817 abstract "Recent evidence indicates that arrest of mammalian cells at the G2/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to γ-radiation. Prior treatment of cells with 2 mm caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity.In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC50 of approximately 200 μm. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G2/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells. Recent evidence indicates that arrest of mammalian cells at the G2/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to γ-radiation. Prior treatment of cells with 2 mm caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity.In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC50 of approximately 200 μm. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G2/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells. ionizing radiation ataxia-telangiectasia ataxia-telangiectasia-mutated polyacrylamide gel electrophoresis glutathione S-transferase phosphate-buffered saline bovine serum albumin kinase-dead gray Most effective anti-cancer therapies are genotoxic agents that damage DNA and kill dividing cells. In addition to apoptosis, DNA damage induced by ionizing radiation (IR)1 or other insults triggers cell cycle checkpoint activation and subsequent cell cycle arrest, augmenting the ability of cells to repair damaged DNA (1.O'Connor P.M. Cancer Surv. 1997; 29: 151-182PubMed Google Scholar, 2.Elledge S.J. Science. 1996; 274: 1664-1672Crossref PubMed Scopus (1761) Google Scholar). The checkpoint could contribute to the development of drug resistance, a formidable limitation in current cancer treatment, and consequently agents that override cell cycle checkpoints could be used to sensitize cells to killing by genotoxic drugs (3.Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2309) Google Scholar). Proof of this concept has arisen from studies with caffeine, which causes the disruption of DNA damage checkpoints (including the G2/M checkpoint), and sensitizes tumor cells to ionizing radiation and other genotoxic agents (4.Lau C.C. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2942-2946Crossref PubMed Scopus (283) Google Scholar, 5.Schlegel R. Pardee A.B. Science. 1986; 232: 1264-1266Crossref PubMed Scopus (267) Google Scholar). However, the mechanism of action of caffeine and its molecular targets remain unclear. One possible target is phosphodiesterase, but a more potent phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, is unable to abrogate checkpoint function in many systems (5.Schlegel R. Pardee A.B. Science. 1986; 232: 1264-1266Crossref PubMed Scopus (267) Google Scholar), suggesting that alternative targets exist for caffeine. For mammalian cells, arrest in G2 phase is due largely to maintenance of inhibitory phosphorylations on Cdc2 (6.Nurse P. Cell. 1997; 91: 865-867Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Dephosphorylation of Cdc2 at these sites is catalyzed by the dual specificity phosphatase Cdc25 (7.Dunphy W.G. Kumagai A. Cell. 1991; 67: 189-196Abstract Full Text PDF PubMed Scopus (446) Google Scholar, 8.Gautier J. Solomon M.J. Booher R.N. Bazan J.F. Kirschner M.W. Cell. 1991; 67: 197-211Abstract Full Text PDF PubMed Scopus (687) Google Scholar, 9.Strausfeld U. Labbe J.C. Fesquet D. Cavadore J.C. Picard A. Sadhu K. Russell P. Doree M. Nature. 1991; 351: 242-245Crossref PubMed Scopus (443) Google Scholar). Recent evidence indicates that the G2/M DNA checkpoint involves inactivation and possibly translocation of Cdc25C into the cytoplasm akin to what has been demonstrated for fission yeast Cdc25 (10.Lopez-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (505) Google Scholar, 11.Kumagai A. Dunphy W.G. Genes Dev. 1999; 13: 1067-1072Crossref PubMed Scopus (255) Google Scholar, 12.Yang J. Winkler K. Yoshida M. Kornbluth S. EMBO J. 1999; 18: 2174-2183Crossref PubMed Scopus (205) Google Scholar). This is at least partially mediated by phosphorylation at serine 216 in Cdc25C and is likely to involve binding of 14-3-3 proteins to this phosphorylation site (13.Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1187) Google Scholar, 14.Dalal S.N. Schweitzer C.M. Gan J. DeCaprio J.A. Mol. Cell. Biol. 1999; 19: 4465-4479Crossref PubMed Scopus (240) Google Scholar). Expression of a Cdc25C mutant where alanine is substituted for serine 216 (S216A) induces premature entry into mitosis by overriding a γ-radiation-induced DNA damage checkpoint (13.Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1187) Google Scholar). During the normal progression through the cell cycle, serine 216 phosphorylation may also contribute to the negative regulation of Cdc25C because Cdc25C is phosphorylated at this site in interphase but not during mitosis when Cdc25 is active (13.Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1187) Google Scholar, 15.Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1123) Google Scholar). cTAK1, a cytoplasmic kinase that phosphorylates Cdc25C specifically at serine 216 in vitro could be involved in the negative regulation of Cdc25C in interphase cells (16.Ogg S. Gabrielli B. Piwnica-Worms H. J. Biol. Chem. 1994; 269: 30461-30469Abstract Full Text PDF PubMed Google Scholar, 17.Peng C.Y. Graves P.R. Ogg S. Thoma R.S. Byrnes M.J.r. Wu Z. Stephenson M.T. Piwnica-Worms H. Cell Growth Differ. 1998; 9: 197-208PubMed Google Scholar). On the other hand, the nuclear kinases Chk1 (13.Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1187) Google Scholar, 15.Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1123) Google Scholar) and Chk2/Cds1 (18.Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1084) Google Scholar, 19.Blasina A. Van de Weyer I. Laus M.C. Luyten W.H.M.L. Parker A.E. McGowan C.H. Curr. Biol. 1999; 9: 1-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 20.Brown A.L. Lee C.H. Schwarz J.K., N, M. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (237) Google Scholar, 21.Chaturvedi 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., D., W.J. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar), which can phosphorylate Cdc25C at serine 216 in vitro, could contribute to the maintenance of this phosphorylation in cells arrested at G2/M in response to DNA damage. So far, only Chk2/Cds1 kinase activity has been shown to increase dramatically in a checkpoint-dependent manner (18.Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1084) Google Scholar, 19.Blasina A. Van de Weyer I. Laus M.C. Luyten W.H.M.L. Parker A.E. McGowan C.H. Curr. Biol. 1999; 9: 1-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 20.Brown A.L. Lee C.H. Schwarz J.K., N, M. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (237) Google Scholar, 21.Chaturvedi 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., D., W.J. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar). However, it is still unclear how DNA damage affects Cdc25C serine 216 phosphorylation, nor do we know if inhibition of the Chk1 or Chk2/Cds1 pathways alters this phosphorylation. Accumulating data suggest that ataxia-telangiectasia-mutated (ATM) kinase is a proximal component of DNA damage-induced cell cycle checkpoint pathway (22.Lavin M.F. Khanna K.K. Int. J. Radiat. Biol. 1999; 75: 1201-1214Crossref PubMed Scopus (123) Google Scholar). ATM must play an important role in the G2/M checkpoint because ATM-deficient cells fail to arrest in the G2 phase of cell cycle prior to mitosis after DNA damage (23.Beamish H. Williams R. Chen P. Lavin M.F. J. Biol. Chem. 1996; 271: 20486-20493Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). It has been demonstrated that radiation-induced Chk2/Cds1 activation is ATM-dependent (18.Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1084) Google Scholar, 19.Blasina A. Van de Weyer I. Laus M.C. Luyten W.H.M.L. Parker A.E. McGowan C.H. Curr. Biol. 1999; 9: 1-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 20.Brown A.L. Lee C.H. Schwarz J.K., N, M. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (237) Google Scholar, 21.Chaturvedi 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., D., W.J. