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• W2023763994 abstract "DNA damage induced by radiation or DNA-damaging agents leads to apoptosis and cell cycle arrest. However, DNA damage-triggered signal transduction involved in these cellular responses is not well understood. We previously demonstrated an important role for SHP-2, a ubiquitously expressed SH2 domain-containing tyrosine phosphatase, in the DNA damage-induced apoptotic response. Here we report a potential role for SHP-2 in a DNA damage-activated cell cycle checkpoint. Cell cycle analysis and the mitotic index assay showed that following DNA damage induced by cisplatin or γ-irradiation, the G2 (but not S) arrest response was diminished in SV40 large T antigen-immortalized embryonic fibroblast cells lacking functional SHP-2. Notably, reintroduction of wild-type SHP-2 into the mutant cells fully restored the DNA damage-induced G2 arrest response, suggesting a direct role of SHP-2 in the G2/M checkpoint. Further biochemical analysis revealed that SHP-2 constitutively associated with 14-3-3β, and that Cdc25C cytoplasmic translocation induced by DNA damage was essentially blocked in SHP-2 mutant cells. Additionally, we showed that following DNA damage, activation of p38 kinase was significantly elevated, while Erk kinase activation was decreased in mutant cells, and treatment of SHP-2 mutant cells with SB203580, a selective inhibitor for p38 kinase, partially restored the DNA damage-induced G2 arrest response. These results together provide the first evidence that SHP-2 tyrosine phosphatase enhances the DNA damage G2/M checkpoint in SV40 large T antigen immortalized murine embryonic fibroblast cells. DNA damage induced by radiation or DNA-damaging agents leads to apoptosis and cell cycle arrest. However, DNA damage-triggered signal transduction involved in these cellular responses is not well understood. We previously demonstrated an important role for SHP-2, a ubiquitously expressed SH2 domain-containing tyrosine phosphatase, in the DNA damage-induced apoptotic response. Here we report a potential role for SHP-2 in a DNA damage-activated cell cycle checkpoint. Cell cycle analysis and the mitotic index assay showed that following DNA damage induced by cisplatin or γ-irradiation, the G2 (but not S) arrest response was diminished in SV40 large T antigen-immortalized embryonic fibroblast cells lacking functional SHP-2. Notably, reintroduction of wild-type SHP-2 into the mutant cells fully restored the DNA damage-induced G2 arrest response, suggesting a direct role of SHP-2 in the G2/M checkpoint. Further biochemical analysis revealed that SHP-2 constitutively associated with 14-3-3β, and that Cdc25C cytoplasmic translocation induced by DNA damage was essentially blocked in SHP-2 mutant cells. Additionally, we showed that following DNA damage, activation of p38 kinase was significantly elevated, while Erk kinase activation was decreased in mutant cells, and treatment of SHP-2 mutant cells with SB203580, a selective inhibitor for p38 kinase, partially restored the DNA damage-induced G2 arrest response. These results together provide the first evidence that SHP-2 tyrosine phosphatase enhances the DNA damage G2/M checkpoint in SV40 large T antigen immortalized murine embryonic fibroblast cells. Genetic stability is maintained by cell cycle checkpoints (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2637) Google Scholar, 2Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1669) Google Scholar). In response to DNA damage, mammalian cells arrest at certain points in the cell cycle. This regulatory mechanism inhibits cell cycle progression until the cell has adequately repaired the DNA damage. For instance, arrest in G1 and S phases prevents damaged DNA from replicating, and arrest in the G2 phase prevents segregation of damaged chromosomes. Failures in cell cycle checkpoints can lead to the acquisition and accumulation of genetic alterations. These changes may result in the activation of oncogenes and/or the inactivation of tumor suppressor genes, both of which can