proteinkinasecregulatesinternalinitiationoftranslation ... · rnai analysis—knockdown experiments...

Protein Kinase C Regulates Internal Initiation of Translationof the GATA-4 mRNA following Vasopressin-inducedHypertrophy of Cardiac Myocytes*

Received for publication, September 15, 2006, and in revised form, January 24, 2007 Published, JBC Papers in Press, February 6, 2007, DOI 10.1074/jbc.M608874200

Anushree Sharma‡1, Janine Masri‡, Oak D. Jo‡, Andrew Bernath‡, Jheralyn Martin‡, Alexander Funk‡,and Joseph Gera‡§2

From the ‡Department of Research & Development, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles,California 91343 and the §Department of Medicine, David Geffen School of Medicine, University of California,Los Angeles, California 90048

GATA-4 is a key member of the GATA family of transcrip-tion factors involved in cardiac development and growth aswell as in cardiac hypertrophy and heart failure. Our previousstudies suggest that GATA-4 protein synthesis may be trans-lationally regulated. We report here that the 518-nt long5�-untranslated region (5�-UTR) of the GATA-4 mRNA,which is predicted to form stable secondary structures (�65kcal/mol) such as to be inhibitory to cap-dependent initia-tion, confers efficient translation to monocistronic reportermRNAs in cell-free extracts. Moreover, uncapped GATA-45�-UTR containing monocistronic reporter mRNAs continueto be well translated while capped reporters are insensitive tothe inhibition of initiation by cap-analog, suggesting a cap-independent mechanism of initiation. Utilizing a dicistronicluciferase mRNA reporter containing the GATA-4 5�-UTRwithin the intercistronic region, we demonstrate that thisleader sequence confers functional internal ribosome entrysite (IRES) activity. The activity of the GATA-4 IRES is unaf-fected in trans-differentiating P19CL6 cells, however, isstrongly stimulated immediately following arginine-vaso-pressin exposure of H9c2 ventricular myocytes. IRES activityis then maintained at submaximal levels during hypertrophicgrowth of these cells. Supraphysiological Ca2� levels dimin-ished stimulation of IRES activity immediately followingexposure to vasopressin and inhibition of protein kinase Cactivity utilizing a pseudosubstrate peptide sequence blockedIRES activity during hypertrophy. Thus, our data suggest amechanism for GATA-4 protein synthesis under conditionsof reduced global cap-dependent translation, which is main-tained at a submaximal level during hypertrophic growth andpoint to the regulation of GATA-4 IRES activity by sarco(ER)-reticular Ca2� stores and PKC.

The family of GATA transcription factors regulates differen-tiation, growth, and survival of a wide range of cell types (1).GATA-4 is a zinc finger-containing transcription factor, whichis expressed, in various mesoderm-derived tissues such as thelung, liver, gonad, and gut where it regulates tissue-specificgene expression (2, 3). Consistent with the observed expressionpatterns, targeted disruption ofGATA-4 inmice has elucidatedimportant functions in each of these tissues. GATA-4 is alsoessential for proper cardiac morphogenesis and also plays animportant role as one of the transcriptional factors, which reg-ulates the hypertrophic response in cardiac myocytes (4–6).Several studies suggest thatGATA-4 can be regulated at both

the transcriptional level and by phosphomodulation duringhypertrophy (1, 7, 8). In cultured neonatal cardiomyocytes,electrical pacing-induced hypertrophy is associated with a sig-nificant increase in GATA-4 mRNA, suggesting a mechanismwhereby total GATA-4 content is up-regulated during hyper-trophy (9). Increases in GATA-4 phosphorylation mediated byRaf/Mek/Erk signaling subsequent to hypertrophic agonistadministration have also been demonstrated (10). Our previousdata (11) suggested that theGATA-4mRNA is also regulated atthe translational level and we undertook this study to evaluatethe possibility that such control may play a role in augmentingGATA-4 potency during differentiation or hypertrophy in car-diac myocytes.Most mRNAs contain 5�-UTRs3 that are relatively unstruc-

tured and typically less than 100 nucleotides in length, whichallows efficient cap-dependent translational initiation (12).However, the leaders of some cellular mRNAs are long, highlystructured, and can contain multiple upstream AUG or CUGcodons such that scanning ribosomes are unlikely to effi-ciently initiate translation. In a number of these mRNAs,translation initiation is mediated by cap-independent mech-anisms via an internal ribosome entry site (13). IRES-medi-ated translation initiation can occur during a variety of physio-logical conditions and has been reported to promote initiation

* This work was supported in part by Grant CA109312 from the National Insti-tutes of Health and by an award from the Veterans Administration. Thecosts of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Present address: Dept. of Immunology and Pathology, Washington Univer-sity School of Medicine, St. Louis, MO.

2 To whom correspondence should be addressed. Tel.: 818-895-9416; Fax:818-895-9554; E-mail: [email protected].

3 The abbreviations used are: UTR, untranslated region; IRES, internal ribo-some entry site; ORF, open reading frame; uORF, upstream open readingframe; LUC, luciferase; mTOR, mammalian target of rapamycin; ITAF, IREStrans-acting factor; eIF-2�, eukaryotic translation initiation factor 2�; AVP,arginine vasopressin; ER, endoplasmic reticulum; BiP, immunoglobinheavy chain binding protein; PKC, protein kinase C; Cdk1, cyclin-depend-ent kinase 1; �-MHC, �-myosin heavy chain; RACE, rapid amplification ofcDNA ends; nt, nucleotide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 13, pp. 9505–9516, March 30, 2007Printed in the U.S.A.

MARCH 30, 2007 • VOLUME 282 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9505

