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Role of Deleted in Breast Cancer 1 (DBC1) Protein in SIRT1 Deacetylase Activation Induced by Protein Kinase A and AMP-activated Protein Kinase*

  • Veronica Nin
    Footnotes
    Affiliations
    From the Department of Anesthesiology and Kogod Aging Center, Mayo Clinic, Rochester, Minnesota 55905
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  • Carlos Escande
    Footnotes
    Affiliations
    From the Department of Anesthesiology and Kogod Aging Center, Mayo Clinic, Rochester, Minnesota 55905
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  • Claudia C. Chini
    Affiliations
    From the Department of Anesthesiology and Kogod Aging Center, Mayo Clinic, Rochester, Minnesota 55905
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  • Shailendra Giri
    Affiliations
    Department of Experimental Pathology, and Mayo Clinic, Rochester, Minnesota 55905
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  • Juliana Camacho-Pereira
    Footnotes
    Affiliations
    From the Department of Anesthesiology and Kogod Aging Center, Mayo Clinic, Rochester, Minnesota 55905
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  • Jonathan Matalonga
    Affiliations
    From the Department of Anesthesiology and Kogod Aging Center, Mayo Clinic, Rochester, Minnesota 55905
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  • Zhenkun Lou
    Affiliations
    Division of Oncology Research, Department of Oncology, Mayo Clinic, Rochester, Minnesota 55905
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  • Eduardo N. Chini
    Correspondence
    To whom correspondence should be addressed: Laboratory of signal transduction, Department of Anesthesiology and Kogod Aging Center, Mayo Clinic, Rochester, MN, 55905. Tel.: 507-255-0992; Fax: 507-255-7300;
    Affiliations
    From the Department of Anesthesiology and Kogod Aging Center, Mayo Clinic, Rochester, Minnesota 55905
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant DK-084055 from the NIDDK. This work was also supported by grants from the American Federation of Aging Research and the Mayo Foundation and by the Strickland Career Development Award.
    This article contains supplemental Figs. S1–S5.
    1 Both authors contributed equally to this work.
    2 Author contribution to this manuscript is part of Ph.D. research.
    3 Supported by American Heart Association Postdoctoral Fellowship Award 11POST7320060.
    4 Supported by Fundaçao de Amparo À Pesquisa do Estado do Rio de Janeiro, Brazil.
Open AccessPublished:May 02, 2012DOI:https://doi.org/10.1074/jbc.M112.365874
      The NAD+-dependent deacetylase SIRT1 is a key regulator of several aspects of metabolism and aging. SIRT1 activation is beneficial for several human diseases, including metabolic syndrome, diabetes, obesity, liver steatosis, and Alzheimer disease. We have recently shown that the protein deleted in breast cancer 1 (DBC1) is a key regulator of SIRT1 activity in vivo. Furthermore, SIRT1 and DBC1 form a dynamic complex that is regulated by the energetic state of the organism. Understanding how the interaction between SIRT1 and DBC1 is regulated is therefore essential to design strategies aimed to activate SIRT1. Here, we investigated which pathways can lead to the dissociation of SIRT1 and DBC1 and consequently to SIRT1 activation. We observed that PKA activation leads to a fast and transient activation of SIRT1 that is DBC1-dependent. In fact, an increase in cAMP/PKA activity resulted in the dissociation of SIRT1 and DBC1 in an AMP-activated protein kinase (AMPK)-dependent manner. Pharmacological AMPK activation led to SIRT1 activation by a DBC1-dependent mechanism. Indeed, we found that AMPK activators promote SIRT1-DBC1 dissociation in cells, resulting in an increase in SIRT1 activity. In addition, we observed that the SIRT1 activation promoted by PKA and AMPK occurs without changes in the intracellular levels of NAD+. We propose that PKA and AMPK can acutely activate SIRT1 by inducing dissociation of SIRT1 from its endogenous inhibitor DBC1. Our experiments provide new insight on the in vivo mechanism of SIRT1 regulation and a new avenue for the development of pharmacological SIRT1 activators targeted at the dissociation of the SIRT1-DBC1 complex.
      Background: DBC1 is a key regulator of SIRT1 activity, although it is unknown how the SIRT1-DBC1 interaction is regulated.
      Results: PKA and AMPK activate SIRT1 by disrupting the interaction between SIRT1 and DBC1.
      Conclusion: We provide mechanistic evidence on how the SIRT1-DBC1 complex is regulated.
      Significance: The SIRT1-DBC1 complex constitutes a target for the development of drugs to activate SIRT1.