ultimately lead to tumorigenesis. However, the precise mechanisms of cell cycle checkpoints and the signaling components involved are not fully understood. In many cases, DNA damage-triggered signaling pathways induce cell cycle arrest by inhibiting the activities of the cyclin-dependent kinases that are required to drive cell cycle progression. The biochemical details of the G1 checkpoint are relatively well understood. A delay in the G1 phase results largely from the activation of p53 and consequent transcriptional induction of the cyclin-dependent kinase inhibitor p21Cip1 (3Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1948) Google Scholar, 4Brugarolas J. Chandrasekaran C. Gordon J.I. Beach D. Jacks T. Hannon G.J. Nature. 1995; 377: 552-557Crossref PubMed Scopus (1150) Google Scholar). By comparison, the DNA damage-induced G2/M checkpoint is more complex. The G2/M transition is regulated by Cdc2 kinase and cyclin B1 as part of the maturation promoting factor that determines entry into mitosis. It has been demonstrated that the G2 arrest is largely dependent on inhibitory phosphorylation of Cdc2 at tyrosine 15 (Tyr15) and threonine 14 (Thr14) and is therefore likely to result from changes in the activities of the opposing kinases and phosphatases that act on Cdc2. Among these upstream regulators, Cdc25C has been identified as crucial for the activation of Cdc2 by dephosphorylating its inhibitory tyrosine sites (5Strausfeld 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, 6Weinert T. Science. 1997; 277: 1450-1451Crossref PubMed Scopus (116) Google Scholar). Dephosphorylation of Cdc2 by Cdc25C and association with cyclin B1 results in rapid entry into mitosis whereas phosphorylation of negative regulatory sites on Cdc2 by Wee1/Myt1 kinases and cyclin B1 degradation or export to the cytoplasm block entry into mitosis. Following DNA damage, Cdc25C is phosphorylated by checkpoint kinases Chk1 and 2, which are activated by ATM and its related kinase ATR (7Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1189) Google Scholar, 8Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1124) Google Scholar, 9Furnari B. Rhind N. Russell P. Science. 1997; 277: 1495-1497Crossref PubMed Scopus (475) Google Scholar, 10Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (195) Google Scholar, 11Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1087) Google Scholar, 12Matsuoka 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 (694) Google Scholar). Upon phosphorylation, Cdc25C binds to 14-3-3 adaptor proteins and is thereby sequestered in the cytoplasm (7Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1189) Google Scholar, 13Lopez-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (505) Google Scholar). Separation of Cdc25C from the nucleus then results in elevated Cdc2 phosphorylation and a reduced Cdc2 kinase activity. As a result, cells arrest in the G2 phase. However, in addition to the ATM/Chk1, 2/Cdc25C/Cdc2 pathway, other mechanisms contributing to the G2/M checkpoint also exist. p21Cip1, a major downstream effector of p53 and p73 transcription factors, contributes mainly to the G1 and S arrests (3Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1948) Google Scholar, 4Brugarolas J. Chandrasekaran C. Gordon J.I. Beach D. Jacks T. Hannon G.J. Nature. 1995; 377: 552-557Crossref PubMed Scopus (1150) Google Scholar); however, its role in inducing the G2 arrest has also been reported, i.e. cells deficient in p21Cip1 are unable to maintain stable G2 arrest when exposed to DNA-damaging agents (14Taylor W.R. Stark G.R. Oncogene. 2001; 20: 1803-1815Crossref PubMed Scopus (1293) Google Scholar, 15Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2538) Google Scholar). More recently, several signaling enzymes important for growth factor and cytokine-induced signal transduction, such as Erk, 1The abbreviations used are: Erk, extracellular signal-regulated kinase; WT, wild type; Ab, antibody; PI, propidium iodide; FACS, fluorescence-activated cell sorting. p38, and Akt kinases, have also been found to be involved in the regulation of the G2/M transition of the cell cycle. For example, Erk kinases have been shown to be required for normal G2/M progression (16Guadagno T.M. Ferrell Jr., J.E. Science. 