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for several mRNAs during cell-cycle progression, differentia-tion, apoptosis, and during stress responses (14–18).Previously, we identified several mRNAs which either

increased or continued to be well translated during conditionswhen cap-dependent translation initiation was inhibited (11,19). One of these mRNAs, GATA-4, increased its translationalefficiency markedly following exposure of cells to the mTORinhibitor rapamycin. Treatment with rapamycin results in theglobal inhibition of cap-dependent translation via a blockade tothe formation of productive eIF4F initiation complexes (20).The human GATA-4 transcript has a 518 nucleotide 5�-UTRand contains 18 upstream initiation codons (21). Thisprompted us to investigate the possibility of whether thismRNA could initiate translation in a cap-independent fashionand contain an IRES within its leader.Because GATA-4 has a prominent role in the regulation of

muscle cell-specific differentiation and IRES-mediatedtranslation may be enhanced during differentiation (22, 23),we analyzed the ability of this leader to mediate internalinitiation during this process. We utilized the P19CL6 cellline model to investigate the possibility that the GATA-4mRNA could initiate IRES-mediated translation during dif-ferentiation. The P19CL6 cell line is a clonal derivative of theP19 embryonal carcinoma cell line which efficiently differ-entiates into beating cardiomyocytes and produces charac-teristic muscle proteins (24). GATA-4 is expressed in thesecells, is induced upon differentiation, and is required totrans-activate muscle-specific genes (25). Additionally,because GATA-4 is known to be required for cardiac hyper-trophy we determined whether IRES-mediated translationinitiation of GATA-4 mRNA could occur following vaso-pressin-induced hypertrophy of H9c2 cardiomyocytes.We report here that the GATA-4 5�-UTR contains IRES

activity, which is stimulated during hypertrophy but not duringdifferentiation. Our data also suggest that GATA-4 IRES activ-ity is enhanced coincident with elevated eIF-2� phosphoryla-tion during hypertrophy. We propose that IRES-mediatedtranslational initiation of GATA-4 mRNA allows continuedprotein synthesis under conditions of reduced cap-dependentinitiation potential during the initial response to vasopressinand continues during hypertrophic growth.Moreover, our datasuggest that sarco (endo)plasmic Ca2� flux may play a role inthe stimulation of GATA-4 IRES activity through a proteinkinase C-dependent pathway during hypertrophy.

EXPERIMENTAL PROCEDURES

Cell Lines and DNA Constructs—All cell lines were obtainedfrom ATCC (American Type Culture Collection) and main-tained in medium supplemented with 10% fetal bovine serum.P19CL6 cell lines expressing the dicistronic reporter mRNAswere generated by co-transfection with pcDNA3.1 andpRGATA-4F followed by G418 selection and subsequentscreening for stable expression by Northern analysis. Stableclones were induced to differentiate under adherent conditionsby plating at a density of 3.7� 105 cells in a 60-mm culture dishwith 1% dimethyl sulfoxide. The medium was changed every 2days. Days of differentiation were numbered consecutively,with the first day of Me2SO treatment as day 0. The protein

kinase C cell permeable myristoylated pseudosubstrate peptide20–28 (12 �M) and nifedipine (1 �M) was obtained from Cal-biochem, and Arg-vasopressin (1 �M) was from Sigma.5�-RACE analysis of the GATA-4 mRNA was performed

on total RNA from H9c2 or HeLa cells using the 5�/3�-RACEkit, 2nd Generation, as described by the manufacturer(Roche Applied Science). To construct the dicistronicreporters, the entire human GATA-4 5�-UTR (accessionnumber NM_002052) was amplified from IMAGE clone5744870 and inserted into the NcoI site of pRF (kindly pro-vided by A. Willis, University of Nottingham, UK). The con-struction of pRmycF has been described previously (19). TheGATA-4 5�-UTR was also inserted into a promoterless ver-sion of pRF (26), (�)pRF (kindly provided by J.-T. Zhang,Indiana University) to generate �(p)RGATA-4F. A 46-bpHindIII-BamHI fragment containing a hairpin structurecontaining an 18-bp stem (�G � �61 kcal/mol) was liber-ated from pSP64-hp7 (27) (kindly provided by L. Maquat,Roswell Park Cancer Institute) and inserted immediatelyupstream of the Renilla ORF in pRGATA-4F to generatephpRGATA-4F. To generate the constructs used in themonocistronic analysis of RNAs in vitro, the firefly luciferaseORF or the GATA-4 5�-UTR-firefly luciferase ORFsequences were amplified from pRGATA-4F and subclonedinto the pSP64 poly(A) vector (Promega) to generate pSPpAand pSPGATA-4pA, respectively. Similarly, the dicistronicconstructs used in the in vitro analyses were generated byamplifying a fragment spanning the Renilla ORF, the inter-cistronic region and the firefly ORF from pRF and subclon-ing this into pSP64 poly(A) to create cRFpoly(A)30.cRGATA-4Fpoly(A)30 and cRp27Fpoly(A)30 were created byamplification of the dicistronic regions of pRGATA-4F andpRp27F (19), respectively, and subcloning into pSP64poly(A) as before. The encephalomyocarditis virus (EMCV)IRES sequences were amplified from pIREShyg (Clontech)and inserted into the NcoI site of cRFpoly(A)30. All con-structs were sequenced to confirm their integrity and primerinformation is available upon request.In Vitro Translation of Mono- and Dicistronic mRNA

Reporters—The moncistronic and dicistronic plasmids werelinearized and used as templates to in vitro transcribe the indi-cated RNAs using SP6 polymerase (19). These mRNAs werecapped and subsequently used to program extracts of the indi-cated cell lines as described previously (19). The translationreactions were performed with the cap analog m7GpppG(Ambion) when indicated.Transient DNA and Dicistronic mRNA Transfections—The

indicated reporter constructs were transfected into cells usingLipofectamine Plus (Invitrogen) and normalized for transfec-tion efficiency by co-transfection with pSV�Gal (Promega).Cells were harvested 18 h following transfection and Renilla,firefly, and �-galactosidase activities were determined (Dual-Glo luciferase and �-galactosidase assay systems, Promega).RNA transfection was performed as previously described (26).Cells were harvested 12 h following RNA transfection, andluciferase activities determined. Relative luciferase values areexpressed as activity per �g of protein.

GATA-4 mRNA IRES Activity

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RNAi Analysis—Knockdown experiments targeting the pRFtranscript were accomplished based on the strategy of VanEden et al. (28). A pool of double-stranded RNAs were synthe-sized (Dharmacon) targeting the Renilla luciferase ORF wereannealed and co-transfected with the indicated mono or dicis-tronic plasmids intoHeLa cells. The targeting sequenceswithinthe Renilla ORF used were 5�-AAAGTTTATGATCCAGAA-CAA-3�and 5�-AACAAAGGAAACGGATGATAA-3�.Metabolic Labeling, Immunoprecipitation, and in Vitro PKC

Activity Assays—P19CL6 or H9c2 cells were pulse-labeled with[35S]methionine/cystine at a final concentration of 100�Ci/ml.Cells were harvested following the indicated treatments andlysates prepared in ice-cold radioimmune precipitation assaybuffer (1� phosphate-buffered saline, 1% Nonidet P-40, 0.5%sodium deoxycholate, 0.1% SDS) with protease inhibitorsadded (Halt™ Protease Inhibitor Mixture EDTA-Free, Pierce).

