The E3 ubiquitin ligase Mule acts through the ATM–p53 axis to maintain B lymphocyte homeostasis A r t i c l e Zhenyue Hao,1,2,3 Gordon S. Duncan,1 Yu-Wen Su,1 Wanda Y. Li,1 Jennifer Silvester,1 Claire Hong,1 Han You,1 Dirk Brenner,1 Chiara Gorrini,1 Jillian Haight,1 Andrew Wakeham,1 Annick You-Ten,1 Susan McCracken,1 Andrew Elia,1 Qinxi Li,1 Jacqui Detmar,5 Andrea Jurisicova,5 Elias Hobeika,6,7 Michael Reth,6,7 Yi Sheng,8 Philipp A. Lang,1 Pamela S. Ohashi,1,2,3,4 Qing Zhong,9 Xiaodong Wang,10 and Tak W. Mak1,2,3,4 1The Campbell Family Institute for Cancer Research and 2Ontario Cancer Institute, University Health Network, Toronto, Ontario M5G 2M9, Canada 3Department of Medical Biophysics and 4Department of Immunology, University of Toronto, Toronto, Ontario M5G 2C1, Canada 5Division of Reproductive Sciences, Department of Obstetrics and Gynecology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada 6Max Planck Institute of Immunobiology and 7Centre for Biological Signaling Studies, BIOSS, University of Freiburg, 79104 Freiburg, Germany 8Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada 9Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 10Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 Cellular homeostasis is controlled by pathways that balance cell death with survival. Mcl-1 ubiquitin ligase E3 (Mule) is an E3 ubiquitin ligase that targets the proapoptotic molecule p53 for polyubiquitination and degradation. To elucidate the role of Mule in B lymphocyte homeostasis, B cell–specific Mule knockout (BMKO) mice were generated using the Cre– LoxP recombination system. Analysis of BMKO mice showed that Mule was essential for B cell development, proliferation, homeostasis, and humoral immune responses. p53 trans- activation was increased by two- to fourfold in Mule-deficient B cells at steady state. Genetic ablation of p53 in BMKO mice restored B cell development, proliferation, and homeostasis. p53 protein was increased in resting Mule-deficient mouse embryonic fibro- blasts (MEFs) and embryonic stem (ES) cells. Loss of Mule in both MEFs and B cells at steady state resulted in increased levels of phospho–ataxia telangiectasia mutated (ATM) and the ATM substrate p53. Under genotoxic stress, BMKO B cells were resistant to apop- tosis, and control MEFs exhibited evidence of a physical interaction between Mule and phospho-ATM. Phospho-ATM, phospho-p53, and Brca1 levels were reduced in Mule- deficient B cells and MEFs subjected to genotoxic stress. Thus, Mule regulates the ATM– p53 axis to maintain B cell homeostasis under both steady-state and stress conditions. The homeostasis of any cellular compartment is maintained by complex pathways that balance survival and apoptosis. For lymphocytes, pro- survival signaling pathways are often countered by the intrinsic pathway of apoptosis in the mitochondria (Rathmell and Thompson, 2002), such that mutation of genes in this pathway leads to lymphoproliferative disorders (Bouillet et al., 1999; Rathmell et al., 2002; Hao et al., 2005). Mitochondrial apoptosis can be triggered by the activation of p53, a major regulator of cellular homeostasis under both steady-state and stress conditions (Villunger et al., 2003; Green and Kroemer, 2009). Because indiscriminant triggering of p53 activation could kill healthy © 2012 Hao et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ by-nc-sa/3.0/). e n i c i d e M l a t n e m i r e p x E f o l a n r u o J e h T CORRESPONDENCE Zhenyue Hao: zhenyuehao@gmail.com OR Tak. W. Mak: tmak@uhnres.utoronto.ca Abbreviations used:Ab, anti- body;ATM, ataxia telangiectasia mutated; BDKO, B cell–specific double KO; BMKO, B cell– specific Mule KO; CSR, class switch recombination; Dox, doxorubicin; ES, embryonic stem; FO, follicular; MEF, mouse embryonic fibroblast; Mule, Mcl-1 ubiquitin ligase E3; MZ, marginal zone; NP(15)-CG, 4-hydroxy-3-nitrophenylacetyl– chicken -globulin; Td, T de- pendent; Ti,T independent; TS, transitional; VSV, vesicular stomatitis virus. The Rockefeller University Press $30.00 J. Exp. Med. Vol. 209 No. 1 173-186 www.jem.org/cgi/doi/10.1084/jem.20111363 173