Abstract
Insulin/IGF-1 Signaling (IIS) is known to constrain longevity by inhibiting the transcription factor FOXO. How phosphorylation mediated by IIS kinases regulates lifespan beyond FOXO remains unclear. Here, we profile IIS-dependent phosphorylation changes in a large-scale quantitative phosphoproteomic analysis of wild-type and three IIS mutant Caenorhabditis elegans strains. We quantify more than 15,000 phosphosites and find that 476 of these are differentially phosphorylated in the long-lived daf-2/insulin receptor mutant. We develop a machine learning-based method to prioritize 25 potential lifespan-related phosphosites. We perform validations to show that AKT-1 pT492 inhibits DAF-16/FOXO and compensates the loss of daf-2 function, that EIF-2α pS49 potently inhibits protein synthesis and daf-2 longevity, and that reduced phosphorylation of multiple germline proteins apparently transmits reduced DAF-2 signaling to the soma. In addition, an analysis of kinases with enriched substrates detects that casein kinase 2 (CK2) subunits negatively regulate lifespan. Our study reveals detailed functional insights into longevity.
Highlights
Insulin/insulin-like growth factor 1 (IGF-1) Signaling (IIS) is known to constrain longevity by inhibiting the transcription factor FOXO
While DAF-16 is required for IIS-mediated lifespan extension, recapitulating the daf-2 longevity to its fullness requires more than DAF-16 overexpression or nuclear translocation[4]
From WT C. elegans and the IIS mutants—each analyzed in three or four biological replicates with two technical replicates—we identified a total of 15,443 phosphosites with >0.75 PhosphoRS site probability[21], a commonly used threshold to ensure the quality of phosphosite assignment (Supplementary Fig. 1a, b)
Summary
Insulin/IGF-1 Signaling (IIS) is known to constrain longevity by inhibiting the transcription factor FOXO. Comparing the wild-type (WT) and the long-lived daf-2 mutant, a subset of proteins that were markedly upregulated or downregulated in the latter were not found to exhibit corresponding changes in the abundance of their mRNA templates. Changes in these proteins affect known lifespan modulators, such as components of the translational machinery[7,8,9]. Only a handful of phosphosites are known to be involved in lifespan regulation[12,13,14,15,16], and no large-scale studies of IIS-related phosphorylation events are reported for C. elegans. We statistically detected kinases with enriched substrates and found that casein kinase 2 (CK2), whose subunits are encoded by kin-3 and kin-10, acts to limit lifespan
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