Loco signaling pathway in longevity

Abstract

Despite the various roles of regulator of G protein signaling (RGS) protein in the G protein signaling pathway that have been defined, the function of RGS has not been characterized in longevity signaling pathways. We found that reduced expression of Loco, a Drosophila RGS protein, resulted in a longer lifespan of flies with stronger resistance to stress, higher MnSOD activity and increased fat content. In contrast, overexpression of the loco gene shortened the fly lifespan significantly, lowered stress resistance and reduced fat content, also indicating that the RGS domain containing GTPase-activating protein (GAP) activity is related to the regulation of longevity. Interestingly, expressional changes of yeast RGS2 and rat RGS14, homologs to the fly Loco, also affected oxidative stress resistance and longevity in the respective species. It is known that Loco inactivates inhibitory Gαi•GTP protein to reduce activity of adenylate cyclase (AC) and RGS14 interacts with activated H-Ras and Raf-1 kinases, which subsequently inhibits ERK phosphorylation. We propose that Loco/RGS14 protein may regulate stress resistance and longevity as an activator in AC-cAMP-PKA pathway and/or as a molecular scaffold that sequesters active Ras and Raf from Ras•GTP-Raf-MEK-ERK signaling pathway. Consistently, our data showed that downregulation of Loco significantly diminishes cAMP amounts and increases p-ERK levels with higher resistance to the oxidative stress.

Introduction

Recent studies investigating the aging process have shown that the regulatory mechanisms of aging involve various signaling pathways, including targets of rapamycin (TOR)-ribosomal protein S6 kinase (S6K), adenylate cyclase (AC)-protein kinase A (PKA), Ras-Raf-MEK-ERK and insulin/insulin-like growth factor 1 (IGF-1) pathways.^1–6 These signaling pathways are evolutionarily conserved in different species such as yeast, nematodes, fruit flies and mammals.⁷ The G protein signaling system has been also shown to control aging via the G protein-coupled receptor (GPCR), methuselah and its ligand, stunted, in fruit flies.^8,9 The function of the heterotrimeric G protein complex Gα•GTP/Gβγ¹⁰ is regulated by the intrinsic nucleotide exchange factor (GEF) activity of GPCR and the GTPaseactivating protein (GAP) activity of RGS protein.¹¹ However, the role of RGS has not been characterized in longevity signaling pathway, even though many RGS proteins have been identified as activators or repressors with GAP activity in the G protein signaling pathway.^12,13

Loco, a fly RGS protein, consists of RGS, two of Raf-like Ras-binding domain (RBD) and GoLoco domains. The RGS and GoLoco domains function as a GTPase activator (GAP) and a guanine nucleotide dissociation inhibitor (GDI), respectively.¹⁴ The loco gene expresses four distinct isoforms (C1–4) with four different 5′ ends and a common 3′ end that includes all of RGS, RBD and GoLoco domains (http://flybase.org). The Loco protein has been shown to function in neuroblast (NB) asymmetric division¹⁴ and blood-brain barrier formation¹⁵ which are mediated through interactions with several GPCRs and the inhibitory G proteins (Gαi and Gαo). Yeast 2-hybrid screen analyses demonstrated that the Loco protein also interacts with the monomeric G proteins Rap1, Rap2 and Ras1 (small GTPase).¹⁶ Nevertheless, the signaling pathway downstream of Loco has not been studied in detail.

Extended Lifespan with Higher Stress Resistance Mediated by Reduced loco Expression

Between flies aged 1 week (96% survival) and 7 weeks (male: 11%; female: 39%), the expression of loco-C1 and -C2 increased approximately 2.2-fold and 3.6-fold, respectively, which suggested that the expression level of loco-C1 and/or -C2 might be important to the longevity of flies. Indeed, hetero-deficiency of the loco gene (loco^P283 /+, amorphic mutant allele¹⁴) extended mean lifespan by 17–20% in both males and females, despite the fact that homozygous deficiency of loco in flies is lethal.¹⁷ Several long-lived mutant flies were more resistant to stresses such as starvation, oxidation and heat compared with wild-type flies,^2,8 indicating that lifespan and stress resistance are often correlated positively.^18–21 Interestingly, the loco hetero-deficient female flies, which exhibited an extended lifespan, also could survive longer under the stresses than wild-type flies. Given that loco expression was elevated in older flies, these results demonstrate that decreasing expression of the loco gene can increase longevity and enhance stress resistances in D. melanogaster. Consistent with higher resistance to the oxidative stress, activity of manganese-containing superoxide dismutase (MnSOD) was 74% increased in loco heterozygous mutant flies compared with wild-type flies.

