Fig. 5: CDK-1 pT179 and other germline phosphoproteins contribute to lifespan determination.

a Model shows the human CDK1 activity regulated through phosphorylation by WEE1 kinase, CAK kinase, and CDC25 phosphatase. Sequences around worm CDK-1 pT32, pY33, and pT179 are identical to that around human CDK1 pT14, pY15, and pT161. Blue, inhibitory phosphorylation. Red, activating phosphorylation. b Phosphoproteomics data show that phosphorylation on CDK-1 decreased in daf-2 worms. CDK-1 protein levels remained similar in WT and daf-2 worms. c Reduction of CDK-1 activity significantly extended the lifespan of WT worms but did not affect the lifespan of the daf-16(mu86) worms. The ne2257ts[cdk-1-I173F] mutation inactivates CDK-1 at the restricted temperature 22.5 °C. d Germline-restricted degradation of WEE-1.3, a negative regulator of CDK-1, significantly shortened the C. elegans lifespan. Endogenous wee-1.3 and daf-2 were tagged with degron by CRISPR/Cas9. All strains carried the ieSi38[Psun-1::TIR1] allele to induce germline-specific protein degradation upon auxin treatment. Lifespan assays were performed at 20 °C, with 1 mM auxin supplied from adult day 1. e Top-ranking tissues that were significantly (p < 1.0e−15, two-sided Z test) enriched or depleted for the hypo-phosphorylated proteins in the daf-2 mutant. The predicted gene expression scores across tissues or cell types were derived from Kaletsky et al.⁷⁹. Boxplot shows the first quartile, median, and third quartile of tissue-specific expression scores of the hypo-phosphoproteins (n = 166). See statistics in Source data. f Germline proteins that were hypo-phosphorylated in the daf-2 mutant participate in lifespan regulation. Individual RNAi clone of genes was fed to the rrf-1(pk1417) worms from adult day 1 at 20 °C and assayed in two independent trials. L4440 is the empty vector control. Red highlights the pro-reproduction and anti-longevity genes. Blue highlights the anti-reproduction and pro-longevity genes. The diagram of a gonad was adapted from WormBook⁸². The germline-related phenotypes refer to WormBase release WS275. g A model illustrates dual roles of germline phosphoproteins in mediating the effects of IIS on reproduction and lifespan regulation. *p < 0.05, ***p < 0.001, NS not significant, two-sided log-rank test, n > 80 worms per strain. See survival statistics in Supplementary Dataset 4.