GATA-4 protein was immunopre-cipitated overnight at 4 °C with 1�gof anti-GATA-4 antibody and thencollected with protein G-Sepharose(Amersham Biosciences, Piscat-away, NJ). The immunoprecipitatewas washed four times in radioim-mune precipitation assay bufferand fresh protease inhibitors andthe complex pelleted for resuspen-sion in SDS sample buffer. Thesamples were separated by SDS-PAGE, the gels dried and visual-ized using a phosphorimager(Molecular Dynamics, Sunnyvale,CA). Phosphor-densitometry wasperformed using ImageQuant(Molecular Dynamics) software.Collectively, immunoprecipitationcontrols included equal numbers ofcells plated per flask and equalamounts of quantitated total pro-tein lysate per sample with equiva-lent amounts of antibody. PKCactivity was determined using theSignaTECT® Protein Kinase C assaysystem (Promega, Madison, WI) asdescribed by the manufacturer.Briefly, cell lysates were incubatedwith [�-32P]ATP and PKC-biotiny-lated peptide substrate in substratebuffer at 30 °C for 5 min and subse-quently spotted onto SAM biotincapture membrane (Promega).Membranes were washed andincorporated label measured by ascintillation counter.RNA and Protein Analysis—

Northern analysis was performed aspreviously described (19) with RNApurified from the indicated cell linesor in vitro translation reactions. Fol-

lowing extraction with TRIzol, 3 �g of total RNA was loadedper lane in 1.4% formaldehyde-containing agarose gels, sepa-rated by electrophoresis and transferred to nylon membranes.Hybridization to riboprobes specific for the indicated tran-scripts was performed in ULTRAhyb hybridization buffer(Ambion). Blots were visualized by exposure to film or by aphosphorimager. RT-PCRwas performed using the ImProm-IIReverse Transcription System (Promega) according to theinstructions of the manufacturer. Polysome analysis was per-formed as previously described (11). Briefly, cells were lysed inbuffer containing 100 �g/ml cycloheximide at 4 °C. Followingremoval ofmitochondria and nuclei, supernatants were layeredonto 15–50% sucrose gradients and spun at 38,000 rpm for 2 hat 4 °C in a SW 40 rotor (Beckman Instruments). Gradientswere fractionated into eleven 1-ml fractions using an ISCODensity Gradient Fractionator at a flow rate of 3 ml/min. The

FIGURE 1. GATA-4 5�-UTR sequences confer efficient translation of uncapped monocistronic reportermRNAs. A, SP6-based monocistronic constructs. B, in vitro translation of capped or uncapped monocistronicmRNAs in translationally competent HeLa cell extracts. The luciferase activity conferred by capped pSPpA wasnormalized to 100%. C, effects of m7GpppG cap analog on the in vitro translation of pSPpA and pSPGATA-4pAmonocistronic reporter mRNAs. Data shown are the mean and S.D. from four independent translation reac-tions. D, Northern analysis of in vitro translation reactions (time 0 and 60 min during in vitro translation) pro-grammed with the indicated mRNAs and probed with a riboprobe specific for the firefly luciferase codingregion.




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profiles of the gradients were monitored via UV absorbance at260 nm. RNAs from the individual fractions were pooled into anon-ribosomal and monosomal containing pool and a polyso-mal pool. These RNAs (100 ng) were subsequently used in real-time quantitative RT-PCR analyses of the indicated transcriptsusing the QuantiTect SYBR Green RT-PCR kit (Qiagen) in anEppendorf Mastercycler equipped with a realplex2 opticalmodule (Eppendorf AG, Germany). Transcript-specific oligo-nucleotides for the indicated mRNAs were designed to amplify150-bp fragments. Immunoblots were performed using stand-ard procedures. Briefly, cells were lysed in 50 mM Tris, pH 7.4,150 mM NaCl, 1% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 2mM phenylmethylsulfonyl fluoride, 0.5 mM EDTA, 10 �g/mlleupeptin, and 10 �g/ml aprotinin. Samples were resolved on4–20% SDS-PAGE gels, transferred to polyvinylidene difluo-ride (0.2 �m) membranes and probed with antibodies to thefollowing proteins: phospho-eIF-2� and eIF-2� (Cell Signal-

ing), Cdk1 (Abcam), BiP (Imgenex),actin (Sigma), GATA-4, MLC-2,and �-MHC antibodies were allfrom Santa Cruz Biotechnology.

RESULTS

The GATA-4 Leader Confers theEfficient Translation of UncappedmRNA Reporters in TranslationallyCompetent Cell Extracts—To beginto study the translational control ofthe human GATA-4 mRNA thetranscriptional start site had to bedetermined and validated. We used5�-RACE to isolate and characterizepotential leaders. A single 5�-RACEproduct was amplified from totalRNA extracted from either U87-MGorHeLa cells. The sequence was 518nucleotides in length and was con-sistent with the previously pub-lished human 5�-UTR (21). TheGATA-4 leader is 67%G-C rich andcontains 18 upstream initiationcodons, several of which are inappropriate sequence context tosupport initiation by a scanningmechanism. Secondary structureprediction using the MFOLD algo-rithm of Zuker (29), predicted ahighly stable structure with freeenergy values greater than �65kcal/mol.We thus expected that theGATA-4 5�-UTR would be inhibi-tory to cap-dependent ribosomalscanning and may be translated in acap-independent fashion. To deter-mine if this was the case, we fusedthe GATA-4 5�-UTR immediatelyupstream of the firefly luciferaseopen reading frame in a SP6 pro-

moter-based construct to generate pSPGATA-4pA as shown inFig. 1A. In vitro transcribed mRNAs from the pSPGATA-4pAconstruct were either capped or not and used to program trans-lation-competent cell-free extracts (30). As shown in Fig. 1B,uncapped pSPpA in vitro transcribed mRNA was poorly trans-lated as comparedwith capped pSPpAmRNAs.However, whenin vitro transcribed pSPGATA-4pA mRNAs were tested in anidentical manner, uncapped transcripts containing theGATA-4 5�-UTR were translated efficiently at nearly the samelevels as capped pSPGATA-4pA mRNAs or capped pSPpAmRNAs (Fig. 1B). Additionally we tested whether or not thecap-analog m7GpppG would effectively inhibit initiation ofcapped pSPpA or pSPGATA-4pA mRNAs in extracts. As canbe seen in Fig. 1C, pSPpA RNA translation was inhibited by�80% in the presence of cap-analog, while pSPGATA-4pAwastranslated well at a comparable concentration of the analog.Northern analysis showed that similar amounts of pSPpA and

FIGURE 2. The 5�-UTR of GATA-4 enhances translation when cap-dependent initiation is inhibited in cis.A, SV40-based monocistronic constructs. B, luciferase activities of HeLa cells transiently transfected with theindicated constructs. Luciferase activity is relative to the �-galactosidase activity conferred by the pSV-�-galplasmid to control for transfection efficiency. The relative luciferase or �-galactosidase activity conferred by thephpGL construct is normalized to 1. The results shown are the mean and S.D. from four independenttransfections.