When body protein, fat and glycogen contents were measured in flies, only fat (triacylglycerol) content significantly increased by 36% in the loco heterozygous mutant flies, showing that reduced expression of the loco gene resulted in higher stress resistance with the accumulation of fat in the body. In fact, many studies have shown a positive correlation between higher stress resistance and fat accumulation in long-lived flies and C. elegans.^1,2,4,8 Another interesting difference in the loco heterozygous mutant compared with wild-type flies was a 20% decrease in cAMP amount that might result in reduced AC-PKA signaling, which is known to induce higher stress resistance and longer lifespan in yeast and mammals.^3,6,7

Longevity Changed by the Extent of loco Expression

The loco expression in loco heterozygous flies (loco^P283 /+) was 79% of the wild-type flies, higher than the expected 50%. When loco expression was reduced using a UAS-loco-dsRNAi transgene under the control of Gal4 drivers, the flies expressing 78% of loco transcripts extended mean lifespan by 32%. Concomitant with a longer lifespan, these flies also exhibited higher stress resistance and elevated fat content, as shown in the loco^P283 /+ flies which mildly decreased the loco expression. However, when loco expression was reduced to 16%, the flies exhibited only 1.5% increase in mean lifespan. With the fact that loco homozygote is lethal,¹⁷ these data imply that a delicate change in loco expression is important for the regulation of longevity. In contrast, the overexpression of loco-C1 transcripts (7.8-fold) significantly shortened the mean lifespan of male flies by 20% compared with the non-overexpressing loco-C1 flies. Besides the shorter lifespan, overexpression of loco-C1 also reduced stress resistance and fat content in these flies. Taking these data together with the longer lifespan in reduced loco gene expression, we can conclude the longevity of flies is altered by the extent of loco expression.

Evolutionary Conservation of Loco Function in Longevity

The yeast RGS2 protein shares 68% homology in the RGS domain with fly Loco protein and converts active Gpa2•GTP into inactive Gpa2•GDP via GAP activity of the RGS domain.²² Gpa2 regulates the adenylate cyclase (AC) activity of Cry1,^23,24 which synthesizes cAMP from ATP and therefore stimulates cAMP-dependent PKA activity in response to glucose. Changes of yeast RGS2 expression produced different amounts of cAMP²² and altered stress resistance and lifespan in Saccharomyces cerevisiae.²⁵ The mammalian RGS14 protein is 80% homologous with all domains of Drosophila Loco-C1 protein (32% identity and 48% similarity, http://www.ebi.ac.uk/Tools/emboss/align). Interestingly, when the expression of rgs14 gene was reduced to 46% by siRNA duplex in rat fibroblast cells, the resistance to oxidative stress (H₂O₂) increased with higher MnSOD expression similarly as the reduced expression of Loco did in flies. These represent that evolutionarily conserved RGS proteins play important roles in regulating stress resistance and longevity in several different species.

Increase of p-ERK Levels in Reduced loco Expression

The mammalian homolog RGS14 was shown to bind activated H-Ras and Raf-1 kinases through the first RBD domain in HeLa cells.^26,27 These interactions inhibited growth factor PDGF-stimulated MAP kinase activity in a H-Ras dependent manner, which subsequently resulted in decrease of p-ERK levels.²⁷ With a report that Loco protein interacts with the monomeric G proteins Rap1, Rap2 and Ras1 in yeast 2-hybrid screen analyses,¹⁶ the RGS14 data suggest that the Loco protein may regulate stress resistance and longevity of flies through the Ras-Raf-MEK-ERK signaling pathway. Consistently, the reduced loco expression enhanced oxidative stress resistance in flies concomitant with higher p-ERK levels (Fig. 1). The induced ERK phosphorylation in AC-PKA and Ras-Raf-MEK-ERK signaling pathways is known to activate anti-apoptosis, cell survival and anti-oxidative stress mechanisms in yeast and mammals.^6,7

Figure 1.

