The Menin Tumor Suppressor Protein Is Phosphorylated in Response to DNA Damage Joshua Francis1,2, Wenchu Lin1,2, Orit Rozenblatt-Rosen1,2, Matthew Meyerson1,2* 1 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America Abstract Background: Multiple endocrine neoplasia type 1 (MEN1) is a heritable cancer syndrome characterized by tumors of the pituitary, pancreas and parathyroid. Menin, the product of the MEN1 gene, is a tumor suppressor protein that functions in part through the regulation of transcription mediated by interactions with chromatin modifying enzymes. Principal Findings: Here we show menin association with the 59 regions of DNA damage response genes increases after DNA damage and is correlated with RNA polymerase II association but not with changes in histone methylation. Furthermore, we were able to detect significant levels of menin at the 39 regions of CDKN1A and GADD45A under conditions of enhanced transcription following DNA damage. We also demonstrate that menin is specifically phosphorylated at Ser394 in response to several forms of DNA damage, Ser487 is dynamically phosphorylated and Ser543 is constitutively phosphorylated. Phosphorylation at these sites however does not influence the ability to interact with histone methyltransferase activity. In contrast, the interaction between menin and RNA polymerase II is influenced by phosphorylation, whereby a phospho-deficient mutant had a higher affinity for the elongating form of RNA polymerase compared to wild type. Additionally, a subset of MEN1-associated missense point mutants, fail to undergo DNA damage dependent phosphorylation. Conclusion: Together, our findings suggest that the menin tumor suppressor protein undergoes DNA damage induced phosphorylation and participates in the DNA damage transcriptional response. Citation: Francis J, Lin W, Rozenblatt-Rosen O, Meyerson M (2011) The Menin Tumor Suppressor Protein Is Phosphorylated in Response to DNA Damage. PLoS ONE 6(1): e16119. doi:10.1371/journal.pone.0016119 Editor: Kerstin Borgmann, University Medical Center Hamburg-Eppendorf, Germany Received August 6, 2010; Accepted December 8, 2010; Published January 14, 2011 Copyright: 2011 Francis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Caring for Carcinoid Foundation (M.M.) and a Ruth L. Kirschstein National Research Service Award Post-Doctoral fellowship F32DK77424 (J.F.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: matthew_meyerson@dfci.harvard.edu Introduction Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene is a tumor suppressor protein whose mechanism of action is not completely understood [1,2]. Germline mutations of MEN1 result in an autosomal dominant syndrome characterized by tumors of the endocrine pancreas, the anterior pituitary, and the parathyroid glands [3]. Somatic mutations of the MEN1 gene have also been described in neuroendocrine tumors and arise when the second wild-type allele has undergone a loss of heterozygosity [4,5,6]. While MEN1 mutations are only tumori- genic within neuroendocrine lineages, menin is expressed in most tissues, at all stages of development and likely has a universal function [7,8]. Sequence analysis of menin protein reveals a high degree of conservation amongst metazoans; however, no obvious functional domains have yet been identified [9]. Menin has been reported to interact with a broad spectrum of proteins, most of which participate in the regulation of transcription (see reviews for details [10,11]. An important insight into understanding the tumor suppressor functions of menin occurred when it was found that menin interacts with a SET1-like histone methyltransferase complex containing KMT2A (MLL) and KMT2D (MLL4 originally known as MLL2) [12,13]. Men1+/- mice that have undergone loss of the wild-type allele within the pancreatic islet develop islet cell hyperplasia and insulinomas, a phenotype that mirrors the human disease [14]. In these islets, there is a failure to recruit the histone methyltrans- ferase complex to the cyclin-dependent kinase inhibitor genes CDKN2C (p18) and CDKN1B (p27) leading to an attenuation of transcription and an increase in proliferation [14]. Surprisingly, menin is also required in the formation for MLL-fusion induced leukemias, where it is thus acting in a pro-oncogenic manner [15]. Genome-wide chromatin studies have found menin to be associated with promoter and coding regions of hundreds of genes suggesting a more general function in transcription [16,17]. While these results are compelling, studies by several laboratories have shown that a portion of MEN1-associated missense point mutants retain the ability to interact with KMT2A/KMT2D suggesting an additional function or functions for menin tumor suppressor activity outside of histone methylation [12,18]. Recently, menin was found to function with SKIP and c-Myc in HIV-1 Tat- mediated transcription which was independent of KMT2A (MLL) [19]. The DNA damage response is an intricate cellular system that coordinates the repair of DNA damage, the activation of cell-cycle checkpoints to facilitate repair, and apoptosis in order to eliminate PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16119