Quantitative Phosphoproteomics Reveals Signaling Mechanisms Associated with Rapid Cold Hardening in a Chill-Tolerant Fly

Rapid cold hardening
(RCH) is a physiological adaptation in which
brief chilling (minutes to hours) significantly enhances the cold
tolerance of insects. RCH allows insects to cope with sudden cold
snaps and diurnal variation in temperature, but the mechanistic basis
of this rapid stress response is poorly understood. Here, we used
phosphoproteomics to identify phosphorylation-mediated signaling events
that are regulated by chilling that induces RCH. Phosphoproteomic
changes were measured in both brain and fat bodies, two tissues that
are essential for sensing cold and coordinating RCH at the organismal
level. Tissues were chilled ex vivo, and changes in phosphoprotein
abundance were measured using 2D electrophoresis coupled with Pro-Q
diamond labeling of phosphoproteins followed by protein identification
via LC–MS/MS. In both tissues, we observed an abundance of
protein phosphorylation events in response to chilling. Some of the
proteins regulated by RCH-inducing chilling include proteins involved
in cytoskeletal reorganization, heat shock proteins, and proteins
involved in the degradation of damaged cellular components via the
proteasome and autophagosome. Our results suggest that phosphorylation-mediated
signaling cascades are major drivers of RCH and enhance our mechanistic
understanding of this complex phenotype.