unlabeled whole cell lysate, and these differences in approach and cell culture conditions may cause variations in the measurement of protein. This suggests that DNA-damage may either stabilize menin protein levels or disrupt the pathways involved in menin degradation. MEN1-associated missense menin mutants show altered Ser394 and Ser487 phosphorylation A major question in the menin field is the link between missense point mutations and their loss of various menin functions— in particular, some but not all of these mutants lack the ability to interact with KMT2A/KMT2D [12]. To determine whether or not MEN1-associated missense point mutants are phosphorylated, we screened a panel of mutants for the ability to undergo Ser487, Ser543 and Ser394 phosphorylation. Flag-tagged point mutants Menin Is Phosphorylated in Response to DNA Damage were immunoprecipitated from transiently transfected 293T cells following treatment with Adr or UV. As shown in Figure 6A, the mutants P12L, L22R, and A309P fail to achieve the level of Ser394 phosphorylation that is observed in the wild type menin and the H139D and A242Vmutants. In addition, the P12L mutant also displayed altered Ser487 phosphorylation following UV treatment (Figure 6A). Interestingly, the MEN1-associated mutants found to be phosphorylated at Ser394, H139D and A242V, are also the tumor mutants that have been shown to not have the ability to interact with histone methyltransferase activity (HMTase) [12]. To examine whether or not Ser394 and/or Ser487 phosphorylation impacts the ability of menin to interact with HMTase activity, Men12/2 mouse embryonic fibroblasts (MEFs) stably expressing wild type menin, or phospho-deficient menin mutants were Figure 6. MEN1 missense point mutants display altered phosphorylation. (A) Flag IPs from293T cells transfected with Flag-wildtype menin, or Flag missense mutants following treatment with 0.05uM Adr for 18h, or 30 minutes after 25J/m2 UV. IPs were immunoblotted with phospho-Ser394, phospho-Ser487, phospho-Ser543 or total menin. (B) Menin IPs from Men12/2 MEFs expressing wildtype or phospho-deficient point mutants were incubated with histone H3 and the methyl donor 3H-SAM to assay for histone methyltransferase activity. Reactions were resolved on 15% SDS-PAGE, amplified, dried and fluorographed. (C) Flag IPs from 293T cells transfected with phospho-deficient point mutants 30 minutes following treatment with 25 J/m2 UV or 18 h following 0.05 uM Adr, were resolved and immunoblotted with phospho-menin antibodies or phospho-RNAPII antibodies. doi:10.1371/journal.pone.0016119.g006 PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e16119