Previously, we have shown that ASK1-interacting protein 1 (AIP1, also known as DAB2IP), a novel member of the Ras-GAP (Ras-GTPase-activating protein) protein family, opens its conformation in response to tumor necrosis factor (TNF), allowing it to form a complex with TRAF2-ASK1 that leads to activation of ASK1-JNK/p38 signaling in endothelial cells (EC). In the present study, we show that a TNF-inducible 14-3-3-binding site on AIP1 is critical for the opening of its conformation and for the AIP1-mediated TNF signaling. Ser-604, located in the C-terminal domain of AIP1, was identified as a 14-3-3-binding site. TNF treatment of EC induces phosphorylation of AIP1 at Ser-604 as detected by a phospho-specific antibody, with a similar kinetics to ASK1-JNK/p38 activation. 14-3-3 associates with an open, active state of AIP1 assessed by an in vitro pulldown assay. Mutation of AIP1 at Ser-604 (AIP1-S604A) blocks TNF-induced complex formation of AIP1 with 14-3-3. TNF treatment normally induces association of AIP1 with TRAF2-ASK1. The interactions with TRAF2 and ASK1 do not occur with AIP1-S604A, suggesting that phosphorylation at this site not only creates a 14-3-3-binding site but also opens up AIP1, allowing binding to TRAF2 and ASK1. Overexpression of AIP1-S604A blocks TNF-induced ASK1-JNK activation. We further show that RIP1 (the Ser/Thr protein kinase receptor-interacting protein) associates with the GAP domain of AIP1 and mediates TNF-induced AIP1 phosphorylation at Ser-604 and JNK/p38 activation as demonstrated by both overexpression and small interfering RNA knockdown of RIP1 in EC. Furthermore, RIP1 synergizes with AIP1 (but not AIP1-S604A) in inducing both JNK/p38 activation and EC apoptosis. Our results demonstrate that RIP1-mediated AIP1 phosphorylation at the 14-3-3-binding site Ser-604 is essential for TNF-induced TRAF2-RIP1-AIP1-ASK1 complex formation and for the activation of ASK1-JNK/p38 apoptotic signaling.