1998; 282: 1312-1315Crossref PubMed Scopus (82) Google Scholar, 17Wright J.H. Munar E. Jameson D.R. Andreassen P.R. Margolis R.L. Seger R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11335-11340Crossref PubMed Scopus (158) Google Scholar) and DNA damage-induced G2/M arrest (18Tang D. Wu D. Hirao A. Lahti J.M. Liu L. Mazza B. Kidd V.J. Mak T.W. Ingram A.J. J. Biol. Chem. 2002; 277: 12710-12717Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). p38 kinase has also been shown to be involved in the G2/M DNA damage cell cycle checkpoint (19Bulavin D.V. Higashimoto Y. Popoff I.J. Gaarde W.A. Basrur V. Potapova O. Appella E. Fornace Jr., A.J. Nature. 2001; 411: 102-107Crossref PubMed Scopus (460) Google Scholar, 20Wang X. McGowan C.H. Zhao M. He L. Downey J.S. Fearns C. Wang Y. Huang S. Han J. Mol. Cell. Biol. 2000; 20: 4543-4552Crossref PubMed Scopus (238) Google Scholar). Therefore, it appears that multiple pathways contribute to the regulation of the G2/M checkpoint following genotoxic stress. SHP-2, a SH2 domain-containing tyrosine phosphatase, is ubiquitously expressed in a variety of tissues and cell types, and has been demonstrated to be involved in diverse signaling pathways, including those initiated by growth factors, cytokines, and insulin (21Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (463) Google Scholar, 22Qu C.K. Biochim. Biophys. Acta. 2002; 1592: 297-301Crossref PubMed Scopus (109) Google Scholar). In most circumstances, SHP-2 plays a positive role in transducing the signal relay from receptor tyrosine kinases, whereby its phosphatase activity has been shown to be required (23Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar, 24Tang T.L. Freeman Jr., R.M. O'Reilly A.M. Neel B.G. Sokol S.Y. Cell. 1995; 80: 473-483Abstract Full Text PDF PubMed Scopus (308) Google Scholar, 25Bennett A.M. Hausdorff S.F. O'Reilly A.M. Freeman R.M. Neel B.G. Mol. Cell. Biol. 1996; 16: 1189-1202Crossref PubMed Scopus (226) Google Scholar), even though the biochemical significance of its catalytic activity remains ill-defined. The N-terminal SH2 domain (N-SH2) plays a critical role in mediating SHP-2 function. A targeted N-terminal deletion of SHP-2 (amino acids 46–110 including the N-SH2) results in a loss-of-function mutation for SHP-2. As a result of this mutation, homozygous mutant (SHP-2Δ/Δ) embryos die at midgestation with multiple developmental defects (26Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (404) Google Scholar, 27Qu C.K. Yu W.M. Azzarelli B. Cooper S. Broxmeyer H.E. Feng G.S. Mol. Cell. Biol. 1998; 18: 6075-6082Crossref PubMed Scopus (107) Google Scholar). Essential roles for SHP-2 in the regulation of a variety of signal transduction pathways and cellular processes such as cell proliferation, differentiation, adhesion, and migration have been characterized by using this SHP-2 gene knockout model and the mutant fibroblast cell lines derived from SHP-2Δ/Δ mutant embryos through SV40 large T antigen immortalization (27Qu C.K. Yu W.M. Azzarelli B. Cooper S. Broxmeyer H.E. Feng G.S. Mol. Cell. Biol. 1998; 18: 6075-6082Crossref PubMed Scopus (107) Google Scholar, 28Qu C.K. Shi Z.Q. Shen R. Tsai F.Y. Orkin S.H. Feng G.S. Mol. Cell. Biol. 1997; 17: 5499-5507Crossref PubMed Scopus (150) Google Scholar, 29Shi Z.Q. Lu W. Feng G.S. J. Biol. Chem. 1998; 273: 4904-4908Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 30Yu D.H. Qu C.K. Henegariu O. Lu X. Feng G.S. J. Biol. Chem. 1998; 273: 21125-21131Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 31Qu C.K. Yu W.M. Azzarelli B. Feng G.