Dynamic Phosphorylation of Apoptosis Signal Regulating Kinase 1 (ASK1) in Response to Oxidative and Electrophilic Stress

Apoptosis
signal-regulating kinase 1 (ASK1) is a critical cellular
stress sensor that senses diverse reactive chemotypes and integrates
these chemical signals into a single biological pathway response.
It is unknown whether ASK1 senses all stressors in the same way or
if unique stress-specific mechanisms detect distinct chemotypes. In
order to answer this question, we treated ASK1-expressing cells with
two distinct stress activators, H2O2 and 4-hydroxy-2-nonenal
(HNE), and monitored the phosphorylation state of ASK1. Phosphorylation
is an important regulator for the activity of ASK1, and we hypothesized
that these two chemically distinct molecules may produce differences
in the phosphorylation state of ASK1. Shotgun mass spectrometry and
manual validation identified 12 distinct ASK1 phosphosites. Targeted
parallel reaction monitoring assays were used to track the phosphorylation
dynamics of each confirmed site in response to treatment. Eleven phosphosites
exhibited dynamic response to one or both treatments. Six of these
sites were identified in both H2O2- and HNE-treated
cells, and four of these exhibited a consistent response between the
two molecules. The results confirm that different chemotypes produce
distinct phosphorylation patterns in concert with activation of a
common MAPK pathway.