Apoptosis signal-regulating kinase 1 (ASK1) is a key sensor kinase in the mitogen-activated protein kinase (MAPK) pathway that determines cellular response to a variety of stressors and has been implicated in several disease states. ASK1 is regulated by a large protein complex, termed the signalosome, which dynamically assembles around ASK1 to either retain the kinase in its inactive form in a non-stressed cell or to promote activity in response to oxidants and other stress factors. We examined the effect of the prototypical lipid electrophile, 4-hydroxy-2-nonenal (HNE), on ASK1 activity and signalosome composition in comparison to activation by H2O2. We generated cell lines stably expressing tandem-affinity tagged ASK1 and its putative signalosome members ASK2 and ASK3. ASK2 is a reported component of the ASK1 signalosome, whereas the recently described ASK3 was identified in our initial shotgun proteomic screen of the complex. Cells were treated with differing concentrations of HNE and H2O2 along with ethanol (vehicle control) or were left untreated in order to monitor the dynamics of signalosome assembly in response to increasing concentration of stressors. The treated cells were harvested and the ASK proteins were immunoprecipitated using both single-affinity and tandem-affinity tags. Each immunoprecipitate was analyzed by SDS-PAGE fractionation and liquid chromatography-tandem mass spectrometry followed by bioinformatic analysis to determine the signalosome protein composition. The activation state of the ASK1 MAPK pathway was assessed by western blot analyses of phosphorylated ASK1 and downstream proteins MKK6, MKK7, JNK, and p38 to compare HNE and H2O2 evoked responses. These combined inventories provide the basis to define signalosome compositions for studies of the effects of electrophiles and oxidants on ASK signalosome dynamics. (Supported by NIH Grant ES013125)

Analysis of the apoptosis signal-regulating kinase signalosome by mass spectrometry

248th National Meeting (2014)

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