THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 257, No. 4, Issue of Februaly 25, pp. 2022-2028. 1982 Printed in U.S.A.

N6,02’-Dibutyryl Cyclic AMP and Glucose Regulate the Amount of Messenger RNA Coding for Hepatic Phosphoenolpyruvate Carboxykinase (GTP)*

(Received for publication, August 3, 1981, and in revised form, October 15, 1981)

Elmus G. Beale, James L. Hartley, and Daryl K. Grannert From the Departments of Internal Medicine and Biochemistry, Diabetes and Endocrinology Research Center, University of Iowa, and the Veterans Administration Hospital, Iowa City, Iowa 52240

Rat liver phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA was purified to 25% of total mRNA activity (>50-fold enrichment) by polysome immuno- precipitation. This preparation was used as template for the synthesis of cDNA that was subsequently cloned in Escherichia coli. The resulting clones were screened by in situ hybridization and by hybrid-selected trans- lation of phosphoenolpyruvate carboxykinase mRNA. The cDNA insert of one plasmid, pPC2, was com- plementary to phosphoenolpyruvate carboxykinase mRNA as determined by these screening procedures. pPC2 cDNA was 760 base pairs in length and a partial restriction enzyme map was constructed. pPC2 was labeled with 32P by nick translation and was used as a hybridization probe to quantitate phosphoenolpyru- vate carboxykinase mRNA following N6,02’-dibutyryl cAMP (Bt2cAMP) injection or glucose feeding. Bt2cAMP increased whereas glucose decreased the level of hy- bridizable phosphoenolpyruvate carboxykinase mRNA and in all cases the changes were proportional to the in vitro translational activities measured in a reticulocyte lysate system. The half-life of phosphoenolpyruvate carboxykinase mRNA sequences was measured by an indirect procedure involving their quantitation, by hy- bridization assay, during deinduction and induction. The half-life was approximately 10-40 min during dein- duction by glucose or during induction stimulated by Bt2cAMP. Our data indicate that cAMP enhances some step in the generation of phosphoenolpyruvate car- boxykinase mRNA.

Numerous examples of regulation of the synthesis of specific eukaryotic proteins by cyclic AMP have been elucidated in recent years (1) yet the mechanism(s) involved in this process has remained elusive. One of the proteins studied most inten- sively is hepatic P-enolpyruvate carboxykinase,’ an enzyme

* This work was supported by National Institutes of Health Grants AM20858 and AM25295 (Diabetes and Endocrinology Research Cen- ter), and Veterans Administration Research Funds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

+. A Veterans Administration Medical Investigator. T o whom cor-

istration Hospital, Iowa City, IA 52240. respondence should be addressed at, Room 3E-19, Veterans Admin-

’ The abbreviations used are: P-enolpyruvate carboxykinase, phos- phoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32); mRNAPEP‘:K, mRNA coding for P-enolpyruvate carboxykinase; BtZcAMP, N6,02’- dibutyryl CAMP; EGTA, ethylene glycol bis(S-aminoethyl ether)- N, N, N ’ , N’-tetraacetic acid; poly(A)+RNA, polyadenylated RNA; Hepes, 4-(2-hy&oxyethyl)-1-piperazineethanesulfonic acid; bp, base pair.

that catalyzes a rate-limiting step in gluconeogenesis. The synthesis of this enzyme is affected by several different agents that regulate gluconeogenesis. Fasting, glucagon (via CAMP), and glucocorticoids are inducers whereas insulin and dietary glucose are deinducers (2). The action of glucose may involve inhibition of glucagon secretion, leading to lowered hepatic cAMP levels (3,4). Studies of the regulation of P-enolpyruvate carboxykinase are therefore important for understanding the regulation of gluconeogenesis and the mechanisms of action of a number of apparently diverse hormones.

The observation that P-enolpyruvate carboxykinase could be induced by cyclic AMP in the face of inhibition of RNA synthesis by agents such as actinomycin D, coupled with similar observations made with tyrosine aminotransferase, contributed to the hypothesis that cyclic AMP induces the synthesis of specific proteins by enhancing the translation of a fixed amount of mRNA (1). The possibility that cyclic AMP could increase the amount of an mRNA was not widely entertained. In the past 4 years, assays of mRNA activity, using in vitro translational systems, have occasioned a com- plete reevaluation of how cAMP regulates the synthesis of specific proteins. These experiments have shown that the activities of the mRNAs coding for P-enolpyruvate carboxy- kinase (5-7), tyrosine aminotransferase (8, 9), alkaline phos- phatase (lo), and lactate dehydrogenase-5 isozyme (11) are increased by cAMP in proportion to the increase in synthesis of these specific proteins. These observations preclude trans- lation being the primary site of action of cyclic AMP but, since the in vitro translational activity of a messenger RNA can theoretically be changed by affecting its rate of transcrip- tion, processing (splicing of exons), modification (capping, polyadenylation, methylation), degradation, or activation from a pre-existing pool of inactive messenger RNA sequences, a number of possible sites of action remain. These steps can be systematically explored using specific complementary DNAs that hybridize with the respective mRNA sequences. This approach has heretofore been difficult because of the relative scarcity of these mRNAs, which even when maximally induced generally constitute less than 0.5% of the total mRNA population. We now report the cloning of a specific DNA, complementary to mRNAPEPLK, that we have used as a hy- bridization probe to quantitate mRNAPEPcK. Our results indicate that BtzcAMP increases and glucose decreases the absolute amount of this mRNA, presumably by affecting some step(s) in the generation (transcription, processing, or modi- fication) of translationally active mRNAPEPCK. An account of this work has been presented in abstract form.2

E. G. Beale, J. L. Hartley, and D. K. Granner (1981) 63rd Annual Meeting of the Endocrine Society, Cincinnati, OH, Abstract 472.