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar), although it is unclear whether activation of Chk1 is also ATM-dependent. It remains to be shown that ATM directly phosphorylates Chk2/Cds1. To study how caffeine abrogates the G2/M checkpoint, it will be necessary to examine how caffeine affects the known G2/M checkpoint signal transduction pathway involving the phosphorylation of serine 216 on Cdc25C. We present evidence here showing that caffeine inhibits ATM-dependent activation of Chk2/Cds1 in vivo. In vitro, caffeine did not inhibit Chk2/Cds1 directly but instead inhibited ATM at an IC50 of approximately 200 μm. During the preparation of this manuscript, similar findings that caffeine can inhibit ATM kinase activity were reported (24.Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar, 25.Blasina A. Price B.D. Turenne G.A. McGowan C.H. Curr. Biol. 1999; 9: 1135-1138Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Our study provides further mechanistic details of how this inhibition can lead to the abrogation of the G2/M checkpoint. Using a recently generated phospho-specific antibody against serine 216, we demonstrate that activation of the DNA damage checkpoint is correlated with an increase in serine 216-phosphorylated Cdc25C in the nucleus and further, that prior treatment of cells with caffeine inhibits such a change. In addition, we provide evidence showing that ATM phosphorylates Chk2/Cds1 at threonine 68 in the N terminus, also in a caffeine-sensitive manner. Finally, caffeine is shown to inhibit ATM-dependent Chk2/Cds1 phosphorylationin vivo. Using a phospho-specific antibody against threonine 68, we found that Chk2/Cds1 is phosphorylated at this site in vivo in cells after IR exposure, and this phosphorylation is inhibited by prior treatment of cells with caffeine. These results are consistent with a model in which caffeine abrogates the G2/M checkpoint by targeting the ATM-Chk2/Cds1 pathway by inhibiting ATM. To make a kinase-dead Chk2, aspartic acid 347 was converted to alanine by site-directed mutagenesis. Full-length wild type and kinase-dead (KD) Chk2 (D347A) were subcloned into pFASTBAC as GST fusion protein (Invitrogen) for baculovirus expression. Recombinant Chk2-GST proteins were purified by a glutathione-Sepharose affinity step followed by a gel filtration column. Full-length kinase-dead (KD) Chk2 and threonine 68 to alanine mutant, N terminus (amino acids 1–222), and C terminus of Chk2 (amino acids 206–543, with D347A mutation) were also subcloned into pGEX-5X for expression in bacteria and subsequently purified as GST fusion proteins as described above. Phosphopeptides of the following sequence were synthesized: phospho-S216, GLYRSP-pS-MPENLNR; phospho-T68, SSLETVS-pT-QELYS, and coupled to KLH. Phosphospecific antibodies to the KLH-coupled peptides were obtained and purified. Anti-Chk2 was described in Chaturvedi et al. (21.Chaturvedi 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., D., W.J. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar). Anti-ATM was purchased from Oncogene Research Products (Cambridge, MA), anti-Cdc25C (C-20) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-phospho-S15 of p53 from New England Biolabs (Beverly, MA). MRC-5 cells were plated in microchamber slides overnight and irradiated the next day with 20 Gy of IR and then incubated at 37 °C for 30 min before fixing and staining. Untreated cells and cells processed with secondary antibody alone were included as controls. The cells were fixed for 10 min in PBS-buffered paraformaldehyde, 2% sucrose solution, followed by permeabilization for 10 min in cold buffer containing 0.5% Triton X-100 in 20 mm Hepes, pH 7.4, 3 mm MgCl2, 50 mm NaCl, and 300 mm sucrose (26.Scully R. Chen J. Ochs R.L. Keegan K. Hoekstra M. Feunteun J. Livingston D.M. Cell. 1997; 90: 425-435Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar). They were then blocked in 3% bovine serum (BSA) in PBS-Tween 20 and incubated in primary antibody at 1:3000 dilution in PBS-BSA for 1 h at 4 °C before washing three times with PBS-BSA. The slides were incubated in fluorescein isothiocyanate-labeled secondary antibody in PBS-BSA for 1 h, washed three times with PBS-BSA, twice with PBS alone, and then dry-mounted. The slides were seen under an inverted florescence microscope (OLYMPUS BX-60). Whole cell extracts were prepared as described (21.Chaturvedi 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., D., W.J. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar). Nuclear and cytoplasmic extracts were prepared using a kit from Pierce. Cells were grown in 100-ml flasks and treated with caffeine (2 mm) overnight and irradiated with 20 Gy. They were then removed by scraping and collected by centrifugation, and the pellets were lysed in extraction buffer to obtain the cytoplasmic fractions in the supernatant. The remaining pellets containing the nuclear fraction were then washed thoroughly three times with PBS and lysed in extraction buffer to obtain the nuclear extract. Chk2 kinase assays (21.Chaturvedi 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., D., W.J. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar) and ATM kinase assays (27.Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281 (1179): 1677Crossref PubMed Scopus (1704) Google Scholar) were performed as described. ATM was immunoprecipitated by adding anti-ATM antibody to total cell lysates and incubating with protein G-Sepharose for 2 h at 4 °C. Kinase assays were performed on the beads by incubating the resulting complexes in kinase buffer containing 10 mm Hepes (pH 7.5), 50 mmβ-glycerophosphate, 50 mm NaCl, 10 mmMgCl2, 10 mm MnCl2, 1 mm dithiothreitol, 5 μm ATP, 10 μCi of [32P]ATP, and 1 μg of substrate for 30 min at 30 °C, and products were analyzed by SDS-PAGE followed by autoradiography. It has been established that the G2/M DNA checkpoint is at least partially mediated by phosphorylation of serine 216 in Cdc25C (13.Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1187) Google Scholar, 14.Dalal S.N. Schweitzer C.M. Gan J. DeCaprio J.A. Mol. Cell. Biol. 1999; 19: 4465-4479Crossref PubMed Scopus (240) Google Scholar). To study the role of serine 216 in this checkpoint, we raised an antibody to a phosphopeptide surrounding the serine 216 phosphorylation site. To test the specificity of the antibody, we performed an in vitroassay using GST-Chk2 as the active kinase and GST-Cdc25C as the substrate. A kinase-dead Chk2 fusion protein (D347A) was included as a negative control. The assay mixture was resolved on 10% SDS-PAGE and blotted with anti phospho-S216 antibody. As shown in the Western blot (Fig. 1 A, lane 3), the phospho-S216 antibody recognized Cdc25C that was incubated with a wild type Chk2 in the in vitro kinase assay but failed to recognize unphosphorylated Cdc25C, incubated with a kinase-dead mutant (D347A) (Fig. 1 A, lane 2). Moreover, a peptide surrounding serine 216 blocked the antibody recognition (data not shown). These findings indicate that the anti-phospho-S216 antibody recognizes Cdc25C specifically when it is phosphorylated at serine 216 and confirms that Chk2/Cds1 phosphorylates Cdc25C at serine 216in vitro. To determine the intracellular localization of serine 216-phosphorylated Cdc25C and the effect of DNA damage checkpoint activation on serine 216 phosphorylation, biochemical fractionation experiments were performed using asynchronously growing MRC-5 cells before and immediately after IR with or without prior treatment of cells with caffeine. Whole-cell, cytoplasmic, or nuclear extracts were prepared as described under “Experimental Procedures.” Equal amounts of protein in each sample were resolved on 10% SDS-PAGE, transferred to membranes, and blotted with anti-phospho-S216 and anti-Cdc25c antibody, respectively. Serine 216-phosphorylated Cdc25C was detected predominantly in the cytoplasmic extracts and to a lesser extent in the nuclear fraction (Fig. 1 B, lanes 1and 5). As a control for biochemical fractionation, nuclear protein nucleolin was found to be localized in the nuclear fractions (data not shown). Immediately after IR, an increase in serine 216-phosphorylated Cdc25C was observed in the nuclear fraction (Fig.1 B, lane 2), and prior treatment of cells with caffeine inhibited this increase (Fig. 1 B, lane 4). On the other hand, there was no appreciable increase in serine 216 phosphorylation in whole cell lysates (data not shown), consistent with the observation that the majority of serine 216-phosphorylated Cdc25C is in the cytoplasm (Fig. 1 B, lane 5). Caffeine alone did not alter serine 216 phosphorylation in the absence of IR (Fig. 1 B, lane 3). Cdc25C was partitioned between nuclear