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. Author manuscript; available in PMC: 2016 Apr 7.
Published in final edited form as: Cell Rep. 2016 Feb 25;14(9):2059–2067. doi: 10.1016/j.celrep.2016.02.012

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

Philip R McQuary 1,2, Chen-Yu Liao 3, Jessica T Chang 1, Caroline Kumsta 1, Xingyu She 1, Andrew Davis 1, Chu-Chiao Chu 1, Sara Gelino 1,2, Rafael L Gomez-Amaro 4, Michael Petrascheck 4, Laurence M Brill 5, Warren C Ladiges 6, Brian K Kennedy 3, Malene Hansen 1,*
PMCID: PMC4823261  NIHMSID: NIHMS759043  PMID: 26923601

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 a selective effector of rsks-1/S6K-mediated longevity, and 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.

INTRODUCTION

The TOR pathway plays a conserved role in organismal aging, in part by regulating mRNA translation through S6 kinase (S6K). Inhibition of either TOR or S6K reduces the rate of protein synthesis and extends the lifespan of organisms ranging from yeast to mice. Animals with decreased S6K activity also have reduced body and brood sizes (Kapahi et al., 2010). However, the nature of the S6K effectors regulating these phenotypes and the mechanisms by which they contribute to the long lifespan of S6K mutants remain elusive.

In the nematode C. elegans, inhibition of S6K (called RSKS-1) reduces translation rates, delays development, decreases body and brood sizes, and promotes stress resistance and longevity (Hansen et al., 2007; Korta et al., 2012; Pan et al., 2007; Selman et al., 2009). A number of proteins have been reported to function downstream of RSKS-1/S6K in lifespan determination; the transcription factors PHA-4/FOXA (Sheaffer et al., 2008), HIF-1 (Chen et al., 2009), HSF-1 (Seo et al., 2013), and HLH-30/TFEB (Lapierre et al., 2013); the autophagy protein ATG-18/WIPI (Lapierre et al., 2013); and AAK-2, the catalytic α subunit of AMP-activated protein kinase (AMPK) (Selman et al., 2009). However, all of these factors play roles in additional C. elegans longevity paradigms, and effectors selective for the S6K pathway have yet to be identified and investigated.

AMPK is the principal energy sensor in eukaryotes and is thus critical for the maintenance of cellular energy homeostasis (Hardie et al., 2012). AMPK functions as a heterotrimer consisting of catalytic α, regulatory γ, and scaffolding β subunits. In response to low energy status, AMPK is activated via phosphorylation and restores homeostasis by activating a number of energy-conserving pathways (Hardie et al., 2012). C. elegans AAK-2/AMPK also functions as an energy sensor (Apfeld et al., 2004) and plays an important role in several longevity paradigms in addition to S6K reduction, including impaired insulin/IGF-1 signaling (Apfeld et al., 2004) and certain forms of dietary restriction (Greer et al., 2007). Although AMPK is activated in response to S6K deficiency in mice and worms (Aguilar et al., 2007; Selman et al., 2009), the mechanism linking these kinases is unclear.

In this study, we sought to discover novel downstream targets of S6K with potential roles in longevity. We used two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) for in-depth discovery and identification of proteins with differential abundance in long-lived rsks-1/S6K mutants compared with wild-type (WT) animals. From this proteomics dataset, we identified arginine kinase ARGK-1/F44G3.2 as the most significantly enriched in rsks-1/S6K mutants. Arginine kinases are functionally equivalent to mammalian creatine kinases (CKs), collectively referred to as phosphagen kinases, which act as intracellular energy buffers (Ellington, 2001). We show that ARGK-1 plays a critical role in establishing specific phenotypes of rsks-1/S6K mutants, as demonstrated by the requirement for argk-1 for the reduced body size, increased stress resistance, increased AAK-2/AMPK activity, and extended lifespan of these mutants. ARGK-1 displayed a restricted expression pattern, including to a small subset of C. elegans glial cells. Consistently, we observed increased levels of CK in the brains of S6K1−/− mice, suggesting conserved regulation of phosphagen kinases in this longevity paradigm. Collectively, we have identified the arginine kinase ARGK-1 as a possibly selective longevity effector of S6K in C. elegans, highlighting an important role for cellular ATP homeostasis in this conserved longevity model.