2022

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

Phosphoenolpyruvate Carboxykinase mRNA Regulation by CAMP 2023

EXPERIMENTAL PROCEDURES

Materials-RNase-free sucrose was purchased from Bio-Rad and sodium deoxycholate was from BDH. Formamide from Matheson, Coleman, and Bell was deionized with AG 501-X8 mixed bed resin, and with Chelex 100 (Bio-Rad) to a conductivity of less than 25 pmho. Staphylococcus aureus, Cowan strain I (Pansorbin), was from Calbi- ochem. [35S]Methionine, reticulocyte lysate translation kits, methyl- “C-proteins, and EN3HANCE were from New England Nuclear. Labeled nucleotides were from Amersham and nick translation re- agents were from Bethesda Research Laboratories. Restriction en- zymes, terminal transferase, and Klenow DNA polymerase I were purchased from New England Biolabs, Sl nuclease and terminal transferase were from Miles, and oligo(dT)-cellulose, type 3, was from Collaborative Research. Nitrocellulose filters (BA85) were from Schleicher and Schuell. Avian myeloblastosis virus reverse transcrip- tase was obtained from Dr. J. W. Beard, Life Sciences, Inc., St. Petersburg, FL. All other reagents were analytical grade. The rat albumin cDNA plasmid prAlb1 (12) was the generous gift of Dr. John Taylor (Department of Microbiology, Pennsylvania State University, Hershey, PA). Dr. Don Cleveland (Department of Biochemistry and Biophysics, University of California at San Francisco) kindly provided the V8 protease.

Animals-Male CD strain rats, 250-300 g body weight, were sub- jected to a fasting-refeeding cycle followed by a 2-h treatment with B&AMP and theophylline (30 mg each/kg of body weight) as de- scribed previously (5, 7). For mRNAPErCK purification experiments (Fig. l), cycloheximide (3.0 mg/kg body weight) was given 1-2 h before the animals were killed, in order to maximize mRNAFsPcK levels (6).

Polysome Isolation-Magnesium-precipitated polysomes were prepared from less than 30 g of rat liver essentially as described by MacGillivray et al. (13). We routinely obtained 100-150 AYW units of polysomes/g of liver with Az,/Ap, ratios of 1.6 to 1.8. Polysomes were stored at -70 “C at a concentration of 100-200 AZW units/ml in 10 mM Hepes pH 7.5, 0.15 M NaCl, 5 mM MgCL, 0.5% Triton X-100, 0.5% sodium deoxycholate, 100 pg/ml heparin.

Potysome Immunoprecipitation-Polysomes synthesizing P-enol- pyruvate carboxykinase were immunoprecipitated by a modification of the procedure described by Gough and Adams (14). Polysomes were thawed and diluted to 15 APW units/ml with 10 mrvr Hepes pH 7.5, 0.15 M NaCl, 5 mM MgClz, 100 pg/ml heparin. Sheep anti-P- enolpyruvate carboxykinase (7), made nuclease-free as described by Palacios et al. (15), was added to give a final concentration of 50 to 100 pg of IgG/ml. This solution was gently stirred for 1 h at 2 “C and then 0.2 M EGTA was added to achieve a final concentration of 2 mM. Subsequently, a 10% (v/v) suspension of S. aureus Cowan strain I cells (washed as described by Kessler (16) and suspended in 50 mM Tris, pH 7.4, 5 mM MgC12, 2 mM EGTA, 0.3 M NaCl) was added at a ratio of 0.2-0.5 ml of cells/mg of IgG. Following a lo-min incubation at 2 “C, the cells were collected by centrifugation through a discon- tinuous sucrose gradient (14) and treated with 5 ml of 1.5% sodium dodecyl sulfate, 10 mM Tris-HCI, pH 7.5, 20 mM EDTA, 0.15 M NaCl for 3 min at 60 “C to release bound RNA. This suspension was centrifuged 10 mm at 8000 rpm in a Sorvall HB-4 rotor and RNA was extracted from the supernatant as described below.

Extraction of Poly(A)‘RNA-Polysome extracts were incubated with 0.6 mg/ml proteinase K (Beckman) and then extracted with phenol/chloroform (1:l). Poly(A)‘RNA was then isolated by a single passage over oligo(dT)-cellulose (50 mg in a 4-mm diameter column) as described by Krystosek et al. (17). In analytical experiments (Figs. 3, 4, and 6-9), poly(A)+RNA was prepared as described previously (7).

Translational Assays-Each reaction mixture contained transla- tion kit components as recommended by New England Nuclear, plus 0.4 mM magnesium acetate, 100 mM potassium acetate (in addition to endogenous magnesium and potassium in the lysate) and 30-50 PCi of [?S]methionine. Poly(A)‘RNA was added in a volume of 1.0 ~1 to give a final concentration of 10 to 20 pg/ml and the mixture was incubated for 90 min at 26 “C. Aliquots were removed for immuno- precipitation and trichloroacetic acid precipitation as described pre- viously (7). or for sodium dodecyl sulfate gel electrophoresis (18) and fluorography with EN”HANCE.

Dot-Blot Hybridization Assay-Quantitation of specific RNA se- quences by hybridization assay was as described by Thomas (19). cDNA probes were labeled with [a-“‘P]dATP and [a-=P]dCTP by nick translation (20) to specific activities of 0.5-l x 10” cpm/pg.

Synthesis of Comphzmentary DNA-mRNAPkPcK (10 ag), enriched

to 20-25% of total mRNA (assessed by translational assay) by poly- some immunoprecipitation, was used as template for the synthesis of cDNA first and second strands essentially as described by Wickens et al. (21). Klenow DNA polymerase I was used for the second strand synthesis.

Construction of Recombinant DNA Plasmids and Transforma- tion-The double-stranded cDNA was tailed as described by Roy- choudhury and Wu (22) using 200 units/ml terminal transferase and 0.2 mM dCTP. Similarly, Pst I-digested pBR322 was tailed with dGTP and the DNAs were annealed. Competent Escherichia coli, strain HB-101, prepared as described by Morrison (23) was thawed and the recombinant DNA was added to 100 al of cells. Following a 30-min incubation at 4 “C, the cells were plated on an LB-Agar plate (1% Tryptone, 0.5 NaCl, 0.5% yeast extract, 1.5% agar) with 25 pg/ml tetracycline. A library of transformants was prepared by suspending the transformant clones in 6 ml of 50% glycerol, 50% LB-tetracycline and stored at -20 “C.

