The wild or untreated type condition was set as 100. only partially on their kinase activities. Recent studies on RIP1 and RIP2 have highlighted the importance of ubiquitination as a key process regulating their capacity to activate downstream signaling pathways. In this study, we found that XIAP, cIAP1 and cIAP2 not only directly bind PF-03394197 (oclacitinib) to RIP1 and RIP2 but also to RIP3 and RIP4. We show that cIAP1 and cIAP2 are direct E3 ubiquitin ligases for all four RIP proteins and that cIAP1 is capable of conjugating the RIPs with diverse types of ubiquitin chains, Rabbit Polyclonal to PAK5/6 (phospho-Ser602/Ser560) including linear chains. Consistently, we show that repressing cIAP1/2 levels affects the activation of NF-B that is dependent on RIP1, -2, -3 and -4. Finally, we identified Lys51 and Lys145 of RIP4 as two critical residues for cIAP1-mediated ubiquitination and NF-B activation. Introduction Cells are continuously confronted with stress signals that initiate at the cell surface or within the cell. The ability to transduce the signal and to activate a cellular response, either through transcription-dependent or Cindependent mechanisms, relies on the recruitment of adaptor proteins to the extra- and intra-cellular sensors. Among these adaptors, members of the receptor-interacting protein kinase (RIP) family have emerged as essential mediators of cellular stress. All RIP kinases contain a kinase domain that is conserved between the family members and bear unique domains that allow protein-protein interactions[1]. RIP1, the first identified member of the RIP kinase family, was shown to participate in the signaling pathways activated downstream of several members of the TNF receptor superfamily[2], Toll-like receptors (TLR) 3 and 4[2], [3], and after genotoxic stress[4]. RIP1 can mediate gene transcription through activation of the MAPK and NF-B signaling pathways, and can induce cell death by the formation of death complexes. Similarly, RIP2 (CARDIAK/RICK) was shown to participate in the activation of the MAPK and NF-B signaling pathways downstream of the innate immune pattern recognition receptors NOD1 and NOD2[5], [6], [7], and to be part of a pro-apoptotic complex activated by NOD1[8]. RIP2 is also required for optimal T-Cell Receptor (TCR) signaling and, although controversial, was reported to mediate TLR responses[5], [6], [7], [9]. Mice lacking RIP3 do not develop spontaneous phenotypic manifestations[10], but this kinase has been reported to be a major player in the execution of a caspase-independent type of cell death called necroptosis[11], [12], [13]. In addition, PF-03394197 (oclacitinib) the ectopic expression of RIP3 in cells was shown to induce apoptosis and NF-B activation[14], [15]. RIP4 (PKK/DIK) also induces JNK and NF-B when ectopically expressed[16], [17], but the pathways that lead to activation of RIP4 remain unidentified. Transgenic studies have indicated that this kinase plays a role in proper skin development and inflammatory responses[18], [19]. The ability of RIP1 and RIP2 to transduce stress signals was shown to rely only partially on their catalytic activities. Just like RIP1, the kinase activity of RIP2 is not required for the NF-B response or for the activation of JNK and p38 [9], [20], [21]. Recent studies have highlighted the importance of ubiquitination as a key process regulating RIP1 and RIP2s capacity to activate downstream signaling pathways[22], [23], [24]. Ubiquitination is a post-translational modification involving covalent attachment of ubiquitin, a 76-amino acid polypeptide, to a target protein by a cascade of reactions carried out by the concerted action of the ubiquitin-activating (E1), -conjugating (E2) and -ligating (E3) enzymes[25]. Protein ubiquitination is emerging as a key regulatory mechanism that controls many physiological processes, including protein degradation, cell signaling, DNA damage response, and protein trafficking. This wide range of consequences originates from the ability of ubiquitin to form polymers in which an internal Lys residue of one ubiquitin moiety is attached to the carboxy-terminal residue of another. Primarily, Lys48-linked and Lys63-linked polyubiquitin modifications have been studied, and these linkages are respectively known to be required for proteasomal degradation and signal transmission. Recently, attention has been paid to a new type of ubiquitin chain that plays a role in NF-B activation C the linear ubiquitin chains, arising from attachment of the C-terminal Gly to the N-terminal Met. So far, LUBAC is the only E3 ubiquitin ligase complex reported to conjugate substrates with linear ubiquitin chains[26], [27]. Inhibitor of apoptosis proteins (IAPs) are phylogenetically conserved proteins characterized by the presence of at least one Baculovirus IAP Repeat (BIR) motif, a zinc-binding structure of approximately 70 amino acid residues that mediates protein-protein interactions[28]. As indicated by their name, the function of these proteins was first believed to be restricted to inhibition of cell death, mostly by direct interference with the PF-03394197 (oclacitinib) proteolytic activities of caspases. However,.