RESULTS

Proteomics Analysis Identifies the Arginine Kinase ARGK-1 as a Putative S6K Effector

To search for new downstream effectors of S6K, we used global proteomic profiling of Day-1 adult rsks-1(sv31) mutants and WT animals (label-free quantification via spectral counting; see Supplemental Experimental Procedures and Table S1 for spectral counts). In this analysis, we observed 339 proteins with spectral counts >1.8-fold more or less abundant in rsks-1(sv31) mutants compared to WT animals (P < 0.05 using a moderate t-test and Welch test). Of these, 139 proteins were putatively increased (Table S2) and 200 proteins were putatively decreased (Table S3) in rsks-1/S6K mutants. Of particular note, the list of proteins more abundant in rsks-1/S6K mutants contained several cytochrome P450s, UGTs, and GSTs, consistent with a role for S6K in the detoxification response (Wang et al., 2010), as well as the autophagy protein ATG-18/WIPI (Tables S1, S2). Using a new translational ATG-18::GFP reporter, we confirmed that rsks-1/S6K animals had increased ATG-18/WIPI levels (Figure S1A), which is consistent with atg-18/WIPI being required for rsks-1/S6K—mediated lifespan extension (Lapierre et al., 2013). However, the majority of the differentially abundant proteins identified have not previously been linked to rsks-1/S6K. Of particular note, GO analysis suggested that a large number of proteins predicted to be associated with biosynthesis of nucleobase-containing compounds were more abundant in rsks-1/S6K mutants compared to WT (Table S2).

The most differentially abundant protein in rsks-1/S6K mutants was ARGK-1/F44G3.2, one of five C. elegans arginine kinases (Fraga et al., 2015). ARGK-1 showed a >35-fold increase in spectral count ratio (Table S2), which arose mainly from undetectable spectral counts in WT animals (Table S1). In contrast, WT and rsks-1(sv31) animals showed comparable argk-1 (and atg-18/WIPI) mRNA levels (Figure S1B), suggesting that argk-1 (and atg-18/WIPI) may be subject to post-transcriptional regulation in rsks-1/S6K mutants. Arginine kinases are members of the phosphagen kinase family, and function to maintain intracellular ATP levels by catalyzing the reversible reaction: ATP + arginine ⇌ ADP + phospho-arginine (Ellington, 2001). Due to its role in energy regulation, we focused the rest of our study on investigating ARGK-1 as a candidate S6K effector.

argk-1 is Required for Body Size, Thermotolerance, and Longevity of rsks-1/S6K Mutants

rsks-1/S6K mutants have previously been shown to have reduced body and brood sizes, increased resistance to environmental stressors, as well as an extended lifespan (Hansen et al., 2007; Korta et al., 2012; Pan et al., 2007; Wang et al., 2010). To investigate whether argk-1 contributes to these phenotypes, we introduced two predicted argk-1 null alleles (ok2993 and ok2973), respectively, into the rsks-1(sv31) mutant. WT animals carrying either of these two alleles behaved similarly and displayed no obvious phenotypes (Figure S2A, data not shown). While rsks-1/S6K mutants are smaller than WT animals (Hansen et al., 2007; Pan et al., 2007; Seo et al., 2013), rsks-1; argk-1 double mutants were similar in size to WT animals (Figure S2A; Day-1 adults). In contrast, rsks-1; argk-1 double mutants displayed reduced brood size (Figure S2B) and developmental delay (data not shown) similar to rsks-1 mutants. Similarly, we observed a partial requirement for argk-1 in rsks-1/S6K–mediated thermotolerance (Table S3). Collectively, our findings suggest an important role for argk-1 in rsks-1/S6K mutants in determining body size and thermotolerance, but not progeny production and developmental timing, thus dissociating these phenotypes.

