THE JOURNAL OF BOLOGCAL CHEMSTRY Vol. 68, No., ssue of August 5, pp. 6664667,99 Printed in U. S. A. Cyloheximide Stabilizes nsulinlike Growth Fatorbinding Protein (GFBP) mrna and nhibits GFBP Transription H4E in Rat Hepatoma Cells (Reeived for publiation, February, 99) Guk T. Ooi& Daniel R. Brown$, DaeShik Suh, Luy Y.H. Tseng, and Matthew M. Rehler From the Growth and Development Setion, Moleular and Cellular Endorinology Branh, National nstitute of Diabetes and Digestive and Kidney Diseases, National nstitutes of Health, Bethesda, Maryland 89 The insulinlike growth fatorbinding proteins The insulinlike growth fators, GF and GF, are (GFBPs) are a family of six proteins that modulate polypeptides hemially related to insulin that are widely the biologial ativity of GF and GF and deter expressed in fetal and adult mammalian tissues (). They mine their bioavailability to tissues. One of the GFBPs, GFBP, is distintive in the dynami response of its levels in human plasma to metaboli hanges. Parallel hanges our in GFBP mrna have a broad array of biologial ativities, inluding the stimulation of mitogenesis, differentiation, the expression of differentiated funtions, and insulinlike metaboli ativity (). GF and GF at by binding to the GF reeptor and and GFBP transription in rat liver. Using the well possibly the GFmannose 6phosphate and insulin reepdifferentiated H4E rat hepatoma ell line as a model tors (). The biologial ativities of the GFs are modulated system, we demonstrated previously that GFBP by the GFbinding proteins (GFBPs), family a of six related transription is positively regulated by dexamethasone proteins that speifially bind GF and GF, whih deterand negatively regulated by insulin. We now examine mine their bioavailability to tissues (). the effet of the protein synthesis inhibitor, ylohex One of the GFBPs, GFBP, is distintive in that its imide, on the hormonal regulation of GFBP gene levels in plasma are dynamially regulated by hanges in expression. Preinubation of H4E ells with.7 metaboli state. Plasma GFBP is inreased by fasting (4, ~ Lyloheximide M for.5 h did not prevent the indu 5) and diabetes (6,7) and rapidly normalized by refeeding and tion of GFBP mrna and GFBP transription insulin treatment, respetively. Although transloation of (determined in nulear runon assays) by dexametha GFBP from plasma to the extravasular spae may ontribute to this regulation (8), sone. By ontrast, yloheximide treatment abolished evidene from rat model systems suggests that rapid regulation of GFBP transription and the derease in GFBP mrna indued by insulin. GFBP mrna abundane play a ritial role. GFBP nsulin rapidly dereased GFBP transription in the absene of yloheximide (>5% inhibition in min) and aused a similar derease in ells pretreated with yloheximide. Cyloheximide alone also dereased GFBP transription. Similar results were observed with a seond protein synthesis inhibitor, anisomyin, whih also prevented the insulinindued derease in GFBP mrna without abolishing the insulinindued inhibition of GFBP transription. These results suggest that although insulin dereases GFBP gene transription in the presene of protein synthesis inhibitors, GFBP mrna levels are maintained beause of stabilization of the mrna. Stabilization was demonstrated diretly in atinomyin Dtreated ells, transription is inreased in diabeti rat liver and normalized within h following insulin treatment (9, ). nsulin aused a similar rapid derease in hepati GFBP mrna, indiating that the rapid inhibition of GFBP transription is aompanied by rapid turnover of GFBP mrna in uiuo. n order to study the moleular events involved in the regulation of GFBP gene expression, we have used a rat hepatoma ell line, H4E, that was established from the well differentiated Reuber H5 hepatoma (). This ell line expresses many hepati proteins, is responsive to gluoortioids and insulin, and synthesizes GFBP as its predominant GFBP (, ). GFBP mrna abundane in H4 E ells is inreased by the syntheti gluoortioid, dexamethasone, and dereased by insulin (4,5). These hanges our predominantly at the level of gene transription, with the inhibitory effet of insulin being dominant over the stimula where the tln of GFBP mrna inreased from to h in the presene of yloheximide; insulin did not affet GFBP mrna turnover. Thus, yloheximide tory effet of dexamethasone (4, 5). sensitive labile proteins ontribute to the maintenane The regulation of gene transription by gluoortioids of basal GFBP promoter ativity and the rapid turn involves the binding of a ligandativated gluoortioid over of GFBP mrna, whih determine the dynami reeptor to a speifi DNA element in the promoter, the regulation of GFBP gene expression. gluoortioid response element (GRE) (68). n some ases, suh as the phosphoenolpyruvate arboxykinase The osts of publiation of this artile were defrayed in part by (PEPCK) (8) and the alaid glyoprotein (9, ) prothe payment of page harges. This artile must therefore be hereby marked aduertisement in aordane with 8 U.S.C. Setion 74 moters, the response is modified by the funtional interation solely to indiate this fat. $To whom orrespondene and reprint requests should be addressed National nstitutes of Health, NDDK, Bldg., Rm. 8D4, Bethesda, MD 89. Tel.: 49648; Fax: 446. Supported in part by the Summer Student Researh Program of the Juvenile Diabetes Foundation. The abbreviations used are: GF, insulinlike growth fator; GFBP, insulinlike growth fatorbinding protein; GRE, gluoortioid response element; PEPCK, phosphoenolpyruvate arboxykinase; BSA, bovine serum albumin; Mops, (Nmorpholino)propanesulfoni aid; RE, iron response element; kb, kilobase(s). 6664
Cyloheximide Stabilizes GFBP mrna 6665 of the GRE with other elements in the hormone response unit after.5 h ompared with ontrol ells (data not shown). Anisomyin that bind aessory transription fators (8). The fators was used at a final onentration of pm (.65 pgml), whih inhibited [H]leuine inorporation by 97% after h (9). Cylohexresponsible for transriptional regulation by insulin are less imide pretreatment had little effet on GFBP mrna levels; in well understood (). Several potential insulin regulatory experiments, GFBP mrna in H4E ells inubated with yloelements have been proposed (4), but the fators binding heximide for.5 h was44 f 4% (mean S.E.) of the level in to these sites have not been onviningly identified. nontreated ontrol ells (data not shown). One approah to disseting the pathways of transriptional Northern Blot HybridizationTotal RNA was prepared using the regulation has been through the use of protein synthesis aid guanidine thioyanatephenolhloroform method (). The RNA (5 pg) was frationated by eletrophoresis on.5% agaroseinhibitors (5, 6). The indution of gene transription by formaldehyde gels in Mops buffer and blotted to nylon membranes gluoortioids is