      Introduction

      SIRT1 is an NAD+-dependent deacetylase that regulates gene expression and protein function by deacetylating lysine residues in proteins. It has been shown to regulate many aspects of cell and tissue metabolism, including liver gluconeogenesis (
      • Liu Y.
      • Dentin R.
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      • Cole P.
      • Yates 3rd, J.
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      • Guarente L.
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      A fasting inducible switch modulates gluconeogenesis via activator/coactivator exchange.
      ,
      • Rodgers J.T.
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      • Haas W.
      • Gygi S.P.
      • Spiegelman B.M.
      • Puigserver P.
      Nutrient control of glucose homeostasis through a complex of PGC-1α and SIRT1.
      ), insulin secretion (
      • Bordone L.
      • Motta M.C.
      • Picard F.
      • Robinson A.
      • Jhala U.S.
      • Apfeld J.
      • McDonagh T.
      • Lemieux M.
      • McBurney M.
      • Szilvasi A.
      • Easlon E.J.
      • Lin S.J.
      • Guarente L.
      Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic β cells.
      ,
      • Moynihan K.A.
      • Grimm A.A.
      • Plueger M.M.
      • Bernal-Mizrachi E.
      • Ford E.
      • Cras-Mneur C.
      • Permutt M.A.
      • Imai S.
      Increased dosage of mammalian Sir2 in pancreatic β cells enhances glucose-stimulated insulin secretion in mice.
      ,
      • Schenk S.
      • McCurdy C.E.
      • Philp A.
      • Chen M.Z.
      • Holliday M.J.
      • Bandyopadhyay G.K.
      • Osborn O.
      • Baar K.
      • Olefsky J.M.
      Sirt1 enhances skeletal muscle insulin sensitivity in mice during caloric restriction.
      ), insulin sensitivity (
      • Schenk S.
      • McCurdy C.E.
      • Philp A.
      • Chen M.Z.
      • Holliday M.J.
      • Bandyopadhyay G.K.
      • Osborn O.
      • Baar K.
      • Olefsky J.M.
      Sirt1 enhances skeletal muscle insulin sensitivity in mice during caloric restriction.
      ), fatty acid oxidation (
      • Gerhart-Hines Z.
      • Rodgers J.T.
      • Bare O.
      • Lerin C.
      • Kim S.H.
      • Mostoslavsky R.
      • Alt F.W.
      • Wu Z.
      • Puigserver P.
      Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1α.
      ), and adipogenesis (
      • Picard F.
      • Kurtev M.
      • Chung N.
      • Topark-Ngarm A.
      • Senawong T.
      • Machado De Oliveira R.
      • Leid M.
      • McBurney M.W.
      • Guarente L.
      Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-γ.
      ). Although the literature regarding the physiological processes regulated by SIRT1 is vast, our knowledge about how this key enzyme is regulated in the cellular context is scarce. In this regard, several possible regulatory mechanisms have been described.
      One of the proposed mechanisms of SIRT1 regulation involves alterations in the intracellular concentration of NAD+. Because SIRT1 enzymatic activity is dependent on NAD+ (
      • Imai S.
      • Armstrong C.M.
      • Kaeberlein M.
      • Guarente L.
      Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase.
      ), changes in the concentration of this nucleotide can lead to changes in SIRT1 activity. Indeed, modification of the two main enzymes responsible for the control of intracellular NAD+ levels, namely NamPT (
      • Revollo J.R.
      • Grimm A.A.
      • Imai S.
      The regulation of nicotinamide adenine dinucleotide biosynthesis by Nampt/PBEF/visfatin in mammals.
      ) and CD38 (
      • Aksoy P.
      • White T.A.
      • Thompson M.
      • Chini E.N.
      Regulation of intracellular levels of NAD: a novel role for CD38.
      ,
      • Barbosa M.T.
      • Soares S.M.
      • Novak C.M.
      • Sinclair D.
      • Levine J.A.
      • Aksoy P.
      • Chini E.N.
      The enzyme CD38 (a NAD glycohydrolase, EC 3.2.2.5) is necessary for the development of diet-induced obesity.
      ,
      • Chini E.N.
      CD38 as a regulator of cellular NAD: a novel potential pharmacological target for metabolic conditions.
      ), can lead to changes in SIRT1 activity (
      • Barbosa M.T.
      • Soares S.M.
      • Novak C.M.
      • Sinclair D.
      • Levine J.A.
      • Aksoy P.
      • Chini E.N.
      The enzyme CD38 (a NAD glycohydrolase, EC 3.2.2.5) is necessary for the development of diet-induced obesity.
      ,
      • Revollo J.R.
      • Grimm A.A.
      • Imai S.
      The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells.
      ,
      • Aksoy P.
      • Escande C.
      • White T.A.
      • Thompson M.
      • Soares S.
      • Benech J.C.
      • Chini E.N.
      Regulation of SIRT 1 mediated NAD dependent deacetylation: a novel role for the multifunctional enzyme CD38.
      ). However, the specificity of this mechanism seems low as there are several other NAD+-consuming enzymes in the cell. Moreover, it remains unknown whether global changes in NAD+ are reflected by similar changes in the nuclei where most SIRT1 is localized.
      Several authors have shown that SIRT1 can be regulated at the transcriptional level (
      • Picard F.
      • Kurtev M.
      • Chung N.
      • Topark-Ngarm A.
      • Senawong T.
      • Machado De Oliveira R.
      • Leid M.
      • McBurney M.W.
      • Guarente L.
      Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-γ.
      ,
      • Chen D.
      • Bruno J.
      • Easlon E.
      • Lin S.J.
      • Cheng H.L.
      • Alt F.W.
      • Guarente L.
      Tissue-specific regulation of SIRT1 by calorie restriction.
      ,
      • Haigis M.C.
      • Sinclair D.A.
      Mammalian sirtuins: biological insights and disease relevance.
      ). Although this mechanism could certainly explain long term changes in SIRT1 activity, it does not account for transient changes in its activity. Several post-transcriptional modifications can also affect SIRT1 activity. In this regard, it has been described that SUMOylation (
      • Yang Y.
      • Fu W.
      • Chen J.
      • Olashaw N.
      • Zhang X.
      • Nicosia S.V.
      • Bhalla K.
      • Bai W.
      SIRT1 sumoylation regulates its deacetylase activity and cellular response to genotoxic stress.
      ) and phosphorylation by several kinases (
      • Gao Z.
      • Zhang J.
      • Kheterpal I.
      • Kennedy N.
      • Davis R.J.
      • Ye J.
      Sirtuin 1 (SIRT1) protein degradation in response to persistent c-Jun N-terminal kinase 1 (JNK1) activation contributes to hepatic steatosis in obesity.
      ,
      • Gerhart-Hines Z.
      • Dominy Jr., J.E.
      • Blttler S.M.
      • Jedrychowski M.P.
      • Banks A.S.
      • Lim J.H.
      • Chim H.
      • Gygi S.P.
      • Puigserver P.
      The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD+.
      ,
      • Guo X.
      • Williams J.G.
      • Schug T.T.
      • Li X.
      DYRK1A and DYRK3 promote cell survival through phosphorylation and activation of SIRT1.
      ,
      • Nasrin N.
      • Kaushik V.K.
      • Fortier E.
      • Wall D.
      • Pearson K.J.
      • de Cabo R.
      • Bordone L.
      JNK1 phosphorylates SIRT1 and promotes its enzymatic activity.
      ,
      • Sasaki T.
      • Maier B.
      • Koclega K.D.
      • Chruszcz M.
      • Gluba W.
      • Stukenberg P.T.
      • Minor W.
      • Scrable H.
      Phosphorylation regulates SIRT1 function.
      ,
      • Zschoernig B.
      • Mahlknecht U.
      Carboxy-terminal phosphorylation of SIRT1 by protein kinase CK2.
      ) can increase SIRT1 activity. The kinases cyclin-dependent kinase 1 (
      • Sasaki T.
      • Maier B.
      • Koclega K.D.
      • Chruszcz M.
      • Gluba W.
      • Stukenberg P.T.
      • Minor W.
      • Scrable H.
      Phosphorylation regulates SIRT1 function.
      ), casein kinase, (
      • Zschoernig B.
      • Mahlknecht U.
      Carboxy-terminal phosphorylation of SIRT1 by protein kinase CK2.
      ,
      • Kang H.
      • Jung J.W.
      • Kim M.K.
      • Chung J.H.
      CK2 is the regulator of SIRT1 substrate-binding affinity, deacetylase activity and cellular response to DNA damage.
      ), and the c-Jun N-terminal kinase (JNK) (
      • Nasrin N.
      • Kaushik V.K.
      • Fortier E.
      • Wall D.
      • Pearson K.J.
      • de Cabo R.
      • Bordone L.
      JNK1 phosphorylates SIRT1 and promotes its enzymatic activity.
      ) have been shown to directly phosphorylate SIRT1. On the other hand, it has been reported that the cAMP-dependent protein kinase (PKA) activates SIRT1 indirectly (
      • Gerhart-Hines Z.