We next compared the lifespans of rsks-1 and rsks-1; argk-1 double mutants. Whereas rsks-1(sv31) single mutants were considerably longer lived than WT animals (Hansen et al., 2007), the presence of either of the two argk-1 deletion alleles abolished this lifespan extension (Figure 1A; Table S4). Similarly, the lifespan of rsks-1(sv31) animals fed bacteria expressing argk-1 dsRNA (i.e., subjected to argk-1 RNAi) during adulthood was comparable to that of WT animals (Figure 1B; Table S4). Notably, argk-1 deletion alleles or argk-1 adult-only RNAi had no significant effects on the lifespan of WT animals (Table S4). These observations indicate an important role for argk-1 in rsks-1/S6K–mediated lifespan extension. We next asked if argk-1 plays a role in other conserved longevity paradigms, i.e., reduced insulin/IGF-1 signaling (e.g., the insulin/IGF-1 receptor mutant daf-2 (Kenyon et al., 1993)), dietary restriction (e.g., the feeding-impaired mutant eat-2 (Lakowski and Hekimi, 1998)), and reduced mitochondrial respiration (e.g., the ubiquinone synthesis mutant clk-1 (Felkai et al., 1999)). In three independent trials, we found that argk-1 RNAi had no significant effect on the lifespans of these long-lived mutants (Figures 1C, 1E; Table S4), and daf-2; argk-1 and eat-2; argk-1 double mutants had comparable lifespans to daf-2 and eat-2 single mutants, respectively (Table S4). Thus, argk-1 is possibly a selective longevity effector in rsks-1/S6K animals.

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

Figure 1

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(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.

argk-1 is Required for Activation of AAK-2/AMPK in rsks-1/S6K Mutants

Given that phosphagen kinases play central roles in buffering intracellular energy levels, we considered whether argk-1 might function together with the known rsks-1/S6K longevity effector, AMP-activated kinase (AMPK). This energy-sensing kinase is functionally conserved in C. elegans (Apfeld et al., 2004), and the α-subunit aak-2/AMPK is required for the long lifespan of rsks-1(ok1255) mutants (Selman et al., 2009). Moreover, these mutants have increased levels of phosphorylated (active) AAK-2/AMPK (Selman et al., 2009; Chen et al., 2013). We therefore examined the effects of argk-1 deletion on AAK-2/AMPK phosphorylation in rsks-1(sv31) mutants. Indeed, levels of phosphorylated AAK-2/AMPK were significantly reduced in rsks-1; argk-1 double mutants compared to rsks-1 single mutants and were similar to the levels in WT animals and argk-1 single mutants (Figures 2A, 2B). We cannot exclude the possibility that the observed increase in phosphorylated AAK-2/AMPK levels in rsks-1(sv31) mutants could reflect a change in total AAK-2/AMPK protein levels. However, our proteomics dataset revealed no significant difference in the relative abundance of AAK-2/AMPK spectral count ratios in rsks-1/S6K compared to WT animals (Table S1), arguing against such an explanation.

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

Figure 2

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(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.

To further investigate the role of ARGK-1 in rsks-1/S6K mediated AAK-2/AMPK activation, we also examined phosphorylation of acetyl-CoA carboxylase (ACC), an enzyme that is important for the biosynthesis and oxidation of fatty acids and is regulated by AMPK-mediated phosphorylation (Hardie et al., 2012). We observed a robust (~18-fold) increase in the abundance of a ~200 kD band, likely corresponding to phosphorylated POD-2/ACC, in rsks-1 animals compared to either WT or argk-1 animals (Figures 2C, 2D). In contrast, rsks-1; argk-1 double mutants showed a partial induction of ACC phosphorylation (Figures 2C, 2D). Here too, we note the possibility that the differences in phosphorylated ACC levels may reflect differences in the levels of ACC total protein. In this case, however, we were unable to address this further because we did not detect POD-2/ACC in our proteomics analysis.

Taken together, these analyses suggest that ARGK-1 is important for full activation of AAK-2/AMPK, suggesting that the two enzymes may function together in rsks-1/S6K animals.

