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 binds preferentially to dephosphorylated ASK1 at Ser-967. (a) The 1...
AIP1 binds preferentially to dephosphorylated ASK1 at Ser-967. (a) The 14-3-3 binding site (pSer-967) in ASK1 is not critical for AIP1 association. AIP1 was cotransfected with vector control, ASK1, or ASK1-S967A, a mutant defective in 14-3-3 binding. Interaction of AIP1 and ASK1 was examined by immunoprecipitation with anti-ASK1 followed by Western blot with anti-FLAG. S/A, ASK1-S967A. (b) Phosphatase treatment increases AIP1-ASK1 complex. Cell lysates containing ASK1 and AIP1-N were incubated with alkaline phosphatase (10 U PPase/40 μg cell lysate) at room temperature for 1 hour. Treated lysates were used IP by anti-ASK1, followed by Western blot with anti-FLAG. A GST–14-3-3 pull-down assay was used as a control for phosphatase treatment. Bound ASK1 was detected by Western blot with anti-FLAG. (c) Peptide specificity. ASK1 was used in a GST–14-3-3 pull-down assay in the presence of peptide ASK1-S or ASK1-pS (0.3 mM). Bound ASK1 was detected by Western blot with anti-FLAG. (d) Peptide competition assay for AIP1-ASK1 interaction. Cell lysates containing ASK1 and AIP1-N were used for immunoprecipitation assay as described in a in the presence of peptide ASK1-S or ASK1-pS (0.3 mM). Interaction of AIP1 and ASK1 was examined by immunoprecipitation with anti-ASK1, followed by Western blot with anti-FLAG.