      • Dominy Jr., J.E.
      • Blttler S.M.
      • Jedrychowski M.P.
      • Banks A.S.
      • Lim J.H.
      • Chim H.
      • Gygi S.P.
      • Puigserver P.
      The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD+.
      ), the effects being mediated by an unidentified kinase. In addition, it has been proposed that AMP-dependent protein kinase (AMPK)
      The abbreviations used are: AMPK
      AMP-activated protein kinase
      DBC1
      deleted in breast cancer 1
      AICAR
      5-amino-1-β-d-ribofuranosylimidazole-4-carboxamide
      6-MB-cAMP
      N6-monobutytyl-cAMP
      EPAC
      exchange protein activated by cAMP
      cpt-cAMP
      8-(4-chlorophenylthio)-adenosine 3,5′-cycle monophosphate-cAMP
      MEF
      mouse embryonic fibroblast
      CA
      constitutively active
      ANOVA
      analysis of variance.
      modulates NAD+ intracellular levels and consequently SIRT1 activity (
      • Cant C.
      • Gerhart-Hines Z.
      • Feige J.N.
      • Lagouge M.
      • Noriega L.
      • Milne J.C.
      • Elliott P.J.
      • Puigserver P.
      • Auwerx J.
      AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity.
      ). Interestingly, some of the effects of PKA appear to be mediated by AMPK (
      • Yin W.
      • Mu J.
      • Birnbaum M.J.
      Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis in 3T3-L1 adipocytes.
      ,
      • Wu H.M.
      • Yang Y.M.
      • Kim S.G.
      Rimonabant, a cannabinoid receptor type 1 inverse agonist, inhibits hepatocyte lipogenesis by activating liver kinase B1 and AMP-activated protein kinase axis downstream of Gαi/o inhibition.
      ). Moreover, PKA activation can lead to a fast activation of AMPK in several tissues and cell models (
      • Yin W.
      • Mu J.
      • Birnbaum M.J.
      Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis in 3T3-L1 adipocytes.
      ,
      • Kimball S.R.
      • Siegfried B.A.
      • Jefferson L.S.
      Glucagon represses signaling through the mammalian target of rapamycin in rat liver by activating AMP-activated protein kinase.
      ,
      • Williamson D.L.
      • Kubica N.
      • Kimball S.R.
      • Jefferson L.S.
      Exercise-induced alterations in extracellular signal-regulated kinase 1/2 and mammalian target of rapamycin (mTOR) signalling to regulatory mechanisms of mRNA translation in mouse muscle.
      ,
      • Djouder N.
      • Tuerk R.D.
      • Suter M.
      • Salvioni P.
      • Thali R.F.
      • Scholz R.
      • Vaahtomeri K.
      • Auchli Y.
      • Rechsteiner H.
      • Brunisholz R.A.
      • Viollet B.
      • T.P.
      • Wallimann T.
      • Neumann D.
      • Krek W.
      PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis.
      ).
      In addition, SIRT1 is regulated by protein-protein interactions. Recently, we and others demonstrated that in vivo SIRT1 is largely associated with its endogenous inhibitor deleted in breast cancer 1 (DBC1) (
      • Escande C.
      • Chini C.C.
      • Nin V.
      • Dykhouse K.M.
      • Novak C.M.
      • Levine J.
      • van Deursen J.
      • Gores G.J.
      • Chen J.
      • Lou Z.
      • Chini E.N.
      Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice.
      ,
      • Zhao W.
      • Kruse J.P.
      • Tang Y.
      • Jung S.Y.
      • Qin J.
      • Gu W.
      Negative regulation of the deacetylase SIRT1 by DBC1.
      ,
      • Kim J.E.
      • Chen J.
      • Lou Z.
      DBC1 is a negative regulator of SIRT1.
      ). DBC1 is a nuclear protein that, in addition to SIRT1, binds to several nuclear receptors and enzymes, including the estrogen receptors α (
      • Trauernicht A.M.
      • Kim S.J.
      • Kim N.H.
      • Boyer T.G.
      Modulation of estrogen receptor α protein level and survival function by DBC-1.
      ) and β (
      • Koyama S.
      • Wada-Hiraike O.
      • Nakagawa S.
      • Tanikawa M.
      • Hiraike H.
      • Miyamoto Y.
      • Sone K.
      • Oda K.
      • Fukuhara H.
      • Nakagawa K.
      • Kato S.
      • Yano T.
      • Taketani Y.
