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O P E N A C C E S S S O U R C E : Science Advances
Abstract
Vaccinia virus–related kinase (VRK) is an evolutionarily conserved nuclear protein kinase. VRK-1, the single Caenorhabditis elegans VRK ortholog, functions in cell division and germline proliferation. However, the role of VRK-1 in postmitotic cells and adult life span remains unknown. Here, we show that VRK-1 increases organismal longevity by activating the cellular energy sensor, AMP-activated protein kinase (AMPK), via direct phosphorylation. We found that overexpression of vrk-1 in the soma of adult C. elegans increased life span and, conversely, inhibition of vrk-1 decreased life span. In addition, vrk-1 was required for longevity conferred by mutations that inhibit C. elegans mitochondrial respiration, which requires AMPK. VRK-1 directly phosphorylated and up-regulated AMPK in both C. elegans and cultured human cells. Thus, our data show that the somatic nuclear kinase, VRK-1, promotes longevity through AMPK activation, and this function appears to be conserved between C. elegans and humans.
INTRODUCTION
Mitochondria are essential subcellular organelles for cellular energy production [reviewed in (1)]. Mitochondria also play important functions in a wide array of other cellular processes, ranging from cellular signaling to apoptosis. In addition, mitochondria play crucial roles in organismal aging, and functional declines in mitochondria are associated with age-related diseases [reviewed in (2)]. However, mild inhibition of mitochondrial respiration has been shown to promote longevity in multiple species [reviewed in (3)]. In Caenorhabditis elegans, the genetic inhibition of mitochondrial respiration genes, including isp-1 (Rieske iron-sulfur protein in complex III) and clk-1 (coenzyme Q biosynthesis enzyme crucial for electron transport), prolongs life span. Inhibition of mitochondrial respiration also increases life span in Drosophila and mammals. Therefore, life-span extension by reduced mitochondrial respiration is conserved, and the elucidation of the molecular mechanism in C. elegans may enhance our understanding of human aging and longevity.
Adenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK), a critical cellular energy sensor that increases life span in multiple species [reviewed in (4)], is one of the factors required for the enhanced longevity caused by inhibition of mitochondrial respiration in C. elegans (5–7). AMPK is activated by an elevated AMP/adenosine 5′-diphosphate (ADP)–to–adenosine 5′-triphosphate (ATP) ratio that results from reduced cellular energy, leading to increases in catabolic processes and inhibition of anabolic processes [reviewed in (8)]. Activation of AMPK results from phosphorylation at residue Thr172 in its catalytic α subunit by upstream kinases. Liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) are two kinases that function to activate AMPK in mammals [reviewed in (9)]. Transforming growth factor–β–activated kinase 1 (TAK1) has also been shown to phosphorylate AMPK in vitro [reviewed in (9)]. However, the upstream regulators of AMPK that facilitate its life span–extending effects remain incompletely understood.
The vaccinia virus–related kinase (VRK) family consists of three serine-threonine protein kinases (VRK1 to VRK3) in mammals, which are related to casein kinases (10–12). Among these three, the best characterized is VRK1, a cell cycle regulator that is abundant in proliferative tissues (13). Unlike mammals, C. elegans has a single VRK ortholog, VRK-1, whose function in cell proliferation is relatively well established (14–16). Strong loss-of-function mutations of the C. elegans vrk-1 gene results in sterility, and reduced vrk-1 function leads to mislocalization of barrier-to-autointegration factor 1 (BAF-1), a phosphorylation target of VRK-1, resulting in severe mitotic defects (15, 17, 18). vrk-1 is also required for germ cell proliferation, likely through its ability to regulate the p53-like protein, C. elegans p53-like-1 (CEP-1) (16), and plays important roles in the development of vulva and uterus (17, 18). However, it remains unknown whether VRK-1 acts in postmitotic cells or has a role in adult life span.
In this study, we sought to elucidate the role of VRK-1 in regulation of adult life span in C. elegans. We found that overexpression of VRK-1::GFP (green fluorescent protein), which was detected in the nuclei of cells in multiple somatic tissues, including the intestine, increased life span. Conversely, genetic inhibition of vrk-1 decreased life span. We further showed that vrk-1 was essential for the increased life span of mitochondrial respiratory mutants. We demonstrated that VRK-1 was responsible for increasing the level of active and phosphorylated form of AMPK (p-AMPK). In addition, we found that mammalian VRK1 directly phosphorylated AMPK at Thr172, resulting in its increased activity. Together, these data indicate that the nuclear protein kinase, VRK-1, which acts in somatic cells, promotes longevity by increasing the activity of AMPK through phosphorylation.