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8528-8533Crossref PubMed Scopus (85) Google Scholar). Using the SV40 large T antigen-immortalized SHP-2Δ/Δ embryonic fibroblast cell lines, we have recently demonstrated that SHP-2 plays an important role in DNA damage-induced cell death, and that it enhances the cellular apoptotic response to DNA damage by promoting activation of nuclear kinase c-Abl (32Yuan L. Yu W.M. Yuan Z. Haudenschild C.C. Qu C.K. J. Biol. Chem. 2003; 278: 15208-15216Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). During the course of that work, we inadvertently noticed that the DNA damage-induced G2 arrest response was diminished in SHP-2 mutant cells. We therefore investigated the potential role of SHP-2 phosphatase in DNA damage-induced cell cycle checkpoint. These results suggest that SHP-2 enhances the DNA damage G2/M checkpoint by modulating Cdc25C cytoplasmic translocation and the MAP kinase pathways. Cell Lines and Reagents—Wild-type (WT) and SHP-2Δ/Δ mutant embryonic fibroblast cell lines were derived from day 9.0–9.5 embryos through SV40 large T antigen immortalization (29Shi Z.Q. Lu W. Feng G.S. J. Biol. Chem. 1998; 273: 4904-4908Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 30Yu D.H. Qu C.K. Henegariu O. Lu X. Feng G.S. J. Biol. Chem. 1998; 273: 21125-21131Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 33Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (190) Google Scholar, 34You M. Flick L.M. Yu D. Feng G.S. J. Exp. Med. 2001; 193: 101-110Crossref PubMed Scopus (115) Google Scholar). Rescued cell lines were generated by transduction of WT SHP-2 cDNA into SHP-2Δ/Δ cells through retroviral-mediated gene transfer. All cell lines were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cisplatin and propidium iodide (PI) were purchased from Sigma. Nocodazole, caffeine, SB203580, and anti-Cdc2 antibody (Ab) were obtained from Calbiochem (La Jolla, CA). Anti-SHP-2, -SV40 large T antigen, -Erk, -phospho-Erk, -Cdc25C, -14-3-3β, -histone H1, and -cyclin B1 Abs were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p38, -phospho-p38, -phospho-Cdc2 (Tyr15), and -phospho-Chk1 (Ser345) Abs were purchased from Cell Signaling Technology (Beverly, MA). The Cdc2 kinase assay kit, anti-phosphohistone H3, and anti-Chk1 Abs were obtained from Upstate Biotechnology (Lake Placid, NY). Cell Cycle Analysis—Cells were harvested and fixed in 70% ethanol. Fixed cells were treated with RNase A (20 μg/ml) at 37 °C for 30 min, washed with phosphate-buffered saline, and then stained with PI (50 μg/ml in phosphate-buffered saline). Cellular DNA content was analyzed with fluorescence-activated cell sorting (FACS) analysis using BD-LSR flowcytometry (BD Biosciences). The cell cycle profiles were determined with the CELLQuest™ software (BD Biosciences). Mitotic Index Assay—Cells grown in slide chambers were treated with cisplatin (5 μm) for various time periods. Treated cells were then fixed in methanol for 10 min and stained with 5% Giemsa. In some experiments, cells were stained by anti-phosphohistone H3 Ab that specifically detects mitotic nuclei. Mitotic cells in late prophase, metaphase, anaphase, and telophase were identified under the fluorescence microscope and expressed as a fraction of the total cells counted. At least 3000 cells were counted in each preparation. Cell Synchronization—Fibroblast cells were synchronized in the G0/G1 phase by serum deprivation for 48 h before experiments. To synchronize cells at the G1/S boundary, asynchronously growing cells were treated with thymidine (2 mm) for 16 h, then thymidine (24 μm), and deoxycydine (24 μm) for 8 h, and finally thymidine (2 mm) for additional 16 h. Synchronized cells were released from the block for experiments by rinsing twice with phosphate-buffered saline and changing medium to complete growth medium. Immunoprecipitation and Immunoblotting Analysis—Cells were lysed in radioimmune precipitation assay buffer (50 mm Tris-HCl pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm NaF, 2 mm Na3VO4, and 1 mm phenylmethylsulfonyl fluoride). Whole cell lysates (500 μg) were immunoprecipitated with 1 μg of purified Abs as indicated. Immunoprecipitates were washed three times with HNTG buffer (20 mm Hepes pH 7.5, 150 mm NaCl, 1% glycerol, 0.1% Triton X-100, and 1 mm Na3VO4) and resolved by SDS-PAGE followed by immunoblotting with the indicated Abs. Cdc2 Kinase Assay—Cdc2 