Lifespan Regulation by Insulin Signaling Through Phosphorylation of Proteins Beyond FOXO

Insulin/IGF-1 Signaling (IIS) constrains longevity by inhibiting the transcription factor FOXO. Beyond FOXO, little is known about how phosphorylation—as mediated by IIS kinases— regulates lifespan. Here, we profiled IIS-dependent phosphorylation changes in a large-scale quantitative phosphoproteomic analysis of wild-type and three IIS mutant C. elegans strains. Our state-of-the-art analysis experimentally identified more than 15,000 phosphosites, among which 448 were differentially phosphorylated in the long-lived daf-2/insulin receptor mutant. We developed a machine-learning-based tool for systematically ranking the likely functional importance of phosphosites to guide candidate selection for follow-up validation. We show that AKT-1 pT492 inhibits DAF-16/FOXO and compensates the loss of daf-2 function, that EIF-2α pS49 potently regulates protein synthesis and daf-2 longevity, and that reduced phosphorylation of multiple germline proteins (e.g., CDK-1) apparently transmits a signal representing reduced DAF-2 signaling to the soma. Finally, kinase-substrate analysis and subsequent experimental validation confirm that casein kinase 2 negatively regulates lifespan. Our new benchmark data resource and machine-learning tool enables unprecedented access to detailed functional insights for studies of longevity.