Dynamic Phosphoproteomic Profiling Captured Early Signaling Events for Cold Acclimation in Larval Zebrafish

The acclimation at mild low temperature can improve the ability of cold tolerance and protect tissues from stress-induced injury in many species, but mechanisms triggering the signal transduction of cold acclimation remain largely unknown. Here, we deciphered the early signaling events associated with immediate temperature drops in zebrafish larvae. Early alternations in the phosphoproteome of larval zebrafish were captured during cold exposure by using an optimized sampling protocol of denaturing with trichloroacetic acid and a tandem mass tagging-based quantitative phosphoproteomic approach. A total of 12,916 phosphosites on 4931 proteins were identified in the phosphoproteome and 7329 phosphosites on 3391 proteins were quantified with 1490 phosphosites on 1098 proteins differentially regulated by cold exposure. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of upregulated phosphosites identified immediate early phosphorylation events on transmembrane proteins, MAPK and transcription factors, and signal pathways that are essential for cold acclimation in larval zebrafish. Moreover, key kinases activated during cold acclimation of larval zebrafish including Ca²⁺/calmodulin-dependent protein kinase-II, protein kinase C, and protein kinase A were experimentally confirmed. Furthermore, a scaffolding protein AHNAK and many other proteins in AHNAK-mediated signaling pathways were robustly phosphorylated, suggesting their key roles in coordinating cold signal perception and transduction. Together, these findings provide a solid foundation for systematically understanding the signaling events of cold acclimation and a valuable phosphoproteome resource for functional studies of stress responses.