EGF-mediated Akt signaling causes Bmi-1 phosphorylation and up-regulation in NSCs Neurosphere formation and NSC proliferation are dependent on EGF (Epidermal growth factor) through signaling by the EGFR (Epidermal Growth Factor Receptor) tyrosine kinase [17]. Therefore, we investigated if signaling by EGF had functional consequences on the proteasomal degradation of Bmi-1. First, the phosphorylation state of Akt was analyzed to evaluate the activity of the phosphoinositide-3-kinase (PI3-K) signaling pathway after EGF treatment. We observed that Akt is activated shortly after EGF treatment in EGF-starved Atm+/+ NSCs and activation is inhibited by PI3-K inhibitor, LY2949002 (Figure 4A). Because Bmi-1 represses gene expression of CDK inhibitors through H2A ubiquitination and promotes NSC proliferation [18–19], we examined the effects of EGF treatment on H2A ubiquitination. H2A ubiquitination was reduced during EGF-starvation, but was restored when EGF was added back to the culture (Figure 4B) coincided with Akt activation, indicating that H2A ubiquitination by Bmi-1 requires EGF-Akt signaling. Next, we examined whether EGF-Akt signaling affects Bmi-1 levels and found that Akt activation by EGF treatment coincided with Bmi-1 up-regulation. We also investigated if Bmi-1 up-regulation by EGF correlates with phosphorylation by Akt. To verify the phosphorylation status of Bmi-1, we treated the protein extracts with l phosphatase, a Ser/Thr and Tyr phosphatase. We found that phosphorylation of Bmi-1 (slower migration on the gel) was increased by EGF treatment and was eliminated by l phosphatase as evidenced by faster migration on the gel (Figure 4C), indicating that Bmi-1 is a phosphoprotein. Collectively, these results imply that Bmi-1 up- regulation and its function in histone H2A ubiquitination is dependent on Akt signaling in NSCs. We next questioned whether Akt is involved in Bmi-1 phosphorylation, thus we performed an amino acid sequence analysis to identify Bmi-1 phosphorylation sites (http://www.scansite.edu). Since Bmi-1 contains a predicted Akt substrate motif, we decided to determine if Bmi-1 is a substrate for Akt. Bmi-1 purified by immunoprecipitation from NSCs was detected by anti-phospho (Ser/Thr) Akt substrate antibody. However, it was less detectable by anti-phospho (Ser/Thr) Akt substrate antibody in NSCs treated with LY294002 (data not shown), suggesting that Bmi-1 may be a substrate of Akt. In Atm+/+ NSCs, Bmi-1 is highly expressed and localizes in the nucleus. Bmi-1 levels were reduced under EGF-starvation condi- tions or after treatment with LY2949002 (Figure 4D, upper panels), further suggesting Akt-dependent Bmi-1 up-regulation. In the presence of EGF stimulus, Bmi-1 localizes in chromatin of glioma stem cells (GSCs), evidenced by the fact that Bmi-1 in GSCs co- localizes with histone H3 in the immunocytochemistry image (data not shown). However, EGF-starvation significantly abolishes Bmi-1 nuclear accumulation and the LY2949002 partially blocks Bmi-1 nuclear accumulation (Figure 4D, lower panels). These results suggest that Bmi-1 nuclear localization is also an Akt-dependent process. Our results demonstrate that Akt signaling causes Bmi-1 phosphorylation in NSCs resulting in up-regulation of Bmi-1. Bmi-1 is downregulated by p38 signaling during oxidative stress through Akt inhibition We have previously reported that H2O2 highly decreases NSC proliferation, and that the p38 inhibitor, SB203580, restores normal proliferation [6]. In the present study we showed that H2O2 decreased Bmi-1 levels in NSCs (Figure 3D), therefore we asked whether the inhibition of p38 signaling by SB203580 rescues Bmi-1 levels even under oxidative stress conditions. We observed that H2O2 down-regulates Bmi-1 in Atm+/+ NSCs, but SB203580 ROS Accelerates Proteasomal Degradation of Bmi-1 to some extent restores Bmi-1 levels