The small G protein RAS2 is involved in the metabolic compensation of the circadian clock in the circadian model Neurospora crassa
2017; Elsevier BV; Volume: 292; Issue: 36 Linguagem: Inglês
10.1074/jbc.m117.804922
ISSN1083-351X
AutoresNorbert Gyöngyösi, Anita Szőke, Krisztina Ella, Krisztina Káldi,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoAccumulating evidence from both experimental and clinical investigations indicates a tight interaction between metabolism and circadian timekeeping; however, knowledge of the underlying mechanism is still incomplete. Metabolic compensation allows circadian oscillators to run with a constant speed at different substrate levels and, therefore, is a substantial criterion of a robust rhythm in a changing environment. Because previous data have suggested a central role of RAS2-mediated signaling in the adaptation of yeast to different nutritional environments, we examined the involvement of RAS2 in the metabolic regulation of the clock in the circadian model organism Neurospora crassa. We show that, in a ras2-deficient strain, the period is longer than in the control. Moreover, unlike in the WT, in Δras2, operation of the circadian clock was affected by glucose; compared with starvation conditions, the period was longer and the oscillation of expression of the frequency (frq) gene was dampened. In constant darkness, the delayed phosphorylation of the FRQ protein and the long-lasting accumulation of FRQ in the nucleus were in accordance with the longer period and the less robust rhythm in the mutant. Although glucose did not affect the subcellular distribution of FRQ in the WT, highly elevated FRQ levels were detected in the nucleus in Δras2. RAS2 interacted with the RAS-binding domain of the adenylate cyclase in vitro, and the cAMP analogue 8-bromo-cyclic AMP partially rescued the circadian phenotype in vivo. We therefore propose that RAS2 acts via a cAMP-dependent pathway and exerts significant metabolic control on the Neurospora circadian clock. Accumulating evidence from both experimental and clinical investigations indicates a tight interaction between metabolism and circadian timekeeping; however, knowledge of the underlying mechanism is still incomplete. Metabolic compensation allows circadian oscillators to run with a constant speed at different substrate levels and, therefore, is a substantial criterion of a robust rhythm in a changing environment. Because previous data have suggested a central role of RAS2-mediated signaling in the adaptation of yeast to different nutritional environments, we examined the involvement of RAS2 in the metabolic regulation of the clock in the circadian model organism Neurospora crassa. We show that, in a ras2-deficient strain, the period is longer than in the control. Moreover, unlike in the WT, in Δras2, operation of the circadian clock was affected by glucose; compared with starvation conditions, the period was longer and the oscillation of expression of the frequency (frq) gene was dampened. In constant darkness, the delayed phosphorylation of the FRQ protein and the long-lasting accumulation of FRQ in the nucleus were in accordance with the longer period and the less robust rhythm in the mutant. Although glucose did not affect the subcellular distribution of FRQ in the WT, highly elevated FRQ levels were detected in the nucleus in Δras2. RAS2 interacted with the RAS-binding domain of the adenylate cyclase in vitro, and the cAMP analogue 8-bromo-cyclic AMP partially rescued the circadian phenotype in vivo. We therefore propose that RAS2 acts via a cAMP-dependent pathway and exerts significant metabolic control on the Neurospora circadian clock. Circadian rhythms are endogenously generated at the cellular level. Core clock mechanisms control rhythmic expression of a large set of genes, which, in turn, regulate various biological processes, including cell growth, proliferation, and metabolism. An important feature of the circadian clock is the ability to display an endogenous rhythm with a constant period length under different environmental conditions. Among these adaptation mechanisms, the most intensively investigated process is temperature compensation. Although a tight interaction between metabolism and the circadian clock has been shown at almost all levels of organisms (1.Eckel-Mahan K. Sassone-Corsi P. Metabolism and the circadian clock converge.Physiol. Rev. 2013; 93: 107-135Crossref PubMed Scopus (363) Google Scholar, 2.Shi M. Zheng X. Interactions between the circadian clock and metabolism: there are good times and bad times.Acta Biochim. Biophys. Sin. 2013; 45: 61-69Crossref Scopus (17) Google Scholar3.Hurley J.M. Loros J.J. Dunlap J.C. The circadian system as an organizer of metabolism.Fungal Genet. Biol. 2016; 90: 39-43Crossref PubMed Scopus (32) Google Scholar), it is still poorly understood how molecular timekeeping is compensated against changes in nutrient availability. Neurospora crassa belongs to the most extensively examined model systems in the field of circadian research and has served as a useful tool for the investigation of different aspects of circadian regulation, including metabolic compensation of the circadian clock (4.Sancar G. Sancar C. Brunner M. Metabolic compensation of the Neurospora clock by a glucose-dependent feedback of the circadian repressor CSP1 on the core oscillator.Genes Dev. 2012; 26: 2435-2442Crossref PubMed Scopus (37) Google Scholar). Monitoring the conidiation rhythm, the time-dependent formation of asexual spores, allows easy study of the effect of genetic changes or pharmacologic manipulations on the output of the circadian oscillator (5.Borkovich K.A. Alex L.A. Yarden O. 