title: RIP1-mediated AIP1 phosphorylation at a 14-3-3-binding site is critical for tumor necrosis factor-induced ASK1-JNK/p38 activation.
abstract: 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.
authors: Interdepartmental Program in Vascular Biology and Transplantation, Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors: Haifeng
authors: H
authors: Zhang
authors: Rong
authors: R
authors: Zhang
authors: Haifeng
authors: H
authors: Zhang
authors: Yan
authors: Y
authors: Lin
authors: Jiehui
authors: J
authors: Li
authors: Jordan S
authors: JS
authors: Pober
authors: Wang
authors: W
authors: Min
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D051528:Guanylate Kinases; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D046912:Multiprotein Complexes; D011506:Proteins; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; D048670:MAP Kinase Kinase 4; D051528:Guanylate Kinases; C485777:MAGI2 protein, human