RESULTS
Nuclear expression of VRK-1 in somatic tissues extends C. elegans life span
To determine the role of vrk-1 in regulating adult life span, we generated transgenic C. elegans overexpressing vrk-1 fused with the GFP gene (vrk-1::GFP). The VRK-1::GFP was predominantly localized to the nuclei of many cells, including neural, intestinal, and hypodermal cells (Fig. 1A and fig. S1, A and B) (16–18). In addition, we found that VRK-1::GFP was highly expressed throughout the C. elegans life cycle, in the soma of larvae (Fig. 1A and fig. S1, A and B) and fully grown adult worms (Fig. 1B and fig. S1C), comprising postmitotic cells after the cells stopped dividing. Notably, extrachromosomal transgenes that overexpress vrk-1::GFP, which is limited to somatic cells because of transgene silencing in germ cells (19), substantially increased adult life span (Fig. 1, C and D, and fig. S2A). We then confirmed the longevity conferred by overexpression of vrk-1::GFP using an integrated transgenic line (Fig. 1, E and F, and fig. S2B). These data suggest that vrk-1 overexpression specifically in somatic cells promotes the longevity of C. elegans.
Fig. 1 VRK-1 is a nuclear protein that increases worm life span.
(A) VRK-1::GFP was localized in the cellular nuclei of multiple tissues including neurons (asterisks), intestine (arrowheads), and hypodermis (fig. S1B, arrows) at L2 larval stage. Nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). See also fig. S1 (A and B) for magnified images of VRK-1::GFP and cellular nuclei for specific tissues. DIC, differential interference contrast. Photo credit: Sangsoon Park, Pohang University of Science and Technology, South Korea. (B) VRK-1::GFP was expressed in somatic tissues of days 1, 3, 5, and 7 adult worms. Scale bars, 50 μm. Photo credit: Murat Artan, MRC Laboratory of Molecular Biology, UK. (C and D) Four independent lines of extrachromosomal vrk-1::GFP-transgenic worms (vrk-1::GFP O/E Ex) displayed increased life span with [©, fig. S2A, transgenic lines 1 to 4] or without (D, transgenic lines 3 and 4) 5-fluoro-2′-deoxyuridine (FUDR) treatment. odr-1p::RFP © and rol-6D (D) were used as coinjection markers, and odr-1p::RFP © and rol-6D (D) transgenic worms were used as controls, respectively. We found that germline-specific transgenic expression of pie-1p::GFP::vrk-1 (16) had no effect on life span (fig. S2, C and D). VRK-1 tagged with GFP appears to be functional because previous reports have shown that GFP::VRK-1 transgenes rescued the sterility, uterine and uterine seam cell developmental defects, and protruding vulva phenotypes of vrk-1 mutants (16–18). (E and F) An integrated vrk-1::GFP transgenic line (vrk-1::GFP Is) extended life span with [(E), four of five trials] or without [(F), three of three trials] FUDR treatment. Control indicates wild-type N2. (G) vrk-1 RNAi significantly shortened life span. See also fig. S2E for life-span results of vrk-1(RNAi) animals treated with FUDR. (H) vrk-1(ok1181) mutation substantially shortened life span without FUDR treatment. In contrast, hypomorphic vrk-1(x1) mutants had a life span similar to that of wild-type worms (fig. S2, H and I). # indicates life-span results that were obtained with FUDR treatment to prevent progeny from hatching. See also table S1 for values and statistical analysis for the life-span data.
We next investigated the effect of vrk-1 inhibition on the life span of C. elegans. vrk-1 RNA interference (RNAi) and strong loss-of-function mutation vrk-1(ok1181) (fig. S2G) (15–17) shortened C. elegans life span (Fig. 1, G and H, and fig. S2, E and F). These results indicate that vrk-1 is required for the maintenance of normal life span. Together, these data indicate that VRK-1 is necessary and sufficient for longevity.
VRK-1 contributes to the longevity of mitochondrial respiration mutants
We then asked whether VRK-1 played a role in the increased life span conferred by various mutations in C. elegans. RNAi knockdown of vrk-1 largely suppressed the longevity conferred by mutations in mitochondrial respiration genes, isp-1 (Fig. 2A and fig. S3, A to C) and clk-1 (Fig. 2B). Similarly, the vrk-1(ok1181) mutation suppressed the long life span of isp-1 mutants (Fig. 2C). We then found that genetic inhibition of vrk-1 by RNAi or by the deletion mutation, vrk-1(ok1181), decreased the longevity of insulin/insulin-like growth factor 1 receptor daf-2(−) mutants (Fig. 2, D and E). In addition, vrk-1 RNAi shortened the life span of dietary restriction mimetic eat-2(−) (Fig. 2F) and hypoxia-inducible factor 1 (HIF-1)–hyperactive vhl-1(−) (Fig. 2G) mutants as well as RNAi-mediated electron transport chain–inhibited cco-1(RNAi) animals (fig. S3, F to H; see Fig. 2 legends for discussion). Together, these data suggest that vrk-1 contributes to longevity conferred by various longevity interventions and, in particular, is essential for that caused by the inhibition of mitochondrial respiration.