ARGK-1 Overexpression Extends the Lifespan of Wild-Type C. elegans

Because we found ARGK-1 levels to be increased in rsks-1(sv31) mutants in our proteomic analysis, we reasoned that exogenously driven overexpression of ARGK-1 might be sufficient to induce at least some of the phenotypes observed in rsks-1/S6K mutants. To investigate this hypothesis, we created transgenic animals containing extrachromosomal arrays overexpressing argk-1 fused to gfp from the ubiquitous promoter of sur-5, an acetyl-CoA synthetase (sur-5p::argk-1::gfp). These animals expressed a GFP-tagged protein of ~72 kD, the expected size for ARGK-1::GFP (data not shown). Interestingly, we found that sur-5p::argk-1::gfp-overexpressing animals (two independent lines, as well as an integrated strain) lived up to 25% longer than WT animals at both 20°C and 25°C (Figures 3A, 3B; Table S5). Importantly, the lifespan extension conferred separately by ARGK-1 overexpression and rsks-1/S6K deficiency was not additive, because rsks-1(sv31) animals expressing sur-5p::argk-1::gfp were no longer lived than the rsks-1(sv31) single mutants (Table S5). These observations are consistent with the interpretation that the longer lifespans of rsks-1/S6K mutants and ARGK-1-overexpressing animals are mediated by at least partially overlapping mechanisms. In further support of this notion, we observed that the FOXA transcription factor PHA-4 was required for the extended lifespans of sur-5p::argk-1::gfp-overexpressing animals (Table S5), as it is for rsks-1/S6K mutants (Sheaffer et al., 2008). Moreover, we found aak-2/AMPK was required for the long lifespan sur-5p::argk-1::gfp animals (Figure 3B; Table S5), and phosphorylation of both AAK-2/AMPK and its downstream target ACC was significantly increased in the sur-5p::argk-1::gfp animals (Figures 3C, 3D), as observed in rsks-1/S6K mutants (Figure 2). These observations suggest that increased AAK-2/AMPK activity may contribute to the lifespan extension observed in animals overexpressing ARGK-1.

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

Figure 3

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(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.

As rsks-1/S6K mutants, animals ubiquitously overexpressing ARGK-1 were smaller than WT animals (Figure S2C), yet had normal brood sizes (Figure S2D) and showed no developmental delay (data not shown), consistent with argk-1 deletion not affecting these two phenotypes in rsks-1/S6K mutants (Figures S2A, S2B; data not shown). Unexpectedly, these transgenic animals also displayed a small reduction in pharyngeal pumping rates (Figure S3A), a phenotype not observed in adult rsks-1/S6K animals ((Pan et al., 2007) and data not shown). However, the transgenic animals were not food deprived since their food intake was comparable to that of WT animals, as measured by a recently described spectrophotometric assay (Gomez-Amaro et al., 2015) (Figure S3B). Thus, the longer lifespan and small body size observed in rsks-1/S6K mutants were recapitulated in animals ubiquitously overexpressing ARGK-1, which despite a small decrease in pharyngeal pumping appeared to have normal food intake and brood size. Collectively, our analyses indicate a correlation between increased levels of ARGK-1, reduced body size, AAK-2/AMPK activation, and longer lifespan. We propose that ARGK-1 is a critical regulator of these phenotypes in rsks-1/S6K mutants.

ARGK-1 is Expressed in a Subset of Glial Cells in C. elegans

We also constructed and analyzed lines expressing argk-1 tagged with mcherry from an endogenous promoter (argk-1p::arg-1::mCherry). Similar to our findings in animals expressing argk-1 from a ubiquitous promoter, we found that two independent lines of argk-1p::argk-1::mCherry-overexpressing animals lived longer (Table S5), were smaller (Figure S2C), had normal brood sizes (Figure S2D), and showed a small reduction in pharyngeal pumping rates (Figure S3C) compared to WT animals. In contrast to sur-5p::argk-1::gfp transgenics, however, argk-1p::argk-1::mCherry-overexpressing animals had significantly reduced food intake (Figure S3D). We do not know why ARGK-1 overexpression affects pumping rates in C. elegans, and results in reduced food intake in argk-1p::argk-1::mCherry-overexpressing animals. Regardless, we cannot exclude the possibility that the extended lifespan of argk-1p::argk-1::mCherry-overexpressing animals may be due to a form of dietary restriction, and we therefore refrain from drawing conclusions regarding the lifespan analysis of these animals.

