Analysis of the signaling complex localization in the cells revealed that TRAF2 Ser-11 phosphorylation is required for the translocation of the signaling complex from CD40 to the cytoplasm. degradation and activation of the noncanonical NF-B pathway. Paris saponin VII Thus, our results provide new insights into the CD40 signaling mechanisms whereby Ser-11 phosphorylation controls RING domain-dependent subcellular localization of TRAF2 to modulate the spatiotemporal activation of the JNK and NF-B pathways. E3 ligase activity by purified proteins has been demonstrated only for TRAF6 (7, 8). Notably, reconstitution of TRAF2- or TRAF2/TRAF5 (TRAF2/5)-deficient mouse embryonic fibroblasts (MEFs) with a RING domain deletion mutation of TRAF2 (TRAF2-R) fully restores immediate tumor necrosis factor alpha (TNF-)-induced NF-B activation while only partially restoring Paris saponin VII c-Jun N-terminal protein kinase (JNK) activation (9, 10). Thus, the exact role of the TRAF2 RING domain Paris saponin VII in NF-B and JNK activation still remains elusive. Ligated CD40 recruits TRAF2, TRAF3, and TRAF6 through its conserved TRAF-binding sites in the cytoplasmic domain, resulting in activation of the canonical (e.g., the RelA/p50 dimer) and noncanonical (e.g., the RelB/p52 dimer) NF-B pathways, as well as the mitogen-activated protein kinase (MAPK; e.g., JNK) cascade (11). TRAF2 and TRAF6 positively regulate CD40-induced MAPK and canonical NF-B activation, while TRAF2 and TRAF3 negatively regulate noncanonical NF-B. Under unstimulated conditions, an E3 ligase complex consisting of TRAF2, TRAF3, and cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) constitutively targets NF-B-inducing kinase (NIK) for ubiquitination and degradation to Paris saponin VII suppress noncanonical NF-B activation (12, 13). Upon stimulation, the recruitment of the E3 ligase complex to CD40 elicits TRAF2/cIAP1/2-mediated ubiquitination and degradation of TRAF3 within the complex, resulting in NIK protein accumulation and NIK-dependent activation of the IKK homodimer. IKK then directly phosphorylates p100 to trigger its ubiquitination and proteasome-dependent partial processing to p52, allowing the nuclear translocation of the transcriptionally active RelB/p52 dimer (12, 13). CD40 ligation has also been shown to activate phosphoinositide 3-kinase (PI3K), phospholipase C (PLC), and Janus family kinase 3 (Jak3), but the best-characterized signaling pathways are the JNK and canonical and noncanonical NF-B pathways. However, the spatiotemporal control of activation of these pathways is not fully understood (11). Recently, Matsuzawa et al. reported that CD40-induced JNK activation requires degradation of TRAF3 and MEKK1-mediated phosphorylation of a protein within the CD40 complex, which triggers translocation of the effector complex (consisting of MEKK1, TRAF2, cIAP1/2, and IKK) from CD40 to the cytoplasm, where MEKK1 is able to interact with and activate the MEK4/7-JNK cascade (14). However, several earlier studies have shown that inhibition of TRAF2 and/or TRAF3 degradation promotes JNK activation rather than inhibition (15,C19). In addition, it is not known how Paris saponin VII phosphorylation of a protein within the CD40 complex triggers the Rabbit Polyclonal to MAPK3 translocation of the effector complex from CD40 to the cytoplasm. Notably, Chaudhuri et al. reported that CD40 signaling elicits TRAF2 phosphorylation on serine residues and that this phosphorylation inhibits TRAF2 interaction with the CD40 cytoplasmic domain (20, 21). However, TRAF2 phosphorylation sites and their functional significance in the TRAF2-CD40 interaction were not characterized in these studies. We previously reported the identification of two phosphorylation sites (Ser-11 and Ser-55) in TRAF2 and showed that phosphorylation at these sites promotes the secondary but not the immediate phase of JNK and NF-B activation following TNF- stimulation (22, 23). However, the mechanisms by which TRAF2 phosphorylation regulates the secondary phase of the signaling pathways remain unknown. In the present study, we demonstrate that CD40 ligation induces TANK-binding kinase.