unaffeted by inhibition of protein synthesis (Genesreen, Du PontNew England Nulear) as desribedpreviously if the effet is mediated by the stable gluoortioid reeptor (). Ethidium bromide staining onfirmed that the ribosomal RNAs alone or together with stable aessory proteins, but is inhib were intat and that equal amounts of RNA were loaded in eah lane. ited if the response requires the indution of a seond gene Total RNA from day gestation or day neonatal rat liver were inluded in eah blot to ontrol for blottoblot variations in the by gluoortioids (5) or if labile aessory transription hybridization. Where appropriate, hybridized radioativity was norfators are involved. Expression of another group of genes, malized against the ounts hybridized to fetal or neonataliver RNA. variously known as immediate early, early growth response, The rat GFBP DNA probe used for hybridization was generated or primary response genes, is rapidly indued by mitogens in by polymerase hain reation amplifiation of a 67base pair fragthe absene of protein synthesis and is thought to our by ment ontaining part of exons and (orresponding to nuleotides ativation of preexisting transription fators (6). Primary 6 to 557 with respet to the ATG translation initiation site) of the DNA isolated from H4E ells (GenBank aession no. response genes, inluding several nulear transription fators M8979). The rat PEPCK DNA probe was a gift from Dr. Rihard and ytoskeletal proteins, have been identified among the Hanson and orresponds to the.kb Pst fragment of ppcklo mrnas that are transribed following the appropriate stim (). The DNA probes were labeled with [x~'p]~ctp (6 Ci ulus in the presene of a protein synthesis inhibitor suh as mmol; Amersham Corp.) by random priming using a kit obtained yloheximide (6). Mohn et al. (7) isolated DNA lones for from Stratagene (La Jolla, CA). The blots were hybridized at 5 "C GFBP from libraries made from RNA isolated from the as desribed previously (4). Nulear Transription AssaysNuleiwere prepared from onregenerating liver of rats that had been treated with ylohex fluent HE ell ultures as desribed previously (5). Six mm imide and onluded that protein synthesis was not required plates were ombined for eah treatment ondition. Cells were washed for the indution of GFBP transription in this experimen with ieold phosphatebuffered saline, sraped, and olleted by tal model. entrifugation (5 min, 4 "C, 5 rpm). The ell pellet was resus n the present study, we examine the effet of yloheximide pended in mm TrisHC, ph 7.9, ontaining mm NaCl, 5 mm MgC, and mm dithiothreitol. Nonidet P4 was slowly added to a on the indution of GFBP gene expression by dexamethafinal onentration of.5% to lyse the ells. Nulei were olleted sone and its inhibition by insulin in H4E ells to determine by entrifugation (5 min, 4 "C, 5 rpm), resuspended in mm whether de mu protein synthesis is required for the regula TrisHC, ph 7.9, ontaining mm NaC, 5 mm MgClz, mm tion of GFBP gene expression by these hormones. dithiothreitol, and 4% glyerol and stored at 7 "C until use. Nulear runon transription assays were performed as desribed EXPERMENTAL PROCEDURES previously (5). Radioative RNA transripts were prepared using [x~p]ctp and [w'p]gtp(amersham Corp.) and hybridized to MaterialsPorine insulin was obtained from Lilly, dissolved in target DNAs immobilized onto nitroellulose. The rat GFBP DNA mm aeti aid (5 mgml), and stored at "C. Dexamethasone target was a 74base pair fragment (orresponding to nuleotides (Sigma) was dissolved in ethanol to a stok onentration of mm. 6 to 58 of the DNA relative to the translation start site; pg Bovine serum albumin (BSA; radioimmunoassay grade), ylohexisample) made by polymerase hain reation amplifiation. Linearized mide, anisomyin, and atinomyin D were purhased from Sigma. DNA ( pg) from plasmid pgem7zf() (Promega, Madison, W) or Cyloheximide was dissolved in water as a 5 mgml stok solution. filters without DNA were used as targets for bakground hybridiza Anisomyin and atinomyin D were freshly prepared in ethanol at tion. onentrations of mm and mgml, respetively. Quaatitation of Hybridization ResultsHybridized radioativity in Cultivation of H4E CellsThe H4E ell line was obtained the Northern blot and transription runon experiments were quanfrom J. W. Koontz (University of Tennessee, Knoxville, TN) (8). titated by @sanning using a omputerdriven radioativity sanner Cells were grown in 75mZ flasks (Beton Dikinson and Co., (AMBS Sanning System, Automated Mirobiology Systems n., Linoln Park, NJ) as monolayer ultures in RPM 64 (GBCO) San Diego, CA). Results are expressed as ounts hybridized, following supplemented with % fetal bovine serum (Flow Laboratories, lot subtration of bakground radioativity hybridized to an equivalent 9474) and inubated in 95% air, 5% Cor! at 7 "C. Cultures were area of the Northern bot or nulear transript radioativity hybridfed twie weekly and passaged weekly (when they reahed onfluene) ized to filters ontaining either no DNA or plasmid DNA in the same at a ratio of :5 using trypsinedta. Confluent ells, 6 passages inubation. after thawing, were used for all experiments. Hormonal Treatment of CellsFor experiments, ells were plated in mm ulture dishes and grown to onfluene in the presene of RESULTS % fetal bovine serum. Confluent ultures were inubated with Dexamethasone nreased GFBP mrna and GFBP serumfree RPM 64 medium ontaining.% BSA for 64 h, Transription in Both Cyloheximidetreated and Untreated after whih the medium was replaed with fresh serumfree medium and equilibrated for a further 6 h before addition of hormones or H4E CellsGFBP mrna levels were relatively unafprotein synthesis inhibitors. nsulin was added diretly to the medium feted when H4E ells were inubated with yloheximide to give a final onentration of pgml. Some ultures were treated alone for up to 4.5 h (h inubation.5h preinubation) with dexamethasone ( p~ final onentration) for or 6 h before but dereased after 7.5 h (6h inubation.5h preinubathe addition of insulin to inrease the starting levels of GFBP tion) (Fig. ). To avoid toxi effets assoiated with longer mrna. inubations, the time of inubation with yloheximide in nhibition of Protein Synthesis in H4E CellsCyloheximide and anisomyin were added diretly to the media.5 h before the subsequent experiments did not exeed 4.5 h. addition of hormones (preinubation) unless otherwise stated and As reported previously (4, 5), following the addition of remained in the media following hormone addition to minimize dexamethasone, GFBP mrna levels inreased progreshanges assoiated with media hange. Cyloheximide was used at a sively at and 6 h (Fig. ). When H4E ells were pretreated onentration of.7 PM ( pgml), whih inhibited ['4C]leuine inorporation into trihloroaeti aidpreipitable proteins by 9% G. T. Ooi, unpublished results.
