DNA damage induced phosphorylation of p53 at Ser15 is shown after both treatments (Figure 4C, lower panel). These data demonstrate that menin is phosphorylated and dephosphorylated at different rates following c-IR and UV, which may be important in mediating protein-protein interactions. To understand whether or not phosphorylation of Ser394 was dependent upon Ser487 and vice versa, a time course study was performed using 293T cells transiently transfected with Flag- tagged wild type menin, Ser394Ala or Ser487Ala point mutants and subjected to either c-R or UV. Treatment of cells expressing Flag-Ser487Ala with either c-IR or UV led to an increase in detectable phosphorylation of Ser394, however the duration of the signal was attenuated after c-IR while found to be more intense after UV treatment as compared to Flag-tagged wild type menin (Figure S4A). In Flag-Ser394Ala expressing cells, Ser487 phos- phorylation levels were elevated for a more prolonged duration following both c-IR and UV treatment as compared to wild type (Figure S4B). These results suggest that the phosphorylation of Ser394 may also be important in the dephosphorylation of Ser487 and that a complex interplay may exist between the phosphory- lation of Ser394 and Ser487. These results demonstrate that menin undergoes at least three post-translational modifications, with Ser394 phosphorylation being DNA-damage dependent. Both Ser543 and Ser487 phosphorylation were present under normal cell culture condi- tions, however an increase in Ser487 phosphorylation does occur following DNA damage. Menin Ser394 phosphorylation is dependent upon ATM and ATR The DNA damage transducing kinases ATM, ATM- and Rad3- related (ATR) and DNA-Protein Kinase (DNA-PK) are activated after damage and phosphorylate downstream targets containing SQ/TQ recognition motifs. To determine whether menin Ser394 phosphorylation was dependent upon ATM and/or ATR follow- ing DNA damage we utilized cells deficient in these enzymes. Following c-IR treatment, there was no detectable phospho- Ser394 in ATM-deficient GM03189 cells while a strong signal was detected in the ATR-deficient GM18326 cells and control GM03323 cells (Figure 5A, upper panel, lanes 2, 5 and 8). In contrast, UV treatment resulted in a strong induction of Ser394 phosphorylation only in control cells while very weak signals were seen in both ATM-and ATR-deficient cells. Phosphorylation of p53 at Ser15 was also abrogated in the ATM deficient cell line after c-IR and UV, indicating the importance of ATM signaling after both forms of damage (Figure 5A, lower panel). To examine further whether Ser394 phosphorylation was dependent upon ATM, 293T cells were pretreated with the ATM inhibitor KU55933 prior to c-IR or UV treatment. A dramatic decrease in detectable Ser394 phosphorylation was seen in cells treated with KU55933 after c-IR when compared to c-IR alone (Figure 5B, upper panel, lanes 2 and 3). No detectable change was observed after UV treatment in the presence or absence of KU55933. Cells pretreated with KU55933 showed a reduction in p53-Ser15 phosphorylation after c-IR treatment but not UV (Figure 5B, lower panel). Pretreatment with a DNA-PK inhibitor did not influence menin-Ser394 phosphorylation after either c-IR or UV exposure (Figure 5C, upper panel, lanes 4 and 6). These results indicate that c-IR induced menin Ser394 phosphorylation is dependent upon ATM, and that UV-induced menin phosphorylation is dependent upon ATR. To determine if menin half-life is extended following DNA damage, we next treated cells with c-IR and added the protein synthesis inhibitor cycloheximide (CHX). In cells treated with Menin Is Phosphorylated in Response to DNA Damage Figure 5. Menin phosphorylation is dependent upon ATM and ATR. (A) Menin IPs from control GM03323, ATM deficient GM03189 and ATR deficient GM18326 lymphoblastoid cells were performed 90 minutes after exposure to 1000 Rads c-IR or 30 minutes after 25J/m2 UV. IPs were resolved and immunoblotted with phospho-Ser394 or total menin antibodies. Whole cell extracts from each cell type were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). (B) 293T cells were pretreated with 10 uM KU55933 for 2 hours prior to treatment with 1000 Rads c-IR or 25J/m2 UV. Menin IPs were performed, resolved and immunoblotted for phospho-Ser394 or total menin. The same whole cell extracts were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). (C) 293T cells were pretreated with 10 uM DNA-PK inhibitor II for 2 hours prior to treatment with 1000 Rads c-IR or 25J/m2 UV. Menin IPs were performed, resolved and immunoblotted for phospho-Ser394 or total menin. The same whole cell extracts were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). doi:10.1371/journal.pone.0016119.g005 CHX alone, menin had a half-life of approximately 45 minutes, while cells treated with c-IR and CHX, menin half-life was extended for at least 2 hours (Figure S4C). These data differ from the half-life reported by Yaguchi et al [31]. The likely explanation is because the previous study analyzed either transfected menin and/or analysis of methionine-labeled protein rather than PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16119