RESEARCH ARTICLE Differential proteome profiles in E2F2-deficient T lymphocytes Mikel Azkargorta 1 , Jesus M. Arizmendi 1 , Felix Elortza 2 , Nere Alkorta 2 , Ana M. Zubiaga 3 and Asier Fullaondo 3 1 Department of Biochemistry and Molecular Biology, University of the Basque Country, Leioa, Spain 2 Proteomics Unit, CIC bioGUNE, Derio, Spain 3 Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Leioa, Spain E2F transcription factors are important regulators of proliferation, differentiation and apoptosis. We have previously shown that E2F2 2/– mice develop late-onset autoimmune features, similar to systemic lupus erythematosus. E2F2-deficient T lymphocytes exhibit enhanced T cell receptor (TCR)-stimulated proliferation, which is presumably responsible for causing autoimmunity in E2F2-deficient mice. The comparison of E2F2 2/– and wild-type T lymphocyte expression profiles by 2-DE followed by MS identification has revealed a set of deregulated proteins involved in TCR- mediated signaling, cell survival and stress responses. The deregulation of these proteins may account for the hyperproliferative phenotype that characterizes E2F2 2/– T cells. Our work shows that proteomic analysis of gene-knockout strains can be a useful methodology to study the functional role of specific genes. Received: June 13, 2005 Revised: October 3, 2005 Accepted: October 18, 2005 Keywords: 2-DE / ATP synthase / Autoimmune disease / Cell cycle S42 Proteomics 2006, 6, S42–S50 1 Introduction E2F transcription factors (E2F1–8) are important regulators of proliferation, differentiation and apoptosis. They are known to play an important role in the control of cell cycle progression in connection with the retinoblastoma (Rb) family of proteins (Rb, p107 and p130), critical elements of the G1/S checkpoint [1]. Ectopic expression of E2F1–3, so- called “activating” E2Fs, results in transcriptional activation of genes involved in DNA replication (cdc6, orc1, mcm’s) and cell-cycle control (cycE, cycA, cdc2, cdc25A), suggesting that E2Fs are critical for the G1/S transition and for the correct progression through the cell cycle [2–4]. Recently, DNA microarray analyses have extended the list of potential E2F target genes. A number of genes involved in DNA damage checkpoint and repair, chromosome maintenance and seg- regation, mitotic checkpoint, apoptosis, differentiation and development have been identified [5], implying that the role of E2F transcription factors in cellular homeostasis is more complex than it was originally thought to be. The generation of mouse strains carrying targeted mutations for individual E2F genes has been instrumental to gain insight into the functions of these transcription factors. The characterization of these knockout mice has shown that loss of individual E2Fs can have distinct developmental and physiological consequences, implying the existence of spe- cific target genes for each E2F [6–8]. We have previously shown that E2F2 is required for immunological tolerance. Interestingly, E2F2-deficient T lymphocytes exhibit enhanced T cell receptor (TCR)-stimulated proliferation and a lower activation threshold, leading to the accumulation of memory T lymphocytes and the development of auto- immunity [7]. Contrary to what was expected for an “activat- Correspondence: Dr. Jesus M. Arizmendi, Department of Bio- chemistry and Molecular Biology, Faculty of Science and Tech- nology, University of the Basque Country, 48940 Leioa, Spain E-mail: jm.arizmendi@ehu.es Fax: 134-94-6013500 Abbreviations: Rb, retinoblastoma; ROS, reactive oxygen spe- cies; TCR, T cell receptor; WT , wild-type DOI 10.1002/pmic.200500438  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com