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The transcription factors White Collar 1 (WC-1) 3The abbreviations used are: WC, White Collar; FRQ, frequency; WCC, White Collar complex; LL, constant light; DD, constant dark; ANOVA, analysis of variance; FGSC, Fungal Genetics Stock Center; LD, light/dark; 8-Br-cAMP, 8-bromo-cyclic AMP; TP, total protein. 3The abbreviations used are: WC, White Collar; FRQ, frequency; WCC, White Collar complex; LL, constant light; DD, constant dark; ANOVA, analysis of variance; FGSC, Fungal Genetics Stock Center; LD, light/dark; 8-Br-cAMP, 8-bromo-cyclic AMP; TP, total protein. and WC-2 and the negative factor Frequency (FRQ) represent the core clock components. Other important regulators of the molecular oscillator include kinases, phosphatases, exosome components, and factors controlling the chromatin status (e.g. Refs. 8.Cha J. Zhou M. Liu Y. 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Metabolic compensation of the Neurospora clock by a glucose-dependent feedback of the circadian repressor CSP1 on the core oscillator.Genes Dev. 2012; 26: 2435-2442Crossref PubMed Scopus (37) Google Scholar), glucose generally increases the rate of protein synthesis and, thus, translation of WC-1, the limiting subunit of the WCC. Glucose-dependent suppression of wc-1 transcription by CSP1, however, compensates for the translation elevation, resulting in glucose-independent expression of the WCC and, thus, stabilization of the period. Recently, PERIOD 1 (PRD-1), an RNA helicase, was also found to control cycle length in the presence of glucose (26.Emerson J.M. Bartholomai B.M. Ringelberg C.S. Baker S.E. Loros J.J. Dunlap J.C. period-1 encodes an ATP-dependent RNA helicase that influences nutritional compensation of the Neurospora circadian clock.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 15707-15712Crossref PubMed Scopus (16) Google Scholar, 27.Adhvaryu K. Firoozi G. Motavaze K. 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A circadian output in Drosophila mediated by neurofibromatosis-1 and Ras/MAPK.Science. 2001; 293: 2251-2256Crossref PubMed Scopus (172) Google Scholar). In Neurospora, activating mutation of RAS-1 (present in the band (bd) strain) enhances the conidiation rhythm and light-induced transcription of the positive factor WC-1 (46.Belden W.J. Larrondo L.F. Froehlich A.C. Shi M. Chen C.H. Loros J.J. Dunlap J.C. The band mutation in Neurospora crassa is a dominant allele of ras-1 implicating RAS signaling in circadian output.Genes Dev. 2007; 21: 1494-1505Crossref PubMed Scopus (129) Google Scholar). Moreover, we recently showed that the phase of conidiation is more sensitive to temperature changes in the bd strain than in the WT (47.Gyöngyösi N. Nagy D. Makara K. Ella K. Káldi K. Reactive oxygen species can modulate circadian phase and period in Neurospora crassa.Free Radic. Biol. Med. 2013; 58: 134-143Crossref PubMed Scopus (22) Google Scholar). 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Finally, it was recently shown that RAS signaling affects the circadian clock in the suprachiasmatic nucleus of mice, acting, at least partially, via ERK and glycogen synthase kinase, thereby fine-tuning the circadian period (54.Serchov T. Jilg A. Wolf C.T. Radtke I. Stehle J.H. Heumann R. Ras activity oscillates in the mouse suprachiasmatic nucleus and modulates circadian clock dynamics.Mol. Neurobiol. 2016; 53: 1843-1855Crossref PubMed Scopus (11) Google Scholar). Our goal in this study was to investigate whether RAS2 is involved in metabolic compensation of the circadian clock of N. crassa. Our results indicate that stability of the molecular clock function against changes in glucose availability is dependent on RAS2. We show that both phosphorylation and subcellular localization of FRQ are dependent on RAS2. We propose that RAS2, acting via a cAMP-dependent pathway, links the molecular oscillator to the glucose-sensing pathway and therefore is an important component of the nutritional compensation of the circadian period. To study the possible interplay between the RAS2-mediated signal transduction pathway and the circadian clock, we aimed to examine the circadian behavior of the ras2 deletion strain. Δras2 (FGSC 12467) was generated during the Neurospora Genome Project (55.Colot H.V. Park G. Turner G.E. Ringelberg C. Crew C.M. Litvinkova L. Weiss R.L. Borkovich K.A. Dunlap J.C. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10352-10357Crossref PubMed Scopus (860) Google Scholar) and had a phenotype similar to that of the smco7 mutant described earlier (56.Kana-uchi A. Yamashiro C.T. Tanabe S. Murayama T. A ras homologue of Neurospora crassa regulates morphology.Mol. Gen. Genet. 1997; 254: 427-432Crossref PubMed Scopus (51) Google Scholar). On agar slants, Δras2 formed less aerial hyphae than the WT and, accordingly, produced relatively few conidia, located primarily in a crescent form at the upper top of the medium (Fig. 1A). When inoculated in liquid medium, Δras2 grew slowly compared with the WT and produced mycelia dominantly in colonial form (small balls) instead of mycelial mats (supplemental Fig. S1A). To confirm that deletion of ras2 is responsible for the morphological defects of the mutant, we generated a strain expressing a tagged form of RAS2 under the control of the cpc-1 promoter in the Δras2 background. Comparable ras2 transcript levels were detected in Δras2, cpc-1-ras2 and the WT, and an anti-FLAG antibody recognized a protein band with the expected molecular mass (29.5 kDa) in the total cell lysates of Δras2, cpc-1-ras2 but detected no signal in the lysates prepared from either Δras2 or WT cells (Fig. 1B). Expression of RAS2FLAG rescued the morphological defects observed in the mutant, indicating that the fusion protein is functionally active and that the altered morphology was a consequence of ras2 deficiency (Fig. 1A and supplemental Fig. S1A). To analyze the conidiation rhythm of Δras2, race tube assays were performed under constant conditions (Fig. 1C). Similar to the smco7 mutant (56.Kana-uchi A. Yamashiro C.T. Tanabe S. Murayama T. A ras homologue of Neurospora crassa regulates morphology.Mol. Gen. Genet. 1997; 254: 427-432Crossref PubMed Scopus (51) Google Scholar), Δras2 had a reduced growth rate compared with the WT. In accordance with literature data (7.Gyöngyösi N. Káldi K. Interconnections of reactive oxygen species homeostasis and circadian rhythm in Neurospora crassa.Antioxid. Redox. Signal. 2014; 20: 3007-3023Crossref PubMed Scopus (25) Google Scholar, 46.Belden W.J. Larrondo L.F. Froehlich A.C. Shi M. Chen C.H. Loros J.J. Dunlap J.C. The band mutation in Neurospora crassa is a dominant allele of ras-1 implicating RAS signaling in circadian output.Genes Dev. 2007; 21: 1494-1505Crossref PubMed Scopus (129) Google Scholar), WT Neurospora did not display a conidiation rhythm on minimal medium but showed sustained banding when the reactive oxygen species generator menadione was present. In Δras2, however, no conidiation rhythm was detected, even when high concentrations (100 μm) of menadione were applied, suggesting that deletion of ras2 affects the rhythmic output. Because entrained conditions generally support conidiation rhythm (47.Gyöngyösi N. Nagy D. Makara K. Ella K. Káldi K. Reactive oxygen species can modulate circadian phase and period in Neurospora crassa.Free Radic. Biol. Med. 2013; 58: 134-143Crossref PubMed Scopus (22) Google Scholar), we incubated race tube cultures in 12/12-h light/dark cycles. Under these conditions, Δras2 also displayed sustained banding for several days (supplemental Fig. S1B). However, when the phase of conidiation was thoroughly analyzed and compared with that of the WT, a significant delay was observed in Δras2, suggesting that RAS2 activity also affects clock function under entrained conditions (Fig. 1D). In Δras2, cpc-1-ras2, the phase of banding was similar to the phase detected in the WT, indicating that expression of RAS2FLAG in the mutant background results in rescue of the clock function. To further analyze the effect of ras2 deficiency on clock function, we generated a Δras2 strain that expressed luciferase under the control of the frq promoter. In the first experiments, we used a medium containing no glucose and followed the rhythm of frq promoter activity in constant darkness (Fig. 2A, top panel). Although a robust rhythm was detected in both the Δras2 and the WT background, the circadian period was significantly longer in the mutant than in the control strain, suggesting that RAS2-mediated signaling interacts with the molecular clock (Fig. 2B). As expected from earlier data (4.Sancar G. Sancar C. Brunner M. Metabolic compensation of the Neurospora clock by a glucose-dependent feedback of the circadian repressor CSP1 on the core oscillator.Genes Dev. 2012; 26: 2435-2442Crossref PubMed Scopus (37) Google Scholar), addition of glucose to the medium did not affect either the robustness or period of the rhythm in the WT (Fig. 2, A, bottom panel, and B). In the ras2-deficient strain, however, a dampening in the amplitude was observed, and the period was more than 2 h longer compared with the WT. These data indicate that compensation of the oscillator function against glucose requires the action of RAS2. Next, we examined whether expression of ras2 is controlled by the circadian clock in the WT. As shown in Fig. 3, although frq levels displayed rhythmic changes in our samples, ras2 mRNA did not oscillate, indicating that ras2 is not a clock-controlled gene in Neurospora. In the
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