Screening for Clones Containing P-enolpyruvate Carboxykinase cDNA-Plasmid screening was carried out at 1000-2000 colonies/ filter as described by Hanahan and Messelson (24). Single-stranded “‘P-cDNAs for use as hybridization probes were synthesized as de- scribed by St. John and Davis (25), using either 20% mRNAPsPcK (prepared by polysome immunoprecipitation as described above) or poly(A)‘RNA isolated from the liver of a fasted-glucose-fed rat, which was devoid of detectable mRNAPsPcK (less than 0.001% of total mRNA activity). Plasmids suspected of containing P-enolpyruvate carboxykinase cDNAs were isolated as described by Kahn et al. (26) and were screened by hybrid-selected translation by the method of Ploegh et al. (27) with the following changes. Eco RI-digested plas- mids were extracted with phenol/chloroform (1:l) and then spotted onto nitrocellulose paper as described by Thomas (19). Hybridization was carried out for 2-3 h and, following elution of the bound RNA, 5 pg of yeast tRNA (Bethesda Research Laboratories) was added as carrier. Translation, immunoprecipitation, and polyacrylamide gel electrophoresis of translation products were done as described above.

Restriction Mapping-Restriction endonuclease mapping was done essentially as described by Boseley et al. (28) using partial digestion and agarose electrophoresis. The unique Cla I site at posi- tion 23 in pBR322 was labeled using [a-J”P]dCTP and DNA polym- erase I (29). The labeled ends were separated asymmetrically by complete digestion with BamH I.

RESULTS

Purification of mRNAPEPCK-In BtzcAMP and cyclohexi- mide-treated rats, mRNAPEPcK constitutes less than 0.5% of total hepatic mRNA as measured by in vitro translational assay. It was therefore essential to enrich mRNAPEPCK to the highest degree possible to maximize the probability of suc- cessfully cloning a P-enolpyruvate carboxykinase cDNA. The procedure we used was to immunoprecipitate polysomes containing mRNAPcPcK with an antiserum directed against P-enolpyruvate carboxykinase (see “Experimental Procedures”). Fig. 1 shows the results of a purification exper- iment. The purified mRNAPEPCK directed the synthesis of a major protein which co-migrated with authentic P-enolpyru- vate carboxykinase (lane 3) and was immunoreactive with P- enolpyruvate carboxykinase antiserum (data not shown). Quantitative densitometry of the fluorogram revealed that mRNAP”P”K activity was 0.4% (lane 2) and 25% (lane 3) of total hepatic mRNA activity before and after purification, respectively. The recovery of mRNApnPcK varied from lo- 30%, and 5-10 pg of purified RNA was routinely recovered from 20-30 g of liver. The apparent purity of mRNAPEPCK obtained by this procedure is similar to that described for immunoglobulin K chain (14), prothrombin (30), and trypa- nosome surface antigen (31) messenger RNAs purified by similar solid phase procedures.

Cloning and Identification of P-enolpyruvate Carboxyki- nase cDNA-mRNAPkpCK, purified as described in Fig. 1, was used as a template for the synthesis of double-stranded cDNA which was then inserted at the Pst I site of the cloning vector pBR322. Recombinant DNA plasmids were used to transform

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

2024 Phosphoenolpyruvate Carboxykinase mRNA Regulation by CAMP

" e92

1 2 3 FIG. 1. Fluorography of in vitro translation products.

Poly(A)+RNA was extracted from rat liver polysomes before and after immunoprecipitation of polysomes with P-enolpyruvate car- boxykinase antiserum. Translation reactions were conducted using reticulocyte lysates programmed with 250 ng of RNA and the [""SI- methionine-labeled products were analyzed by sodium dodecyl sul- fate-polyacrylamide gel electrophoresis and fluorography. Details of these procedures are described in the text. The figure shows the products synthesized with no added RNA (lane I ) , with RNA ex- tracted from total hepatic polysomes (lane 2). and with RNA isolated from immunoprecipitated polysomes (lane 3) . The arrows denote molecular weight markers and P-enolpyruvate carboxykinase (PEPCK) determined from standards on a separate lane (not shown). The standards and their molecular weights, shown at the right, are: phosphorylase b (92,000). bovine serum albumin (69,000), ovalbumin (46,000), carbonic anhydrase (30,000). and p-lactoglobulin A (18,000). BPB, bromphenol blue.

E. coli strain HB-101. One thousand seven hundred tetracy- cline-resistant transformants were obtained and pooled to make a cDNA library which was plated on nitrocellulose filters. Replicates were made (24) and screened by differential in situ hybridization (25) using '"P-labeled probes that were synthesized from mRNAs which were either devoid of or enriched in mRNA"E'"'K. In the experiment shown in Fig. 2, approximately 70 colonies hybridized to the enriched probe whereas fewer than 10 colonies hybridized to the control probe. This result suggested that those colonies that hybrid- ized strongly with the enriched probe, but not with the control probe, contained DNAs complementary to mRNA"E':'"'K.

The number of positive clones was lower than expected from the apparent purity of the mRNA and could be due to

several factors. First, the purity of the mRNA could have been overestimated by the translational analysis (Fig. 1). Second, we made no attempt to enhance hybridization by altering the time or stringency of hybridization. Finally, trans- formation could occur without the presence of a significant insert. After the initial cloning, we selected 15 clones, isolated plasmid DNAs from 10-ml cultures OT each, and determined the insert sizes by electrophoresis in agarose. Nine of the 15 plasmids had inserts that were less than 100 bp long. A minimum 30-bp dG-dC tail length on each end leaves less than 40 bp Cor the cDNA, some of which might be oligo(dA- dT). Six plasmids had inserts that were 400-1200 bp in length. Thus, 60% of the transformed clones had virtually no cDNA inserts.