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pSPGATA-4pAmRNAs of the expected size could be detectedin the in vitro translation reactions (Fig. 1D). These data sug-gested that the GATA-4 5�-UTR sequences present in the pSP-GATA-4pA construct do not affect steady-state mRNA levels,or are likely to contain splice sequences. Thus, the 5�-UTR ofGATA-4 can confer efficient translation of uncapped reportermRNAs.The Leader of the GATA-4 mRNA Enhances Translation in

Vivo When Cap-dependent Translation Is Inhibited in cis—Be-cause we had previously observed that the GATA-4mRNAwaswell translated under conditions of reduced eIF-4F complexformation (11), we sought to determine whether a similar affect

could be seen if its translation wereinhibited in cis. We introduced astable hairpin structure (�G � �61kcal/mol) (27) immediately distal tothe SV40 promoter sequences inpGL to generate phpGL (Fig. 2A).This cis-acting structural elementreduced the translation of theparental construct by 85% whentransfected intoHeLa cells (Fig. 2B).To assess whether the GATA-45�-UTR could mediate translation ifcap-dependent translation was in-hibited in this manner, we insertedthe GATA-4 5�-UTR between thehairpin and the translation startsite generating phpGATA-4L.This construct expressed 5-foldmore luciferase activity in HeLacell transfectants then the paren-tal construct phpGL suggestingthat the GATA-4 5�-UTR can pro-mote translation initiation whencap-dependent scanning is re-duced (Fig. 2B). To addresswhether cryptic promoters werepresent within the hairpin orGATA-4 5�-UTR sequences weremoved the SV40 promotersequences from phpGATA-4L togenerate �(p)hpGATA-4L andtested HeLa transfectants forluciferase activity. As can be seen(Fig. 2B), no significant luciferaseactivity was detected suggestingthat these sequences, in the con-text of the monocistronic reporterplasmid, do not support promoteractivity.Dicistronic Reporter mRNAs Con-

taining the GATA-4 5�-UTR withinthe Intercistronic Region Demon-strate IRES Activity—To determinewhether the GATA-4 5�-UTR couldfunction as an IRES in vivo, weintroduced it into the intercistronic

region within the dicistronic mRNA reporter plasmid pRF togenerate pRGATA-4F (Fig. 3A). This plasmid contains theRenilla luciferase ORF as the first cistron and the firefly lucif-eraseORF as the second downstream cistron. In all the cell linestransfectedwith the pRGATA-4F construct downstream fireflyluciferase activity was 6–10-fold higher as compared with val-ues obtained for the control plasmid pRF (Fig. 3B). As a positivecontrol, transfection with a construct baring the c-myc IRES inthese lines also yielded firefly luciferase activities which werecomparable to those obtained with pRGATA-4F. Renilla lucif-erase levels were comparable in all the cell lines tested, suggest-ing that the steady-state mRNA levels and the relative levels of

FIGURE 3. The 5�-UTR of GATA-4 functions as an IRES in a dicistronic mRNA. A, schematic diagram ofdicistronic constructs containing the indicated sequences within the intercistronic region of plasmid pRF. TheIRES from the c-myc leader was used as a positive control (61, 62). B, relative firefly and Renilla luciferaseactivities of pRF, pRGATA-4F and pRmycF after transient transfection into the indicated cell lines (see legends).For each cell line the relative firefly or Renilla luciferase activities conferred by pRF are normalized to 1. Theresults shown are the mean and S.D. from four independent transfections. C, Northern blot analysis of mRNAisolated from H9c2 cells transiently transfected with no DNA or the pRF, pRmycF, or pRGATA-4F plasmids andhybridized with a riboprobe specific for sequences within the firefly luciferase coding region. D, RT-PCR analysisof H9c2 cells transiently transfected with pRGATA-4F plasmid. Specific primers (black arrows in A) were used toamplify a fragment (3.5 kb) spanning the entire firefly ORF, the intercistronic region containing the GATA-45�-UTR sequences and the Renilla ORF. �RT (lane 1) indicates the addition of reverse transcriptase while, �RT(lane 3) indicates a sample in which it was omitted.




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cap-dependent translation of the dicistronic constructs weresimilar. Furthermore, Northern analysis revealed a single stableluciferasemRNA species of the expected size for each construct(Fig. 3C).In an effort to more carefully examine the possibility of

monocistronic RNAs containing the firefly luciferase ORFbeing generated in vivo from these dicistronicmRNAs via aber-rant splicing events we attempted to detect splice variants byRT-PCR. The integrity of the mRNAs was determined usingprimers spanning the Renilla and firefly cistrons (shown in Fig.3A). As shown in Fig. 3D, a single RNA species of the expectedmolecular mass was amplified fromHeLa cells transfected withpRGATA-4F.No other RNAswere observed suggesting that noaberrant splicing was taking place. These results support thenotion that the GATA-4 5�-UTR can initiate translation inter-nally and that the firefly luciferase activity observed is not likelybecause of cap-dependent initiation of shorter monocistronicreporter transcripts.Ribosomal Readthrough Does Not Occur in Dicistronic Con-

structs Containing the GATA-4 5�-UTR—To determine if theGATA-4 5�-UTR could function in a dicistronic construct byfacilitating termination codon readthrough of the RenillaORFthrough the intercistronic region we inserted the hairpin struc-ture (Fig. 2A) upstream of the Renilla ORF within the dicis-tronic construct pRGATA-4F to generate phpRGATA-4F (Fig.4A). If the GATA-4 5�-UTR sequences were mediating ribo-somal readthrough of the Renilla termination codon, inhib-

iting translation of the upstreamORF should also result in a pro-portional reduction in firefly lucif-erase translation. We transfectedeither pRGATA-4F or phpRGATA-4Fin the indicated cell lines andassessed the relative levels of lucif-erase activity in extracts preparedfrom these cells. As shown in Fig.4B, introduction of the stable hair-pin reduced Renilla luciferase activ-ity by �80% as compared with cellstransfected with the control con-struct in the cell lines tested. How-ever, the downstream firefly lucifer-ase ORF remained well translatedand luciferase activity did notchange significantly comparing celllines transfected with the hairpin-containing construct to those trans-fected with the control dicistronicconstruct lacking the hairpin struc-ture (Fig. 4B). To control for thepossibility of activating a crypticpromoter within the GATA-45�-UTR sequences in the context ofthe dicistronic reporter plasmid wealso inserted the GATA-4 5�-UTRsequences in a promoterless versionof pRF and determined luciferaseactivity in HeLa cells transfected

with this construct �(p)RGATA-4F. As with the monocis-tronic promoterless control, (Fig. 2B) no significant luciferaseactivity was detected (Fig. 4B). These data support the notionthat the GATA-4 5�-UTR does not stimulate readthrough ofthe Renilla termination codon.Dicistronic RNA Transfections and Renilla Luciferase ORF

