Two Coordinated Mechanisms Underlie Tumor Necrosis Factor Alpha-Induced Immediate and Delayed IB Kinase Activation

Tumor necrosis factor alpha (TNF-)-induced NF-B activation has been believed to depend on TRAF2- and cIAP1-mediated RIP1 ubiquitination. However, recent findings have challenged the notion that these proteins play essential roles in NF-B acti-vation. Here, by assessing the kinetics and amplitude of IB kinase (IKK) activation, we report that TNF--induced immediate and robust activation of IKK requires K63-linked and linearly linked ubiquitination of RIP1 and that in the absence of RIP1 ex-pression, TRAF2 and cIAP1 cooperatively induce delayed IKK activation by recruiting LUBAC to TNFR1. Knockdown of HOIP (a component of LUBAC) in RIP1-deficient cells completely impairs the recruitment and activation of IKK but does not affect K63-linked ubiquitination of TRAF2 and recruitment of TAK1 to TNFR1, suggesting that the K63-linked ubiquitin chain is not capable of recruiting IKK in vivo. We also demonstrate that TRAF2 and cIAP1 together, but not either one alone, directly cata-lyze linearly linked ubiquitination of RIP1. Importantly, in embryonic hepatocytes, TNF- activates NF-B through a RIP1-independent pathway. Thus, our findings clarify molecular details of this important signaling mechanism by providing evidence for the existence of two phases of IKK activation: the immediate phase, induced by TRAF2/cIAP1-mediated ubiquitination of RIP1, and the delayed phase, activated by TRAF2/cIAP1-dependent recruitment of LUBAC. Tumor necrosis factor alpha (TNF-) is a pleiotropic cytokinethat can exert opposing biological effects—proinflammatory and proapoptotic — by activating the NF-B and caspase 8 path-ways, respectively (1, 2). The cytokine acts through two distinc