C. elegans S6K Mutants Require a Creatine-Kinase-like Effector for Lifespan Extension

Abstract

Deficiency of S6 kinase (S6K) extends the lifespan of multiple species, but the underlying mechanisms are unclear. To discover potential effectors of S6K-mediated longevity, we performed a proteomics analysis of long-lived rsks-1/S6K C. elegans mutants compared to wild-type animals. We identified the arginine kinase ARGK-1 as the most significantly enriched protein in rsks-1/S6K mutants. ARGK-1 is an ortholog of mammalian creatine kinase, which maintains cellular ATP levels. We found that argk-1 is possibly a selective effector of rsks-1/S6K-mediated longevity and that overexpression of ARGK-1 extends C. elegans lifespan, in part by activating the energy sensor AAK-2/AMPK. argk-1 is also required for the reduced body size and increased stress resistance observed in rsks-1/S6K mutants. Finally, creatine kinase levels are increased in the brains of S6K1 knockout mice. Our study identifies ARGK-1 as a longevity effector in C. elegans with reduced RSKS-1/S6K levels.

Figure 1. argk-1 is Selectively Required for the Long Lifespan of rsks-1/S6K Mutants

(A) Lifespan analysis of wild-type (WT, N2), argk-1(ok2993), rsks-1(sv31), and argk-1(ok2993); rsks-1(sv31) double mutants. Four replicate experiments and four additional experiments assaying the argk-1(ok2973) allele were performed, see Table S4 for details and statistical analysis. (B–E) Lifespan analysis of WT, rsks-1(sv31) (B), insulin/IGF-1 receptor daf-2(e1370) (C), dietary-restricted eat-2(ad1116) (D), and mitochondrial clk-1(e2519) animals (E) fed bacteria expressing control or argk-1 dsRNA from Day-1 of adulthood. Three replicate experiments were performed, as well as analysis of daf-2; argk-1 and eat-2; argk-1 double mutants, see Table S4. All lifespan experiments were performed at 20°C.

Figure 2. argk-1 is Required for Increased AMPK Activity in rsks-1/S6K Mutants

(A) Phosphorylation of AMP-activated kinase (AMPK) was assessed in wild-type (WT, N2), rsks-1(sv31), argk-1(ok2993), and rsks-1(sv31); argk-1(ok2993) animals by Western blotting using an antibody against mammalian phospho-AMPKα Thr 172. β-actin was used as a loading control. (B) Quantification of the phospho-AMPK signal (normalized to the loading control) averaged from three independent Western blots (as in A). Error bars are SEM. *P < 0.05; ***P < 0.001 by one-way ANOVA. (C) Phosphorylation of acetyl-CoA carboxylase (ACC) was assessed in the strains indicated in (A) by Western blotting using an antibody against mammalian phospho-ACC Ser 59. β-actin was used as a loading control. The band at ~200 kDa is likely to be POD-2/ACC (see Supplemental Experimental Procedures). (D) Quantification of the phospho-ACC signal (normalized to the loading control) averaged from three independent Western blots (as in C). Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA. All lysates were from Day-1 adults raised at 20°C.

Figure 3. aak-2/AMPK Contributes to Lifespan Extension Induced by Overexpression of ARGK-1

(A) Lifespan analysis of transgenic animals overexpressing argk-1 from the ubiquitous sur-5 promoter (sur-5p::argk-1::gfp) and their non-transgenic siblings (WT) at 25°C. P < 0.0001, log-rank test. Nine replicate experiments of two independent lines were performed at 20°C and 25°C, see Table S5. (B) Lifespan analysis of WT or aak-2(ok524) animals and their transgenic siblings expressing sur-5p::argk-1::gfp at 20°C. Four replicate experiments were performed at 20°C or 25°C, see Table S5. (C) Phosphorylation of AMPK and ACC was assessed in transgenic animals expressing sur-5p::argk-1::gfp and non-transgenic siblings (WT). β-actin was used as a loading control. (D) Quantification of phospho-AMPK (left) and phospho-ACC (right) signals (normalized to the loading control) averaged from three independent Western blots (as in C). Error bars are SEM. *P < 0.05 by one-way ANOVA. All lysates were from Day-1 adults raised at 20°C. (E) Expression pattern of ARGK-1 in the head region of Day-1 adult C. elegans expressing mCherry-tagged ARGK-1 from a 1.1 kb endogenous promoter (argk-1p::argk-1::mCherry). A reporter for the potassium-chloride cotransporter KCC-3 tagged with GFP was used to aid in cell identification. The merged panel shows partial overlap of the ARGK1::mCherry and KCC-3::GFP signals. See also Figure S3F for co-localization with an itx-1p:;gfp reporter. The pharyngeal bulb is visible in the differential interference contrast (DIC) image. Scale bar, 50 µm.

Figure 4. Creatine Kinase Expression is Increased in the Cerebellums of S6K^−/− Mice

(A) Levels of creatine kinase (CK-B, brain isoform), the mammalian homolog of ARGK-1, were assessed in S6K1^+/+ (n = 6) and S6K1^−/− (n = 5) mice cerebellar tissue lysates. Males and females of age 5–8 weeks were analyzed. (B) CK-B levels (normalized to GADPH) from (A) was quantified. Error bars, ± SEM. *P<0.05, unpaired, two-tailed Student’s t-test. See Figure S4 for measurements in hippocampus and skeletal muscle.