Dynamic Regulation of Apoptosis Signal-Regulating Kinase 1

Abstract

Apoptosis signal-regulating kinase 1 (ASK1) is a critical sensor of cellular stress that is capable of integrating several chemically distinct signals into a single response pathway and is believed to play a role in several human pathologies. ASK1 is believed to be regulated both by protein-protein interactions (as a multiprotein complex termed the signalosome) and phosphorylation of several key residues. These two mechanisms have predominantly been studied after activation of ASK1 by H2O2, which represents only one of the several stressors that is known to trigger signaling through the ASK1 pathway. In this dissertation, I examined both regulatory mechanisms of ASK1 in response to activation with 4-hydroxy-2-nonenal (HNE), which is chemically and mechanistically distinct from H2O2. I tested the hypothesis that there is a consensus two-state signalosome that dynamically assembles around ASK1 in response to activation using targeted mass spectrometry assays. I performed absolute quantitation assays on known ASK1-interacting proteins and reported the first stoichiometric estimate for the ASK1 signalosome composition. My data suggests that this complex is stably composed of ASK1, ASK2, and 14-3-3 proteins in a 2:2:1 ratio. Fourteen other protein-protein associations with ASK1 were detected as dynamic in response to HNE treatment, but appear to be transient in nature. Thus it is likely that the ASK1 signalosome is actually composed of a stable core component that transiently associates with other proteins as needed, resulting in the concurrent presence of many different signalosomes in the cell as opposed to two distinct multiprotein complexes. In order to determine if ASK1 senses different stressors by the same mechanism, I treated ASK1 cells with HNE and H2O2 and monitored the dynamic changes in the phosphorylation state of ASK1. I detected unique phosphosites for each of the stressors that exhibited concentration-dependent responses, indicating that ASK1 senses these stressors by different mechanisms. I also detected a core set of phosphorylations on ASK1 that were consistent between both treatments, which likely represent the residues necessary for general regulation of activity. The use of these approaches to monitor protein interaction and phosphorylation state dynamics can be extended to study most other multiprotein complexes with a higher degree of confidence than methods currently commonly employed.