Hirao and T. implying an essential role for ATR in the DNA replication checkpoint (14), and mouse embryonic stem cells lacking Chk1 have a defective G2/M DNA damage checkpoint (15). Furthermore, in response to UV, HU, and IR, Chk1 is found to be phosphorylated on Ser345, and the phosphorylation in response to UV is ATR dependent (15). ATR and Chk1 disruptions both lead to peri-implantation embryonic lethality in mice (14C16). Thus, Chk1 is regulated by ATR (15). ATM-dependent G1 arrest in response to IR is achieved by activation of p53 (17), which acts as a sequence-specific DNA-binding transcription factor to induce transcription of the Cdk inhibitor p21CIP1/WAF1 (18, 19). Activation of p53 in response Lexacalcitol to DNA damage is accomplished by both increasing its protein levels and by increasing its transcriptional activity. The increase in p53 levels in response to DNA damage is caused by decreased degradation of p53, and phosphorylation of Ser20 on p53 is required for the decrease in p53 degradation (20C22). Recently, Chk2 was found to phosphorylate Ser20 on p53 (10, 23, 24), and we have found that Chk2 is required for increased stability of p53 in response to IR (10), providing a possible mechanism for regulation of p53 stability by Chk2. Transcriptional activation of p53 is mediated by acetylation and phosphorylation of p53. Acetylation of p53 by histone acetyltransferases Lexacalcitol CBP/p300, which function as a coactivator for p53-mediated transcription (25, 26), is shown to enhance sequence-specific DNA binding of p53 (27). Phosphorylation of Ser15 on p53 was found to increase the interaction with CBP/p300 and acetylation of p53 (28). ATM phosphorylates Ser15 (29, 30). However, Ser15 is phosphorylated in an ATM-independent manner when cells are exposed to UV, indicating that a kinase distinct from ATM phosphorylates Ser15. A candidate kinase is ATR, which phosphorylates Ser15 when Lexacalcitol cells are exposed to IR but in an ATM-independent manner when cells are treated with UV or HU. Furthermore, Chk2 was found to be regulated by phosphorylation of the N-terminal Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD). Materials and Methods Plasmids. Full-length Chk2 cDNA with for 10 min, cell extracts Lexacalcitol were incubated with anti-Chk2 antibodies and protein ACSepharose (Amersham Pharmacia) for 1 h at 4C. The precipitated Sepharose beads were washed four times with 20 mM Tris?HCl buffer (pH 8.0) containing 100 mM NaCl, 0.1% Triton X-100, 10 mM NaF, 1 mM Na3VO4, 2 g/ml aprotinin, 2 g/ml leupeptin, 2 g/ml antipain, and 1 mM PMSF and then applied to SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% nonfat-milk in 50 mM Tris?HCl (pH 7.5), 100 mM NaCl, and 0.05% Tween 20, the membranes were incubated with anti-phospho-Thr68 antibodies and 100 g/ml non-phospho-Chk2-peptides (ETVSTQELYS), and then horseradish peroxidase-conjugated anti-rabbit IgG antibodies (Promega), followed by detection by enhanced chemiluminescence (Amersham Pharmacia). To reprobe with anti-Chk2 antibodies, membranes were washed twice with 50 mM glycine (pH 2.0) and then twice with 50 mM Tris?HCl (pH 7.5), Rabbit polyclonal to TLE4 100 mM NaCl, 0.05% Tween 20 before blocking with 5% nonfat-milk in 50 mM Tris?HCl (pH 7.5), 100 mM NaCl, 0.05% Tween 20. ATM/ATR Kinase Assay. ATM kinase assay with immunoprecipitated ATM from normal and AT lymphoblast cells was carried out as described (29). ATM-WT, ATM-KD, ATR-WT, and ATR-KD proteins were expressed as FLAG-tagged protein in 293T cells by transfection of the expression vector for ATM (28) or ATR (29). DNA transfection into 293T cells was performed with lipofectamine and Opti-MEM (GIBCO/BRL). Forty-eight hours after transfection, cells were lysed by sonication in 50 mM Tris?HCl buffer (pH 7.5) containing 50 mM -glycerophosphate, 150 mM NaCl, 10% glycerol, 1% Tween 20, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 2 g/ml aprotinin, 2 g/ml leupeptin, 2 g/ml antipain, and 1 mM DTT. After centrifugation at 10,000 for 10 min, FLAG-tagged.
