ROS Accelerates Proteasomal Degradation of Bmi-1 Figure 5. Bmi-1 is downregulated by p38 signaling during oxidative stress through inhibiting Akt. (A) Atm+/+ NSCs were pretreated for 2 hours with either LY2949002 (LY, 10 mM), PD98059 (PD, 10 mM), or SB203580 (SB, 10 mM), and then treated for 4 hours with 100 mM H2O2. Protein samples were analyzed by Western blotting fo levels of phospho-p38, p38, phospho-Akt, Akt, and Bmi-1. (B) Atm+/+ NSCs were pretreated for 2 hours with SB203580 (SB, 10 mM) and then treated for 4 hours with 100 mM H2O2. Total RNA were purified and analyzed by quantitative RT-PCR analysis for bmi-1, p21, p16, and p19 expression. Probing for gapdh was used as an internal control. (C) Atm+/+ NSCs were treated for 4 hours with okadaic acid (OA, PP2A inhibitor) or 100 mM H2O2. Protein samples (30 mg) were treated in vitro with l phosphatase for 1.5 hours and analyzed by Western blotting for levels of phospho-p38 and p38, phospho-Akt, Akt, and Bmi-1. (D) Atm+/+ NSCs were treated with 100 mM H2O2 for times indicated in the presence or absence of 10 mM MG132, and analyzed by Western blotting for levels of phospho-p38 and p38, phospho-Akt, Akt, and Bmi-1. doi:10.1371/journal.pone.0016615.g005 NSCs (Figure 6B). These results suggest that Bmi-1 down- regulation in NSCs occurs by proteasomal degradation and Akt signaling protects Bmi-1 from proteasomal degradation. We analyzed an Akt activation profile in Atm+/+ and Atm-/- NSCs after exposure to H2O2. We found that Atm-/- NSCs, but not Atm+/+ NSCs, failed to activate Akt after exposure to H2O2 (data not shown), demonstrating that ATM is required for the activation of Akt. In the absence of ATM, Bmi-1 phosphorylation by Akt is reduced, which might accelerate the p38-dependent degradation of Bmi-1. Notably, treatment of Atm-/- NSCs with SB203580 restored Akt activation and normal proliferation [6], which implies that in Atm-/- NSCs, Bmi-1 degradation may be accelerated by chronic p38 activation. To address this hypothesis we treated Atm+/+ and Atm-/- NSCs with cycloheximide, to block de novo synthesis of Bmi-1 and the half-life of Bmi-1 was compared by Western blotting analysis. Next, to confirm that Bmi-1 degradation is linked to p38 signaling in Atm-/- NSCs, we treated Atm-/- NSCs with SB203580 and calculated the half-life of Bmi-1 as compared to untreated NSCs. We observed that the half-life of Bmi-1 in Atm-/- NSCs was shortened compared to Atm+/+ NSCs, but SB203580 extended the turnover of Bmi-1 in Atm-/- NSCs (Figure 6C). We also found that Bmi-1 levels and H2A ubiquitination are reduced in Atm-/- NSCs compared to Atm+/+ NSCs. Inhibition of p38 signaling by SB203580 significantly restored these levels in Atm-/- NSCs (Figure 6D). Taken together with the up-regulation of p38 signaling and the down-regulation of Akt signaling in Atm-/- NSCs (Figure 1A), these results demonstrate that in Atm-/- NSCs p38-mediated proteasomal degradation of Bmi-1 is accelerated compared to normal NSCs. Discussion The majority of previous investigations examining A-T associated neurodegeneration have focused on neurons and PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16615