kinase activity was assessed by using the Cdc2 kinase assay kit from Upstate Biotechnology following the manufacturer's instruction. Cell lysates (200 μg) were immunoprecipitated with 1 μg of anti-cyclin B1 Ab. Immunoprecipitates were washed and assayed for the kinase activity by using histone H1 as the substrate. A mixture solution containing histone H1 (20 μg/reaction), 5 μCi of [γ-32P]ATP, and PCK, PKA, and PKI inhibitor mixture (1:5 dilution) in kinase assay buffer was added into each immunoprecipitation product and incubated at 30 °C for 20 min. An aliquot of reaction mixture (25 μl) was spotted onto a piece of P81 filter paper. The filters were washed three times with 0.75% phosphoric acid and once with acetone. 32P incorporation into histone H1 was then measured by liquid scintillation counting. DNA Damage-induced Cell Cycle Response Is Decreased in SHP-2 Δ/Δ Murine Embryonic Fibroblast Cells—During the course of our previous studies defining the role of SHP-2 in DNA damage-induced apoptosis (32Yuan L. Yu W.M. Yuan Z. Haudenschild C.C. Qu C.K. J. Biol. Chem. 2003; 278: 15208-15216Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), we noticed a difference in the cell cycle profiles between normally growing, SV40 large T antigen-immortalized, WT and mutant embryonic fibroblast cells carrying the amino acids 46–110 deletion of SHP-2 (SHP-2Δ/Δ). An increase in the percentage of mutant cells in the G2/M phase was observed (Fig. 1B). This change in the cell cycle of SHP-2 mutant cells does not appear to be due to a defect in cell cycle parameters, since reintroduction of WT SHP-2 into SHP-2Δ/Δ cells (rescued cells) completely corrected cell cycle profiles. As Erk kinase activity has been shown to be required for the G2/M transition of the cell cycle (16Guadagno T.M. Ferrell Jr., J.E. Science. 1998; 282: 1312-1315Crossref PubMed Scopus (82) Google Scholar, 17Wright J.H. Munar E. Jameson D.R. Andreassen P.R. Margolis R.L. Seger R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11335-11340Crossref PubMed Scopus (158) Google Scholar) and SHP-2 plays a positive role in the growth factor and cytokine-induced Erk pathway (23Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar, 24Tang T.L. Freeman Jr., R.M. O'Reilly A.M. Neel B.G. Sokol S.Y. Cell. 1995; 80: 473-483Abstract Full Text PDF PubMed Scopus (308) Google Scholar, 25Bennett A.M. Hausdorff S.F. O'Reilly A.M. Freeman R.M. Neel B.G. Mol. Cell. Biol. 1996; 16: 1189-1202Crossref PubMed Scopus (226) Google Scholar), the prolonged G2/M phase is presumably attributed to the decreased Erk kinase activity in the mutant cells under normal culture conditions. More interestingly, in response to the treatment of the DNA damaging chemotherapeutic drug cisplatin, the percentage of WT cells in the G2/M phase was significantly increased (G2/M arrest), suggesting activation of the cell cycle G2/M checkpoint. However, the DNA damage-induced G2/M arrest response in SHP-2Δ/Δ cells is diminished (Fig. 1B), indicating a defect in the G2/M checkpoint control in mutant cells. Notably, the G2/M arrest response to cisplatin treatment was fully restored in the rescued cell line (Fig. 1B), suggesting that the diminished cell cycle response to DNA damage in mutant cells is attributed directly to loss of SHP-2 function. It is noteworthy that the defect in DNA damage cell cycle control caused by the SHP-2 mutation appears to be specific for the G2/M but not S checkpoints, since the DNA damage-induced response of the arrest in the S phase is not altered in SHP-2 mutant cells (Fig. 1C). The above experiments were conducted with asynchronized cells. To better determine the role of SHP-2 in the cell cycle response to DNA damage, WT, SHP-2Δ/Δ, and rescued cells were synchronized at the G1/S boundary as described under “Experimental Procedures,” and cell cycle responses of the synchronized cells to DNA damage were then examined. Similar to the asynthronized cells, synchronized SHP-2Δ/Δ cells did not show a significant increase in the percentage of G2/M following DNA damage (Fig. 1D), further confirming the