Knockdown Demonstrate GATA-4 5�-UTR IRES Activity—Ourdata to this point indicated the presence of an IRES within theGATA-4 5�-UTR sequences. However, in the light of severalrecent reports (31–33) describing cryptic promoter activity oraberrant splicing events leading to the generation of monocis-tronic firefly ORF containing transcripts, we decided to trans-fect the in vitro transcribed, capped, and polyadenylated dicis-tronic RNAs indicated in Fig. 5A. This approach avoids thepossibility of nuclear events altering reporter mRNA structureand requires only the cytoplasmic transfection of the tran-scripts.We generated plasmids, which produced the dicistronicRNAs, cRFpolyA30, cRgata-4polyA30, cRp27polyA30, andcRemcvFpolyA30 in vitro and transfected them into HeLa cellsby liposomal encapsulation (26). Twelve hours following trans-fection, cell extracts were prepared, and luciferase activitydetermined. As shown in Fig. 5B, the downstreamcistron fireflyluciferase activity wasmarkedly higher (�10–12-fold increase)in extracts prepared from cells transfected with the dicistronicmRNAs containing the cellular IRESs cRgata-4polyA30 andcRp27polyA30 mRNAs, and much higher in cells transfectedwith the dicistronic mRNA containing the viral (EMCV) IRES

FIGURE 4. GATA-4 5�-UTR sequences do not facilitate ribosomal readthrough in dicistronic plasmids.A, schematic diagram of plasmids used in transient transfections. �(p)RGATA-4F is identical to pRGATA-F otherthen removal of the SV40 promoter sequences. A stable hairpin structure was introduced upstream of theRenilla start to generate phpRGATA-4F. B, relative firefly and Renilla luciferase activities in extracts of theindicated transiently transfected cells with the indicated plasmids. pSV-�-gal was used as a control to normal-ize for transfection efficiencies. The relative firefly and Renilla luciferase conferred by the pRGATA-4F plasmidwas normalized to 1 for each cell line. The results shown are the mean and S.D. from four independenttransfections.




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cRemcvFpolyA30 mRNA, as compared with the control RNA,cRFpolyA30.In addition, we also attempted to knockdown Renilla lucifer-

ase expression by siRNA-mediated transfection. If downstreamfirefly luciferase expression was solely derived from dicistronicRNAs, not monocistronic firefly luciferase transcripts as aresult of cryptic promoter activity or aberrant splicing events,one would expect that siRNAs targeting the coding region ofthe Renilla ORF should inhibit firefly expression proportion-ately (28, 31). Thus, siRNAs targeting the Renilla ORF were

co-transfected into cells with the indicated constructs in Fig. 6.As can be seen, controls in which siRNAs were co-transfectedwith monocistronic Renilla and firefly luciferase constructsresulted in a specific reduction in Renilla luciferase activitywhile firefly activity was relatively unchanged. However, whenthe dicistronic control pRFwas co-transfected with the siRNAstargeting the Renilla ORF, we observed a 90% reduction inRenilla activity concomitant with a reduction of firefly lucifer-ase by a comparable amount, suggesting that both proteins aretranslated from the same dicistronic mRNA. Similarly, whencells were co-transfected with the siRNAs and either pRF con-taining the GATA-4 5�-UTR or the IRES sequences from theencephalomyocarditis virus within the intercistronic region,Renilla and firefly luciferase activities were reduced compara-bly. This suggested that the luciferase activities we observedwere derived from dicistronic mRNAs and taken together,these data support the notion that the GATA-4 5�-UTR con-tains a bona fide IRES.GATA-4 IRES Activity Is Not Stimulated during Differentia-

tion of Cardiomyocytes—Because GATA-4 plays a prominentrole in differentiation (34) and IRES activity has been reportedto be stimulated during this process (22), we examinedwhetherGATA-4 IRES activity was enhanced in P19CL6 cardiomyo-cytes induced to differentiate. P19CL6 clones which stablyexpressed the pRGATA-4F mRNA were initially examined forendogenous GATA-4 up-regulation as well as other proteinsknown to have increased expression following the induction ofdifferentiation in this cell line. Shown in Fig. 7A, GATA-4expression is increased by day 4 of culture in differentiationmedium (DM) and is relatively high by day 12 in DM. Steady-state GATA-4mRNA levels remained unchanged in differenti-ation media (Fig. 7B), supporting the notion that GATA-4 isregulated at the translational level. We then examined newGATA-4 protein synthesis in 35S-metabolically labeled P19CL6cells during differentiation. As shown in Fig. 7C, new GATA-4protein synthesis increased �4-fold in cells in differentiationmedia by day 12 relative to cells maintained in growthmedium.Increased expression of the cardiac contractile isoforms ofmyosin light and heavy chain proteins was also observed coin-cident with the induction of GATA-4 expression (Fig. 7A). ThepRGATA-4F mRNA was also found to be full-length as deter-

mined byNorthern analysis andwasunaffected by culture in DM (notshown).The phosphorylation of eIF-2�

has been shown to be required forIRES activity under several differentconditions including differentia-tion, although the underlyingmech-anisms are not clearly understood(15, 22, 54, 55). Thus, we deter-mined the phosphorylation status ofeIF-2� following the induction ofdifferentiation in P19CL6 cellsexpressing pRGATA-4F mRNA. Asshown in the inset of Fig. 7D, eIF-2�was significantly phosphorylated by24 h after the induction of differen-

FIGURE 5. Transfection of in vitro transcribed dicistronic mRNAs contain-ing the GATA-4 5�-UTR sequences demonstrates IRES activity. A, sche-matic diagrams of dicistronic mRNAs transiently transfected into HeLa cells. B,relative firefly and Renilla luciferase activities of extracts prepared from cellstransfected with the indicated dicistronic mRNAs. The relative firefly andRenilla luciferase activities of cRFpoly(A)30 were normalized to 1. The resultsshown are the mean and S.D. from four independent transfections.