We next analyzed these transgenic animals to learn more about the expression pattern of ARGK-1. Using confocal microscopy, we detected weak ARGK-1 expression in a small number of cells located mainly in the head and tail, but predominantly close to the anterior pharyngeal bulb (Figure 3E, and data not shown). This restricted expression pattern was consistent with essentially undetectable numbers of ARGK-1 spectral counts in WT animals (Table S1). To identify ARGK-1-expressing cells, we examined double-transgenic animals also expressing GFP fused to KCC-3, a sodium potassium chloride cotransporter and marker for glial cells of C. elegans (Tanis et al., 2009). As in other organisms, C. elegans glia surround and support neurons and play important roles in their protection and nourishment, and possibly in intercellular signaling (Oikonomou and Shaham, 2011). We observed co-localization of ARGK-1 fluorescence in several KCC-3-positive glia (Figure 3E), likely positioned in the inner and outer labial sensilla next to sensory neurons connecting to the outside environment since we observed overlap with an itx-1p::gfp transcriptional reporter that highlights inner and outer labial socket cells (Figure S3E) (Haklai-Topper et al., 2011). The overlap in ARGK-1 and KCC-3 expression was particularly interesting because mammalian KCC3 has been reported to colocalize with CK, the mammalian ortholog of ARGK-1, in cultured cells as well as in mouse brains (Salin-Cantegrel et al., 2008). We note that it is possible that ARGK-1 is expressed at additional sites in C. elegans since animals carrying a transcriptional GFP reporter (argk-1p::gfp) showed noticeable GFP signal in additional cells besides the inner and outer labial socket cells, including in the intestine (Figure S3F). Irrespectively, we conclude that ARGK-1 is expressed in glia in C. elegans, highlighting the possibility that ARGK-1 could play a role in neuronal function.

Expression of Creatine Kinase is Elevated in S6K1-deficient Mice

Mice deficient in S6K1, like C. elegans rsks-1/S6K mutants, are longer lived than WT animals (Selman et al., 2009). To address whether CK, the mammalian ortholog of ARGK-1, is similarly regulated by S6K1, we examined a newly created S6K1−/− line (Figures S4A, S4C). Since we found ARGK-1 to be expressed in glia in C. elegans, we examined CK expression in the cerebellum of mice, an area of the brain in which glial cells are known to participate in synaptic transmission (Pfrieger, 2009). Extracts of cerebellums from young (5–8 weeks old) S6K−/− and S6K+/+ mice were subjected to Western blot analysis of CK-B, an isoform of CK prominently expressed in the brain. Interestingly, we found that CK-B levels were significantly increased in cerebellar extracts from both female and male S6K1−/− mice compared to WT (Figures 4A, 4B). While statistical significance was not reached, trends towards elevated CK levels was also observed in the hippocampus as well as in skeletal muscle from S6K1−/− mice compared to WT (Figures S4D, S4E). These observations raise the possibility that, like ARGK-1 in C. elegans rsks-1/S6K mutants, CK may also be subject to regulation by S6K in mice.

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

Figure 4

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(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.

DISCUSSION

Inhibition of S6K is a conserved longevity paradigm, but little is known of the intermediary effectors that link the loss of S6K activity with lifespan-extending pathways. Through a proteomics analysis, we identified the arginine kinase ARGK-1 as a novel and selective rsks-1/S6K longevity effector in C. elegans. ARGK-1 appears to act in concert with the energy sensor AMPK, another rsks-1/S6K longevity effector, thus highlighting an important role for energy regulation in this paradigm. CKs are the mammalian functional orthologs of arginine kinases, and we observed that CK expression is increased in the cerebellums of S6K−/− mice, highlighting the possibility that S6K may similarly regulate CKs in higher organisms.

Our study provides direct evidence linking the protein family of phosphagen kinases to longevity and to S6K. In support of this, we observed (i) ARGK-1 to be more abundant in rsks-1/S6K mutants than in WT animals (by spectral counts), (ii) ubiquitous overexpression of ARGK-1 is sufficient to extend C. elegans lifespan, and (iii) argk-1 is required for lifespan extension in the rsks-1/S6K mutants but does not appear to play a role in other longevity paradigms. While we have not been able to confirm an increase in ARGK-1 levels in rsks-1/S6K mutants, our observations collectively indicate that reduction of RSKS-1/S6K engages ARGK-1 to extend C. elegans lifespan, and identify ARGK-1 as the first selective longevity effector of RSKS-1/S6K. ARGK-1 is one of five arginine kinases in C. elegans (Fraga et al., 2015), and five isoforms of CK are expressed in different cell types in mammals. Although none of these enzymes have previously been linked to longevity, supplementation with creatine equivalent to arginine in metazoans improves healthspan and longevity in mice and provides beneficial effects to mice and humans with Parkinson disease (Matthews et al., 1999; Bender et al., 2008; Bender et al., 2006; Hass et al., 2007). Moreover, we observed CK-B significantly increased in the cerebellum of both female and male S6K−/− mice, yet only female S6K−/− mice are longer lived (Selman et al., 2009). Collectively, these observations warrant further experiments to clarify the role of arginine and creatine kinases in lifespan determination, including in sex-specific contexts.