Cylo Dex GFBP mrna Cyloheximide Stabilizes 6666.5 6 (h) B B W U BB F ~ ~ FG.. Effet of yloheximide o n steadystate levels of GFBP mrna in basal and dexamethasonestimulatedells. Confluent H4E ells were onditioned in serumfree medium ontaining.% BSA for 4 h, after whih the medium was hanged to freshserumfree medium. Someultures were pretreatedwith yloheximide (.7 PM) for.5 h, after whih dexamethasone (final onentration of PM) was added (time ) to yloheximidetreated and untreated ultures. Control ultures didreeive not yloheximide or dexamethasone,butotherwise were handled identially. Total RNA was prepared,.5,, and 6 h after the timeof dexamethasone addition from dupliate plates for eah sample. The autoradiograph shows Northern hlot hyhridization of the isolated total RNAs using a rat GFBP DNA probe. Radioativity hybridized to RNA from ontrol ells (Cylo, Der) didnothange over the 6 h of the experiment (not shown). Only the region of the autoradiograph showing the single.55kh GFBP mrna band is shown. For ease of omparison, the time yloheximidetreated samples are shownfor (Cyle, Der) andnontreated boththedexamethasonetreated (Cyle, Dex) time ourses. with yloheximide for.5 h before adding dexamethasone, the dexamethasoneindued inrease in GFBP mrna was still observed (Fig. ).n the experiment shown, the inrease ourred earlier (at.5 and h) in yloheximidetreated ells than inells that did notreeive yloheximide, but thismore rapid responsewas not observed inotherexperiments. GFBP mrnadereased in dexamethasonetreated ells after 7.5 h of yloheximide treatment (6 h after dexamethasone addition), as observed in ells that had been inubated with yloheximide alone for 7.5 h. Wereported previously that dexamethasoneinreased GFBP mrna in H4E ells by inreasing transription without affeting GFBP mrna stability (4, 5). The fat thatgfbpmrna wasindued by dexamethasonein yloheximidetreated ells suggested that inhibition of protein synthesis did not affet the indution of GFBP transription by dexamethasone. To test this diretly, nulear runon transription assays were performed on ultures that had been pretreatedwith yloheximide for.5 hbefore adding dexamethasonefor h. Although yloheximide treatment alone dereased basal GFBP transription (see below), dexamethasone inreased GFBP gene transription 45fold in nulei from both yloheximidetreated and untreated ultures (Fig. ). These results indiate that ongoing protein synthesis is not neessary forthe indutionof GFBP transription by gluoortioids. Cyloheximide Abolished the nhibition of GFBP mrna by nsulinwe reported previously that insulin treatmentof H4E ells auseda rapid derease in GFBP mrna (5). T o determine the effets of yloheximide on the inhibition of GFBP mrnaby insulin, half of the ultureswere pretreated with yloheximide for.5 h, after whih insulin was added to all ultures without a hange in media (Fig. ). n ells that did not reeive yloheximide, insulin treatment dereased GFBP mrna 65% after.5 h and 95% after h. n ontrast, the derease in GFBP mrna following insulin treatment wasabolishedin ultures that had been pretreated with yloheximide. Cyloheximide also abolished the inhibition of GFBP mrna by insulin in H4E ells thathad been stimulated previously withdexamethasone (data not shown). Cylo Cylo GFBP pgem GFBP pgem Cont h E [ l A B C D E F FG.. Dexamethasone inreases GFBP gene transription in yloheximidepretreated and untreated ells. Confluent H4E ells were equilibrated for 6 h in serumfree medium ontaining.% RSA. Onehalf of the ultures were pretreated with yloheximide for.5 h after whih dexamethasone was added diretly to some ultures without a media hange. Nulei were prepared a t the time of dexamethasone addition (Cont h ) and from ultures that did ( D e r h ) and did not (Cont h ) reeive dexamethasone for h. Radioative elongated RNA transripts were prepared from two preparations of nulei for eah experimental treatment a t eah time point asdesribed under Experimental Proedures. n theleft panel (Cylo), eah horizontal row represents hybridization to the same transript preparation from ells not treated with yloheximide; in the right panel (Cyle), eah row was hybridized to the same transript preparation from ells that had been inubated with yloheximide. Transripts ( X lo6 pmhybridization) were hybridized to filters ontaining rat GFBP (A, R, D, E ) or pgem7 plasmid (C, F ) DNA as target or to filters not ontaining any DNA (not shown). Themeanbakground radioativityusing pgem7 DNA was not signifiantly different from that hybridized to filters that did not ontain DNA (797? and 77 f 86 ounts, respetively). Cyloheximide treatment alone dereased hasal GFBP transription to 8 and %of ontrol a t.5 and 4.5 h,respetively, in this experiment. nsulin Rapidly Dereased GFBP Gene Transription in H4E CellsWe haveshown previously thatgfbp transription was dereased after h of insulin treatment of H4E ells (5). T o examine the time ourse of this inhibition, ells were preinubated with dexamethasone for h to partially indue GFBP transripts. GFBP transription ontinued to inrease during the additional 8min inubation withdexamethasone (Fig. 4).nontrast, in ultures that reeived insulin, a major derease (75%) in GFBP transription was observed min after addition of insulin, the firsttimepoint examined. After8min,transription in insulintreated ells was only 5% of that in ells that did not reeive insulin. Thetl of this inhibition was 9 min. nsulin Dereased GFBPTransriptionin Cyloheximidepretreated CellsWe next asked whetheryloheximide abolished the inhibition of GFBP mrna by insulin by abrogating the inhibition of GFBP gene transription by insulin. H4E ells were treated with dexamethasone for 6 h and then preinubated with or without yloheximide for.5 h, after whih insulin was added for.5 h. nsulin dereased GFBP transription by 9% inyloheximidetreated and untreated ells (Figs. 5 and 6, Table ). Despite this inhibitionof transription, in the same experiment(figs. 5 and 6), steadystate levels of GFBP mrna were not dereased by insulin in yloheximidetreated ells. n these experiments, we also noted that treatment with yloheximide alone partially dereased GFBP gene transription in a timedependent manner (Table ). GFBP transription was dereased 64% after.5 h and 89% after h of yloheximide treatment. As the inhibition of GFBP transription indued by yloheximide was less omplete thantheinhibitionaused by insulin, it was possible to