FIGURE 6. Effects of siRNA targeting the upstream Renilla cistron on luciferase expression from dicis-tronic mRNAs containing the GATA-4 5�-UTR. The indicated plasmids were transfected with or without thesiRNA pool targeting the Renilla ORF into HeLa cells. pRL � pFL indicates co-transfection of monocistronicRenilla or firefly plasmids. The relative amounts of firefly and Renilla luciferase activities are shown as a percent-age relative to the activities in the absence of Renilla siRNA, which were set to 100%. The results shown are themean and S.D. from four independent transfections.




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tiation and was only slightly detectable by 48 h.We then exam-ined luciferase activity in lysates from cells induced to differen-tiate at various time points as compared with culturesmaintained in growth medium. As shown in Fig. 7D, there wasno significant increase in the ratio of firefly:Renilla luciferaseactivities in cells expressing the pRGATA-4F mRNA at anytime following the induction of differentiation. Similarly, incells stably expressing the control pRF mRNA, no changes infirefly:Renilla luciferase ratios were observed during differenti-ation. These data suggested that IRES-mediated initiation of

GATA-4 mRNA does not appear to occur in differentiatingP19CL6 cardiomyocytes.GATA-4 IRES Activity Is Stimulated during Vasopressin-in-

duced Hypertrophy of Cardiomyocytes—Because many IRESsaremost active during times of cellular stress and when generalmRNA translation initiation is inhibited (13, 35), we examinedwhether GATA-4 IRES activity was stimulated following theinduction of hypertrophy in cardiac myocytes. We initiallyexamined GATA-4 expression in H9c2 stimulated to undergohypertrophy by vasopressin exposure (36). Treatment of H9c2with vasopressin at concentrations of 10 nM or greater and atphysiologic extracellular Ca2� concentrations is biphasic and isknown to result in a rapid suppression of overall protein syn-thesis in association with phosphorylation of eIF-2� reachingmaximum inhibition by 30 min of exposure to the hormone.This initial reduction in protein synthesis is followed by a 1.2–1.5-fold stimulation of protein synthesis by 12–16 h of exposure(37). Significant hypertrophic growth occurs by 24 h followingvasopressin exposure with no change in cell number or cellcycle distribution (36, 37). Thus, we examined the affects ofvasopressin on GATA-4 expression at early and later timepoints. As shown in Fig. 8A, treatment of H9c2 cells with vaso-pressin resulted in a �10–12-fold increase in GATA-4 proteinlevels as early as 30 min following exposure to the hormone.The molecular chaperone BiP, a protein whose expression isknown to be stimulated following vasopressin-induced hyper-trophy (38, 39), demonstrated increasing expression, whileCdk1 protein content was inhibited within 1 h of exposure con-sistent with hypertrophic reprogramming.Northern analysis ofsteady-state GATA-4 mRNA abundance demonstrated only a�2-fold increase in transcript levels (Fig. 8B) by 120 min fol-lowing vasopressin. While these data were suggestive of trans-lational control of the GATA-4 mRNA following vasopressintreatment, we examined new GATA-4 protein synthesis fol-lowing exposure to the hormone. As shown in Fig. 8C, newGATA-4 protein synthesis was stimulated �7-fold relative tolevels in unstimulated control cells within 30 min of exposure.Increased synthesis was then maintained at �4–5-fold overunstimulated levels during hypertrophic growth. Polysomalanalysis of GATA-4 mRNA also demonstrated increased poly-somal association of GATA-4 mRNA by 30 min of vasopressintreatment (Fig. 8D) with 51% of the mRNA found in polysomalfractions as compared with 12% at time 0 of vasopressin treat-ment. A determination of the steady-state mRNA levels bysumming the nonribosomal, monosomal, and polysomal sig-nals also showed a �2.5-fold increase in mRNA abundance ofGATA-4 mRNA following vasopressin exposure (not shown).To determinewhetherGATA-4 IRES-mediated translation ini-tiation was also increased following vasopressin exposure, wetransiently transfected H9c2 cells with pRGATA-4F andexposed transfectants to vasopressin and assessed luciferaseactivities at various time points following exposure. Asshown in Fig. 8E, firefly luciferase activity was significantlyincreased within 30 min of vasopressin exposure and main-tained high levels of activity consistent with the increasesseen in new GATA-4 synthesis, translational efficiency andtotal GATA-4 protein expression following vasopressintreatment. No significant change in the firefly:Renilla ratios

FIGURE 7. GATA-4 IRES activity is not enhanced during differentiation.A, expression of GATA-4, MLC-2, �-MHC and actin in P19CL6 cardiomyo-cytes induced to differentiate (GM, growth media; DM, differentiationmedia). B, Northern analysis of GATA-4 mRNA and 18 S rRNA in P19CL6 cellstransferred to differentiation medium for the indicated time points. C, newGATA-4 protein synthesis in P19CL6 cells following transfer to differentiationmedia at the indicated time points. Fold increase in immunoprecipitatedGATA-4 from P19CL6 cells in DM relative to GM is shown. Cells were pulsedwith [35S]methionine/cystine during the last 3 h of culture in either DM or GM.Data presented are the mean and S.D. of three independent experiments.D, relative fold change in firefly:Renilla ratio following the induction of differ-entiation in P19CL6 cardiomyocytes stably expressing pRGATA-4F. The basallevel ratio of firefly:Renilla was normalized to 1 prior to the addition of differ-entiation medium. Circles denote values obtained from cells expressing thepRGATA-4F mRNA, while triangles denote values obtained from cells express-ing the control pRF mRNA in growth medium (open) and differentiationmedium (closed). Three independent stable transformants were tested, andthe mean and S.D. luciferase activities are shown. Inset, immunoblot analysisof eIF-2� phosphorylation on Ser-51 and total eIF-2� levels following theinduction of differentiation in P19CL6 cardiomyocytes.




