Human ubiquitin specific protease 31 is a deubiquitinating enzyme implicated in activation of nuclear factor-nB Christos Tzimas a,c , Gianna Michailidou a , Minas Arsenakis a , Elliott Kieff b , George Mosialos c, * , Eudoxia G. Hatzivassiliou a,c, * a Department of Biology, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece b Infectious Disease Division, Brigham and Women’s Hospital, 181 Longwood Ave, Boston, MA 02115, USA c Institute of Immunology, Biomedical Sciences Research Center Al. Fleming, 34 Al. Fleming Str., Vari 16672, Greece Received 4 March 2005; received in revised form 16 March 2005; accepted 16 March 2005 Available online 26 April 2005 Abstract TRAF2 mediates activation of the transcription factors NF-nB and AP1 by TNF. A yeast two-hybrid screen of a human cDNA library identified a ubiquitin specific protease homologue (USP31) as a TRAF2-interacting protein. Two cDNAs encoding for USP31 were identified. One cDNA encodes a 1035-amino acid long isoform of USP31 (USP31, long isoform) and the other a 485-amino acid long isoform of USP31 (USP31S1, short isoform). USP31 and USP31S1 share a common amino terminal region with homology to the catalytic region of known deubiquitinating enzymes. Enzymatic assays demonstrated that USP31 but not USP31S1 possess deubiquitinating activity. Furthermore, it was shown that USP31 has a higher activity towards lysine-63-linked as compared to lysine-48-linked polyubiquitin chains. Overexpression of USP31 in HEK 293T cells inhibited TNFa , CD40, LMP1, TRAF2, TRAF6 and IKKh-mediated NF-nB activation, but did not inhibit Smad-mediated transcription activation. In addition, both USP31 isoforms interact with p65/RelA. Our data support a role for USP31 in the regulation of NF-nB activation by members of the TNF receptor superfamily. D 2005 Elsevier Inc. All rights reserved. Keywords: NF-nB signaling; TNF; TNFR; TRAF; Ubiquitin proteasome system; Ubiquitin specific protease 1. Introduction TRAFs constitute a family of proteins that have been implicated in signal transduction by members of the TNF receptor superfamily including TNFRI, TNFRII, CD40, CD30, CD27, LThR and the EBV oncoprotein LMP1 [1]. Six mammalian TRAF molecules have been identified so far. Among them, TRAF2, TRAF5 and TRAF6 have the ability to activate the NF-nB and AP1 signaling path- ways, upon overexpression [1]. TRAF-mediated activa- tion of the NF- nB pathway is dependent on the activation of the IKK complex, whereas AP1 activation by TRAFs is mediated by MAPKs including JNK and p38 [2,3]. TRAFs are characterized by the presence of a conserved carboxyl terminal region known as the TRAF domain, which interacts with the cytoplasmic domains of TNF receptors. The amino terminal region of TRAFs is rich in cysteine and histidine residues arranged in putative zinc-finger motifs. With the exception of TRAF1, TRAFs contain an amino terminal RING finger motif, which has been implicated in the activation of NF- nB and MAPKs. 0898-6568/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2005.03.017 Abbreviations: AP1, activator protein 1; DTT, dithiotreitol; DUB, deubiquitinating enzyme; EBV, Epstein-Barr virus; IKK, InB kinase; JNK, Jun-kinase; LMP1, latent membrane protein 1; LThR, lymphotoxin h receptor; MAPK, mitogen activated protein kinase; NP-40, Nonidet P-40; NF-nB, nuclear factor-kappaB; TAK1, transforming growth factor-beta activated protein kinase; TNF, tumor necrosis factor; TNFR, TNF receptor; TRAF, tumor necrosis factor receptor associated factor; Ub, Ubiquitin; USP, ubiquitin specific protease. * Corresponding authors. Institute of Immunology, Biomedical Sciences Research Center Al. Fleming, 34 Al. Fleming Str., Vari 16672, Greece. Tel.: +30 2109656703; fax: +30 2109653934. E-mail addresses: mosialos@fleming.gr (G. Mosialos), chatzivasiliou@fleming.gr (E.G. Hatzivassiliou). Cellular Signalling 18 (2006) 83 – 92 www.elsevier.com/locate/cellsig