The adaptor and signaling proteins TRAF2, TRAF3 and cIAP1 and cIAP2 were suggested to inhibit alternative nuclear factor kappa B (NF-κB) signaling in resting cells by targeting NF-κB inducing kinase (NIK) to ubiquitin-dependent degradation, thus preventing processing of the NF-κB2 precursor protein p100 to release p52. However, the respective functions of TRAF2 and TRAF3 in NIK degradation and activation of alternative NF-κB signaling has remained elusive. We now show that CD40 or BAFF receptor activation resulted in TRAF3 degradation in a cIAP1-cIAP2-and TRAF2-dependent way due to enhanced cIAP1, cIAP2 TRAF3-directed ubiquitin ligase activity. Receptor-induced activation of cIAP1 and cIAP2 correlated with their K63-linked ubiquitination by TRAF2. Degradation of TRAF3 prevented association of NIK with the cIAP1-cIAP2-TRAF2 ubiquitin ligase complex, which resulted in NIK stabilization and NF-κB2-p100 processing. Constitutive activation of this pathway causes perinatal lethality and lymphoid defects.

SummaryCytokine signaling is thought to require assembly of multi-component signaling complexes at cytoplasmic segments of membrane-embedded receptors, in which receptor-proximal protein kinases are activated. Indeed, CD40, a tumor necrosis factor receptor (TNFR) family member, forms a complex containing adaptor molecules TRAF2 and TRAF3, ubiquitin conjugating enzyme Ubc13, cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/2), IκB kinase regulatory subunit IKKγ (also called NEMO) and mitogen-activated protein kinase (MAPK) kinase kinase MEKK1 upon ligation. TRAF2, Ubc13, and IKKγ were required for complex assembly and activation of MEKK1 and MAPK cascades. However, these kinases were not activated unless the multi-component signaling complex translocated from CD40 to the cytosol upon c-IAP1/2-induced degradation of TRAF3. This twostage signaling mechanism may apply to other innate immune receptors, accounting for spatial and temporal separation of MAPK and IKK signaling.Cytoplasmic segments of cytokine, growth factor and antigen receptors serve as assembly sites for complexes containing adaptor proteins, protein kinases, and other signaling factors (1,2). Ligand-induced assembly of such complexes is thought to concentrate and subsequently activate protein kinases on the receptor, thereby triggering a plethora of effector pathways that control metabolism, proliferation and survival. Amongst cytokine receptors, the tumor necrosis factor receptor (TNFR) family contains members of biomedical importance that bind trimeric ligands (3,4). TNFR engagement results in assembly of multi-component receptor-associated signaling complexes that activate inhibitor of NF-κB (IκB) kinase (IKK) and mitogen-or stressactivated protein kinase (MAPK/SAPK) cascades (3,5). Key players in TNFR signaling are TNF receptor-associated factors (TRAF), serving as E3 ubiquitin ligases and adaptors that recruit and activate protein kinases that act at the apex of effector pathways (3,5-7). These protein kinases include transforming growth factor-β activated kinase 1 (TAK1) and MAP or extracellular signal-regulated kinase kinase kinase 1 (MEKK1), which in turn activate IKK (8), and the MAPK/SAPKs JNK and p38 (5,9). Most TRAF proteins contain an N-terminal RING finger required for signal transduction and formation of K63-linked polyubiquitin chains conjugated through the C-terminal glycine of the added ubiquitin moiety and K63 of the †To whom correspondence should be addressed.

ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a critical role in the events involved in initiating T-cell responses by the antigen receptor. Here we review the structure of ZAP-70, its regulation, its role in development and in disease. We also describe a model experimental system in which ZAP-70 function can be interrupted by a small chemical inhibitor.