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was observed in control cellstransfected with the pRF plasmidfollowing exposure to vasopressin.GATA-4 IRES activity remainedsignificantly higher, �50% of themaximal value during the hyper-trophic growth phase followinghormone stimulation. These experi-ments suggested that GATA-4 IRESactivity was stimulated during vaso-pressin-inducedhypertrophicgrowthof H9c2 cardiomyocytes.Protein Kinase C Activity Is

Required for GATA-4 IRES Activityduring Hypertrophy—Several reportshave implicated PKC activity inthe induction of translation of spe-cific mRNAs following vasopres-sin treatment in H9c2 cardiomyo-cytes (37, 38). Thus, we examinedwhether PKC activity was requiredfor GATA-4 IRES activity duringthe hypertrophic response to vaso-pressin. To inhibit PKC activity weutilized a cell-permeable myris-toylated pseudosubstrate fromPKC, inhibitor 20–28 (37, 40). Asshown in Fig. 9A, H9c2 cellsexposed to vasopressin displayedmarked activation of PKC within30 min of exposure. However,PKC activity was inhibited (�95%)by the pseudosubstrate peptide ata concentration of 12 �M. H9c2cells were transiently transfectedwith pRGATA-4F and treatedwith vasopressin in the presenceor absence of the PKC inhibitorand luciferase activities deter-mined at various time points. Asshown in Fig. 9B, the ratio of fireflyto Renilla luciferase activityincreased �9-fold within 30 minof vasopressin exposure in theabsence of inhibitor 20–28 pep-tide. This relative increase in fire-fly luciferase activity subsequentlydiminished by �50% of maximalvalues coincident with thedephosphorylation of eIF-2� (Fig.9C) and the induction of cap-de-pendent protein synthesis by 1 hfollowing exposure to vasopressin.We note however, that theobserved decrease in GATA-4IRES activity from its maximalactivity at 30 min following vaso-pressin exposure to its submaxi-

FIGURE 8. GATA-4 IRES activity is stimulated during vasopressin-induced hypertrophy. A, expressionof GATA-4, Cdk1, BiP, phospho-eIF-2�, and actin following AVP exposure of H9c2 cardiomyocytes atvarious time points. B, steady-state GATA-4 mRNA levels in H9c2 cells following AVP exposure at theindicated time points. 18 S rRNA levels are shown below. Values in parentheses below represent foldincreases in GATA-4 mRNA abundance at the indicated time points relative to untreated cells as deter-mined by densitometry. C, new GATA-4 protein synthesis in H9c2 cells exposed to AVP at the indicatedtime points. Cells were pulsed with [35S]methionine/cystine for the last 20 min of treatment for measure-ments of early time points (left panel), and the last 3 h of treatment for the later time points (right panel).Extracts were prepared and GATA-4 immunoprecipitated. The corresponding densitometric values areshown as fold increase relative to control untreated cells. The results shown are the mean and S.D. of threeseparate experiments. D, polysomal association of GATA-4 mRNAs following AVP treatment. Representa-tive polysome profiles are shown above and fractions were pooled into nonribosomal-monosomal (N,open bars) and polysomal (P, closed bars) RNA fractions at 0, 0.5, and 6 h following H9c2 cell exposure toAVP, then subjected to real-time quantitative RT-PCR analysis to quantify the indicated mRNA levels in thetwo fractions. Measurements were done in quadruplicate, and the mean and S.D. are shown. E, H9c2 cellswere transiently transfected with pRGATA-4F (open bars) or pRF (closed bars) and treated with AVP for theindicated time points. Results are expressed as the relative fold change in firefly:Renilla ratio in the pres-ence of AVP as compared with ratios obtained in the absence of AVP. The results shown are the mean andS.D. of four individual experiments.




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mal value would reflect decay of the firefly luciferasereporter. This apparent decrease in luciferase activity is con-sistent with its relatively short protein half-life in vivo (�2 h)(41). The ratio of firefly to Renilla luciferase activity onlyminimally increased following exposure to vasopressin in

cells pretreated with the PKCpseudosubstrate peptide. Theseresults suggested that PKC activitywas required for the induction ofGATA-4 IRES activity followingvasopressin exposure. The GATA-4increase in protein abundance fol-lowing vasopressin exposure wasalso inhibited by treatment with thePKC inhibitor (Fig. 9D). Similarly,BiP expression was also dependenton PKC activity following vasopres-sin exposure as had been demon-strated previously (38).Supra-physiological Ca2� Levels

Reduce Vasopressin-induced GATA-4IRES Activity—Because the contin-ued synthesis of several proteinshas been demonstrated to besensitive to ER-associated Ca2�

mobilization in H9c2 cells follow-ing vasopressin induced hypertro-phy (37, 39), we tested whetherelevated extracellular Ca2� levelsaffected GATA-4 IRES activityand protein levels following vaso-pressin exposure. These high Ca2�

levels are expected to diminish ERCa2� depletion following hormo-nal challenge (37). As shown inFig. 9B, treatment of cells trans-fected with pRGATA-4F withvasopressin in the presence of 3mM Ca2� resulted in an �90%reduction in firefly:Renilla lucifer-ase activity ratio by 30 min as com-pared with controls containingphysiological Ca2� levels. Vasopres-sin-mediated induction of GATA-4protein levels were also significantlyreduced in the presence of elevatedCa2� (Fig. 9C). In contrast, whenwepretreated cells with the Ca2� chan-nel blocker nifedipine, which blocksplasmalemmal Ca2� entry and hasbeen previously demonstrated toreduce Ca2� content of H9c2 cellsby �40% at the concentration used(37), vasopressin treatment stillresulted in the comparable stimu-lation of GATA-4 IRES activity(Fig. 9B) and induction of proteinlevels (Fig. 9D) as had previously

been observed with the hormone alone. These data supportthe notion that the increased IRES activity and proteinexpression of GATA-4 in H9c2 cells following vasopressintreatment is dependent on the mobilization of Ca2� seques-tered by the ER.

FIGURE 9. Vasopressin-induced GATA-4 IRES activity requires PKC and is inhibited by supraphysiologicalCa2� levels. A, AVP-induced PKC activity is inhibited by a pseudosubstrate peptide inhibitor 20 –28 (PS). H9c2cells were exposed to AVP for 30 min in the absence or presence of the PKC inhibitor at the indicated concen-trations. The results shown are the mean and S.D. of three independent experiments. B, H9c2 cells weretransiently transfected with pRGATA-4F and exposed to AVP alone or with indicated inhibitors or mediumcontaining elevated Ca2� for the indicated time points. The relative fold change in firefly:Renilla ratio is shownobtained following the indicated treatments as compared with luciferase values obtained with no treatments.The results shown are the mean and S.D. of four individual experiments. C, kinetics of eIF-2� phosphorylationin H9c2 cells following exposure to AVP for the indicated time points. Immunoblot was probed with an anti-body specific for serine 51 phosphorylated eIF2� and total eIF2�. D, steady-state expression levels of GATA-4,BiP, Cdk1, and actin in H9c2 cells under the conditions as in B. Immunoblots in C and D are representative of fourindependent experiments with similar results. E, translational regulation of the GATA-4 IRES may involve aPKC-induced or activated ITAF following binding of vasopressin to V1 receptors (V1R) and subsequent activa-tion of PKC.