potential role of SHP-2 in DNA damage-induced cell cycle regulation. Remarkably, the role of SHP-2 in G2/M checkpoint control is not specific to cisplatin-induced DNA damage; the γ-irradiation-induced G2/M arrest response is also diminished in SHP-2 mutant cells (Fig. 2). Collectively, these results demonstrate an important role of SHP-2 phosphatase in the DNA damage-induced G2/M checkpoint in SV40 large T antigen-immortalized murine embryonic fibroblast cells. Since SHP-2Δ/Δ cells showed a defect in the G2/M arrest response, we next wanted to determine whether this defect was specific to DNA damaging treatment. WT, SHP-2Δ/Δ, and rescued cells were treated with nocodazole, a compound that disrupts nuclear microtubules and arrests the cell cycle in mitosis (35Hayne C. Tzivion G. Luo Z. J. Biol. Chem. 2000; 275: 31876-31882Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). As shown in Fig. 3A, all cell types were efficiently blocked in the G2/M phase of the cell cycle. To further validate the potential role of SHP-2 in the DNA damage-induced G2/M checkpoint, we treated cells with cisplatin in conjunction with caffeine, a specific inhibitor of ATM/ATR kinase, which has been demonstrated to play an important role in the DNA damage G2/M checkpoint (2Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1669) Google Scholar). Cell cycle analyses showed that although caffeine treatment alone did not disturb cell cycle distribution, combined caffeine treatment abolished cisplatin-induced G2/M arrest in WT as well as rescued cells. However, this drug did not show any effect in SHP-2 mutant cells (Fig. 3B). To precisely determine whether the increase of G 2 /M cells observed in our cell cycle analyses resulted from a delay in G2 or M phases, the mitotic index assay (which directly determines the fraction of cells in mitosis) was conducted. In response to DNA damage, mitotic activity of WT cells was quickly and progressively decreased, indicating a G2 delay in the cell cycle. Although mitosis of SHP-2 mutant cells was also decreased shortly following DNA damage, this G2 delay could not be sustained, cells re-entered mitosis 2 h after DNA damage. Remarkably, rescued cells showed very similar changes in mitotic behavior as the WT control (Fig. 4A). This data suggests that in response to DNA damage, WT and rescued cells arrest in G2 phase and that without functional SHP-2 phosphatase, the G2 delay in SHP-2Δ/Δ mutant cells was significantly decreased and shortened. To more rigorously examine the cell cycle response of SHP-2 mutant cells to DNA damage, we synchronized the cells in the G0/G1 phase or at the G1/S boundary by serum deprivation or thymidine treatment (see “Experimental Procedures”), respectively. Synchronized cells were released and treated with cisplatin for 4 and 8 h. Mitotic cells were counted and compared with the untreated cells that were released from synchronization for the same periods of time. Consistent with the data obtained from asynchronized cells (Fig. 4A), mitotic activity of synchronized WT cells was significantly decreased following DNA damage. By contrast, this response in synchronized SHP-2 mutant cells was diminished (Fig. 4, B and C), further confirming the role of SHP-2 in DNA damage cell cycle regulation. As the G2/M transition of the cell cycle is mainly controlled by Cdc2 kinase, and phosphorylation of Tyr15 and Thr14 in Cdc2 negatively regulates its kinase activity, we next examined the phosphorylation status of Tyr15 in Cdc2 in response to DNA damage by using a specific Ab. As shown in Fig. 5A, Cdc2 (Tyr15) phosphorylation was gradually induced in WT cells following DNA damage. By contrast, phosphorylation of this site in SHP-2Δ/Δ cells was slightly induced and then quickly decreased to the basal level. This observation is fully consistent with the mitotic index data (Fig. 4). Furthermore, we examined Cdc2 kinase activity by using histone H1 as the substrate. As demonstrated in Fig. 5B, after DNA damage (8 h), Cdc2 kinase activity was markedly decreased in WT as well as rescued cell lines. However, no