We found that argk-1 was also important for some but not all of the phenotypes we examined in C. elegans rsks-1/S6K mutants. Specifically, argk-1 was required, in addition to lifespan, for the reduced body size and increased stress resistance, but not for the reduction in progeny production and developmental delay observed in rsks-1/S6K mutants. In turn, animals ubiquitously overexpressing ARGK-1 were smaller and longer lived, but these animals generally had normal brood sizes and developmental rates. Thus, we observed a correlation between small body size, increased stress resistance and longer lifespan, but no correlation to reduced progeny production or developmental delay. It is possible that argk-1 exerts its physiological effects by reversing the reduced mRNA translation rates observed in rsks-1/S6K mutants (Hansen et al., 2007; Pan et al., 2007); however, since brood size or developmental rates, two very metabolically demanding outputs, were not significantly changed, this may not be the case. Instead, ARGK-1 might be important for regulation of the intracellular energy status of specific cells in rsks-1/S6K mutants. In particular, we found ARGK-1 to be expressed in a small subset of glia adjacent to multiple sensory neurons in the inner labial sensilla and outer labial sensilla. These neurons connect to the outside environment, raising the interesting possibility that specific glia play a role in communicating with the exterior to ensure the long lifespan of rsks-1/S6K mutants, potentially via a circuit that also includes the intestine (since a transcriptional argk-1 reporter also showed expression in this tissue). In such a hypothetical scenario, ARGK-1 might ensure adequate ATP levels for specific enzymes in ARGK-1-expressing cells, as has been proposed for CK-B and KCC3 in mouse brains (Salin-Cantegrel et al., 2008). Such ATP-dependent enzymes might be part of a metabolic pathway important for the longevity observed in rsks-1/S6K mutants. Identification of the proximal targets and the site-of-action of ARGK-1 will be helpful in exploring the exact mechanism by which RSKS-1/S6K modulates longevity via ARGK-1 in C. elegans.

In support of a central role for ARGK-1 in energy regulation in rsks-1/S6K mutants, we observed that argk-1 was both necessary and sufficient to activate another known RSKS-1/S6K-longevity effector, AAK-2/AMPK. Consistent with a link between these two energy-regulating systems, we found that aak-2/AMPK was activated in and was required for the long lifespan of animals ubiquitously overexpressing ARGK-1. These observations suggest a model in which reduced levels of RSKS-1/S6K leads to induction of ARGK-1, which in turn is critical for full activation of AMPK. Although AAK-2/AMPK is ubiquitously expressed in C. elegans (Mair et al., 2011), it is not clear whether the interaction between ARGK-1 and AMPK in rsks-1/S6K mutants is direct or indirect. In this regard, it is interesting to note that CK and AMPK can cross-regulate each other’s activity in mammalian muscle cells (Ponticos et al., 1998). Further experiments are needed to fully elucidate how ARGK-1 and AAK-2/AMPK act together to extend the lifespan of C. elegans rsks-1/S6K mutants, and whether a similar link is conserved in S6K1-deficient mice.

In addition to ARGK-1, our proteomics analysis identified 138 other proteins that were putatively more abundant and 200 putatively less abundant in young rsks-1/S6K mutants compared to WT animals. While most of these proteins have not previously been linked to rsks-1/S6K, many proteins with roles in xenobiotic responses or in detoxification were found to be more abundant, consistent with the observation that rsks-1/S6K mutants, similar to other translation mutants, are more stress resistant than WT animals (Hansen et al., 2007; Pan et al., 2007; Wang et al., 2010). Such translation mutants include C. elegans with reduced levels of the translation initiation factor IFG-1/eIF4G, which achieve stress resistance at least in part by mechanisms involving differential mRNA translation (Rogers et al., 2011). Our study suggests that similar regulatory mechanisms may exist in rsks-1/S6K mutants, since we observed ARGK-1 and ATG-18/WIPI proteins, but not their corresponding mRNA levels, to be more abundant in rsks-1/S6K mutants compared to WT animals. Future studies are needed to determine the exact mechanism by which ARGK-1 and ATG-18/WIPI are regulated in rsks-1/S6K mutants. Likewise, it will be interesting to explore whether other differentially abundant proteins identified in our proteomic analysis, like ARGK-1, represents novel longevity determinants.

