RIP1-mediated AIP1 phosphorylation at a 14-3-3-binding site is critical for tumor necrosis factor-induced ASK1-JNK/p38 activation.
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.
Mesh Terms:
14-3-3 Proteins, Amino Acid Substitution, Animals, Apoptosis, Carrier Proteins, Cattle, Cells, Cultured, Endothelial Cells, Enzyme Activation, Humans, MAP Kinase Kinase 4, MAP Kinase Kinase Kinase 5, MAP Kinase Signaling System, Multiprotein Complexes, Mutation, Missense, Phosphorylation, Protein Binding, Protein Processing, Post-Translational, Proteins, Receptor-Interacting Protein Serine-Threonine Kinases, TNF Receptor-Associated Factor 2, Tumor Necrosis Factor-alpha, p38 Mitogen-Activated Protein Kinases
14-3-3 Proteins, Amino Acid Substitution, Animals, Apoptosis, Carrier Proteins, Cattle, Cells, Cultured, Endothelial Cells, Enzyme Activation, Humans, MAP Kinase Kinase 4, MAP Kinase Kinase Kinase 5, MAP Kinase Signaling System, Multiprotein Complexes, Mutation, Missense, Phosphorylation, Protein Binding, Protein Processing, Post-Translational, Proteins, Receptor-Interacting Protein Serine-Threonine Kinases, TNF Receptor-Associated Factor 2, Tumor Necrosis Factor-alpha, p38 Mitogen-Activated Protein Kinases
J. Biol. Chem.
Date: May. 18, 2007
PubMed ID: 17389591
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