      Repression of estrogen receptor β function by putative tumor suppressor DBC1.
      ), the androgen receptor (
      • Fu J.
      • Jiang J.
      • Li J.
      • Wang S.
      • Shi G.
      • Feng Q.
      • White E.
      • Qin J.
      • Wong J.
      Deleted in breast cancer 1, a novel androgen receptor (AR) coactivator that promotes AR DNA-binding activity.
      ), the transcription factor BRCA1 (
      • Hiraike H.
      • Wada-Hiraike O.
      • Nakagawa S.
      • Koyama S.
      • Miyamoto Y.
      • Sone K.
      • Tanikawa M.
      • Tsuruga T.
      • Nagasaka K.
      • Matsumoto Y.
      • Oda K.
      • Shoji K.
      • Fukuhara H.
      • Saji S.
      • Nakagawa K.
      • Kato S.
      • Yano T.
      • Taketani Y.
      Identification of DBC1 as a transcriptional repressor for BRCA1.
      ), and the deacetylase HDAC3 (
      • Chini C.C.
      • Escande C.
      • Nin V.
      • Chini E.N.
      HDAC3 is negatively regulated by the nuclear protein DBC1.
      ).
      SIRT1 and DBC1 form a dynamic complex in cells and in vivo (
      • Escande C.
      • Chini C.C.
      • Nin V.
      • Dykhouse K.M.
      • Novak C.M.
      • Levine J.
      • van Deursen J.
      • Gores G.J.
      • Chen J.
      • Lou Z.
      • Chini E.N.
      Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice.
      ). Moreover, the binding between SIRT1 and DBC1 is regulated by the energetic state of the organism (
      • Escande C.
      • Chini C.C.
      • Nin V.
      • Dykhouse K.M.
      • Novak C.M.
      • Levine J.
      • van Deursen J.
      • Gores G.J.
      • Chen J.
      • Lou Z.
      • Chini E.N.
      Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice.
      ). So far it is unknown which are the molecular pathways that modulate the interaction between SIRT1 and DBC1 and consequently SIRT1 activity in vivo.
      Here, we show that the activation of the cAMP/PKA pathway leads to SIRT1 activation through an AMPK-dependent mechanism. Furthermore, this activation is DBC1-dependent and involves dissociation of the SIRT1-DBC1 complex. We propose that AMPK activation, either pharmacological or induced by PKA, results in the dissociation of SIRT1 from DBC1 and activation of SIRT1. Our results provide insight into the mechanisms that regulate the interaction between SIRT1 and DBC1 and may lead to newer pharmacological approaches to activate SIRT1.

      DISCUSSION

      The beneficial effects of SIRT1 activation have been studied extensively (
      • Guarente L.
      Franklin H. Epstein Lecture: sirtuins, aging, and medicine.
      ). Mounting evidence from independent groups shows that SIRT1 activation leads to protection against metabolic syndrome, cardiovascular diseases, and cancer (
      • Guarente L.
      Franklin H. Epstein Lecture: sirtuins, aging, and medicine.
      ) among other diseases. However, a much more complex issue is how to achieve SIRT1 activation in vivo. We have recently shown that, in vivo, most SIRT1 is bound to its regulator DBC1 and that this interaction is dynamic and can be displaced (
      • Escande C.
      • Chini C.C.
      • Nin V.
      • Dykhouse K.M.
      • Novak C.M.
      • Levine J.
      • van Deursen J.
      • Gores G.J.
      • Chen J.
      • Lou Z.
      • Chini E.N.
      Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice.
      ). In fact, we also showed that when DBC1 is absent and therefore SIRT1 is more active mice are protected against metabolic syndrome (
      • Escande C.
      • Chini C.C.
      • Nin V.
      • Dykhouse K.M.
      • Novak C.M.
      • Levine J.
      • van Deursen J.
      • Gores G.J.
      • Chen J.
      • Lou Z.
      • Chini E.N.
      Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice.
      ). It therefore becomes of key importance to understand how the SIRT1-DBC1 complex is regulated because it may lead to the development of new strategies to activate SIRT1 pharmacologically.
      In this work, we attempted to characterize in detail the mechanism involved in the regulation of the interaction between SIRT1 and DBC1 and therefore SIRT1 activity. We found that SIRT1 activity is positively regulated by the protein kinases PKA and AMPK. We showed that PKA activation leads to a fast and transient SIRT1 activation. This activation was AMPK-dependent, involved the dissociation of SIRT1 from DBC1, and occurred independently of changes in NAD+ levels.