Cylo.,.5.5 (h) Time (rnin) 4 8 Cyloheximide Stabilizes GFBP mrna 6667 5 R Dex ns h n?= 8 L v 4 Hours FG.. Effet of yloheximide on the inhibition of GFBP mrna by insulin. Confluent H4E ells were inubated in serumfree medium ontaining.% BSA for 6 h and refed with fresh serumfree medium. After h, yloheximide was added to onehalf of the ultures. After.5h pretreatment, insulin was added to all of the ultures (time ) and the inubation ontinued. Total RNA was prepared from dupliate ultures,.5, and h after insulin addition. Top, autoradiograph showing Northern blot hybridization using a rat GFBP DNA probe. Treatment times refer to the duration of exposure of the ells to insulin. Bottom, the hybridized radioativity shown in the autoradiograph above was quantitated by @sanning. Mean f S.D. are plotted at different times after insulin addition. Open irles, ells that reeived yloheximide; losed irles, ells not treated with yloheximide. appreiate a further inhibition of transription upon addition of insulin to yloheximidetreated ells. Cyloheximide Prolongs the Halflife of GFBP mrna in H4E CellsThe fat that insulin inhibited GFBP transription without dereasing GFBP mrna levels in yloheximidetreated H4E ells suggested that yloheximide treatment stabilized GFBP mrna. This was demonstrated diretly by measuring the halflife of GFBP mrna following the addition of atinomyin D to inhibit new RNA synthesis. Confluent ells were inubated overnight with dexamethasone to inrease the level of GFBP mrna at the start of the experiment. Some ultures were pretreated with yloheximide for.5 h, after whih atinomyin D was added 4 6 8 Minutes FG. 4. nsulin rapidly dereases GFBP gene transription in dexamethasonestimulated H4E ells. Confluent H4 E ells were inubated with PM dexamethasone for h, after whih insulin was added to some of the ultures. Nulei were prepared,, 4, and 8 min after insulin addition (Dex ns) and from ultures that did not reeive insulin (Dex) at and 8 min. Radioative RNA transripts were prepared as desribed under Experimental Proedures. Two preparations of nulei (6 platespreparation) were examined for eah experimental treatment at eah time point and hybridized to nitroellulose filters ontaining immobilized rat GFBP DNA as target. Top panel, autoradiograph showing hybridization of dupliate samples of radioative RNA transripts (.8 X lo6 pm) to GFBP target DNA. Cultures not treated with to all ultures to inhibit RNA synthesis (time O), and the insulin were examined only at the start and end of the experiment ( remaining GFBP mrna quantitated at different times and 8 min). Bottom panel, the hybridized radioativity was quantitated by @sanning. Results are the mean? S.D. of the dupliate after atinomyin D addition. Some ultures reeived insulin samples. Bakground radioativity hybridized to filters ontaining at the same time as atinomyin D. pgem7 DNA ( f 7 ounts) was not signifiantly different GFBP mrna dereased by first order kinetis in both from radioativity hybridized to filters without DNA (55 f 4 yloheximidepretreated and untreated ells (Fig. 7). The ounts) and has been subtrated. halflife of GFBP mrna in nonyloheximidetreated ells was h and is omparable in the absene and presene GFBP mrna in yloheximidetreated ells, however, was of insulin. This is onsistent with the observations that insulin dereased GFBP mrna predominantly through inhibition of transription (,5) and that insulin did not alter inreased to 57 h, indiating that the turnover of GFBP mrna was dereased. Effets of Anisomyin on GFBP Transription and the degradation of GFBP mrna (). The halflife of GFBP mrna in H4E CellsTo establish that the
Cyloheximide Stabilizes GFBP mrna 6668 TransriDtion :C&o GFBP Cont.5 h Contoh ns.5 h in GFBP mrna seen in ells that did not reeive anisomyin wasgreatly redued in anisomyintreatedells. nsulin treatment for hdereased GFBP mrna by 9% in ells that did not reeive anisomyin, ontrasted with only an 4% derease in anisomyintreatedells (Table ). These results suggest that anisomyin,like yloheximide,stabilizes GFBP mrna. The inhibition of PEPCK mrna by nsulin in H4E Cells s Abolished by CyloheximideHormonal regulation of the PEPCK gene in H4E ellsresembles that of the GFBP gene; both PEPCK gene transription and PEPCK mrna aumulation are dereasedby insulin and inreased by dexamethasone (8).For omparison with GFBP, we determined the effet of yloheximide on the inhibition of PEPCK mrna by insulin in H4E ells. n the abseneof yloheximide, as expeted, insulin treatment for.5 h dereased PEPCK mrna to% of the levels seen in ontrol ells (Fig. 9). n ells that hadbeen pretreated withyloheximide for.5 h, the insulinindued inhibition of PEPCK mrna was abolished. Sine PEPCK gene transription was inhibited by insulin alone or in ombination with yloheximide (5),these results suggest that yloheximide stabilizes PEPCK mrna as itdoes GFBP mrna. Blank pgem7 [R [ [ Cvlo ns.5 h, A C B E D F Northern Conl h "' Cyle Cylo ns.5 h Conl.5 h y r l 4 5 6 7 8 9 DSCUSSON The well differentiated H4E rathepatoma ell line seretes GFBP as its predominant GFBP () and exhibits ene and equilibrated for 6 h in serumfree medium ontaining.% hormonal regulation of GFBP mrna similar to that seen BSA. Cultures were then refed with fresh serumfree medium ontaining p~ dexamethasone and inubated for 6 h. Onehalf of the in uiuo (). n H4E ells, GFBP mrna is inreased by ultures were pretreated with yloheximide for.5 h, at whih time dexamethasone and dereased by insulin (4, 5), refleting (5). T o determinethe insulin was added to some of the ultures. Nulei were prepared hangesingfbptransription and.5 h after insulin addition. Radioative elongated RNA tran ellular mehanisms responsible for this regulation, we have sripts were preparedand hybridized to filters ontaining rat GFBP used protein synthesis inhibitors in onjuntion with these or pgem7 plasmid DNA or to filters not ontaining any DNA. For hormones to assess their effets on the synthesis, stability, omparison, total RNA was prepared from tripliate plates of yloheximidepretreated and untreated ells and.5 h after insulin and steadystatelevels of GFBP mrna. Dexamethasone inreases steadystate GFBP mrna in treatment and analyzed by Northern blotting. Top panel, autoradiograph of elongated RNA transripts hybridizing to nitroellulose H4E ells by inreasing gene transription withoutaffetfilters ontaining immobilized GFBP DNA (lunes A and B ), ing mrna turnover (4,5). Cyloheximide pretreatment for pgem7 DNA (lunes C and D),and no DNA (blank; lunes E and F)..5 h suppressedproteinsynthesis by >9%, but did not Eah horizontal row represents hybridization of transripts from a prevent the indution of GFBP mrna by dexamethasone single preparation of nulei to dupliate filters ontaining eah DNA target. Transripts from dupliate preparations of nulei were ex a t.5 and h. Sine yloheximide toxiity ours between amined for eah experimental treatment. The input radioativity was 4.5 and 7.5 h before the peak indution of GFBP mrna the same for all transripts (. X O6 pm). Similar results were by dexamethasone is reahed a t h (5), it is not possible obtained in two other experiments. Bottom p a n e l, autoradiograph of to evaluate the effet of yloheximide on the magnitude or a Northern blot showing steadystate GFBP mrna in ells from duration of the inrease in GFBPmRNA. the same experiment shown above. Only the region of the autoradidexamethasone also indued GFBP transription in the ograph showing the single.55kb GFBP mrna band is shown. presene of yloheximide, indiating that de nouo protein synthesis was not required