in H2O2-treated NSCs. Restorative effects were not observed after treatment with the PI3- K inhibitor LY2949002, nor ERK1/2 inhibitor PD98059 (Figure 5A). Since p21 expression was selectively increased by H2O2-treatment in Atm+/+ NSCs (Figure 3E), we also questioned whether p38 inhibition by SB203580 has an effect on p21 expression in H2O2-treated NSCs. As expected, no distinct differences in bmi-1, p16, and p19 expression were observed in H2O2-treated NSCs. p21 expression significantly increased in response to H2O2 treatment, but the p38 inhibitor, SB203580, highly reduced p21 expression (Figure 5B), further indicating that during oxidative stress Bmi-1 down-regulation and its functional consequence are a p38 dependent process. The existence of cross talk between p38 MAPKs and the PI3K/Akt and/or ERK survival pathways has also been described in other cellular systems. For example, activation of p38 MAPKs inhibits the PI3K/Akt and/or ERK pathways through activation of PP2A phosphatases [20–22]. In the present study, we showed Akt pathway stabilizes Bmi-1 (Figure 4). However, negative regulation of Akt by p38 in NSCs and the relevance of this cross talk in the Bmi-1 regulation are unclear. Therefore, we decided to investigate whether negative regulation of Bmi-1 by p38 could be linked to Akt and/or PP2A regulation. We treated Atm+/+ NSCs with okadaic acid, a PP2A inhibitor or with l phosphatase, a Ser/Thr and Tyr phosphatase. We expected that okadaic acid would increase Akt phosphorylation because PP2A is known to dephosphorylate Akt. However we observed that okadaic acid decreased Akt phosphorylation levels. In contrast, okadaic acid triggered phosphorylation of both p38 and Bmi-1. This result indicates that p38 negatively regulates Akt via PP2A in NSCs, and that Bmi-1 is phosphorylated by an Akt-independent mechanism. On the other hand, treatment of protein extract with l phosphatase dephosphorylated p38 and Akt, as well as Bmi-1 (Figure 5C). These results suggest that there might be an inhibitory cross talk between p38 and Akt as they show opposite activation events by okadaic acid and implies the relevance of Akt inhibition by PP2A in the down-regulation of Bmi-1 induced by p38 activation. Interestingly, okadaic acid treatment has similar effects on p38, Akt, and Bmi-1 with H2O2 treatment, eliciting p38 and Bmi-1 phosphorylation and Akt dephosphorylation (compare with Figure 5D, at 1 hour). These results suggest that although Bmi-1 contains no known p38 phosphorylation motif (http://www. scansite.edu), p38 may also phosphorylate Bmi-1 when Akt is unable to phosphorylate Bmi-1 under oxidative stress conditions. To further link p38/Akt to Bmi-1 stability, we asked whether the proteasome inhibitor MG132 prevents H2O2-induced p38 phos- phorylation and restores levels of Akt phosphorylation and Bmi-1 expression. We treated Atm+/+ NSCs with H2O2 for 1 or 7 hours. Bmi-1 levels were decreased at 1 hour and barely detected at 7 hours. In contrast, MG132 stabilized Akt phosphorylation, coinciding with Bmi-1 up-regulation and phosphorylation (Figure 5D). These observations indicate that the Akt-dependent phosphorylation of Bmi-1 results in its up-regulation by inhibiting the proteasome-dependent degradation process. Interestingly, MG132 downregulates p38 activation levels (Figure 5D) and SB203580 restored Bmi-1 levels in H2O2-treated Atm+/+ NSCs (Figure 5A). Collectively, these results demonstrate that during oxidative stress, Bmi-1 is downregulated by p38-mediated proteasomal degradation through Akt inhibition. p38 signaling leads to proteasomal degradation of Bmi-1 in Atm-/- NSCs We compared the phosphorylation status of Bmi-1 in protein extracts from Atm+/+ and Atm-/- NSCs after treatment with l phosphatase. Bmi-1 phosphorylation was unimpaired regardless of PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e16615