appreciable change in Cdc2 kinase activity is detected in SHP-2 mutant cells. It is important to emphasize that re-introduction of WT SHP-2 in SHP-2Δ/Δ cells completely rescued the response of Cdc2 kinase activity to DNA damage, again suggesting a direct role for SHP-2 in DNA damage-induced cell cycle response, and that although the truncated SHP-2 with a low expression level exists in the mutant cells, the SHP-2 mutation appears to be a loss-of-function mutation rather than a gain-of-function mutation. Defective G2 /M Checkpoint in SHP-2 Mutant Cells—To elucidate why the G2 arrest response induced by DNA damage is attenuated in the mutant cells lacking functional SHP-2, we attempted to dissect the molecular mechanism by which SHP-2 modulates the G2/M checkpoint. Previous studies have demonstrated that several pathways contribute to the DNA damage-induced G2/M arrest, among which the ATM, ATR/Chk1, 2/Cdc25C/Cdc2 pathway is well characterized (2Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1669) Google Scholar, 14Taylor W.R. Stark G.R. Oncogene. 2001; 20: 1803-1815Crossref PubMed Scopus (1293) Google Scholar). To determine whether this pathway is targeted by the SHP-2Δ/Δ mutation, we examined the phosphorylation status of Chk1 kinase (Ser345) that has been shown to be critical for its kinase activity (10Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (195) Google Scholar) and thereby downstream signaling, by using a specific anti-phospho-Chk1 (Ser345) Ab. As shown in Fig. 6A, phosphorylation of Chk1 is comparably induced by DNA damage in WT and SHP-2 mutant cells. This data, together with our previous observation that phosphorylation of p53Ser 15 by ATM/ATR kinase was not changed in SHP-2 mutant cells (32Yuan L. Yu W.M. Yuan Z. Haudenschild C.C. Qu C.K. J. Biol. Chem. 2003; 278: 15208-15216Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), suggest that Chk kinase activation is not affected by the SHP-2 mutation. We next examined the downstream processes of the ATM, ATR/Chk1, 2/Cdc25C/Cdc2 pathway. Previous studies have shown that Cdc2 phosphorylation and thereby kinase activity are regulated by Cdc25C, and that both phosphorylation (activation) induced by Chk kinases and cytoplasmic translocation by association with 14-3-3β are essential for Cdc25C activity on Cdc2 kinase (7Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1189) Google Scholar, 13Lopez-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (505) Google Scholar). Since Chk1 activation is not affected in SHP-2 mutant cells, we focused on the Cdc25C translocation response. Nuclear extracts were prepared from G0/G1 synchronized cells and subjected to quality control as we previously reported (32Yuan L. Yu W.M. Yuan Z. Haudenschild C.C. Qu C.K. J. Biol. Chem. 2003; 278: 15208-15216Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Anti-Cdc25C immunoblotting showed that in response to DNA damaging treatment, Cdc25C in the WT nucleus was decreased, suggesting its cytoplasmic translocation. By contrast, DNA damage-induced Cdc25C translocation was essentially blocked in SHP-2Δ/Δ mutant cells. Remarkably, this response is efficiently restored in the rescued cells (Fig. 6B). It is important to mention that the Cdc25C level in the cytoplasm was not significantly changed in the three cell types following DNA damage (data not shown). This is because a large amount of the Cdc25C is localized in the cytoplasm of the cell lines; small amounts of translocated nuclear Cdc25C did not make a significant difference in the Cdc25C level in the cytoplasm. To further elucidate the molecular mechanism by which SHP-2 modulates DNA damage-induced Cdc25C translocation, we examined potential interactions between SHP-2 and 14-3-3/Cdc25C. As shown in Fig. 6C, 14-3-3β was constantly detected in the anti-SHP-2 immunocomplex, and reci" @default.
• W2023763994 created "2016-06-24" @default.
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• W2023763994 date "2003-10-01" @default.
• W2023763994 modified "2023-09-28" @default.
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