for the indution. This independstabilization of GFBP mrnaby yloheximide was due to ene from ongoing protein synthesis suggests that suffiient the inhibition of de m u o protein synthesis and not to other quantities of the requisite transription fators (i.e. the glueffets, analogous experiments were performed using a stru oortioid reeptor and possibly aessory fators (8))are turally distint, more speifi inhibitor of protein synthesis, present before and during dexamethasone treatment. anisomyin (4) (Fig. 8). Anisomyin inhibits a different step Gluoortioid indution of gene expression that is indein translation elongation (peptide bond formation) than y pendent of de nouo protein synthesis also has been reported loheximide (transloation of thenasenthainfromthe for the p (5), mouse mammary tumor virus (6) growth and ribosomal A site to thep site) (4). Dexamethasoneindued hormone genes (7), whereas gluoortioid effets on other H4E ells were pretreated with anisomyin for.5 h, after liver genes suh as albumin (8), tryptophan,dioxygenase whih insulin was added for a further.5 h. Nulear runon (9), amiroglobulin (4), PEPCK (5), tyrosine aminotransriptionexperimentsindiatedthatinsulininhibited transferase (4), and aaidglyoprotein (9,) require de GFBP transription to similar extents in anisomyinprenouo protein synthesis. n the aaid glyoprotein promoter, treated and untreatedells, 5 and 7%, respetively (Fig. a DNA element that onferred timedependent yloheximide 8). Treatment with anisomyin alone also dereased GFBPsensitivity was mapped nuleotides downstream from the transription by 7 & 8% at.5 h and 4 & % at h GREandpresumablyinteratedwith a preexisting labile (results not shown). fator that was neessary for gluoortioid indution in adalthough insulin dereased GFBP transription in both dition to the gluoortioid reeptor (9, ). Gluoortioid anisomyintreated and untreated ells, the resulting derease responsiveness in the PEPCK promoter mapped to a single FG.5. nsulin inhibits GFBP gene transription in yloheximidetreated H4E ells. Cells were grown to onflu
Cyloheximide Stabilizes GFBP m R N A 6669 Northern Transription FG. 6. Quantitative analysis of GFBP gene transription and steadystate GFBP mrna abundane in yloheximidetreatedand Cylo Cylo Cylo 8 nontreated ells.5 h following insulin treatment.the hyhridized radioativity shown in Fig. 5 was analyzed by Bsanning. Solid bar, no insulin treatment; hathed bar, insulintreated. Left, results are the mean f S.D. of hybridizations of dupliatefilters fromtwo preparations of nulei. The mean pcem7 and blank ounts for hybridizations were not signifiantly different (95 f 65 and 85,respetively; n = 4). The bakground radioativity (blank filters) for eah hybridization inubation was subtrated. Right, resultsarethe mean S.D. of tripliate hybridizations. The bakground radioativity orresponding to an equivalent area of the blothas been subtrated(meanbakground is 58 9; n = ). n 6 C. 4 nl..n x i nsulin TARLE r T Cylo T 8 6 4 4 nsulin whether theaessory fators interating with sites AF and or AF need to be ontinuously synthesized and whether they are responsiblefor inhibition of the indution of PEPCK transription by protein synthesis inhibitors (5). nsulin rapidly dereasesgfbp mrna by inhibiting transription (5) without affeting the halflife of GFBP mrna (). The inhibition of GFBP gene transription ( t l of 9 min) is quite rapid and onsiderably faster than the tl of ytoplasmi GFBP mrna ( h), indiating that the primary effet of insulin on GFBP regulation is at the transriptionallevel (4). Thisis one of the most rapid inhibitory effets of insulin on gene transription that have been desribed. Transription of the PEPCK gene in H4E ells was signifiantly inhibited 5 min after addition of Perent hybridization" insulin and was redued to basal levels after 5 min (4, Experiment 44). The indution ofs (45) and @atin (46) gene trancyloheximide Cyloheximide sription also was rapid, ourring within 5min after insulin. <Oh addition..4.7 Unlike the indution of GFBP mrna by dexametha 8 5 sone, whih was unaffeted by yloheximide, the derease in Mean S.E. 7.5 9..6 C.5 GFBP mrna levels following insulin treatment was abola (insulininsulin) X. ished in ells that had been pretreated with yloheximide. Hybridized ounts were lower than bakground. Sine insulin inhibited GFBP transriptionin the absene of yloheximide, we performed nulear runon experiments TABLE to determine whetheryloheximide abolished this transriptimedependent inhibition of GFBP gene transription by tional regulation. To the ontrary, insulin inhibited GFBPvloheximide Hybridized radioativity was ompared in yloheximidetreated transription in yloheximidetreated ells asit did in and untreated ells at.5 and h (.5h preinubation, and.5h untreated ells. Similar results were obtained using a struinubation). Results shown are from the three experiments presented turally unrelated inhibitor of protein synthesis, anisomyin. in Table. The perent of total hybridized ounts in yloheximideanisomyin inhibits a different step in translation elongation treated ells was ompared with that in ells not treated with ylothan yloheximide and is less likely to have effets unrelated heximide (taken as %) for the respetive times. to the inhibition of protein synthesis (4). The preservation Perent hybridization" of GFBP mrna levels in insulintreated ells in the presexperiment.5 h h ene of protein synthesis inhibitors despite inhibition of transription suggested that the turnoverof GFBP mrna had.9 9. 6. 9.6 been slowed. This was demonstrated diretly using atino.8 8. myin D to blok transription,measuringthe deay of Mean f S.E. 6...8 f.6 GFBP mrna over time. Cyloheximide treatment pro(yloheximideyloheximide) X. longed the halflife of GFBP mrna from to h. The respetive turnover rates were not altered by insulin, nuleotide region whih onsisted of two gluoortioid onsistent with the previousonlusion thattheprimary regulation of GFBP mrna by insulin is at the level of reeptorbindingsitesand two bindingsites foraessory fators(afand AF) (8). t remains to be determined transription (5,). The persistene f GFBP mrna nhibition of GFBP gene transription by insulin a t.5 h in yloheximidepretreated and untreated ells H4E ells were stimulated with dexamethasone for 6 h, after whih onehalf of the ultures were treated with yloheximide for.5 h. nsulin was added to some ultures and nulei prepared and.5 h after insulin addition. Radioative elongated RNA transripts were prepared and hybridized to filters ontaining rat GFRP or pgem7 plasmid DNA as target, or blank filters that did not ontain DNA. Radioativity was quantitated by 8sanning. Results shown are the hyhridized ounts in insulintreated ultures ompared with the respetive ultures (yloheximidepretreated or untreated) not reeiving insulin (taken as %). nall experiments, the bakground radioativityhybridized to the filters ontaining pcem7 plasmid DNA has been suhtrated. The orresponding autoradiographs for experiment are shown in Fig. 5.
