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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AIP1 enhances TNF-α–induced ASK1 activity. (a and b) AIP1 enhances TNF-α...
AIP1 enhances TNF-α–induced ASK1 activity. (a and b) AIP1 enhances TNF-α–induced ASK1 and JNK activation. BAECs were transfected with vector control (–) or AIP1 constructs (AIP1-F, -N, or -C). Cell lysates were used to determine protein expression by Western blot with anti-FLAG (a) ASK1 and JNK activation were measured by an in vitro kinase assay (b). The relative ASK1 and JNK activities (setting TNF-α–treated vector control as 1.0) are indicated below each lane. (c) AIP1 enhances ASK1-induced EC apoptosis. BAECs were transfected with vector control (–) or AIP1 constructs (AIP1-F, AIP1-N, and AIP1-C) in the absence or presence of ASK1. Forty-eight hours after transfection, cells were stained with DAPI, and apoptotic cells with nucleus fragmentation were counted under a fluorescence microscope. The apoptotic rate is shown. Data presented are mean of three independent experiments. N/C, AIP1-N + AIP1-C. (d) AIP1 inhibits TNF-α–induced ERK activation. ECs were transfected with AIP1 constructs followed by TNF-α stimulation (10 ng/ml for 15 minutes). ERK activation was determined by Western blot with phospho-ERK antibody. Total ERK was determined by Western blot with anti-ERK. Similar results were obtained from two additional independent experiments. (e) ASK1 binding and the GAP activity of AIP1 are required for AIP1-enhanced ASK1 activation. AIP1 constructs were transfected into BAECs in the presence of HA-tagged ASK1. Protein expression was determined by Western blot with anti-HA (for ASK1) or anti-FLAG (for AIP1). ASK1-induced JNK activity was determined as described in b. Data shown are representative of three similar experiments.