In conclusion, our study reports the arginine kinase ARGK-1 to be the first selective effector relevant to the lifespan extension observed in C. elegans rsks-1/S6K mutants. This mechanism likely involves regulation of AMPK, highlighting an important role for energy metabolism in this longevity paradigm. Future experiments will reveal to which extend this longevity pathway plays a conserved role in aging.

EXPERIMENTAL PROCEDURES

C. elegans Strains

C. elegans strains (Table S7) were maintained as described in Supplemental Experimental Procedures. Two argk-1 mutant alleles were obtained from the CGC and were outcrossed at least four times.

Lifespan Analysis

Lifespan analysis was carried out as previously described (Hansen et al., 2005). Data were analyzed using STATA software (StataCorp), and P values were calculated using the Mantel-Cox log-rank test.

Western Blot Analysis

See Supplemental Experimental Procedures for details on preparation of C. elegans and of mice samples, and for immunoblotting with specific antibodies.

Confocal Imaging

See Supplemental Experimental Procedures for details on mounting and imaging of transgenic C. elegans.

Supplementary Material

supplemental table S1
supplemental table S2
supplemental table S3
Supplemental information

Acknowledgments

We thank members of the Hansen lab for insightful discussions; Drs. S. Tuck and A. O’Rourke for comments on the manuscript; Dr. D. Wolf for assistance with proteomics; Drs. J.-L. Li and R. Williams for assistance with bioinformatics; Drs. K. Motamedchaboki and A.R. Campos for assistance with data submission to PeptideAtlas; Dr. L. Lapierre for initial QPCR analysis, Dr. D. Fraga for sharing data ahead of publication; Dr. S. Shaham for providing the LX1116 strain and for feedback on data; Dr. S. Chalasani for feedback on data; Dr. W. Mair for sharing unpublished reagents; Drs. Z. Ronai, R. Bodmer, and A. Dillin for input on the study; and the Caenorhabditis Genetic Center (supported by NIH grant P40 OD010440) for providing argk-1 strains. PRM was funded by an NIH/NIA NRSA fellowship (F31 AG039222); C-YL was a Glenn/AFAR Postdoctoral Fellow; BKK was supported by NIH/NIA grant R01 AG035336 and an Ellison Medical Foundation Senior Scholar in Aging award; MH was supported by NIH/NIA grants R01 AG038664 and R01 AG039756 and by a Glenn Award for Research in Biomedical Mechanisms of Aging from the Glenn Foundation for Medical Research. This work was also supported by the NCI under award number 5P30CA030199.

Footnotes

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AUTHOR CONTRIBUTIONS

PRM, C-YL, BKK and MH designed the experiments. PRM, C-CC, SG, AD, XS, and MH created new C. elegans strains. LMB designed and performed the 2DLC-MS/MS analysis and compiled a list of differentially expressed proteins that was processed by Jian-Liang Li and Roy Williams in the institute’s bioinformatics cores. C-CC analyzed ATG-18 levels and performed all heat-shock assays, and PRM analyzed the data. JTC and XS carried out confocal imaging analyses. CK carried out QPCR analyses. AD and MH conducted single lifespan repeats, and AD analyzed the data. RGA carried out the food-intake experiments in MP’s lab. C-YL carried out the mouse experiments in BK’s lab, which made and characterized the S6K−/− mice in collaboration with WCL’s lab. PRM conducted all other experiments and analyzed the data. PRM and MH wrote the manuscript with input from co-authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplemental table S1
NIHMS759043-supplement-supplemental_table_S1.xls (2.1MB, xls)
supplemental table S2
NIHMS759043-supplement-supplemental_table_S2.xlsx (18.7KB, xlsx)
supplemental table S3
NIHMS759043-supplement-supplemental_table_S3.xlsx (23.4KB, xlsx)
Supplemental information
NIHMS759043-supplement-Supplemental_information.pdf (840.7KB, pdf)

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