Twelve plasmids from colonies that were positive by differ- ential hybridization were further characterized by hybrid-se- lected translation (27). One plasmid, pPC2, appeared to hy- brid-select mRNA"E'"'" In . a preliminary screen (data not shown). pPC2 was then tested in the hybrid selection experi- ment shown in Fig. 3. In this experiment, pPC2, the vector plasmid pBR322, or the plasmid prAlbI containing a rat albumin cDNA (12), were bound to nitrocellulose filters and then hybridized to 50 pg of total poly(A)'RNA isolated from a Bt?cAMP-treated rat. pBR322 and prAlbI were used to assess the specificity of hybrid selection. The filters were washed extensively and the mRNA hybridized to the bound plasmid was eluted in boiling water and translated in a retic- ulocyte lysate system. The proteins synthesized in vitro were analyzed by polyacrylamide gel electrophoresis and fluorog- raphy. A large number of proteins were synthesized under these conditions, (cf lane I with lanes 2-5), indicating that some RNA was nonspecifically adsorbed by the filters. In order to determine if P-enolpyruvate carboxykinase and al- bumin were present in these protein mixtures, aliquots from the translation reaction were immunoprecipitated using anti- P-enolpyruvate carboxykinase, anti-rat albumin, or nonim- mune serum. These immunoprecipitations showed specific selection of mRNA"""('" by only the cDNA plasmid pPC2 (cf lanes 6-8), and specific selection of albumin mRNA by only prAlBI (cf lanes 10-12). When both pPC2 and prAlbI DNAs were bound to the same filter, both P-enolpyruvate carboxy- kinase and albumin mRNAs were selected (lanes 9 and 13). Proteins smaller than P-enolpyruvate carboxykinase and al- bumin were also immunoprecipitated by the corresponding antisera and are discussed below. No proteins were precipi-

- -

FIG. 2. Screening by differential colony hybridization. The purified mRNAl":l"'" described in Fig. 1 was used as template for the synthesis and cloning of recombinant cDNA as described in the text. Replicate cultures of the transformed bacteria were grown on nitro- cellulose filters and then subjected to in situ hybridization using single-stranded cDNA probes synthesized using poly(A)'RNA devoid

activity (autoradiogram A ) or enriched in mRNA"El"'" as in Fig. 1 (autoradiogram B ) . The three sets of dots enclosed by ovals on the edges of each autoradiogram are key marks used to align and identify hybridizing colonies on the original filter. Procedures are described under "Experimental Procedures."

of mHNAl'El'('"

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

Phosphoenolpyruvate Carboxykinase mRNA Regulation by CAMP 2025

ments of P-enolwruvate carboxvkinase. The observations

-92

-Albumin 'PEEK

-46

-30

a * 18

I 'PI 1 2 3 4 5 0 I O 3 IUII I L I . .

Total Anti-PEPCK Anti-Alb

FIG. 3. Screening by hybr id selection. A recombinant plasmid presumed to contain sequences complementary to mHNA""""" (pPC2) was tested by hybrid-selected translation as described under "Experimental Procedures." pBH322 and a recombinant DNA plas- mid containing rat serum albumin cDNA (prAlbI) were used to assess the specificity of hybrid selection. The figure shows fluorograms derived from ""S-labeled peptides synthesized in citro in lysates programmed with hybrid-selected RNAs. Two-pl aliquots were used for analysis of total translational products and 7.5-p1 aliquots were used for the immunoprecipitated samples. Lane I shows total trans- lational products without added RNA. Products of the in cifro translation of hybrid-selected RNAs are shown for pBH322 (lanes 2. 6, and IO), pPC2 (lanes 3, 7, and I 1 ). prAlbI (lanes 4.8, and 12), and a mixture of pPC2 and prAlbI (lanes 5, 9, and 13). Total translation products are shown in lanes 2-5. Aliquots of translation products were precipitated with antisera to 1'-enolpyruvate carboxykinase (Anti-PEPCK, lanes 6-9) or to albumin (Anti-Alb; lanes 10-13). The mobilities of mefhyl-"C-protein standards and their molecular weights (xlO-.') are shown at the right. The standards are the same as those in Fig. 1.

tated by nonimmune serum (data not shown). Since serum albumin mRNA was very abundant (20 to 50 times the trans- lational activity of mRNA""'"'"), the small amount of this message which appeared to have been selected by both the cloning vector pBR322 (lane 10) and by pPC2 (lane 1 1 ) was attributed to nonspecific adsorption.

To test the possibility that the smaller proteins that im- munoprecipitate with anti-P-enolpyruvate carboxykinase and with anti-rat serum albumin (Fig. 3) might be fragments of the respective proteins, we compared the peptide cleavage products of P-enolpyruvate carboxykinase and albumin with the smaller proteins. The procedure employed digestion with S. aureus V8 protease as described by Cleveland et al. (32). RNA recovered from a hybrid selection experiment was trans- lated and the "S-labeled products were immunoprecipitated with anti-P-enolpyruvate carboxykinase or anti-albumin sera. They were subjected to two-dimensional polyacrylamide gel electrophoresis in which proteins were digested in situ with V8 protease between the first (sodium dodecyl sulf&te tube gel in 8.75% acrylamide) and the second (sodium dodecyl sulfate slab gel in 15% acrylamide) dimensions. The fluoro- grams of gels from such an experiment are shown in Fig. 4. I t is evident that the smaller proteins share peptides in common with their respective parent substances. The peptide pattern of P-enolpyruvate carboxykinase labeled in vivo and then purified by immunoprecipitation is shown in the inset in A. This pattern and the molecular weights of the peptides are identical with those derived from the in vitro translation of the hybrid-selected RNA (A) . Further, the peptide patterns of P-enolpyruvate carboxykinase, albumin, and tyrosine ami- notransferase (data not shown) are distinctly different from each other. I t therefore seems probable that the smaller proteins which appear with P-enolpyruvate carboxykinase upon translation of hybrid-selected mRNA (Fig. 3) are frag-

