DNA damage-induced G2/M checkpoint in SV40 large T antigen-immortalized embryonic fibroblast cells requires SHP-2 tyrosine phosphatase.
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 gamma-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 14-3-3beta, 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.
Yuan
Liangping
L
Department of Hematopoiesis, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855, USA.
Yu
Wen-Mei
WM
Qu
Cheng-Kui
CK
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D002110:Caffeine / Q000494:pharmacology; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D002945:Cisplatin / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D016195:G2 Phase; D005720:Gamma Rays; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D011233:Precipitin Tests; D021381:Protein Transport; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D013997:Time Factors; D020687:cdc25 Phosphatases / Q000378:metabolism; D018909:src Homology Domains
D000952:Antigens, Polyomavirus Transforming; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D002110:Caffeine; D004247:DNA; C517010:Cdc25c protein, mouse; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D020687:cdc25 Phosphatases; D002945:Cisplatin; D015739:Nocodazole