667 Cyloheximide Stabilizes GFBP mrna 8 6 4 8 6 4 ns Gyl ns ns 'N ns 4 5 6 7 Cylo Time (h) FG. 7. Cyloheximide stabilizes LGFBP mrna in atinomyindtreated H4E ells. Confluent HE ells were inubated with dexamethasone in serumfree medium for 8 h. Onehalf of the ultures were then treated with yloheximide for.5 h. nsulin was then added to half of the yloheximidepretreated ultures and half of the untreated ult~~res. Atinomyin D (5 pgml) was added at the same time as insulin (time zero) to all ultures without a hange of media. Total RNA was harvested,.5,, 4.5, and 6 h after atinomyin D addition and analyzed by Northern blot hybridization using the rat GFBP DNA probe. Hybridized radioativity was quantitated by ( sanning after orretion for bakground radioativity (<% of time zero) and was plotted at different times. Cirles, yloheximidetreated squares, not treated with yloheximide; open symbols with dashed lines, insulintreated; solid symbols, solid lines, not treated with insulin. All points are the mean S.D. of tripliate determinations exept for the zero time point of the ells that did not reeive insulin or yloheximide whih are dupliates. Similar results were obtained in two other experiments. after insulin treatment in yloheximidetreated H4E ells, therefore, primarily reflets stabilization of GFBP mrna, whih overomes the inhibition of transription by insulin (and yloheximide). Cyloheximide also stabilized GFBP mrna in human HepG hepatoarinoma ells, although mrna turnover in the absene of the protein synthesis inhibitor was onsiderably slower (tllz h) than in H4TE ells (47). Protein synthesis inhibitors may affet GFBP mrna stability diretly (by inhibiting the translation of GFBP mrna or the stability of polyribosomes formed from GFBP mrna) or by inhibiting the translation of proteins involved in the degradative pathway. Preedents for both types of effets have been desribed. The degradation of histone (48) and @tubulin (49) mrnas is diretly oupled with their translation. Histone mrna is only degraded when ribosomes are loated within nuleotides of a ' stem and loop struture, whihmaybe the leavage site for a ribosomebound ribonulease (48). Degradation of @tubulin mrna by a ribosomebound nulease only is initiated when a tetrapeptide sequene at the start of the tubulinoding region protrudes from the polyribosome during translation (5). Alternatively, mrnas for PEPCK (5), vitellogenin (5), and tyrosine aminotransferase (5) are seletively stabilized by inhibitors of protein synthesis suh as yloheximide that prevent the breakdown of the polyribosomemrna omplex (4), but not by protein synthesis inhibitors that redue the density of ribosomes on the mrna suh as patamyin or puromyin. n these ases, assoiation of the mrna with ribosomes, rather than inhibition of translation per se, protets them from degradation. De novo synthesis of a protein involved in the mrna degradation pathway determines the stability of the human transferrin reeptor mrna (54). This was elegantly demonstrated using mutant transferrin reeptors whose mrna had been destabilized by removal of the iron response element (RE) motif in the "untranslated region and by introduing an RE from ferritin in the 5"untranslated region so that translation of transferrin reeptor mrna ould be speifially inhibited by dereased iron availability. ron limitation seletively inhibited translation of transferrin reeptor mrna but did not stabilize the mrna, whereas stabilization was ob served following global inhibition of protein synthesis by yloheximide. Studies of this type will be required to determine whether yloheximide stabilizes GFBP mrna by inhibiting translation of an mrna enoding a rapidly turningover protein in the degradative pathway or of GFBP mrna itself. The fat that stabilization of GFBP mrna was observed in basal, dexamethasonestimulated, and insulininhibited ells suggests that the mehanism of mrna stabilization is similar in eah ondition and that it is not regulated by these hormones. The determinants of mrna stability and the proteins involved in mrna degradation are poorly defined (5557). The 5' ap and ' poly(a) sequenes (whih bind a stabilizing poly(a)binding protein) protet against nulease attak. The poly(a) tail of s mrna is shortened and removed prior to degradation of the mrna. nhibition of protein synthesis dereased both the trunation of the poly(a) tail and subsequent mrna degradation, suggesting that a labile protein is involved in the removal of poly(a) that preedes mrna degradation (58). Most sequenes regulating mrna stability are loated in the 'untranslated region and inlude the iron response element (54,59) and an AUrih sequene (AUUUA)
667 Cyloheximide Stabilizes GFBP m R N A Anis Anis 6oo 5 t Cylo Cylo T 8 T 6.P m N._ D t._ m._ T? Ll L7 > L : 7 l o o 4 OL t nsulin t nsulin FG. 9. Cyloheximide abolishes the inhibition of PEPCK in anisomrna by insulin in H4E ells. The Northern blot shown in myintreated and untreated H4E ells. Cells were gown to Fig. was strippedof GFRP prohe and rehyhridized with a EPCK onfluene and equilibrated for 6 h in serumfree medium ontaining DNA probe. Radioativity hybridized to the.6kh PEPCK mrna.% RSA. Cultures were then refed with fresh serumfree medium was quantitated by sanning and the mean i S.D. plotted. Rakontaining p~ dexamethasone and inubated overnight. Onehalf ground radioativitv orresponding to an equivalent area of the blot p ~ for ).5 h. of the ultures were pretreated with anisomyin ( 4 f nsulin was then added to some of the ultures andnulei prepared has heen subtrated. (The mean hakground radioativitv was 44 ounts; n =.) Results.5 h after insulin addition are shown. and.s h after insulin addition. Radioative elongated RNA tranlpft panel, no yloheximide treatment; right panel, yloheximidex pm)tofilters sriptswerepreparedandhybridized(.9 Solid bar, no insulin; hathed bar, with insulin. treated. ontaining immohilized rat GFRP and pgem7 plasmid DNA or to filters not ontaining any DNA (blank). The hybridized radioativity was quantitated by psanning. Results plottedare the mean i into a stable mrna an destabilize it (64), the presene of S.D. of hybridizations of dupliate filters from two preparations of this element