." consistent with this conclusion are: 1) P-enolpyruvate car- boxykinase and the smaller proteins which were seen only when mRNA was hybrid-selected with pPC2, and not with pBR322 or prAlbI, shared common proteolytic peptides; 2) P- enolpyruvate carboxykinase labeled in vivo in rat liver, and then purified, has the same peptide map as that synthesized in vitro; 3) both P-enolpyruvate carboxykinase and the smaller proteins immunoprecipitated with anti-P-enolpyru- vate carboxykinase antibody; and 4) the antiserum used has been previously demonstrated to be specific for P-enolpyru- vate carboxykinase (7). Albumin produced by translation of RNA hybrid-selected by prAlbI showed evidence of degrada- tion exactly analogous to P-enolpyruvate carboxykinase, i.e. smaller proteins appeared only upon hybrid selection with prAlbI, they immunoprecipitated only with anti-albumin an- tibody, and they shared proteolytic peptides in common with albumin. We cannot explain the mechanism by which discrete, small fragments of these specific proteins are formed. Since this happens only during the hybrid selection procedure, and not during a straight in vitro translation assay (cfi Figs. 1 and 3), it would seem that something occurs during hybrid selec- tion that causes termination at selective sites and/or causes selective site degradation of these mRNAs.

Characterization of pPC2-The cDNA insert of pPC2 could be excised with Pst I and was about 760 bp in length (Fig. 5). Since the coding sequence of mRNAPEPCK was ex- pected to be approximately 2000 bp (not including probable 5' and 3' noncoding sequences), pPC2 probably represents less than 40% of theoretical full length. To obtain a restriction map of the insert, pPC2 was digested with Cla I and BamH I which cut 774 and 1126 bp from the insert boundary, respectively, but did not cut the insert. The two Cla I termini, but not the BamH I ends, were labeled with DNA polymerase I ana [a-"'P]dCTP (29) and the long linear DNA containing the insert near the labeled end was partially digested with various restriction enzymes (28). Digestion products were separated by agarose gel electrophoresis and the labeled frag-

I B

1' 67 54 20 I 09 45 35 t t I t t

FIG. 4. Proteolytic digestion and analysis of proteins from hybrid selection. A hybrid selection experiment similar to that described in Fig. 3 was conducted in order to obtain the multiple proteins immunoprecipitated with P-enolpyruvate carboxykinase or albumin antisera. The respective samples were subjected to electro- phoresis in 2-mm diameter tube gels with 8.75% acrylamide. These gels were layered onto slab gels of 15%' acrylamide and overlayered with a solution containing 25 pg/ml S. aureus V8 protease as described by Cleveland et al. (32). Proteolytic digestion occurred during the stacking procedure of the second dimension. Following electrophore- sis, the slab gel was subjected to fluorography to visualize the ""S- containing peptides. A, fluorogram of peptides obtained following hybrid selection with pPC2 and immunoprecipitation with anti-1'- enolpyruvate carboxykinase. B, peptides following hybrid selection with prAlbI and immunoprecipitation with anti-rat serum albumin. Only the central portions of each gel are shown. The same molecular weight markers used in Figs. 1 and 3 were used here. Molecular weights of the parent proteins (horizontal dimension) and the V8- peptides (vertical dimension) were estimated from a standard curve and are shown in both A and B. The inset in A shows the V8 peptides generated from 1'-enolpyruvate carboxykinase that had been labeled with [:'%]methionine in uivo and then electrophoresed on a 15% polyacrylamide gel.

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

2026 Phosphoenolpyruvate Carboxykinase mRNA Regulation by CAMP

ments were detected by autoradiography. Of the 17 endonu- cleases tested, eight cut within the cDNA (Fig. 5), while Aua 11, BamH I, BstN I, Cla I, Eco RI, HincII, HindIII, Msp I, and Sal I did not cut in the insert. A notable feature was the apparent absence of restriction enzyme sites in the region between 0 and 225 bp. Nine of the restriction enzymes used to construct the map have 4-bp recognition sequences and should cut an average of once every 256 bp of random sequence DNA. If pPC2 is derived from the poly(A)' region of mRNA""'"'K, pPC2 would be complementary to the 3'-end of mRNA1'"PCK and consist largely of noncoding sequence. I t would thus not be surprising that none of the enzymes tested cut in the 0 to 225-bp region. Indeed, pPC2 did not hybrid-arrest the trans- lation of mRNA""':'"'K (data not shown), suggesting that little or no coding sequence was present (33).

Quantitation by Hybridization-In order to use the cloned cDNA to measure mRNA"E1"'K levels by hy- bridization, it was necessary to establish appropriate condi- tions for such an assay. mRNA"l"'K was quantitated by a dot- blot hybridization procedure (19) whereby samples of hepatic poly(A)'RNA from various test conditions are bound to nitro- cellulose filters. The cloned insert was isolated from pPC2, labeled with "P by nick translation, and then allowed to hybridize with the mRNAr'"l'"K bound to the filters. Unhy- bridized DNA was washed off and an autoradiogram of the

mRNA!'!.''c"

Pst Hf A HfD D A Hf HaPsI 1 1 1 1( 1 1 I ( (

R 1

T 53 S9 1 I 1

Imp 0 d l 0 2 0.3 0.4 0.5 016 0.7 OB FIG. 5. Restriction map of pPC2. The restriction map of the

cDNA insert was determined as described in the text. The end closest to the Eco R1 site in pBR322 is designated as 0 on the cDNA. A scale showing kilobase pairs is shown below the map. Symbols: A, Alu I; D, Dde I; Ha, Hae 111; Hf, HinfI; R, Rsa I; S9. Sau96 I; S3. Sau3A I; T, Tag I. No sites were found for Acta 11, BamH I. BstN I, Cla I, Eco KI, HincII, HindIII, Msp I, and Sal I.