Fig. 2 Inhibition of VRK-1 suppresses the longevity of mitochondrial respiration mutants.
(A and B) vrk-1 RNAi suppressed the long life span of isp-1(qm150) [isp-1(−)] (A) and clk-1(qm30) [clk-1(−)] (B) mutants. vrk-1 RNAi partially but substantially suppressed the longevity of isp-1(−) mutants without FUDR treatment (fig. S3A). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis confirmed that vrk-1 RNAi decreased vrk-1 mRNA level for the life-span assays (fig. S3, B and C). Different from mitochondrial respiration mutants, longevity caused by inhibition of mitochondrial respiration using cco-1 RNAi knockdown (39, 40) was indiscriminately decreased by vrk-1 RNAi (fig. S3F). Our data are consistent with previous reports showing that mutation and RNAi of mitochondrial electron transport chain components distinctly promote longevity by acting with different factors [reviewed in (3)]. © A strong loss-of-function vrk-1(ok1181) mutation suppressed the long life span of isp-1(−) mutants without FUDR treatment. In addition, vrk-1(x1) mutation substantially reduced the long life span of isp-1(−) mutants (fig. S3D). (D and E) vrk-1 RNAi (D) or vrk-1(ok1181) mutation (E) substantially reduced the life span of daf-2(e1370) [daf-2(−)] mutants. (F and G) Knockdown of vrk-1 shortened the life span of eat-2(ad1116) [eat-2(−)] (F) and vhl-1(ok161) [vhl-1(−)] (G) mutants, similarly to that of wild type. Hypomorphic vrk-1(x1) mutation did not reduce the long life span of daf-2(−) (fig. S3E), eat-2(−) (fig. S3I), or osm-5(−) (fig. S3J) animals. # indicates life-span results obtained with FUDR treatment to prevent progeny from hatching. See also table S1 for values and statistical analysis for life-span data.
VRK-1 regulates expression of AMPK target genes
We next investigated the mechanism by which VRK-1 contributed to the longevity of mitochondrial respiration mutants. To this end, we first determined whether inhibition of nuclear protein VRK-1 affected gene expression in isp-1(−) mutants by performing mRNA sequencing (mRNA-seq) analysis. We identified 1589 up-regulated and 199 down-regulated genes in isp-1(−) mutants versus wild-type worms (fold change > 2 and < 0.5, P < 0.001; Fig. 3A and data file S1). Among these differentially expressed genes (DEGs), the expression of 328 up-regulated genes and 22 down-regulated genes was dependent on vrk-1 (Fig. 3A and data file S1). Gene Ontology (GO) analysis indicated that genes involved in diverse biological processes were enriched among vrk-1–dependent up-regulated genes in isp-1(−) mutants, whereas those involved in lipid transport were enriched among the down-regulated genes (Fig. 3B).
Fig. 3 Genes up-regulated by isp-1(−) in a vrk-1–dependent manner overlap with AMPK–up-regulated genes.
(A) vrk-1 RNAi suppressed the isp-1(−)–mediated induction and repression of a subset of genes. Black dots indicate mean FPKM (fragments per kilobase of transcript per million mapped reads) of genes that were up- or down-regulated by isp-1(−) under a control RNAi condition [fold change, >2 (top) or <0.5 (bottom); P < 0.001]. Red dots indicate mean FPKM of the genes in isp-1(−) treated with vrk-1 RNAi compared to wild type (WT) treated with control RNAi. See also data file S1. (B) Biological process GO terms that were enriched among genes up- or down-regulated by isp-1(−) in a vrk-1–dependent manner (***P < 0.001). UV, ultraviolet. (C to F) Genes up-regulated by isp-1(−) in a vrk-1–dependent manner were enriched for genes up-regulated by AMPK. Genes that were induced in isp-1(−) animals in a vrk-1–dependent fashion were analyzed to calculate overlaps and representation factor (RF) with published transcriptome data by using WormExp (*P < 0.05 and ***P < 0.001) (see Materials and Methods). aak-2 O/E1 and aak-2 O/E2: genes up-regulated by aak-2 overexpression from two different transcriptome data (C, D, and F). CA-aak-2: genes up-regulated by constitutively active (CA) aak-2 (C and E). atfs-1(gof): an atfs-1(et18) gain-of-function mutant ©. spg-7(RNAi): a condition that induces mitochondrial stress and activates ATFS-1. ATFS-1 target genes: genes whose promoter regions bind ATFS-1 in spg-7(RNAi) animals ©. skn-1(RNAi)1 and skn-1(RNAi)2: genes down-regulated by skn-1(RNAi) from two different transcriptome data ©. “WT vs. skn-1(RNAi): oxidative stress” indicates genes down-regulated by skn-1(RNAi) under oxidative stress conditions. See also fig. S4 for details.
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