      While this article was in preparation, Gerhart-Hines et al. (
      • Gerhart-Hines Z.
      • Dominy Jr., J.E.
      • Blttler S.M.
      • Jedrychowski M.P.
      • Banks A.S.
      • Lim J.H.
      • Chim H.
      • Gygi S.P.
      • Puigserver P.
      The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD+.
      ) also showed SIRT1 activation by PKA. These authors propose that SIRT1 activation by PKA involves phosphorylation of SIRT1 and changes in the affinity of SIRT1 for NAD+. Although our findings do not exclude this possibility, we showed that SIRT1 must dissociate from DBC1 to be activated by PKA. Also, a recent paper shows that the polyphenol resveratrol induces cellular SIRT1 activation via activation of the cAMP/EPAC pathway and AMPK (
      • Park S.J.
      • Ahmad F.
      • Philp A.
      • Baar K.
      • Williams T.
      • Luo H.
      • Ke H.
      • Rehmann H.
      • Taussig R.
      • Brown A.L.
      • Kim M.K.
      • Beaven M.A.
      • Burgin A.B.
      • Manganiello V.
      • Chung J.H.
      Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases.
      ). Therefore, our group and two independent groups have shown that an increase in cAMP leads to SIRT1 activation. Our study provides further information about the specific mechanism of SIRT1 activation, which is mediated by modulation of the SIRT1-DBC1 interaction.
      Recent publications, including this one, have reported changes in SIRT1 activity that are independent of alterations in the concentration of NAD+ (
      • Gerhart-Hines Z.
      • Dominy Jr., J.E.
      • Blttler S.M.
      • Jedrychowski M.P.
      • Banks A.S.
      • Lim J.H.
      • Chim H.
      • Gygi S.P.
      • Puigserver P.
      The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD+.
      ,
      • Escande C.
      • Chini C.C.
      • Nin V.
      • Dykhouse K.M.
      • Novak C.M.
      • Levine J.
      • van Deursen J.
      • Gores G.J.
      • Chen J.
      • Lou Z.
      • Chini E.N.
      Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice.
      ). Since our first observation of an increase in SIRT1 activity in vivo without detectable changes in NAD+ concentration (
      • Escande C.
      • Chini C.C.
      • Nin V.
      • Dykhouse K.M.
      • Novak C.M.
      • Levine J.
      • van Deursen J.
      • Gores G.J.
      • Chen J.
      • Lou Z.
      • Chini E.N.
      Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice.
      ), evidence has accumulated to prove that it is possible to induce SIRT1 activation without detectable changes in intracellular NAD+ levels (
      • Gerhart-Hines Z.
      • Dominy Jr., J.E.
      • Blttler S.M.
      • Jedrychowski M.P.
      • Banks A.S.
      • Lim J.H.
      • Chim H.
      • Gygi S.P.
      • Puigserver P.
      The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD+.
      ). During physiological processes, it may be necessary to activate SIRT1 and not other NAD+-consuming enzymes. However, an increase in the cytoplasmic and nuclear levels of NAD+ could result in higher activity of several sirtuins and the poly(ADP-ribosyl) polymerases. Therefore, one can foresee that there must be an alternative, specific mode of SIRT1 regulation.
      The results presented here provide mechanistic insight into the PKA-induced SIRT1 activation. Our results point to AMPK as a kinase that is downstream of PKA. In support of this notion, we observed that forskolin induces a transient activation of AMPK that parallels the increase in SIRT1 activity, that the AMPK inhibitor compound C abolishes the effect of forskolin in SIRT1 activity, and that in AMPK KO MEFs SIRT1 is insensitive to cAMP elevations. In line with our observations, others have also reported that PKA can transiently activate AMPK (
      • Yin W.
      • Mu J.
      • Birnbaum M.J.
      Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis in 3T3-L1 adipocytes.
      ,
      • Wu H.M.
      • Yang Y.M.
      • Kim S.G.
      Rimonabant, a cannabinoid receptor type 1 inverse agonist, inhibits hepatocyte lipogenesis by activating liver kinase B1 and AMP-activated protein kinase axis downstream of Gαi/o inhibition.
      ,
      • Kimball S.R.
      • Siegfried B.A.
      • Jefferson L.S.
      Glucagon represses signaling through the mammalian target of rapamycin in rat liver by activating AMP-activated protein kinase.
      ,
      • Williamson D.L.
      • Kubica N.
      • Kimball S.R.
      • Jefferson L.S.
      Exercise-induced alterations in extracellular signal-regulated kinase 1/2 and mammalian target of rapamycin (mTOR) signalling to regulatory mechanisms of mRNA translation in mouse muscle.
      ,
      • Djouder N.
      • Tuerk R.D.
      • Suter M.
      • Salvioni P.
      • Thali R.F.
      • Scholz R.
      • Vaahtomeri K.
      • Auchli Y.
      • Rechsteiner H.
      • Brunisholz R.A.
      • Viollet B.
      • T.P.
      • Wallimann T.
      • Neumann D.
      • Krek W.
      PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis.
      ,
      • Collins S.P.
      • Reoma J.L.
      • Gamm D.M.
      • Uhler M.D.
      LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo.
      ). This activation seems to occur through the kinase LKB1, which activates AMPK by phosphorylation of serine 172 (
      • Hardie D.G.
      New roles for the LKB1→AMPK pathway.
      ). PKA phosphorylates LKB1 directly at serine 431 (
      • Collins S.P.
      • Reoma J.L.
      • Gamm D.M.
      • Uhler M.D.
      LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo.
      ,
      • Sapkota G.P.
      • Kieloch A.
      • Lizcano J.M.
      • Lain S.
      • Arthur J.S.
      • Williams M.R.
      • Morrice N.
      • Deak M.
      • Alessi D.R.
      Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90RSK and cAMP-dependent protein kinase, but not its farnesylation at Cys433, is essential for LKB1 to suppress cell growth.
      ), and phosphorylation at this site is needed for some of the LKB1 functions (
      • Sapkota G.P.
      • Kieloch A.
      • Lizcano J.M.
      • Lain S.
      • Arthur J.S.
      • Williams M.R.
      • Morrice N.
      • Deak M.
      • Alessi D.R.
      Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90RSK and cAMP-dependent protein kinase, but not its farnesylation at Cys433, is essential for LKB1 to suppress cell growth.
      ,
      • Shelly M.
      • Cancedda L.
      • Heilshorn S.
      • Sumbre G.
      • Poo M.M.
      LKB1/STRAD promotes axon initiation during neuronal polarization.
      ). Another possible mechanism that may lead to AMPK activation by the cAMP/PKA pathway is through regulation of cAMP degradation. Once cAMP levels increase in cells, the level of this second messenger quickly returns to basal levels due to its degradation by phosphodiesterases. Phosphodiesterases degrade cAMP into 5′-AMP (
      • Lugnier C.
      Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents.
      ), and therefore its action could result in a fast accumulation of AMP. Therefore, it seems plausible that agents that activate the cAMP/PKA pathway could result in activation of AMPK either by activation of LKB1, an increase in AMP levels, or both.
      In this work, we present evidence that SIRT1 is phosphorylated upon AMPK activation. However, it remains unknown which kinase is responsible for SIRT1 phosphorylation upon PKA and AMPK activation. Our data identified serines 47, 605, and 615 as key residues involved in the regulation of the interaction between SIRT1 and DBC1. We provide evidence of phosphorylation of serine 47, but it remains to be elucidated whether serines 605 and 615 are also phosphorylated when PKA and AMPK are activated. As mentioned before, two independent groups failed to detect SIRT1 phosphorylation by AMPK (
      • Cant C.
      • Gerhart-Hines Z.
      • Feige J.N.
      • Lagouge M.
      • Noriega L.
      • Milne J.C.
      • Elliott P.J.
      • Puigserver P.
      • Auwerx J.
      AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity.
      ,
      • Greer E.L.
      • Oskoui P.R.
      • Banko M.R.
      • Maniar J.M.
      • Gygi M.P.
      • Gygi S.P.
      • Brunet A.
      The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor.
      ), which suggests that the regulation of this process is extremely complex. However, sequence analysis of human SIRT1 suggests that SIRT1 could be a target for AMPK phosphorylation. The consensus sequence for AMPK phosphorylation as first proposed by Carling and Hardie (
      • Carling D.
      • Hardie D.G.
      The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase.
      ) consists of an arginine in the −2, −3, or −4 position and a hydrophobic residue at −1 relative to the serine or threonine target of phosphorylation. Later on, a study by Dale et al. (
      • Dale S.
      • Wilson W.A.
      • Edelman A.M.
      • Hardie D.G.
      Similar substrate recognition motifs for mammalian AMP-activated protein kinase, higher plant HMG-CoA reductase kinase-A, yeast SNF1, and mammalian calmodulin-dependent protein kinase I.
      ) described the importance of a leucine at positions −5 and +4 (although other hydrophobic amino acids are accepted too) besides the basic amino acid at −3 or −4. More recently, another study identified that AMPK favors a serine with a valine or arginine at −2 (
      • Gwinn D.M.
      • Shackelford D.B.
      • Egan D.F.
      • Mihaylova M.M.
      • Mery A.
      • Vasquez D.S.
      • Turk B.E.
      • Shaw R.J.
      AMPK phosphorylation of raptor mediates a metabolic checkpoint.
      ). However, it is also important to note that not all known substrates for AMPK have the perfect consensus sequence but a variation of it. FOXO3 for example is phosphorylated by AMPK at Ser-413, which is not flanked by a leucine at position −5 or +4, and Ser-588 that lacks a basic residue at −3 or −4 (
      • Greer E.L.
      • Oskoui P.R.
      • Banko M.R.
      • Maniar J.M.
      • Gygi M.P.
      • Gygi S.P.
      • Brunet A.
      The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor.
      ). Using the above data as a reference, we searched the human SIRT1 sequence for serines and threonines in potential AMPK consensus sequence. Interestingly, the serine residue in position 605, one of the sites that is involved in SIRT1 activation by PKA, is flanked by a lysine at −4, a leucine at −5, and a valine at −2 in addition to a hydrophobic amino acid at −1, which is very close to the optimal sequence for AMPK phosphorylation. This fact suggests that further effort should be made to determine whether SIRT1 is a direct target for AMPK.
      Of interest is that Kang et al. (
      • Kang H.
      • Suh J.Y.
      • Jung Y.S.
      • Jung J.W.
      • Kim M.K.
      • Chung J.H.
      Peptide switch is essential for Sirt1 deacetylase activity.
      ) recently found that the amino acids 631–655 in SIRT1 (what they call the essential for SIRT1 activity region) are necessary for SIRT1 activation. Moreover, these authors proposed that this region is important for the regulation of SIRT1 by DBC1. Interestingly, two of the amino acids, 605 and 615, which are involved in the PKA/AMPK-induced activation of SIRT1, are in close proximity to this region. Therefore, amino acids 605 and 615 may play a role in the activation of SIRT1 by PKA and AMPK by regulating the essential for SIRT1 activity region of SIRT1.
      Serine 47 in SIRT1 has been shown to be target of JNK1 (
      • Nasrin N.
      • Kaushik V.K.
      • Fortier E.
      • Wall D.
      • Pearson K.J.
      • de Cabo R.
      • Bordone L.
      JNK1 phosphorylates SIRT1 and promotes its enzymatic activity.
      ). Furthermore, Gao et al. (
      • Gao Z.
      • Zhang J.
      • Kheterpal I.
      • Kennedy N.
      • Davis R.J.
      • Ye J.
      Sirtuin 1 (SIRT1) protein degradation in response to persistent c-Jun N-terminal kinase 1 (JNK1) activation contributes to hepatic steatosis in obesity.
      ) recently showed that JNK1 leads to SIRT1 phosphorylation and a fast increase in SIRT1 activity upon glucose treatment that correlates very well with the time course that we observed for PKA-dependent SIRT1 activation. It could be that AMPK and JNK1 are acting in conjunction to promote SIRT1 dissociation form DBC1.
      Finally, we want to highlight how important it is to understand how the interaction between SIRT1 and DBC1 is regulated to study the regulation of SIRT1. The fact that in the absence of DBC1 the cAMP/PKA and AMPK pathways are incapable of activating SIRT1 suggests that a possible phosphorylation of SIRT1 per se is not enough to sustain an increase in its activity. Furthermore, our observations place the SIRT1-DBC1 complex as a key physiological target for SIRT1 regulation.
      In summary, our results provide a novel mechanism of SIRT1 activation by a cAMP/PKA/AMPK/DBC1-dependent pathway. It also provides the evidence that the interaction between SIRT1 and DBC1 can be regulated by endogenous cell signaling pathways and opens the possibility that other signals may also promote SIRT1 activation through the dissociation of the SIRT1-DBC1 complex. For instance, it was recently shown that the ataxia telangiectasia mutated kinase (ATM) phosphorylates DBC1 and increases the interaction between SIRT1 and DBC1 (
      • Yuan J.
      • Luo K.
      • Liu T.
      • Lou Z.
      Regulation of SIRT1 activity by genotoxic stress.
      ). Finally, mounting evidence indicates that modulation of SIRT1 activity can be achieved without the nonspecific changes in global cellular NAD+ levels. The understanding of specific mechanisms of SIRT1 activation as described here may provide a clearer picture about the regulation of cellular SIRT1 and its physiological consequences.

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