24.0

Poly (A)* RNA per Spot (ng) FIG. 6. Quantitation of mRNA"E"'K by dot-blot hybridiza-

tion. Poly(A)'RNA was isolated from a fasted, glucose-fed, BtncAMP-treated rat. Various dilutions were applied to nitrocellulose filters and analyzed by dot-blot hybridization (19) using the inserted cDNA from pPC2 that was labeled with ,"I' by nick translation ( A ) . The same RNA samples were also hybridized to the inserted cDNA from prAlbI (12) that was also labeled with ''21' by nick translation ( B ) . Following hybridization and washing, autoradiograms were pre- pared on X-Omat AH x-ray film. The autoradiograms, shown in the insets, were scanned using a densitometer (I'ransidyne General Cor- poration) equipped with an integrator. The results are plotted as nanograms of RNA/spot uersus autoradiogram image density ex- pressed in integrator units (which are arbitrary since they depend upon the sensitivity setting of the densitometer). The vertical (error) bars designate the range of the duplicate spots. The experimental details are described under "Experimental Procedures."

FIG. 7. Hybridization analysis of hepatic mRNAPEKK from fasted, glucose-fed, and BtzcAMP-treated rats. Nine male rats (100 g, C1) strain) were fasted 20 h; then 6 were fed 500 mg of glucose by stomach tube. Two hours later, 3 of the glucose-fed rats were injected intraperitoneally with saline and 3 were injected with Bt2cAMP plus theophylline (3 mg each/100 g body weight). All rats were killed 2 h after the injections and hepatic poly(A)'RNA was extracted (7) and analyzed by dot-blot hybridization as described in Fig. 6, using '"1'-pPC2 insert ( A ) or "'P-prAlbI insert (B) . The autoradiograms shown above are: 1, RNAs from fasted rats; 2, RNAs from fasted, glucose-fed rats; and 3, RNAs from fasted, glucose-fed rats treated with Bt2cAMP. Each dot contained 250 ( A ) or 10 ng ( B ) of poly(A)'RNA. The quantity of specific mHNAs and the autoradi- ogram image densities are directly proportional under these hybridi- zation conditions (Fig. 6).

filter was prepared. The results of such an experiment are shown in Fig. 6. The autoradiogram image densities are pro- portional to the quantity of poly(A)'RNA spotted on the filters when less than 200-300 ng were probed with "P-pPC2 insert and less than 8-10 ng were probed with "'P-prAlbI insert. The RNA used for this characterization was isolated from a fasted, glucose-refed, BtcAMP-treated rat and, thus, mRNAPE1"'K was maximally induced. The difference in sensi- tivities between the two probes probably reflects the approx- imate 50-fold greater abundance of albumin mRNA as com- pared to mRNAP"l'CK. Neither probe bound to RNAs isolated from tissues that do not make P-enolpyruvate carboxykinase or albumin (data not shown). Moreover, neither probe bound to tRNA or rRNA, and the addition of up to 30 pg of rRNA to poly(A)'RNA applied to a single spot had no effect on the quantity of probe hybridized (data not shown). These results indicate that the dot-blot procedure can be used to determine relative concentrations of specific mRNAs.

Effect of Bt2cAMP a n d Glucose on mRNAPEPCK Leuels- We have employed this assay to assess the effect of Bt2cAMP and glucose on mRNAP""CK levels in rat liver. The autoradi- ogram, shown in Fig. 7A, demonstrates the dramatic changes that occur as a result of feeding glucose to fasted rats, and treatment of fasted-refed rats with BtcAMP. Glucose feeding decreased whereas Bt2cAMP injection increased mRNAP"'"'K. This result could be obtained as a result of specific changes in the degree of polyadenylation of mRNAPEPCK. This possibility was eliminated since virtually no hybridizable mRNA""'"K can be detected in poly(A)-RNA and this result is unaffected by BtcAMP and glucose (data not shown). In contrast, the mRNA coding for albumin did not change in these same RNA samples (Fig. 7B). This measurement, made using the cDNA insert from prAlbI which is complementary to albumin mRNA (12), provided an internal control for the hybridization reac- tion since the activity of albumin mRNA is not affected by glucose feeding (3) or by Bt2cAMP during the short treatment times used here.3 This observation, coupled with the fact that

E. G . Beale, J . L. Hartley, and D. K. Granner, unpublished observations.

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

Phosphoenolpyruuate Carboxykinase mRNA Regulation by CAMP 2027

glucose and BbcAMP do not affect the total translational activity of poly(A)+RNA (3,5-7,9), indicates that these effects on ~ R N A ~ ~ ~ ~ ~ are quite specific.

If the activity of mRNAPEPCK is principally determined by changes in the quantity of specific RNA sequences, different treatment conditions should result in proportional changes in mRNAPEPCK quantitated by translational and hybridization assays. The results of such a comparison are shown in Fig. 8. Glucose feeding decreased hybridizable mRNAPEPCK from 0.50 & 0.01 to 0.06 k 0.02 arbitrary units and the subse- quent treatment with BbcAMP increased the quantity of mRNAPEPCK to 0.80 & 0.01 units. The corresponding transla- tional activities of mRNAPEPCK in these same control, glucose- fed, and glucose-fed plus CAMP-treated animals were 0.19 & 0.02%, 0.01 f 0.005%, and 0.24 f 0.04%, respectively. These proportionate changes in mRNAPEPCK measured by transla- tion or by hybridization have been seen in three separate experiments. Thus, a mechanism by which either Bt2cAMP or glucose acts by changing the activity of a constant amount of mRNAPEPCK is excluded. These observations led us to conclude that regulation occurs in the generation or degra- dation of this specific mRNA.

mRNAPEPCK Turnover-We have previously presented ev- idence that BhcAMP treatment has no detectable effect on the rate of inactivation of mRNAPEPCK (7). We have repeated similar experiments here but mRNAPEPCK was quantitated by

Control Glwose Glutose +Bt2cAMP

FIG. 8. Comparison of mRNApEPCK quantitated by hybridi- zation and by translation assays. The image densities of an autoradiogram of the dot-blot shown in Fig. 7 were quantitated as described in Fig. 6. The in vitro translational activities of the same RNAs were measured using the rabbit reticulocyte lysate system. The hybridized RNA is shown by the open bars. The translational activity of mRNAPEPCK is shown by the hatched burs and is expressed as a percentage of the total mRNA activity (i.e. (radioactivity incor- porated into phosphoenolpyruvate carboxykinase/radioactivity incor- porated into trichloroacetic acid-insoluble material) X 100). The vertical bars designate the standard error of the mean of the three RNA samples in each treatment group.