does not neessarily make the mrna unstable nulei. The hakground radioativity (blank) for eah hybridization under all onditions(56).a seond instabilityelement is inubation has been subtrated. (The mean pgem7 and blank ounts forhybridizationwere 9 i 74 ( n = ) and 867 f 4 ( n = 4), present in the proteinoding region of fos (64, 65) and respetively.) Left panel, no anisomyin treatment; right panel, anim y (66) whih in the ase of my (66) is suffiient by itself somyintreated. Solid bar, no insulin addition: hathed bar, insulin to destabilize the mrna. treated. Resultsof the Northern blot hybridization in this experiment Cyloheximide has been used to selet growthrelated imare presented in Tahle (experiment ). FG. 8. nsulin dereases GFBP transription mediate early or primaryresponse genes that are ativatedin thetransition from restingto yling ells (6,67).The TABLE ll premise of this seletion strategy is that transriptionof these nhibition of GFRP rnrna by insulin in thepresenr and absene genes does not require new protein synthesis but ours by of anisornyin ativation of preexisting transription fators (for example, Confluent H4E ellsinserumfreemediumwerestimulated by phosphorylation (68)). n fat, inhibition of protein synwith dexamethasone overnight, after whih onehalf of the ultures thesis superindued the mrnasfor several growthrelated p~ anisomyin for.s h. nsulin was added were preinubated with immediateearly genes suh as fos, beause atransient to onehalf ofthe ultures and the inubation ontinued. Total mrna by inreased was prepared from dupliate ultures h after insulin addition. The inrease in transription wasaompanied RNA was analyzedby Northern blot hybridization using an GFRP mrna stability (6, 697). For my, the posttransrip DNA probe. Hybridized radioativity was quantitated hy.santional effets predominated (66). Mohn et al. (7) isolated a ning. Resultsof three experiments are shown. Hybridized radioativ lone for GFBP from a DNA library established from the is ompared with the respetive ultures ity in insulintreated ultures regenerating liver of yloheximidetreated rats and desig(anisomyinpretreated or untreated)thatdidnotreeiveinsulin nated it an immediate early gene. n H4E ells, however, (taken as %).n all experiments, the bakground radioativity yloheximide partially inhibits GFBP gene transription orresponding to a blank area on the blot has been subtrated. in a timedependent manner, suggesting that ontinued synperent hvhridization Experiment thesis of a labile protein is neessary to maintain normal Anisomyin Anisomyin levels of GFBP transription under basal, dexamethasone 6.. stimulated, and dexamethasonestimulatedinsulininhibited 9.8 4.5 onditions. l.i. 7.4 A similar argument may be made for the PEPCK gene.4 i76. 4.6 C 4 Mean i S.E. whih also is expressed in yloheximidetreated regenerating (insulininsulin) X. rat liver (7). Bortoff and Messina (5) have reported that yloheximide (78 PM, 6 min) inhibits transription of the PEPCK gene in the presene or absene of insulin. Cylohexseen in many lymphokines, ytokines, and protoonogenes imide abolished the derease in PEPCK mrna indued by (suh as granuloytemarophage olonystimulating fator, fos, and my) (6). Human (6, 6) and rat (6) GFBP insulin in H4E ells (Fig. 9) and in the related FTOB suggest have 5 and 4 AUUUA motifs in their untranslated region, rat hepatoma ell line (5). Together these results respetively.although introdution of an AUrih element that stabilization of PEPCK mrna predominatesover tran~~~~
667 Cyloheximide Stabilizes GFBP mrna sriptional inhibition in yloheximidetreated ells. Stabilization of PEPCK mrna by yloheximide also has been observed in rat liver (7, 74). For reasons that are not apparent, Sasaki et al. (4) obtained different results; yloheximide treatment of H4E ells (.7 pm, 6 min) did not affet PEPCK transription under basal, CAMPindued, or CAMPinduedinsulininhibited onditions, nor did it abolish the derease in PEPCK mrna aused by insulin (.5h pretreatment with.7 pm yloheximide, followed by h treatment with insulin). Messina and oworkers have observed that protein synthesis inhibitors mimi the effet of insulin on the transription of several genes (suh as fos (45), p (5), atubulin (46), tyrosine aminotransferase (4), PEPCK (5), albumin (44), and?atin (46)) in HE ells and suggested that these effets may be mediated through protein kinase C, sine both insulin and yloheximide inrease diaylglyerol and protein kinase C ativity in some experimental systems (7577). n support of this proposal, phorbol esters mimi the effets of both insulin and protein synthesis inhibitors in H4E ells in induing (fos (45), p (5), @atin(46) and atubulin (46)) or inhibiting (PEPCK (78) and albumin (44)) gene transription. Our results with GFBP are onsistent with this proposal to the extent that insulin and yloheximide both derease GFBP gene transription. Whether this derease is mediated by protein kinase C remains to be eluidated. n summary, we have shown that inhibition of protein synthesis affets the regulation of rat GFBP gene expres sion at both transriptional and posttransriptional levels. Protein synthesis inhibitors derease GFBP transription in basal, dexamethasoneindued, or dexamethasoneinduedinsulininhibited onditions, suggesting that ongoing protein synthesis is needed for basal promoter ativity. These inhibitory effets were opposed and outweighed under all onditions by stabilization of GFBP mrna. Further haraterization of the labile proteins responsible for these effets on GFBP transription and GFBP mrna stability will be required to understand the mehanisms by whih the dynami regulation of GFBP mrna is ahieved in uiuo. AknowledgmentsWe thank John MClaskey for many stimulating disussions in the early phases of this projet and Leon Bah, Yves Boislair, Alexandra Brown, Charles T. Roberts, Jr., and Yvonne Yang for ritial review of the manusript. REFERENCES. Rehler, M. M., and Nissley, S. P. (99) in Handbook Exp. Phrm. (Sporn, M. B., and Roberts, A. B., eds) Vol. 95, pp. 667, SpringerVerlag, Berlin. Rehler. ~,~ M. M. (99) Vitam. Horm. 47. 4 ~~. Clemmons, D. R. (99) Growth Re&,%%7 4. Cotterill, A. M., Cowell, C. T., Baxter, R. C., MNeil, D., and Silinik, M. (988) J. Clin. Endorinol. & Metab. 