176 | Chapter 7 specifically recognizing the phosphorylation of the Ser215 residue of FOXO1. However, in our study, we found that DNA damage did not have any effect on MAPKAPK5 protein levels. We further hypothesized that the activated ATM may possibly recruit other proteins that are able to modulate the stability of FOXO1. For instance, it has been demonstrated that silent information regulator 2 (Sir2) binds and deacetylates FOXO1 at Lys242, Lys245, and Lys262 residues acetylated by cAMP-response element-binding protein (CREB)-binding protein (CBP)63. The acetylation of FOXO1 results in decreased DNA binding and expression of FOXO1 target genes64. In addition, in response to DNA damage, ATM was shown to inhibit SIRT1, the mammalian Sir2 orthologue65. We therefore speculate that upon induction of DNA damage, ATM might inhibit SIRT1, leading to increased FOXO1 acetylation, and consequently decreased FOXO1 DNA-binding capacity, thereby reducing the expression of Rag1 and Rag2 mRNA. However, our data did not convincingly show that DNA damage affected FOXO1 acetylation in a human BCR-ABL1+ pre-B-cell line after NCS exposure. To further provide a mechanistic explanation of how FOXO1 regulates RAG1/2 expression in pre-B cells, we investigated signaling pathways involved in the regulation of FOXO1. The PI3K/AKT pathway plays a pivotal role in regulating the activity of the transcription factor FOXO1. In response to pre-B cell receptor (pre-BCR) signals, AKT phosphorylates FOXO1 at Ser256. Phosphorylation at this residue by AKT leads to the nuclear exclusion and subsequent degradation of FOXO1, inhibiting its transcriptional activity and preventing the expression of its target genes. Additionally, AKT can phosphorylate other serine and threonine residues on FOXO1, contributing to the regulation of FOXO1 activity in response to signaling through the PI3K/AKT pathway66. In our study, we observed that DNA damage resulted in ATM-dependent phosphorylation of AKT at Ser473. In response to DSBs, ATM-dependent AKT activation was shown to be critical for downstream transmission of signals to extracellular signal-regulated kinase (ERK), thereby triggering either cell proliferation and survival, or apoptosis, depending on the extent of the DNA damage67. Moreover, we found other downstream AKT targets68 to be phosphorylated in response to NCS-induced DNA damage, such as mTOR on Ser2448. We therefore hypothesized that PI3K/AKT might play a role in DNA damage-induced downregulation of RAG. However, contrary to our hypothesis, AKT inhibition failed to prevent the DNA damage-induced downregulation of RAG. We used 2 different small-molecule AKT inhibitors (GSK690693 and AKT inhibitor VIII), none of which was able to prevent the damage-induced RAG downregulation. In addition, overexpression of the dominant-negative kinase-dead mutant of AKT did not prevent the NCS-induced RAG downregulation either. Though under circumstances that do not involve exposure to DNA damage, the PI3K/AKT pathway has been shown to modulate RAG expression in developing B cells69, we conclude that the DNA damage-induced downregulation of RAG in pre-B cells is not mediated through AKT signaling. In addition to AKT, we investigated the effect of pharmacological inhibition of other kinases that have been reported to regulate FOXO1 activity and subcellular localization, such as p38 mito-
RkJQdWJsaXNoZXIy MTk4NDMw