Figure 2.
ATM phosphorylates p27Kip1. (a) In vitro kinase assays demonstrate ATM phosphorylation of p27Kip1. HEK293 cells were transfected with FLAG-tagged ATM and were irradiated with 15 Gy IR. Two hours after IR treatment, the HEK293 cells were lysed and activated ATM was immunoprecipitated from the lysate with anti-Flag antibody. The immunoprecipitated FLAG-ATM was then incubated with recombinant His-p27Kip1 or His-p53 protein, and γ-32P-ATP in the presence or absence of ATM inhibitor (KU-55933) for 1 hour at 30◦C. 32P-labeled proteins were then separated by SDS-PAGE followed by autoradiography. (b) Radiation-activated ATM phosphorylates p27Kip1 in vitro. HEK293 cells were transfected with FLAG-tagged ATM and were irradiated with 15 Gy IR (2 h recovery). Activated ATM was immunoprecipitated with anti-FLAG antibody and incubated with recombinant His-p27Kip1 in the presence or absence of p27Kip1 for 1 h at 30◦C. The proteins were resolved on an SDS-PAGE, followed by immunoblot analysis using anti-ATM, anti-p27, and anti-p27 pS140 antibodies. (c) Time-course of phosphorylation on p27Kip1 Serine 140 following irradiation. HEK293 cells were transfected with Myc-p27Kip1 and then treated with 10 Gy ionizing radiation (IR). Cells were recovered up to 3 hours from irradiation. After cells were lysed at each indicated time, total lysates were resolved on SDS-PAGE, followed by immunoblot analysis with indicated antibodies. CHK2 T68 and p53 S15 are targets of ATM phosphorylation and serve as markers for activation of the DNA damage response. GAPDH served as a loading control. (d) p27Kip1 S140 phosphorylation is impaired by ATM-specific inhibitor in HEK293 cells. HEK293 cells were transfected with Myc-p27Kip1, treated with 0, 10 or 20 µM of ATM inhibitor KU-55933 and then irradiated to 10 Gy ionizing radiation (IR). CHK2 T68 and p53 S15 are DNA damage-associated targets of ATM phosphorylation. After 0.5 h recovery, immunoblot analyses were performed with the indicated antibodies.