TRAF-mediated modulation of NF-kB AND JNK Activation by TNFR2 Lucía Cabal-Hierro 1 , Montserrat Rodríguez, Noelia Artime, Julián Iglesias, Lorea Ugarte, Miguel A. Prado 2 , Pedro S. Lazo ⁎ Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, 33071 Oviedo, Spain abstract article info Article history: Received 18 July 2014 Accepted 15 August 2014 Available online 22 August 2014 Keywords: TNF receptors Death Receptors TRAF proteins Apoptosis NF-κB JNK Tumor Necrosis Factor Receptor 2 (TNFR2) activates transcription factor κB (NF-κB) and c-Jun N-terminal kinase (JNK). Most of the biological activities triggered by TNFR2 depend on the recruitment of TNF Receptor-Associated Factor 2 (TRAF2) to the intracellular region of the receptor. The intracellular region of TNFR2 contains five highly conserved amino acid sequences, three of which appear implicated in receptor signaling. In this work we have studied the interaction of TNFR2 with TRAF proteins as well as the functional consequences of this interaction. We show that TRAF1, TRAF2 and TRAF3 bind to the receptor through two different binding sites located at con- served modules IV and V of its intracellular region, respectively. We also show that TRAF1 and TRAF3 have oppo- site effects to TRAF2 on NF-κB and JNK activation by TNFR2. Moreover, we show that TNFR2 is able to induce JNK activation in a TRAF2-independent fashion. This new receptor activity relies on a sequence located in the conserved module III. This region is also responsible for the ability of TNFR2 to induce TRAF2 degradation, thus emphasizing the role of conserved module III (amino acids 338–379) on receptor signaling and regulation. We show that only TNFR2 can induce TRAF2 degradation while TRAF1 or TRAF3 is not subjected to this regulatory mechanism and that TRAF1, but not TRAF3, is able to inhibit TRAF2 degradation. © 2014 Elsevier Inc. All rights reserved. 1. Introduction TNF has been established as a main player in the immune processes of the organism [1,2]. In the last few years, the role of the inflammatory reactions on cancer development [3] has highlighted the role of TNF both in tumor development [4–7] and inhibition [8,9]. The study of TNF structure and functions led to the identification of one of the main families of cytokines, the so-called TNF superfamily which exerts its biological effects through its interaction with transmembrane pro- teins which constitute the TNFR superfamily of receptors [10–12]. TNFRs trigger their biological responses upon binding of different adaptor proteins and may lead to final responses as diverse as cell pro- liferation or cell death. The TRAF proteins were initially identified be- cause of their interaction with different TNFRs, regulating them and acting as transducers for different biological pathways [13]. In mam- mals, the TRAF family is constituted of 7 different members. All these proteins contain the TRAF domain, involved in their interaction with the TNFRs and with other TRAF proteins [14,15]. Except for TRAF1, all TRAF proteins contain a RING domain in their N-terminal region. The deletion of this domain in TRAF2, TRAF5 or TRAF6 leads to the genera- tion of dominant negative mutants, indicating that it is essential for the biological activity of the protein [16,17]. More recently, several stud- ies have suggested an E3 ligase activity for this RING thus pointing to a role in ubiquitination involved in the regulation of TNFR signaling [18, 19]. Binding of TRAF proteins to TNFRs occurs through highly defined motifs present on the receptors. Two sequence motifs have been sug- gested to be implicated in TRAF proteins binding. One is constituted by the sequence (P/S/A/T)x(Q/E)E and the other one is represented by the sequence PxQxxD [14]. Both TNF receptors TNFR1 and TNFR2 are transmembrane proteins with high similarity in their extracellular regions although they differ widely in their intracellular domains. TNFR1 contains in its intracellular region a death domain while TNFR2 does not contain a death domain. Instead, it has two different regions implicated in the binding of TRAF proteins [20,21]. Although TNFR1 signaling has been widely character- ized and reviewed [22], TNFR2 activity is less well understood. Most of the biological activities triggered by TNFR2 depend on its interaction Cellular Signalling 26 (2014) 2658–2666 Abbreviations: AIP1, ASK1 interacting protein; AP-1, activating protein 1; BKO, binding knock out; CRMA, cytokine response modifier A; cIAP, cellular inhibitor of apoptosis; DMEM, Dulbecco modified Eagle's medium; FLAG, FLAG peptide; HA, hemagglutinin; HEK, human embryonic kidney; IKK, IκB kinase; IκB, inhibitor of NF-κB; JNK, N-terminal Jun kinase; NF-κB, nuclear factor κB; PEI, polyethylenimine; RANK, receptor activator of NF-κB; TNF, tumor necrosis factor; TNFR, TNF receptor; TRADD, TNF receptor associated protein with death domain; TRAF, TNF receptor associated factor. ⁎ Corresponding author at: Departamento de Bioquímica y Biología Molecular and Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Campus de El Cristo, 33071 Oviedo, Spain. Tel.: +34 98 510 4213; fax: +34 98 510 3157. E-mail address: pslazo@uniovi.es (P.S. Lazo). 1 Present address: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 2 Present address: Department of Cell Biology, Harvard Medical School, Boston, MA, USA. http://dx.doi.org/10.1016/j.cellsig.2014.08.011 0898-6568/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig