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cAMP response element-binding protein (CREB) is importedinto mitochondria and promotes protein synthesisDomenico De Rasmo1, Anna Signorile1, Emilio Roca1and Sergio Papa1,21 Department of Medical Biochemistry, Biology and Physics (DIBIFIM), University of Bari, Italy2 Institute of Biomembranes and Bioenergetics (IBBE), Consiglio Nazionale delle Ricerche, Bari, ItalyIntroductionThe cAMP response element-binding protein (CREB)is a ubiquitous transcription factor in the highereukaryotes that recognizes the DNA consensussequence TGACGTCA, the cAMP response element(CRE) in gene promoters [1,2].Phosphorylation of CREB by cAMP-dependent pro-tein kinase (protein kinase A; PKA), as well as byCa2+-dependent and other protein kinases, inresponse to different cellular signals, promotes tran-scription of CRE-regulated genes [1–4]. Activation ofthe expression of nuclear CRE-regulated genes hasbeen shown to be involved in a variety of cellular pro-cesses, including apoptosis [5,6], oxidative stress [7],neuronal growth, and plasticity [5,8]. In yeast, cAMPwas found to reverse the glucose repression of mito-chondriogenesis [9] and to activate the expression ofmitochondrial genes [10,11] and nuclear genes [12,13]of respiratory chain proteins. In Saccharomyces cerere-visiae, where the RAS ⁄ cAMP ⁄ PKA system appears tobe involved in regulation of the biogenesis of theoxidative phosphorylation system [13], a probable cis-regulatory element on mtDNA, responsible for cAMP-mediated transcription, was identified [11]. In yeastand mammalian cells, the cAMP cascade is involved inthe regulation of mitochondrial dynamics [14] andbioenergetics [15–17].In 1999, findings were presented [18] indicating thatCREB is localized in the inner mitochondrial compart-ment as well as in the nucleus. These observations,based on the use of CREB and phospho-CREB anti-KeywordscAMP cascade; complex I; CREB;mitochondrial protein synthesis; PKACorrespondenceS. Papa, Department of MedicalBiochemistry, Biology and Physics,University of Bari, Policlinico,P.zza G. Cesare, 70124 Bari, ItalyFax: +39 080 5448538Tel: +39 080 5448540E-mail: papabchm@cimedoc.uniba.it(Received 9 March 2009, revised 26 May2009, accepted 4 June 2009)doi:10.1111/j.1742-4658.2009.07133.xThe cAMP response element-binding protein (CREB) is a ubiquitoustranscription factor in the higher eukaryotes that, once phosphorylated,promotes transcription of cAMP response element-regulated genes. Wehave studied the mitochondrial import of CREB and its effect on theexpression of mtDNA-encoded proteins. [35S]Methionine-labelled CREB,synthesized in vitro in the Rabbit Reticulocyte Lysate system using a con-struct of the human cDNA, was imported into the matrix of isolated ratliver mitochondria by a membrane potential and TOM complex-dependentprocess. The imported CREB caused cAMP-dependent promotion of thesynthesis of mitochondrially encoded subunits of oxidative phosphorylationenzyme complexes. Thus, CREB moves from the cytosol to mitochondria,in addition to the nucleus, and, when phosphorylated by cAMP-dependentprotein kinase, promotes the expression of mitochondrial genes.AbbreviationsADU, arbitrary densitometric units; AKAP, A kinase anchoring protein; CAP, chloramphenicol; cPKA, catalytic subunit of cAMP dependentprotein kinase; CRE, cAMP response element; CREB, cAMP response element-binding protein; db-cAMP, dibutyryl cAMP; IBMX,isobutylmethylxanthine; PKA, cAMP-dependent protein kinase (protein kinase A); RRL, Rabbit Reticulocyte Lysate.FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 4325bodies, as well as on mtDNA mobility shift assays,were confirmed by Lee et al. [19] and Ryu et al. [20].Lee et al. also showed that binding of CREB to CREsequences in the D-loop of mtDNA increased the tran-script levels of the ND2, ND5 and ND6 mitochondrialgenes of complex I of the respiratory chain. Ryu et al.[20] found that activation by the antioxidant iron che-lator deferoxamine of PKA, localized in the mitochon-drial matrix [21], promoted CREB binding to themtDNA D-loop. The presence in mitochondria of theCREB factor and, in particular, the use of phospho-CREB antibody to detect this transcription factor inmitochondria were, however, questioned by Plateniket al. [22]. These authors showed that commerciallyavailable antibodies for phospho-CREB crossreactwith the E1 a-subunit of mitochondrial pyruvate dehy-drogenase. Because of the relatively high abundance inmitochondria of pyruvate dehydrogenase, this crossre-activity might overwhelm the reaction of the antibodywith phospho-CREB and produce false-positiveresults. The general physiological relevance of a possi-ble role of CREB and PKA in providing a regulatorymechanism in the expression of mitochondriallyencoded proteins of the respiratory chain prompted usto investigate the mitochondrial import of the CREBprotein, synthesized in vitro, and its effect on theexpression of mitochondrial genes. The resultsunequivocally show that exogenous CREB is importedinto isolated rat liver mitochondria and causes amarked, cAMP-dependent, stimulation of the expres-sion of the mitochondrially encoded subunits of oxida-tive phosphorylation complexes.Results[35S]Methionine-labelled CREB is imported intoisolated mitochondriaMitochondrial import of CREB was investigated byincubating freshly isolated, intact rat liver mitochon-dria with the protein synthesized in the Rabbit Reti-culocyte Lysate (RRL) system with [35S]methionine.Figure 1A,B shows time-dependent mitochondrialuptake of the radioactive CREB, which was largelyresistant to trypsin digestion unless mitochondria weredissolved by Triton X-100 (Fig. 1C, lane 3). Theamount of mitochondrial proteins and mitochondrialintegrity were checked by immunodetection of the39 kDa subunit of the inner membrane complex I, inthe absence and in the presence of trypsin. Mitochon-drial uptake of CREB was promoted by the mitochon-drial membrane potential, as shown by its inhibitionby valinomycin (Fig. 1C, lane 4). The residual radio-active CREB detected in the mitochondrial pellet inthe presence of valinomycin represents the amount ofprotein bound at the mitochondrial outer surface, asshown by its complete digestion by trypsin (Fig. 1C,lane 5). When mitochondria were pretreated withA B C 40030020010000 10203040506070Fig. 1. Import into isolated mitochondria of [35S]methionine-labelledCREB. [35S]Methionine-labelled CREB, synthesized in the RRLtranslation system, was added to isolated rat liver mitochondria.(A, C) Autoradiograms of SDS ⁄ PAGE slabs of the mitochondrialpellet. The RRL gel slab on the left of (A) is an autoradiogram of anamount of the radioactive CREB synthesis mixture correspondingto half of the amount added to mitochondria for the import assay.No precipitable aggregate of radioactive CREB was detectable aftercentrifugation of the RRL CREB synthesis translation mixture in theabsence of added mitochondria. Where indicated, mitochondria,after completion of the import incubation, were treated, beforepelletting, with trypsin (1 lg per 50 lg of mitochondrial protein) for35 min at 0 °C. (C) Import incubation for 60 min: in lane 3, mito-chondria were treated with trypsin in the presence of 0.2% TritonX-100; in lanes 4 and 5, valinomycin (Val) (0.1 lg per mg ofmitochondrial protein) was present during the import incubation.(B) Mean values in arbitrary densitometric units (ADU) (three sepa-rate experiments) of the trypsin-resistant [35S]methionine-labelledCREB radioactivity, detected in the mitochondrial pellet, plotted asa function of the import incubation time. The SDS ⁄ PAGE slabswere also blotted with an antibody against the 39 kDa subunit ofcomplex I. See Experimental procedures and [24] for furtherdetails.CREB and mitochondrial protein synthesis D. De Rasmo et al.4326 FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBStrypsin, before the import assay of radioactive CREB(Fig. 2A, lanes 3 and 4), the amount of importedCREB, resistant to trypsin treatment after completionof the uptake, and that of externally bound CREB,digested by trypsin, were both reduced. This indicatesthat mitochondrial binding and import of CREBinvolve surface components of the outer membraneimport complex. Addition to the mitochondrial importmixture of an antibody against Tom20, an outermembrane receptor of the mitochondrial import sys-tem [23], reduced the binding of [35S]methionine-labelled CREB to the mitochondrial surface (amountdigested by trypsin) and the accumulation in mito-chondria (trypsin-resistant amount) (Fig. 2B, lanes 5and 6). No significant effect was exerted on CREBbinding and import by an antibody against Tom70(Fig. 2B, lanes 3 and 4).The dependence of CREB uptake on Tom20 andmembrane potential indicates that the protein reachesthe inner mitochondrial compartment. Submitochon-drial localization of the imported CREB was directlyverified by separation of mitochondrial subfractions(Fig. 3). After the import incubation and trypsin treat-ment of mitochondria, organelles were swollen in ahypo-osmotic medium to eliminate CREB bound atthe surface. Residual mitochondria and mitoplasts,deprived of the outer membrane and of residual super-ficially bound radioactive CREB, were disrupted bysonication, and the inner membrane fraction was sepa-rated from the matrix content. The radioactive CREBof the mitoplast fraction, still resistant to trypsin inthis fraction, was recovered in the matrix fraction.Mitochondrial subfractionation was checked byimmunochemical detection of marker proteins of theouter membrane (porin), inner membrane (core IIsubunit of the cytochrome bc1complex), and matrix(cyclophilin D) (Fig. 3).Imported CREB promotes expression ofmitochondrial genesThe impact of imported CREB and PKA on theexpression of mitochondrial genes was studied bytesting their effect on the synthesis of [35S]methionine-labelled mitochondrially encoded subunits of oxidativephosphorylation complexes in rat liver mitochondria.Addition of cAMP or dibutyryl cAMP (db-cAMP)A B Fig. 2. Inhibition of [35S]methionine-labelled CREB mitochondrialimport by proteolytic digestion of mitochondrial outer surface com-ponents and by an antibody against Tom20. (A) Lane 1: controlimport of [35S]methionine-labelled CREB. Lane 2: import of the[35S]methonine-labelled CREB followed by trypsin treatment. Lanes3 and 4: CREB import in mitochondria pretreated for 35 min at 0 °Cwith trypsin. Where indicated, mitochondria were also treated withtrypsin after completion of the import incubation. (B) Lanes 1 and2: as in (A). Lanes 3 and 4: mitochondrial import in the presence of3 lg of the antibody against Tom70 (Santa Cruz Biotechnology, CA,USA). Lanes 5 and 6: mitochondrial import in the presence of theantibody against Tom20 (Santa Cruz Biotechnology). Where indi-cated, mitochondria were treated with trypsin after completion ofthe import incubation. Aliquots of the samples were blotted withan antibody against the 39 kDa subunit of complex I. See Experi-mental procedures and [24] for further details.Fig. 3. Submitochondrial localization of imported [35S]methionine-labelled CREB. Mitochondrial import of [35S]methionine-labelledCREB was followed for 60 min as described in Experimental proce-dures and in the legend to Fig. 1. After import of [35S]methionine-labelled CREB, followed by trypsin treatment of mitochondria (Mt),these were spun down and treated to separate mitoplasts (Mp) andinner membrane (I.M.) and matrix (M.) fractions as described inExperimental procedures. Aliquots of the samples were analysed bySDS ⁄ PAGE and autoradiography, or immunoblotted with the anti-bodies specified. Lanes 1 and 2: mitochondria isolated from theimport mixture, before or after trypsin treatment, respectively. Lane3: mitoplast fraction. Lane 4: mitoplast fraction subjected to trypsintreatment. Lane 5: inner membrane fraction. Lane 6: matrix fraction.For other details, see Experimental procedures.D. De Rasmo et al. CREB and mitochondrial protein synthesisFEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 4327caused some stimulation of the overall radioactivityof the gel lane with the [35S]methionine-labelled mi-tochondrially encoded proteins (Fig. 4). In particulardb-cAMP increased the synthesis of ND1 by 80%,of CoxIII ⁄ ATP6 by 47%, and of ND6 by 30%(Fig. 4). The addition of the RRL-synthesized CREBalone resulted in enhancement of the synthesis ofmitochondrial proteins, this effect being stronglypotentiated when CREB was added together withcAMP, db-cAMP or the catalytic subunit of PKA(cPKA), respectively, in the incubation mixture(Fig. 4B, whole gel lane radioactivity of the[35S]methionine-labelled mitochondrial-encoded pro-teins). In particular, the synthesis of ND1, ND6 andCoxIII ⁄ ATP6, was enhanced approximately two-foldby the combination of CREB and cAMP or cPKAAB200250125025012502501250200400010002001000Fig. 4. Effect of CREB, cAMP and cPKA on mitochondrial protein synthesis. Mitochondrial protein synthesis was performed in a rat livermitochondria suspension in the presence of [35S]methionine and cycloheximide plus the RRL mixture without the addition of the cDNACREB construct (lanes 1–4), or the RRL mixture with the cDNA CREB construct and cold methionine (lanes 5–9). The mitochondrial proteinsynthesis control (CTRL) contained: rat liver mitochondria suspension and the RRL mixture without the addition of the cDNA CREB con-struct. (A) SDS ⁄ PAGE autoradiography of [35S]methionine-labelled mitochondrial proteins. Lane 1: control. Lane 2: 50 lM cAMP plus 50 lMIBMX. Lane 3: 50 lM db-cAMP plus IBMX. Lane 4: cPKA (1 Unit per 10 lg of mitochondrial protein). Lane 5: no addition. Lane 6: cAMP plusIBMX. Lane 7: db-cAMP plus IBMX. Lane 8: cPKA. Lane 9: CAP (3 mgÆmL)1). (B) Histograms showing the mean ADU (as percentage of con-trol) of the whole gel lane radioactivity of the [35S]methionine-labelled mitochondrial proteins and of individual protein spot radioactivity. Meanvalues of three separate experiments; **P < 0.01; *P < 0.05. The inset shows autoradiography of [35S]methionine-labelled CREB immuno-precipitated by an antibody against phospho-CREB (Santa Cruz Biotechnology). [35S]Methionine-labelled CREB was synthesized in the RRLtranslation system in the absence (control) or in the presence of 10 Units of cPKA. Translation products were immunoprecipitated by thephospho-CREB antibody. For other details, see Experimental procedures.CREB and mitochondrial protein synthesis D. De Rasmo et al.4328 FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS(Fig. 4B). Evidence that added cPKA catalysed thephosphorylation of CREB is provided by a controlexperiment showing that the radioactive CREB wasimmunoprecipitated by an antibody against phospho-CREB only when cPKA was added to the mixture(Fig. 4, inset).It may be noted that in the experiment presented inthe Fig. 4, not all of the mitochondrially encodedsubunits of the oxidative phosphorylation complexesexhibited labelling by [35S]methionine under the experi-mental conditions used. The experiment presented inFig. 5, in which a higher amount of [35S]methioninewas used in the mitochondrial protein synthesis and adifferent acrylamide gel concentration was applied forSDS ⁄ PAGE, shows more subunits of oxidative phos-phorylation complexes labelled with [35S]methionine.The combined addition of CREB and cAMP or cPKAresulted also, in this case, in a marked enhancement ofthe overall gel lane radioactivity with the [35S]methio-nine-labelled mitochondrially encoded proteins(Fig. 5B). The autoradiogram presented in Fig. 5Ashows that the synthesis of individual mitochondriallyencoded proteins was generally promoted by the addi-tion of CREB with cAMP or cPKA. This stimulatoryeffect on mitochondrially encoded subunits was com-pletely abolished by the addition of H89, a specificinhibitor of PKA.The addition to the mitochondrial protein synthesismixture of the RRL-synthesized NDUFS4 nuclear sub-unit of complex I, used as a control, had no effect onthe synthesis of mitochondrially encoded subunits ofthis and other oxidative phosphorylation complexes(results not shown). Evidence has been presented else-where that PKA-mediated phosphorylation of theNDUFS4 nuclear subunit of complex I [24], as well asof other nuclear-encoded mitochondrial proteins[25–27], promotes the import into mitochondria ofthese proteins. The presence of cPKA or cAMP in theimport mixture had, however, no effect on the uptakeof CREB by isolated mitochondria (results notshown).DiscussionThe present results show that in vitro synthesizedCREB is imported into the mitochondrial matrix by amembrane potential-dependent mechanism. The CREBimported into mitochondria, and therefore resistantto digestion by trypsin unless mitochondria weredissolved by Triton, did not undergo N-terminal pro-cessing as has also been observed for other nuclear-encoded mitochondrial matrix-targeted proteins[26,28]. The inhibition of both mitochondrial surfacebinding and import into mitochondria of radioactiveCREB by the antibody against Tom20 shows, how-ever, that the import of CREB is mediated by theTOM complex involved in the translocation of pro-teins into the matrix space [23,29–31]. The antibodyagainst Tom70, which is an import receptor for inser-tion of hydrophobic imported proteins into the innermembrane, did not reduce the import of CREB. TheCREB mitochondrial import could also be assisted bychaperones, such as the mitochondrial heat shockprotein 70 [19].A B Fig. 5. Effect of H89 on the promotion of mitochondrial proteinsynthesis by CREB plus cAMP or cPKA. Mitochondrial protein syn-thesis was carried out in the presence of the RRL system supple-mented with cold synthesized CREB, as described in the legend toFig. 4. The RRL mixture with cold synthesized CREB was presentin all of the lanes, including the control. The mitochondrial proteinsynthesis control (CTRL) contained rat liver mitochondria suspen-sion and the RRL mixture with the addition of the cDNA CREB con-struct. (A) SDS ⁄ PAGE autoradiography of mitochondrial proteinssynthesized in the presence of [35S]methionine. For the experimen-tal conditions, see legend to Fig. 4. Where indicated, H89 (100 nM)was present during the import incubation. (B) Histograms showingthe mean ADU (as percentage of control) of the whole gel laneradioactivity of the [35S]methionine-labelled mitochondrial proteins.For other details, see Experimental procedures.D. De Rasmo et al. CREB and mitochondrial protein synthesisFEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 4329Externally synthesized CREB, once imported intomitochondria, strongly stimulates the synthesis ofsubunits of oxidative phosphorylation encoded bymitochondrial genes; this requires, as in the case ofnuclear CRE-regulated genes, CREB phosphorylationby PKA [2–4]. The synthesis of all of the mitochondri-ally encoded subunits of oxidative phosphorylationenzyme complexes was, in fact, stimulated by thecombined addition of CREB and cAMP or cPKA.The stimulatory effect was particularly evident(approximately two-fold enhancement) in the case ofthose proteins that were more heavily labelled with[35S]methionine, such as ND1, ND6, and CoxIII ⁄ ATP6(Figs 4 and 5).Phosphorylation of CREB, which is synthesized oncytosolic ribosomes, can take place in vivo in the cytosolbefore its import into mitochondria, and ⁄ or after it isimported into mitochondria. PKA is, in fact, present invarious subcellular regions, including the cytosol andouter and inner mitochondrial compartments [21,32,33].Our results show that, under the experimental condi-tions used, CREB was phosphorylated by the PKA thatwas evidently present in the RRL system used for thein vitro synthesis of CREB and ⁄ or in the mitochondrialsample, as well as by the added catalytic subunit ofPKA. In this last case, no cAMP was obviouslyrequired. The minor promoting effect on mitochondrialprotein synthesis given by the addition of cAMP orcPKA (in separate samples) in the absence of addedCREB results from phosphorylation of CREB presentin the RRL system and ⁄ or in the mitochondrial sample[18–20]. Evidence has been produced showing the exis-tence of a pool of PKA and PKA-anchoring protein(AKAP) localized in the inner mitochondrial compart-ment [21]. Intramitochondrial PKA can be activated bycAMP generated within mitochondria by a carbondioxide ⁄ bicarbonate-regulated soluble adenylyl cyclase[34,35]. Lee et al. [19] have shown that disruption ofCREB activity, by overexpression of a mito-taggednegative dominant CREB, decreases the expression ofmitochondrial genes in transfected cells. It has also beenshown that activation of mitochondrial PKA by theantioxidant deferoxamine results in phosphorylation ofmitochondrial CREB and its binding to the CREsequence in the mitochondrial D-loop DNA [20].In conclusion, the present findings provide unequiv-ocal evidence that the transcription factor CREB isimported into the mitochondrial matrix and promotes,when phosphorylated by PKA, the synthesis of mito-chondrially encoded subunits of oxidative phosphory-lation complexes. Our results also lend support, freefrom the uncertainties involved in immunochemicalanalysis [22], for the presence of CREB in the innermitochondrial compartment, where it can also bephosphorylated by PKA present in the same compart-ment [21]. This is not a surprise, as CREB, in order toexert its effect on mitochondrial protein synthesis, hasto move from the cytosol, where it is synthesized,into mitochondria, where transcription ⁄ translation ofmtDNA-encoded proteins takes place.Positive modulation by CREB of the expression ofnuclear [36,37] and mitochondrial genes of proteins ofthe oxidative phosphorylation system could represent animportant regulatory mechanism for the expression ofthis housekeeping cellular function, thus contributing tothe role of CREB in a variety of cellular processes.Experimental procedurescDNA construct and in vitro translationFull-length human CREB cDNA was generated byRT-PCR, using RNA extracted from primary fibroblastsfrom skin biopsy specimens of control subjects. The CREBcDNA was cloned in the pGEM vector with the T7promoter. Plasmid construction was confirmed by DNAsequencing. In vitro transcription ⁄ translation of CREBcDNA was performed in RRL system (Promega Biotech,Madison, WI, USA) as reported by De Rasmo et al. [24].One microgram of CREB construct was added to 50 lLofPromega standard mixture, containing T7 RNA polymeraseand a standard amino acid mixture with [35S]methionine(20 lCi). Incubation was performed at 30 °C for 90 min.Rat liver mitochondriaMitochondria were isolated from rat liver as described inref. [38].Import assayThe assay was performed as in [24]. Sixteen microlitres of theRRL translation mixture producing [35S]methionine-labelledCREB was added to the import mixture containing210 mm mannitol, 7 mm Hepes (pH 7.4), 0.35 mm MgCl2,2.5 mgÆmL)1BSA, rat liver mitochondria (500 lg of pro-tein), 3 mm ATP, 3 mm GTP, 15 mm malate and 30 mmpyruvate in a final volume 200 lL. After incubation at 30 °Cfor the times specified in the figures, aliquots of the mixturewere transferred to ice-cooled tubes and supplemented withprotease inhibitors (Sigma, St Louis, MO, USA) (1 lL per250 lg of mitochondrial protein). Mitochondria were spundown at 4000 g for 10 min, and supernatant and mitochon-drial proteins were separated by SDS ⁄ PAGE and transferredto a nitrocellulose membrane. Radioactive protein bandswere detected by personal fx at phosphorus imager(Bio-Rad, Milan, Italy) and quantified by versadoc (Bio-CREB and mitochondrial protein synthesis D. De Rasmo et al.4330 FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBSRad). The same samples were also immunoblotted with anantibody against the 39 kDa subunit of complex I of therespiratory chain (Invitrogen, Paisley, UK).Mitochondrial protein synthesisRRL translation medium with unlabelled synthesizedCREB or without CREB was added to the import mixturecontaining a standard amino acid mixture with [35S]methio-nine (20 lCi), 210 mm mannitol, 7 mm Hepes (pH 7.4),0.35 mm MgCl2, 2.5 mgÆmL)1BSA, rat liver mitochondria(125 lg of protein), 3 mm ATP, 3 mm GTP, 15 mm malate,30 mm pyruvate and 50 ngÆmL)1cycloheximide in a finalvolume of 50 lL. Incubation was performed at 30 °C for1 h in the presence, where indicated, of cAMP, cPKA,db-cAMP, isobutylmethylxanthine (IBMX), H89, orchloramphenicol (CAP). The incubation was prolonged for10 min after the addition of unlabelled amino acid mixture.Mitochondria were then spun down at 4000 g for 10 min,and proteins were separated by SDS ⁄ PAGE and transferredto a nitrocellulose membrane. Radioactive protein bandswere detected by personal fx at phosphorus imager(Bio-Rad) and quantified by versadoc (Bio-Rad).Submitochondrial localization of imported[35S]methionine-labelled CREBMitochondrial sublocalization of imported [35S]methionine-labelled CREB was performed essentially as described inref. [39]. After mitochondrial import of [35S]methionine-labelled CREB and trypsin treatment, reisolated mitochon-dria were split in two aliquots and resuspended in 250 mmsucrose, 1 mm EDTA, and 10 mm Mops ⁄ KOH (pH 7.2),or in 1 mm EDTA and 10 mm Mops ⁄ KOH (pH 7.2); thelatter medium was used to obtain mitoplasts by mitochon-drial swelling. After 15 min of incubation on ice, eachsample was split into two, and one of these was againsubjected to trypsin treatment. Mitochondrial and mito-plasts fractions were spun down at 10 000 g for 10 min.After mitoplast sonication, the sample was centrifuged at150 000 g for 15 min. The pellet representing the innermembrane proteins was resuspended in the SDS ⁄ PAGEloading buffer; the supernatant, representing the matrixfraction, was treated with trichloroacetic acid, and the pre-cipitate was resuspended in the SDS ⁄ PAGE loading buffer.All samples were analysed by SDS ⁄ PAGE and autoradio-graphy, or immunoblotted with antibodies against theporin, cyclophilin D and core II subunit of complex III(Invitrogen) as specified in the legend to Fig. 3.AcknowledgementsThis work was supported by the National Project on‘Molecular Mechanisms, Physiology and Pathology ofMembrane Bioenergetics System’, 2005, Ministerodell’Istruzione, dell’Universita`e della Ricerca (MIUR),Italy, the University of Bari, and Research FoundationCassa di Risparmio di Puglia.References1 Brindle P, Nakajima T & Montminy M (1995) Multipleprotein kinase A-regulated events are required for tran-scriptional induction by cAMP. Proc Natl Acad SciUSA 92, 10521–10525.2 Shaywitz AJ & Greenberg ME (1999) CREB: a stimu-lus-induced transcription factor activated by a diversearray of extracellular signals. Annu Rev Biochem 68,821–861.3 Tan Y, Rouse J, Zhang A, Cariati S, Cohen P &Comb MJ (1996) FGF and stress regulate CREB andATF-1 via a pathway involving p38 MAP kinase andMAPKAP kinase-2. EMBO J 15, 4629–4642.4 Sands WA & Palmer TM (2008) Regulating genetranscription in response to cyclic AMP elevation. CellSignal 20, 460–466.5 Riccio A, Ahn S, Davenport CM, Blendy JA &Ginty DD (1999) Mediation by a CREB familytranscription factor of NGF-dependent survival ofsympathetic neurons. Science 286, 2358–2361.6 Bonni A, Brunet A, West AE, Datta SR, Takasu MA &Greenberg ME (1999) Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependentand -independent mechanisms. Science 286, 1358–1362.7 Bedogni B, Pani G, Colavitti R, Riccio A, Borrello S,Murphy M, Smith R, Eboli ML & Galeotti T (2003)Redox regulation of cAMP-responsive element-bindingprotein and induction of manganous superoxide dismu-tase in nerve growth factor-dependent cell survival.J Biol Chem 278, 16510–16519.8 Lonze BE & Ginty DD (2002) Function and regulationof CREB family transcription factors in the nervoussystem. Neuron 35, 605–623.9 Fang M & Butow RA (1970) Nucleotide reversal ofmitochondrial repression in Saccharomyces cererevisiae.Biochem Biophys Res Commun 41, 1579–1583.10 Chandrasekaran K & Jayaraman J (1978) Effect ofcyclic AMP on the biogenesis of cytochrome oxidasein yeast. FEBS Lett 87, 52–54.11 Iqbal J, Ge´rard HC, Rahman MU & Hudson AP(1996) A probable cis-regulatory element on yeastmitochondrial DNA responsible for cAMP-mediatedtranscription. Curr Genet 30, 493–501.12 Neuman-Silberberg FS, Bhattacharya S & Broach JR(1995) Nutrient availability and the RAS ⁄ cyclic AMPpathway both induce expression of ribosomal proteingenes in Saccharomyces cererevisiae but by differentmechanisms. Mol Cell Biol 15, 3187–3196.D. De Rasmo et al. CREB and mitochondrial protein synthesisFEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 433113 Dejean L, Beauvoit B, Bunoust O, Gue´rin B &Rigoulet M (2002) Activation of Ras cascade increasesthe mitochondrial enzyme content of respiratory com-petent yeast. Biochem Biophys Res Commun 293, 1383–1388.14 Cribbs JT & Strack S (2007) Reversible phosphoryla-tion of Drp1 by cyclic AMP-dependent protein kinaseand calcineurin regulates mitochondrial fission and celldeath. EMBO Rep 8, 939–944.15 Pagliarini DJ & Dixon JE (2008) Mitochondrial modu-lation: reversible phosphorylation takes center stage?Trends Biochem Sci 31, 26–34.16 Papa S, De Rasmo D, Scacco S, Signorile A,Technikova-Dobrova Z, Palmisano G, Sardanelli AM,Papa F, Panelli D, Scaringi R et al. (2008) Mammaliancomplex I: a regulable and vulnerable pacemaker inmitochondrial respiratory function. Biochim BiophysActa 1777, 719–728.17 Remacle C, Barbieri MR, Cardol P & Hamel PP (2008)Eukaryotic complex I: functional diversity and experi-mental systems to unravel the assembly process. MolGenet Genomics 280, 93–110.18 Cammarota M, Paratcha G, Bevilaqua LR, Levi deStein M, Lopez M, Pellegrino de Iraldi A, Izquierdo I& Medina JH (1999) Cyclic AMP-responsive elementbinding protein in brain mitochondria. J Neurochem 72,2272–2277.19 Lee J, Kim CH, Simon DK, Aminova LR, AndreyevAY, Kushnareva YE, Murphy AN, Lonze BE, KimKS, Ginty DD et al. (2005) Mitochondrial cyclic AMPresponse element-binding protein (CREB) mediatesmitochondrial gene expression and neuronal survival.J Biol Chem 280, 40398–40401.20 Ryu H, Lee J, Impey S, Ratan RR & Ferrante RJ(2005) Antioxidants modulate mitochondrial PKA andincrease CREB binding to D-loop DNA of the mito-chondrial genome in neurons. Proc Natl Acad Sci USA102, 13915–13920.21 Sardanelli AM, Signorile A, Nuzzi R, De Rasmo D,Technikova-Dobrova Z, Drahota Z, Occhiello A, PicaA & Papa S (2006) Occurrence of A-kinase anchorprotein and associated cAMP-dependent protein kinasein the inner compartment of mammalian mitochondria.FEBS Lett 580, 5690–5696.22 Pla´tenı´k J, Balcar VJ, Yoneda Y, Mioduszewska B,Buchal R, Hynek R, Kilianek L, Kuramoto N,Wilczynski G, Ogita K et al. (2005) Apparent pres-ence of Ser133-phosphorylated cyclic AMP responseelement binding protein (pCREB) in brain mitochon-dria is due to cross-reactivity of pCREB antibodieswith pyruvate dehydrogenase. J Neurochem 95, 1446–1460.23 Brix J, Dietmeier K & Pfanner N (1997) Differentialrecognition of preproteins by the purified cytosolicdomains of the mitochondrial import receptorsTom20, Tom22, and Tom70. J Biol Chem 272,20730–20735.24 De Rasmo D, Panelli D, Sardanelli AM & Papa S(2008) cAMP-dependent protein kinase regulates themitochondrial import of the nuclear encoded NDUFS4subunit of complex I. Cell Signal 20, 989–997.25 Anandatheerthavarada H, Biswas G, Mullick J,Sepuris N, Pain D & Avadhani G (1999) Dual targetingof cytochrome P4502B1 to endoplasmic reticulumand mitochondria involves a novel signal activation bycyclic AMP-dependent phosphorylation at ser128.EMBO J 18, 5494–5504.26 Robin M, Anandatheerthavarada H, Biswas G, SepurisN, Gordon D, Pain D & Avadhani G (2002) Bimodaltargeting of microsomal CYP2E1 to mitochondriathrough activation of an N-terminal chimeric signal bycAMP-mediated phosphorylation. J Biol Chem 277,40583–40593.27 Robin M, Prabu S, Raza H, Anandatheerthavarada H& Avadhani G (2003) Phosphorylation enhances mito-chondrial targeting of GSTA4-4 through increasedaffinity for binding to cytoplasmic Hsp70. J Biol Chem278, 18960–18970.28 Yadava N & Scheffler IE (2004) Import and orientationof the MWFE protein in mitochondrial NADH-ubiqui-none oxidoreductase. Mitochondrion 4, 1–12.29 Neupert W & Brunner M (2002) The protein importmotor of mitochondria. Nat Rev Mol Cell Biol 3, 555–565.30 Papa S, Petruzzella V & Scacco S (2007) Electron trans-port, structure, redox-coupled protonmotive activityand pathological disorders of respiratory chain com-plexes. In Handbook of Neurochemistry and MolecularNeurobiology, Brain Energy Metabolism (Lajtha A,Dienel G & Gibson G, eds), pp. 93–118. SpringerVerlag, Berlin Heidelberg.31 Bolender N, Sickmann A, Wagner R, Meisinger C &Pfanner N (2008) Multiple pathways for sortingmitochondrial precursor proteins. EMBO Rep 9, 42–49.32 Schwoch G, Trinczek B & Bode C (1990) Localizationof catalytic and regulatory subunits of cyclic AMP-dependent protein kinase in mitochondria from variousrat tissues. Biochem J 270, 181–188.33 Wong W & Scott JD (2004) AKAP signalling com-plexes: focal points in space and time. Nat Rev Mol CellBiol 5, 959–970.34 Zippin JH, Chen Y, Nahirney P, Kamenetsky M,Wuttke MS, Fischman DA, Levin LR & Buck J (2003)Compartmentalization of bicarbonate-sensitive adenylylcyclase in distinct signaling microdomains. FASEB J17, 82–84.35 Acin-Perez R, Salazar E, Kamenetsky M, Buck J, LevinLR & Manfredi G (2009) Cyclic AMP produced insidemitochondria regulates oxidative phosphorylation. CellMetab 9, 265–276.CREB and mitochondrial protein synthesis D. De Rasmo et al.4332 FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS36 Scarpulla RC (1997) Nuclear control of respiratorychain expression in mammalian cells. J Bioenerg Bio-membr 29, 109–119.37 Kelly DP & Scarpulla RC (2006) Transcriptional regu-latory circuits controlling mitochondrial biogenesis andfunction. Genes Dev 18, 357–368.38 Ito A, Ogishima T, Ou W, Omura T, Aoyagi H,Lee S, Mihara H. & Izumiya N (1985) Effects ofsynthetic model peptides resembling the extension pep-tides of mitochondrial enzyme precursors on importof the precursors into mitochondria. J Biochem 98,1571–1582.39 Ryan MT, Voos W & Pfanner N (2001) Assayingprotein import into mitochondria. Methods Cell Biol65, 189–215.D. De Rasmo et al. CREB and mitochondrial protein synthesisFEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 4333 . cAMP response element-binding protein (CREB) is imported into mitochondria and promotes protein synthesis Domenico De Rasmo1,. import into mitochondria, and ⁄ or after it is imported into mitochondria. PKA is, in fact, present invarious subcellular regions, including the cytosol and outer
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