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DISCUSSION

Our previous studies implicated a cap-independent mecha-nism of translation initiation for a number of specific tran-scripts under conditions of impaired eIF-4F formation (11). Inthis report we have investigated the mechanism of initiation ofthe GATA-4 mRNA and identified an IRES within its 5�-UTR.Moreover, we have confirmed IRES activity by several criteria,which support the hypothesis that the GATA-4 transcript isable to initiate translation internally such that cap recognitionis not required. We also demonstrate that the GATA-4 mRNAis subject to translational control during differentiation andhypertrophy. We have also demonstrated that GATA-4 IRESactivity is stimulated maximally, coincident with phosphoryla-tion of eIF-2�, and continues during hypertrophic growth ofcardiomyocytes following treatment with the hormone argi-nine vasopressin in a PKC-dependent manner. Additionally,our results suggest that Ca2� mobilization from intracellularstores is also required for maximal IRES activity immediatelyfollowing exposure to the hormone. Our results are consistentwith amodel inwhich vasopressin treatment of cardiomyocytesresults in the initial inhibition of global cap-dependent proteinsynthesis attributable to phosphorylation of eIF-2�, duringwhich time GATA-4 IRES activity is strongly stimulated as aresult of Ca2� release from the sarcoplasmic/endoplasmicreticulum (S(E)R) and activation of PKC. The subsequentrecovery from this inhibition results in an increase in cap-de-pendent protein synthesis, during the hypertrophic growthphase, at which time GATA-4 IRES activity is maintained atsubmaximal levels and is dependent on PKC activity for contin-ued function. Thus, GATA-4 protein synthesis is specificallyinduced during hypertrophic reprogramming and its synthesisis maintained during hypertrophy leading to transactivation ofgenes required for the accumulation of cell size.The observation that Ca2� release from S(E)R stores induces

rapidGATA-4 IRES activity is intriguing. Several Ca2�-bindingproteins are known to interact with RNAs to regulate mRNAstability (42, 43) and indeed we noted a small but significantincrease in the steady-state mRNA levels of GATA-4 followingvasopressin exposure. This increase in abundance may be theresult of effects on transcription ormRNA stability. However, itis also possible that such Ca2�-binding factors may directly orindirectly regulate IRES activity in response to changes in cyto-solic Ca2� levels. Indeed, the IRES-trans acting factors (ITAFs)hnRNP A and hnRNP C are known to bind calmodulin in thepresence of Ca2� (44) and in addition, hnRNP A is also knownto be regulated by PKC phosphorylation which alters its abilityto bind RNA (45, 46). Thus, these data potentially link Ca2�

signaling pathways to ITAF activity. Ongoing studieswill inves-tigate whether hnRNP A regulates GATA-4 IRES activitybecause our experiments show a requirement for PKC activityfor GATA-4 IRES function during hypertrophy.Our data imply that the GATA-4 IRES is also active during

hypertrophic growth when the default cap-dependent initia-tionmechanism is active. Several reports have documented therequirement for mTOR and its downstream effector S6K1 incardiac myocyte hypertrophy during the activation of cap-de-pendent initiation (47–51). Our data support a role for the cap-

independent synthesis of proteins during this process. The acti-vation of cap-dependent initiationmay seem incompatiblewiththe continued IRES-mediated initiation of GATA-4 mRNA weobserved in our experiments. However, currentmodels regard-ing the control of cap-independent protein synthesis suggestthat some IRES-containing mRNAs may be able to initiatetranslation by both cap-dependent and cap-independentmechanisms depending on ensuing conditions and that the twomechanisms may not be mutually exclusive (13, 15). Indeed,several IRES have strict requirements for canonical initiationfactors (15, 52). Furthermore, a cap-dependent IRES hasbeen described within the eIF-4G1 mRNA which is activefollowing poliovirus infection (53). We speculate that duringcardiomyocyte hypertrophic reprogramming a specificITAF(s) is activated which enhances GATA-4 IRES activityand allows continued, albeit, reduced activity while condi-tions for cap-dependent initiation are also permissive.Several studies have demonstrated a role for eIF-2� phos-

phorylation in the regulation of particular IRESs (22, 54, 55).However, the mechanisms by which enhanced eIF-2� phos-phorylation leads to stimulation of IRES activity is not wellunderstood. One possible mechanism may be the ability of aparticular IRES to direct efficient initiation in the absenceof translation-competent ternary complexes (eIF2/GTP/tRNAMet-i) as a result of eIF-2� phosphorylation. Recentreports have described initiation factor-independent transla-tion for viral IRESs (56, 57).Whether any cellular IRESs are ableto initiate in a similar manner is unknown. Another possibilitymay be that eIF-2� phosphorylation indirectly stimulates IRESactivity via induction or activation of an ITAF. During vaso-pressin-induced hypertrophy, stimulation of GATA-4 IRESactivity correlated with enhanced eIF-2� phosphorylation (Fig.9,B andC), however, themechanism bywhich these two eventsmay be linked is unclear. It is possible that eIF-2� phosphoryl-ation results in accumulation or activation of an ITAF, which isrequired for GATA-4 IRES activity as has been postulated forother cellular IRESs whose activity is regulated by eIF-2� phos-phorylation status (54). However, the observation that theGATA-4 IRES did not show elevated activity during P19CL6cell differentiation, even though initially increased eIF-2�phosphorylation was observed, suggests that eIF-2� phospho-rylation alone is insufficient to stimulate GATA-4 IRES activitydirectly. Specific ITAFs required forGATA-4 IRES activitymaynot be present or activated such as to stimulate IRES activityunder this condition. In fact, we examined whether PKC activ-ity was stimulated during differentiation of P19CL6 cells anddid not observe significant activation (not shown). Thus, it ispossible that a PKC-induced or activated ITAF regulatesGATA-4 IRES activity during vasopressin-induced hypertro-phy which is absent during differentiation (Fig. 9E). Futureexperiments will address what additional factors are requiredfor GATA-4 IRES activity under these conditions.

Acknowledgments—We thank Drs. Anne Willis, J.-T. Zhang, andLynne Maquat for providing reagents. We also thank Ardella Sher-wood for excellent administrative assistance. J. G. thanks Dr. AlanLichtenstein for his continued support.




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Alexander Funk and Joseph GeraAnushree Sharma, Janine Masri, Oak D. Jo, Andrew Bernath, Jheralyn Martin,mRNA following Vasopressin-induced Hypertrophy of Cardiac Myocytes

Protein Kinase C Regulates Internal Initiation of Translation of the GATA-4

doi: 10.1074/jbc.M608874200 originally published online February 6, 20072007, 282:9505-9516.J. Biol. Chem.

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proteinkinasecregulatesinternalinitiationoftranslation ... · rnai analysis—knockdown experiments...

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