title: Identification of Novel Nuclear Factor of Activated T Cell (NFAT)-associated Proteins in T Cells.
abstract: Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/αA, NFATc1/βC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.
authors.affiliation: From the German Rheumatism Research Center (DRFZ), Leibniz Institute, 10117 Berlin.
authors.forename: Christian H
authors.initials: CH
authors.lastname: Gabriel
authors.affiliation: the Institute for Theoretical Biology, Charité and Humboldt University Berlin, 10015 Berlin.
authors.forename: Fridolin
authors.initials: F
authors.lastname: Gross
authors.affiliation: From the German Rheumatism Research Center (DRFZ), Leibniz Institute, 10117 Berlin.
authors.forename: Martin
authors.initials: M
authors.lastname: Karl
authors.affiliation: the Leibniz-Institut für Molekulare Pharmakologie, 13125 Berlin.
authors.forename: Heike
authors.initials: H
authors.lastname: Stephanowitz
authors.affiliation: From the German Rheumatism Research Center (DRFZ), Leibniz Institute, 10117 Berlin.
authors.forename: Anna Floriane
authors.initials: AF
authors.lastname: Hennig
authors.affiliation: From the German Rheumatism Research Center (DRFZ), Leibniz Institute, 10117 Berlin.
authors.forename: Melanie
authors.initials: M
authors.lastname: Weber
authors.affiliation: From the German Rheumatism Research Center (DRFZ), Leibniz Institute, 10117 Berlin.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Gryzik
authors.affiliation: the MicroDiscovery GmbH, 10405 Berlin, and.
authors.forename: Ivo
authors.initials: I
authors.lastname: Bachmann
authors.affiliation: From the German Rheumatism Research Center (DRFZ), Leibniz Institute, 10117 Berlin.
authors.forename: Katharina
authors.initials: K
authors.lastname: Hecklau
authors.affiliation: the Institute of Cellular and Molecular Immunology, Georg-August-University of Göttingen, 37073 Göttingen, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Wienands
authors.affiliation: the MicroDiscovery GmbH, 10405 Berlin, and.
authors.forename: Johannes
authors.initials: J
authors.lastname: Schuchhardt
authors.affiliation: the Institute for Theoretical Biology, Charité and Humboldt University Berlin, 10015 Berlin.
authors.forename: Hanspeter
authors.initials: H
authors.lastname: Herzel
authors.affiliation: From the German Rheumatism Research Center (DRFZ), Leibniz Institute, 10117 Berlin.
authors.forename: Andreas
authors.initials: A
authors.lastname: Radbruch
authors.affiliation: the Leibniz-Institut für Molekulare Pharmakologie, 13125 Berlin.
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
authors.affiliation: From the German Rheumatism Research Center (DRFZ), Leibniz Institute, 10117 Berlin, baumgrass@drfz.de.
authors.forename: Ria
authors.initials: R
authors.lastname: Baumgrass
mesh_terms: D006801:Humans; D019169:Jurkat Cells; D013058:Mass Spectrometry; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D050778:NFATC Transcription Factors; C494003:NFATC1 protein, human; C494006:NFATC2 protein, human; D009687:Nuclear Proteins
keywords: AP1 transcription factor (AP1); NFAT transcription factor; RUNX transcription factor; SILAC; bioinformatics; cAMP-response element-binding protein (CREB); lymphocyte; mass spectrometry (MS); protein-protein interaction