67. 88887 5. Bushy, W. H., Snyder, D. K., and Clemmons, D. R. (988) J. Clin. Endorinol. & Metab. 67, 5 6. Brismar, K., Gutniak, M., Povoa, G., Werner, S., and Hall, K. (988) J. Endorinol. nvest.,5996 7. Suikkari, A. M., Koivisto, V. A,, Rutanen, E. M., YkiJarvinen, H., Karonen, S. L., and Seppiila, M. (988) J. Clin. Endorinol. & Metab. 66, 667 8. Bar, R. S., Clernmons, D. R., Boes, M., Bushy, W. H., Booth, B. A,, Dake, B. L., and Sandra, A. (99) Endorinology 7, 7886 9. Ooi, G. T., Tseng, L. Y.H., and Rehler, M. M. (99) Growth Regul., 5. Ooi, G. T., Tseng, L. Y.H., Tran, M. Q., and Rehler, M. M. (99) Mol. Endorinol. 6,98. Pitot. H. C.. Peraino. C.. Morse., P. A,.. and Potter, V. R. (964) Natl. Caner fit. Mohgr.,'94. Yang, Y. W.H., Brown, A. L. Orlowski, C. C., Graham, D. E., Tseng, L. Y.H., Romanus, J. A., and'rehler, M. M. (99) Mol. Endorim[. 4, 98. Unterman, T. G., Oehler, D. T., Gotway, M. B., and Morris, P. W. (99) Endorinology 7, 789797 4. Orlowski, C. C., Ooi, G. T., and Rehler, M. M. (99) Mol. Endorinol. 4, 59599 5. Orlowski, C. C., Ooi, G. T., Brown, D. R., Yang, Y. W.H., Tseng, L. Y.H., and Rehler, M. M. (99) Mol. Endorinol. 5,887 6. Mithell, P. J., and Tjian, R. (989) Siene 45,778 7. Beato, M. (989) Cell 56,544 8. Luas, P. C., and Granner, D. K. (99) Annu. Reu. Biohem. 6, 7. 9. Klein, E. S., Reinke, R., Feigelson, P., and Ringold, G. M. (987) J. Biol. Chem. 6,55. Klein, E. S., DiLorenzo D., Possekert, G., Beato, M., and Ringold, G. M. (988) Mol. Endorinbl., 45. O'Brien, R. M., and Granner, D. K. (99) Biohem. J. 78,6969. Nasrin, N., Erolani, L., Denaro. M., Kong, X. F., Kang,., and Alexander, M. (99) Pro. Natl. Ad. Si. U. S. A. 87, 57577. Keller, S. A., Rosenberg, M. P., Johnson, T. M., Howard, G., and Meisler, M. H. (99) Genes & Dev. 4,6 4. Johnson, T. M., Rosenherg, M. P., and Meisler, M. H. (99) J. Biol. Chem. 68.464468 5. Yamamoto, K. R., and Alherts, B. M. (976) Annu. Rev. Bwhem. 45, 7 746 6. Hershman, H. R. (99) Annu. Rev. Biohem. 6,89 7. Mohn, K. L., Melhy, A. E., Tewari, D. S., Laz, T. M., and Taub, R. (99) Mol. Cell. Biol., 94 8. Koontz, J. W., and wahashi, M. (98) Siene,947949 9. Messina, J. L. (99) J. Bwl. Chem. 66, 79958. Chomzynski, P., and Sahi, N. (987) Anal. Biohem. 6, 5659. Brown, A. L., Graham, D. E., Nissley, S. P., Hill, D. J., Strain, A. J., and Rehler, M. M. (986) J. Biol. Chem. 6, 445. YooWarren, H., Monahan, J. E., Short, J., Short, H., Bruzel, A,, Wynshaw Boris, A., Meisner, H. M., Samols, D., and Hanson, R. W. (98) Pro. Natl. Aad. Si. U. S. A. 8, 65666. Unterman, T. G., Oehler, D. T., Murphy, L. J., and Lason, R. G. (99) Endorrnology 8, 697 4. Vazquez, D. (979) nhibitors of Protein Synthesis, pp. 68, Springer Verlag, Berlin 5. Bortoff, K. D., and Messina, J. L. (99) Mol. Cell. Endorinol. 84,946 6. Ringold, G. M., Yarnamoto, K. R., Bishop, J. M., and Varmus, H. E. (977) Pro. Natl. Aad. Sei. U. S. A. 74, 87988 7. Yamarnoto, K. R. (985) Annu. Rev. Genet. 9, 95 8. Nawa, K., Nakamura, T., Kumatori, A,, Noda, C., and hihara, A. (986) J. Biol. Chem. 6, 6886888 9. Nakamura, T., Niimi, S., Nawa, K., Noda, C., hihara, A., Takagi, Y., Anal, M., and Sakakl, Y. (987) J. Bol. Chem. 6, 777 4. Addison, W. R., and Kurtz, D. T. (986) Mol. Cell. Biol. 6, 446 4. Messina, J. L., Chatterjee, A. K., Strapko, H. T., and Weinstok, R. S. (99) Arh. Biohem. Biophys. 98.566 4. Granner, D. K. (987) Kidney nt., S8S9 4. Sasaki, K., Cripe, T. P., Koh, S. R., Andreone, T. L., Petersen, D. D., Beale, E. G., and Granner, D. K. (984) J. Biol. Chem. 59,5455 44. Messina, J. L. (99) Biohim. Biophys. Ata 7, 5 45. Messina, J. L. (99) J. Biol. Chem. 66, 775 46. Messina, J. L. (99) Mol. Endorinol. 6,9 47. Angervo, M. (99) Biohem. Biophys. Res. Commun. 89, 778 48. Graves, R.A., Pandey, N. B., Chodhoy, N., and Marzluff, W. F. (987) Cell 48,6566 49. Gay, D. A,, Sisodia, S. S., and Cleveland, D. W. (989) Pro. Natl. Ad. Si. U, S. A. 86, 5765767 5. Yen, T. J., Mahlin, P. S., and Cleveland, D. W. (988) Nature 4, 58 585 5. Hua, J., and Hod, Y. (99) Mol. Endorinol. 6, 4844 5. Blume, J. E., and Shapiro, D. J. (989) Nulei Aids Res. 7,994 5. Ernest, M. J. (98) Biohemistry,6766767 54. Koeller, D. M., Horowitz, J. A,, Casey, J. L., Klausner, R. D., and Harford, J. B. (99) Pro. Natl. Ad. Sei. U. S. A. 88, 7778778 55. Nielsen, D., and Shapiro, D. (99) Mol. Endorinol. 4, 95957 56. Atwater, J. A,, Wisdom, R., and Verma,. M. (99) Annu. Reu. Genet. 4, 5954 57. Jakson, R. J., and Standart, N. (99) Cell 6, 54 58. Wilson, T., and Treisman, R. (988) Nature 6,9699 59. Mullner, E. W., and Kuhn, L. C. (988) Cell 5,8585 6. Shaw, G., and Kamen, R. (986) Cell 46,659667 6. Cubbage, M. L., Suwanihkul, A,, and Powell, D. R. (989) Mol. Endorinol. R _,"_ LLLF(F\l "" 6. Julkunen, M., Koistinen, R., AaltoSetala, K., Seppala, M., Janne,. A., and Kontula. K. (988) FEES Lett. 6. 95 6. Murphy, L. J., 'Seneviratne, C., Ballejo, G.; Croze, F., and Kennedy, T. G. (99) Mol. Endorinol. 4,96 64. Shy, A,B., Greenberg, M. E., and Belaso, J. G. (989) Genes & Deu., 67 65. Kabnik K. S., and Housman, D. E. (988) Mol. Cell. Biol. 8.445 66. Wisdom,'R., and Lee, W. (99) Genes & Deu. 6,4 67. Lau, L. F., and Nathans, D. (985) EMBO J. 4,455 68. Hunter, T., and Karin, M. (99) Cell 7, 7587 69. Lau, L. F., and Nathans, D. (987) Pro. Nd. Ad. Sei. U. S. A. 84,8 86 7. Greenberg, M. E., Hermanowski, A. L., and Ziff, E. B. (986) Mol. Cell. Biol. 6, 557 7. Almendral, J. M., Sommer, D., MaDonaldBravo, H., Burkhardt, J., Perera, J., and Bravo, R. (988) Mol. Cell. Blol. 8, 448 7. Mohn, K. L., Laz, T. M., Melhy, A. E., and Taub, R. (99) J. Bid. Chem. 65,949 7. Nelson, K., Cimhala, M. A., and Hanson, R. W. (98) J. Biol. Chem. 55, 859855 74. Cimbala, M. A,, Lamers W. H., Nelson, K., Monahan, J. E., YooWarren, H., and Hanson, R. W.(98) J. Biol. Chem. 57, 769766 75. Farese, R. V., Barnes. D. E.. Davis, J. S., Standaert, M. L., and Pollet, R. J. (984) J: Biol. Chm. 59, 7947 76. Cooper, D. R., Konda, T. S., Standaert, M. L., Davis, J. S., Pollet, R. J., and Farese, R. V. (987) J. Btol. Chem. 6,669 77. Widmaier, E. P., and Hall, P. F. (985) Mol. Cell. Endorinol. 4, 888 78. Chu, D. T., Davis, C. M., Chrapkiewiz, N. B., and Granner, D. K. (988) J. Biol. Chem. 6, 7