and cytoplasmic fractions similarly to serine 216-phosphorylated Cdc25C. No gross relocation of Cdc25C was observed immediately after IR (Fig. 1 B, lanes 2 and6). Our data support a model that an increase in serine 216 phosphorylation in the nucleus is caused by the activation of serine 216 kinase but cannot totally rule out the possibility of nuclear relocation of Cdc25C as an alternative mechanism. This finding implies that caffeine could inhibit the activation of the serine 216 kinase in the nucleus after IR at a concentration at which it can abrogate the G2/M checkpoint. To confirm the cellular localization of phosphorylated Cdc25C after IR, indirect immunofluoresence analysis of normal diploid human fibroblasts (MRC-5) was performed. Actively proliferating cells were γ-irradiated and allowed to recover for 30 min at 37 °C before immunostaining. As a negative control, cells were left untreated or unirradiated. As shown in Fig. 2, punctate staining for serine 216 was obtained; in contrast to the results of biochemical fractionation experiments (Fig. 1 B), serine 216 staining was found to be enriched in the nucleus of both irradiated and unirradiated samples. Cytoplasmic staining was diminished in all the samples. This can be explained by the fact that phosphorylated serine 216 epitope is perhaps not exposed in the cytoplasm due to interaction with 14-3-3 proteins as has been reported previously (13.Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1187) Google Scholar, 14.Dalal S.N. Schweitzer C.M. Gan J. DeCaprio J.A. Mol. Cell. Biol. 1999; 19: 4465-4479Crossref PubMed Scopus (240) Google Scholar). We observed a possible increase in nuclear serine 216 reactivity after IR treatment of cells as these cells stained brighter than the unirradiated cells (Fig. 2). To determine whether the increase in serine 216 phosphorylation observed after radiation is ATM-dependent, we repeated the biochemical fractionation experiment using the A-T cell line AGO4405. No change in anti-phospho-S216 staining was observed in AGO4405 cells after IR exposure (Fig. 1 C, lanes 1 and2), and caffeine had no effect in the A-T cells (Fig.1 C, lanes 3 and 4). These results demonstrate that radiation-induced phosphorylation of Cdc25C on serine 216 in the nucleus is dependent on ATM and is inhibited by caffeine. Which serine 216 kinase does caffeine affect in the nucleus? Both Chk1 and Chk2/Cds1 have been shown to localize to the nucleus and to phosphorylate serine 216 of Cdc25Cin vitro (13.Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1187) Google Scholar, 15.Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1123) Google Scholar) (18.Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1084) Google Scholar, 19.Blasina A. Van de Weyer I. Laus M.C. Luyten W.H.M.L. Parker A.E. McGowan C.H. Curr. Biol. 1999; 9: 1-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 20.Brown A.L. Lee C.H. Schwarz J.K., N, M. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (237) Google Scholar, 21.Chaturvedi 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., D., W.J. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar). Although the phosphorylation state of Chk1 is altered in response to DNA damage (15.Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1123) Google Scholar), it does not correspond to an increase in its kinase activity toward Cdc25C. 2K. K. Khanna, S. Kozlov, and B. Gabrielli, unpublished results. It is still unclear whether Cdc25C is a substrate for Chk1 in vivo. On the other hand, Chk2/Cds1 kinase activity toward Cdc25C increases substantially after IR in an ATM-dependent manner (18.Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1084) Google Scholar, 19.Blasina A. Van de Weyer I. Laus M.C. Luyten W.H.M.L. Parker A.E. McGowan C.H. Curr. Biol. 1999; 9: 1-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 20.Brown A.L. Lee C.H. Schwarz J.K., N, M. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (237) Google Scholar, 21.Chaturvedi 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., D., W.J. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar). To test the possibility that Chk2/Cds1 is a target for caffeine in abrogating the G2/M checkpoint, we determined whether ATM-dependent Chk2/Cds1 activation is inhibited by caffeine. Immunoprecipitate kinase assays were performed using total lysates prepared from normal (MRC-5) and A-T (AGO4405) cells treated with caffeine and then irradiated with 20 Gy of radiation using GST-Cdc25C as a substrate (21.Chaturvedi 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., D., W.J. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar). Lysates from cells that were untreated, treated with caffeine alone, and γ-irradiated alone were included in the assay as controls. As shown in Fig.3 A in normal cells, IR causes activation of Chk2/Cds1 (lane 2), and this activation is inhibited by prior treatment of cells with caffeine (lane 4). However, no such activation was found in A-T cells indicating that caffeine inhibited the ATM-dependent Chk2/Cds1 activation (Fig. 3 A, lanes 6 and 8). The Chk2 activity was also measured by an immunoassay using anti-phospho-S216 antibody (Fig. 3 B). Reaction mixtures identical to those used in the radioactive kinase assay were analyzed on 10% SDS-PAGE, transferred onto nitrocellulose membrane, and blotted with anti-phospho-S216. Serine 216 phosphorylation activity of Chk2 was clearly increased in response to DNA damage (Fig. 3 B,lane 2), and the increase was prevented by prior treatment of cells with caffeine (Fig. 3 B, lane 4). Again no effect of caffeine was found in A-T cells (Fig. 3 B,lane 8). To examine whether Chk2/Cds1 is a direct target of caffeine, an in vitro kinase assay was performed with recombinant Chk2 using GST-Cdc25C as a substrate in the presence or absence of increasing concentrations of caffeine. A kinase-dead mutant of Chk2 (D347A) was included as a negative control in the assay. Caffeine did not inhibit the kinase activity of Chk2 even at a concentration of 2 mm (Fig. 3 C) suggesting that Chk2 is not a direct target; instead, caffeine might inhibit either ATM kinase activity or the pathway leading to Chk2/Cds1 activation. Chk2/Cds1 activation after IR has been shown to depend on ATM kinase activity (18.Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1084) Google Scholar, 19.Blasina A. Van de Weyer I. Laus M.C. Luyten W.H.M.L. Parker A.E. McGowan C.H. Curr. Biol. 1999; 9: 1-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 20.Brown A.L. Lee C.H. Schwarz J.K., N, M. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (237) Google Scholar, 21.Chaturvedi 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., D., W.J. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar). To test the possibility that ATM can phosphorylate Chk2/Cds1 directly, active ATM was immunoprecipitated from normal human lymphoblastoid C3ABR cells that had been either γ-irradiated or mock-treated. ATM nonexpressing human A-T lymphoblast (L3) cells were used as a negative control. Because wild type Chk2 has a high level of autophosphorylation activity (data not shown), kinase-dead Chk2 mutant (D347A) was used as the substrate in the ATM kinase assay. As shown in Fig.4 A, ATM immunoprecipitated from C3ABR phosphorylated Chk2/Cds1 directly, and ATM activity increased by approximately 4-fold after radiation. In negative control A-T cells (L3), there is only background phosphorylation of Chk2. To analyze the location of phosphorylation, we expressed Chk2 as two separate domains: the N-terminal domain, containing amino acids 1–222; and the C-terminal domain (with D347A mutation to inactivate kinase activity), containing amino acids 206–543. As shown in Fig.4 A, most of the phosphorylation was in the N-terminal domain; there was very little in the C-terminal domain (D347A). Similarly constructed wild type C-terminal domain is kinase-active (data not shown), indicating that misfolding is not a cause of the lack of phosphorylation. Analysis of the Chk2/Cds1 sequence within this region revealed a cluster of serines (S) and threonines (T) followed by glutamine (Q) (SQ or" @default.
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• W2070632817 title "Caffeine Abolishes the Mammalian G2/M DNA Damage Checkpoint by Inhibiting Ataxia-Telangiectasia-mutated Kinase Activity" @default.
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