A decrease in loco expression induces ERK phosphorylation. Survival % and p-ERK levels of 2-day-old female flies at 0, 16 and 24 h after oxidative stress (5% sucrose + 20 mM paraquat). Amount of p-ERK protein was normalized with GAPDH (protein gel blot) and p-ERK levels of wild-type (+/+) flies at 0 h was set at 1 fold. Δloco/+: loco^P283 heterozygote.

Based on the previous reports in reference ^{6, 7, 14–16, 26–27} and our results²⁵ including Figure 1, it is likely that the Loco protein regulates stress resistance and longevity by modulating ERK phosphorylation through at least two mechanisms (Fig. 2): first, the RGS domain-dependent inactivation of the inhibitory Gαi protein, which results in the activation of the AC-cAMP-PKA pathway and the inactivation of Raf; and second, the RBD domain-dependent inhibition of active Ras/Raf as a molecular scaffold to sequester their functions. In both cases, p-ERK levels are reduced. An alternative potential mechanism by which Loco inhibits Ras•GTP is to convert Ras•GTP into Ras•GDP by the GAP activity mediated through the RGS domain, as similarly occurs in the regulation of inhibitory Gαi•GTP protein.¹⁴ Deletion analysis of RGS and GoLoco domains²⁵ showed that the GAP activity of the Loco RGS domain may be important for the regulation of the aging process. The RGS proteins (RGS2, Loco and RGS14) may be involved globally in the biological processes of stress resistance and lifespan in several species as upstream regulators of longevity signaling pathway beyond the specific cellular functions.

Figure 2.

Models of Loco signaling pathway with small GTPases. A decrease in Loco expression, an RGS protein in flies, enhances stress resistance and extends lifespan. Vertical arrow: our data;²⁵ line and arrow: hypothesized pathways according to the previous published reports in references 6, 7, 14–16, 26 and 27.

Further Studies of the Loco Signaling Pathway in Longevity

Investigating the physical interaction between Loco and Ras/Raf proteins will be an important next step in characterizing how Loco regulates the Ras•GTP-Raf-MEK-ERK signaling pathway. Another important question to investigate is whether converting Gαi•GTP and/or Ras•GTP into the GDP form by the GAP activity of Loco is necessary for regulating the aging process. Because of the global and epigenetic aspects of the aging process, the entire aging process is not dependent upon a single gene or a signaling pathway.^7,28 Therefore, it is possible that there are more genes and signaling pathways involved in the Loco-mediated regulation of stress resistance and aging besides the proposed AC-PKA and Ras•GTP-Raf-MEK-ERK pathways (Fig. 2). Using genomic (RNA-seq) and/or proteomic (iTRAQ) approaches, the gene expression profiles of flies with different loco gene expression levels can be compared in order to identify genes downstream of loco that are related to the regulation of longevity.

The changes in loco expression affects stress resistance and lifespan accompanied by changes of fat contents, suggesting that the Loco pathway might play important roles in fat body for the regulation of longevity. The fat body is involved in the metabolism and storage of fat in adult flies. Investigation of the fat body-specific modulation of loco expression will reveal how the Loco protein regulates stress resistance and longevity in a tissue-specific manner.

Considering that Loco is required at various developmental stages, including as neuroblast division, glial differentiation, blood-brain barrier formation and reproductive systems,^{14,15,17,29–31} the changes of loco expression during embryonic development may cause detrimental consequences, such as lethality of loco homozygous mutant flies.^17,31 Future studies that investigate adult-specific regulation of loco expression will reveal more directly the function(s) of Loco in adult longevity.

Abbreviations

GAP

GTPase-activating protein

GPCR

G protein-coupled receptor

RBD

Raf-like Ras-binding domain

RGS

regulator of G protein signaling

Extra View to: Lin YR, Kim K, Yang Y, Ivessa A, Sadoshima J, Park Y. Regulation of longevity by regulator of G-protein signaling protein, Loco. Aging Cell. 2011;10:438–447. doi: 10.1111/j.1474-9726.2011.00678.x.