0 4 O B o I TIME (mid

FIG. 9. Time course of hybridizable ,RNAPEPCK following glucose feeding and following BtzcAMP treatment. mRNAPEPCK was quantitated by the dot-blot procedure, described in Fig. 6, follow- ing glucose feeding to fasted rats ( A ) or after Bt2cAMP injection into fasted-refed rats ( B ) . Experimental conditions were as described in Fig. 7 except that the animals were killed at the indicated times following treatment, and BtZcAMP was injected at 45-min intervals.

the dot-blot procedure. The basis of this determination of mRNAPEPCK half-life is that the rate of change from an initial to a final steady state, following either glucose feeding or BtzcAMP treatment, is equivalent to the turnover rate (34). This assumes that the synthesis or degradation rates change rapidly and become constant so that the rates of change of

negative (deinduction) or positive (induction) directions. The time courses of mRNAPEPCK levels following glucose feeding and Bt,cAMP treatment are shown in Fig. 9, A and B, respec- tively. Although the experiments shown here cover time pe- riods of only 2 h, mRNAPEPCK levels remain unchanged 2-4 h following either glucose or BtZcAMP (data not shown). Thus, new steady states have apparently been achieved. Several techniques have been devised to analyze such kinetic data (35, 36). The analysis of Fig. 9 using any one of these procedures indicated that turnover does become fiist order and we esti- mated that the half-life of mRNAPEPcK is 10-40 min. No discernible effect of BtZcAMP on the half-life was noted. Since at least a 20-fold change in mRNAPEPCK stability would be necessary to account for the magnitude of induction by Bt,cAMP, it is unlikely that the regulation of turnover rate is significant under the experimental conditions used here.

m ~ ~ ~ P E P C K approach first order. This is true for changes in

DISCUSSION

Previous studies from several laboratories, including our own, have shown that the synthesis of hepatic P-enolpyruvate carboxykinase is rapidly increased by Bt2cAMP treatment and is decreased by dietary glucose (1-3, 5-7, 37). Alterations in the synthesis of P-enolpyruvate carboxykinase correlate well with changes in mRNAPEPCK activity, as measured by in vitro translational assays (3, 5-7). Heretofore, it has not been possible to directly analyze the various mechanisms which could change the translational activity of mRNApEpCK. We have succeeded in isolating a cDNA, pPC2, directed against a portion of the mRNA coding for P-enolpyruvate carboxy- kinase. The observations supporting this assertion include: 1) pPC2 was generated from poly(A)'RNA in which

ential colony hybridization revealed many clones, including pPC2, that hybridized to a probe that was rich in P-enolpyr- uvate carboxykinase cDNA but not to a probe devoid of these sequences (Fig. 2); and 3) pPC2 selected RNA which pro- grammed the in vitro synthesis of P-enolpyruvate carboxyki- nase and proteins which appeared to be fragments of P- enolpyruvate carboxykinase (Figs. 3 and 4). Ultimate proof, however, awaits a comparison of sequence information of P- enolpyruvate carboxykinase and P-enolpyruvate carboxyki- nase cDNA, neither of which is presently available.

We have used pPC2 as a hybridization probe to show that

ately in response to BtzcAMP injection and dietary glucose. This indicates that an activation-inactivation process involv-

conditions used here. Previous time course measurements of the changes in the in vitro translational activity of mRNAPEPCK following BtzcAMP treatment or glucose feeding indicated a half-life of approximately 20 to 30 min and led to the conclusion that BtzcAMP treatment does not significantly affect the rate of inactivation of mRNAPEPCK (6, 7). The results reported here corroborate and extend this conclusion since the kinetics is very similar whether mRNAP"r"'K is quantitated by in vitro translation assay (3 ,6 ,7) or by hybrid- ization assay (Fig. 9). If Bt2cAMP has no effect on the degra- dation rate of mRNAPEPCK, it must stimulate some step in the biosynthetic pathway (transcription, processing, modification, transport). Clearly, this important point was determined in-

m ~ ~ ~ P E P C K was the major single species (Fig. 1); 2) differ-

m ~ ~ ~ P E P C K concentration and activity change proportion-

ing m ~ ~ ~ P E P C K is not quantitatively important under the

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

2028 Phosphoenolpyruvate Carboxykinase mRNA Regulation by CAMP

directly and must be substantiated by directly quantitating synthesis and turnover with isotopic labeling

procedures using specific cDNAs to affinity purify the radio- active mRNAPEPcK sequences.

Although this is the fiist report of the measurement, by hybridization assay, of the induction by cyclic AMP of a nonabundant mRNA that codes for an enzyme, this effect is not unique. cAMP also increases the mRNA that codes for an abundant protein, albumin, in cultured mouse hepatoma cells (38). While the physiological significance of this effect on albumin is not clear, there are a t present two examples of pretranslational regulation by CAMP, P-enolpyruvate car- boxykinase, and albumin mRNAs. One must therefore con- sider the possibility that cAMP has a nuclear site of action in eukaryotic cells. One would predict from the hypothesis of Kuo and Greengard (39) that a CAMP-dependent protein kinase which phosphorylates some regulatory protein is in- volved. Alternatively, a mechanism similar to that in bacteria (40) may exist in which a CAMP-binding regulatory protein interacts with chromatin and leads to a change in specific mRNA levels. Elucidation of the mechanism(s) involved will likely require direct measurements of each of the steps in

generation. Such studies should now be possible since the rate-limiting step has been the production of a specific cDNA probe.

m ~ ~ ~ P E P C K

m ~ ~ ~ E ’ E P C K

Acknowledgments-We are grateful for the advice given by Kath- ryn Robson, Don Cleveland, Keith Yamamoto, John Donelson, Jim Hargrove, and Tris Parslow. We also thank Steve Koch for technical assistance and Janet Adams for typing the manuscript.

