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.