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Negative Regulation of C/EBPbeta1 by Sumoylation in Breast Cancer Cells
Allison A. Atwood1, Rachel Jerrell2, Linda Sealy1,2*
1 Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America, 2 Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of AmericaAbstract
Sumoylation is a post-translational modification that is oftentimes deregulated in diseases such as cancer. Transcription factors are frequent targets of sumoylation and modification by SUMO can affect subcellular localization, transcriptional activity, and stability of the target protein. C/EBPbeta1 is one such transcription factor that is modified by SUMO-2/3. Nonsumoylated C/EBPbeta1,p52-C/EBPbeta1, is expressed in normal mammary epithelial cells but not breast cancer cell lines and plays a role in oncogene-induced senescence, a tumor suppressive mechanism. Although p52-C/EBPbeta1 is not observed via immunoblot in breast cancer cell lines, higher molecular weight bands are observed when breast cancer cell lines are subjected to immunoblot analysis with a C/EBPbeta1-specificantibody. We show that exogenously expressed C/ EBPbeta1 is sumoylated in breast cancer cells, and that the higher molecular weight bands we observe in anti-C/EBPbeta1 immunoblots of breast cancer cell lines is sumoylated C/EBPbeta1. Phosphorylation oftentimes enhances sumoylation, and phosphorylation cascades are activated in breast cancer cells. We demonstrate that phosphorylation of C/EBPbeta1Thr235by Erk-2 enhances sumoylation of C/EBPbeta1 in vitro. In addition, sumoylated C/EBPbeta1 is phosphorylated on Thr235 and mutation of Thr235 to alanine leads to a decrease in sumoylation of C/EBPbeta1. Finally, using a C/EBPbeta1-SUMO fusion protein we show that constitutive sumoylation of C/EBPbeta1 completely blocks its capability to induce senescence in WI38 fibroblasts expressing hTERT. Thus,sumolylation of C/EBPbeta1 in breast cancer cells may be a mechanism to circumvent oncogene-induced senescence.
Citation: Atwood AA, Jerrell R, Sealy L (2011) Negative Regulation of C/EBPbeta1 by Sumoylation in Breast Cancer Cells. PLoS ONE 6(9): e25205. doi:10.1371/ journal.pone.0025205 Editor: Pan-Chyr Yang, National Taiwan University Hosipital, Taiwan Received July 22, 2011; Accepted August 29,2011; Published September 28, 2011 Copyright: ß 2011 Atwood 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 funded by the National Institutes of Health, NIH GM69634, and the CellBiology and Molecular Sciences training grant to Vanderbilt University School of Medicine. 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: linda.sealy@vanderbilt.edu

Introduction
The post-translational modification sumoylationregulates the function of a growing list of proteins that have roles in a variety of cell processes. Because of this, deregulation of the SUMO pathway has been observed in numerous diseases including neurodegenerative disorders [1] diabetes [2], and cancer [3]. Four members of the SUMO (Small Ubiquitin-like MOdifier) family have been identified, SUMO-1, -2, -3, and -4, all of which share homologywith ubiquitin [4]. SUMO-2 and SUMO-3 only differ from one another by three amino acid residues and are viewed as being functionally identical. SUMO-2/3 are 50% identical to SUMO-1 [5]. Much less is known about SUMO-4 than the first three members of the SUMO family. SUMO proteins are a group of polypeptides that conjugate to the lysine residue within the target four amino acid consensus sequence:...