REFERENCES 1. Wicks, W. (1974) Adu. Cyclic Nucleotide Res. 4,335-438 2. Tilghman, S., Hanson, R. & Ballard, F. (1976) in Gluconeogene-

sis-Its Regulation in Mammalian Species (Hanson, H. & Mehlman, M., eds) pp. 47-91, John Wiley & Sons, New York

3. Kioussis, D., Reshef, L., Cohen, H., Tilghman, S., Iynedjian, P., Ballard, F. & Hanson, R. (1978) J. Biol. Chem. 253, 4327-4332

4. Seitz, H., Muller, M., Nordmeyer, P., Krone, W. & Tarnowski, W. (1976) Endocrinology 99, 1313-1318

5. Iynedjian, P. & Hanson, R. (1977) J. Biol. Chem. 252, 655-662 6. Nelson, K., Cimbala, M. & Hanson, R. (1980) J. Biol. Chem. 255,

7. Beale, E., Katzen, C. & Granner, D. (1981) Biochemistry, 20,

8. Ernest, M. J . & Feigelson, P. (1978) J. Biol. Chem. 253, 319-322

8509-8515

4878-4884

9. Noguchi, T., Diesterhaft, M. & Granner, D. (1978) J. Biol. Chem. 253, 1332-1335

10. Firestone, G. & Heath, E. (1981) J. Biol. Chem. 256, 1396-1403 11. Derda, D., Miles, M., Schweppe, J . & Jungmann, R. (1980) J.

12. Kioussis, D., Hamilton, R., Hanson, R., Tilghrnan, S. & Taylor, J.

13. MacGillivray, R., Chung, D. & Davie, E. (1979) Eur. J. Biochem.

14. Gough, N. & Adams, J. (1978) Biochemistry 17, 5560-5566 15. Palacios, R., Palmiter, R. & Schimke, R. (1972) J. Biol. Chem.

16. Kessler, S. (1975) J. Zmmunol. 115, 1617-1624 17. Krystosek, A,, Cawthon, M. & Kabot, D. (1975) J . Biol. Chem.

18. Laemmli, U. (1970) Nature (Lond.) 227,680-685 19. Thomas, P. (1980) Proc. Natl. Acad. Sei. U. S. A . 77, 5201-5205 20. Kelley, R., Cozzarell, N., Deutscher, M., Lehman, I. & Kornberg,

21. Wickens, M., Buell, G. & Schimke, R. (1978) J . Biol. Chem. 253,

22. Roychoudhury, R. & Wu, R. (1980) Methods Enzymol. 65, 43-62 23. Morrison, D. (1979) Methods Enzymol. 68, 326-328 24. Hanahan, D. & Meselson, M. (1980) Gene 10,63-67 25. St. John, T. & Davis, R. (1979) Cell 16, 443-452 26. Kahn, M., Kolter, R., Thomas, C., Figurski, D., Meyer, R., Re-

maut, E., & Helinski, D. R. (1979) Methods Enrymol. 68, 268- 280

27. Ploegh, H., Orr, J . & Strominger, J . (1980) Proc. Natl. Acad. Sci. U. S. A . 77,6081-6085

28. Boseley, P. G., Moss, T. & Birnstiel, M. L. (1980) Methods Enz~vvmol. 65, 478-494

29. Hartley, J . & Donelson, J. (1980) Nature 286, 860-865 30. MacGillivray, R., Degen, S., Chandra, T., Woo, S. & Davie, E.

(1980) Proc. Natl. Acad. Sei. U. S. A . 77, 5153-5157 31. Shapiro, S. & Young, J. (1981) J. Biol. Chem. 256, 1495-1498 32. Cleveland, D., Fischer, S., Kirschner, M. & Laemmli, U. (1977) J.

33. Ricciardi, It., Miller, J . & Roberts, B. (1979) Proc. Natl. Acad.

34. Berlin, C. & Schimke, R. (1965) Mol. Pharmacol. 1, 149-156 35. Segal, H. & Kim, Y. (1963) Proc. Natl. Acad. Sei. U. S. A. 50,

36. Guyette, W., Matusik, R. & Rosen, J . (1979) Cell 17, 1013-1023 37. Wicks, W., Barnett, C. & McKibbin, J . (1974) Fed. Proc. 33,1105-

38. Brown, P. & Papaconstantinou, J. (1979) J. Biol. Chem.. 254,

39. Kuo, J. & Greengard, P. (1969) Proc. Natl. Acad. Sci. U. S. A . 64,

40. Pastan, I. & Adhya, S. (1976) Bacteriol. Rev. 40,527-551

Biol. Chem. 255, 11112-11121

(1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4370-4374

98,477-485

247, 2316-2321

250, 6077-6084

A. (1970) J. B i d Chem. 245,39-45

2483-2495

Biol. Chem. 252, 1102-1 106

Sei. U. S. A. 76, 4927-4931

912-918

1111

9379-9384

1349-1355

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

E G Beale, J L Hartley and D K Grannercoding for hepatic phosphoenolpyruvate carboxykinase (GTP).

N6,O2'-dibutyryl cycle AMP and glucose regulate the amount of messenger RNA

1982, 257:2022-2028.J. Biol. Chem. 

  http://www.jbc.org/content/257/4/2022Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/257/4/2022.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from