AIP1 mediates TNF-α–induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3
Rong Zhang, Xiangrong He, Weimin Liu, Meng Lu, Jer-Tsong Hsieh, Wang Min
Rong Zhang, Xiangrong He, Weimin Liu, Meng Lu, Jer-Tsong Hsieh, Wang Min
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Article Cardiology

AIP1 mediates TNF-α–induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3

Abstract

TNF-α activates ASK1 in part by dissociating 14-3-3 from apoptosis signal–regulating kinase 1 (ASK1). In the present study, we identified a novel Ras GTPase-activating protein (Ras-GAP) as an ASK1-interacting protein (AIP1). AIP1 binds to the C-terminal domain of ASK1 via a lysine-rich cluster within the N-terminal C2 domain. AIP1 exists in a closed form through an intramolecular interaction between the N-terminus and the C-terminus, and TNF-α induces unfolding of AIP1 leading to association of AIP1 with ASK1. Thus, the N-terminus of AIP1 containing the C2 and GAP domains constitutively binds to ASK1 and facilitates the release of 14-3-3 from ASK1. In contrast to 14-3-3, AIP1 binds preferentially to dephosphorylated ASK1. Recruited AIP1 enhances ASK1-induced JNK activation, and the ASK1 binding and the GAP activity of AIP1 are critical for AIP1-enhanced ASK1 activation. Furthermore, TNF-induced ASK1/JNK activation is significantly blunted in cells where AIP1 is knocked down by RNA interference. These data suggest that AIP1 mediates TNF-α–induced ASK1 activation by facilitating dissociation of inhibitor 14-3-3 from ASK1, a novel mechanism by which TNF-α activates ASK1.

Authors

Rong Zhang, Xiangrong He, Weimin Liu, Meng Lu, Jer-Tsong Hsieh, Wang Min

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TNF-α induces association of AIP1 with ASK1 in ECs. (a) BAECs were trans...
TNF-α induces association of AIP1 with ASK1 in ECs. (a) BAECs were transfected with AIP1-F or AIP1-N followed by treatment with TNF-α (10 ng/ml for 15 minutes). Cell lysates were immunoprecipitated with anti-ASK1, and ASK1 in the immunoprecipitate was determined by Western blot with anti-AIP1. (b) Specificity of AIP1 antibody. A polyclonal antibody against AIP1 was produced by Cocalico Biologicals Inc. by immunizing rabbits with GST–AIP1-PH. 293T cell lysates expressing FLAG-tagged AIP1-F, -N, and -C were used to determine the specificity of anti-AIP1 by Western blot (lanes 1–3). Anti-FLAG was used as a control (lanes 4–6). (c) AIP1 is highly expressed in cultured ECs. AIP1 expression in cell lysates from HUVECs, breast cancer MCF-7 cells, or prostate cancer cell line LNCaP (20 μg of total protein from each sample) was measured by Western blot with anti-AIP1. (d) TNF-α induces association of AIP1 with ASK1, whereas it dissociates 14-3-3 from ASK1. HUVECs were either untreated or treated with TNF-α (10 ng/ml for 15 minutes). Cell lysates were immunoprecipitated with anti-ASK1 followed by Western blot with anti-AIP1 or anti-14-3-3. (e) TRAF2 induces association of AIP1 with ASK1. BAECs were transfected with vector control (VC) or FLAG-tagged TRAF2 (TR2). Association of TRAF2 or AIP1 with ASK1 was determined by immunoprecipitation with anti-ASK1, followed by Western blot with anti-FLAG or anti-AIP1. (f) AIP1 has no effect on TRAF2-ASK1 complex. BAECs were transfected with vector control or AIP1-N, followed by TNF-α treatment (10 ng/ml). Endogenous TRAF2-ASK1 complex was determined by immunoprecipitation with anti-TRAF2 followed by Western blot with anti-ASK1. IP, immunoprecipitate; IB immunoblot.