4 DNA damage response regulates RAG1/2 expression through ATM-FOXO1 | 95 We observed FOXO1 phosphorylation on Ser256 upon NCS treatment (Figure 3A). Because FOXO1 Ser256 phosphorylation has previously been linked to ubiquitin-mediated FOXO1 degradation by Skp2, a subunit of the Skp1/Cul1/F-box protein complex35,36, we examined the level of FOXO1 protein following DNA damage. In response to NCS treatment, decreased levels of FOXO1 protein were detected in BV173 cells by immunoblot analysis (Figure 3A), coinciding with the appearance of a faster migrating band, suggestive of a FOXO1 cleavage product. Of note, this FOXO1 cleavage product was not prevented by pre-treatment with the pan-caspase inhibitor Z-VAD, or the acidic protease inhibitors chloroquine and NH4Cl (data not shown). Furthermore, FOXO1 mRNA levels were modestly but significantly reduced in BV173 cells (80% of DMSO-treated controls, p=0.0003) and in primary mouse WT pre-B cells (61% of DMSO-treated controls, p=0.02) upon induction of DNA damage (Figure 3B). Interestingly, DNA damage-induced FOXO1 phosphorylation and cleavage/degradation were prevented pre-treating BV173 cells with the ATM inhibitor KU55933 (Figure 3A). Similarly, KU55933 prevented the DNA damage-induced decrease of FOXO1 mRNA levels (Figure 3B). DNA damage thus seems to control the availability and expression of FOXO1 in an ATM-dependent manner. The fact that DNA damage had a modest effect on FOXO1 protein stability/cleavage, whereas it greatly impacted on the Erag-binding capacity of FOXO1 suggests that DNA damage regulates RAG1/2 expression primarily by modulating DNA binding of FOXO1 and that FOXO1 degradation/cleavage may be a secondary event. PI3K-AKT signaling is not involved in the regulation of RAG1 expression in response to DNA damage Because DNA damage induced FOXO1 phosphorylation on Ser256, which is an established AKT phosphorylation site that affects FOXO1 protein stability, we also explored the contribution of PI3K-AKT signaling in the regulation of FOXO1 and RAG1 following DNA damage. We found that induction of DNA damage resulted in the ATM-dependent phosphorylation of AKT on Ser473 in BV173 cells (Figure 3A), similar to that reported earlier for other cell types37. Besides FOXO1, mTOR has been suggested to be a direct target of AKT, phosphorylating mTOR on Ser2448 38. We assessed mTOR phosphorylation as a positive control for activation of AKT kinase, and consistently, in our experiments DNA damage-dependent AKT Ser473 phosphorylation coincided with increased mTOR Ser2448 phosphorylation (Figures 3A and 4A). Interestingly, inhibition of AKT kinase activity by increasing amounts of GSK690693 prevented NCS-induced phosphorylation of mTOR, but surprisingly, had no effect on the loss of RAG1 protein (Figure 4A). Pre-treatment with the AKT inhibitor had no appreciable effect on the DNA damage-induced p53 Ser15 phosphorylation. The use of a different structurally unrelated AKT kinase inhibitor, AKT inhibitor VIII, did not prevent the DNA damage-induced loss of RAG1 protein either (Figure 4B).
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