Isolation of a Complementary DNA Encoding the Catalytic Subunit Of

Home , Molecular probe

(CANCER RESEARCH 50. 1675-1680, March 15. 1990] Isolation of a Complementary DNA Encoding the Catalytic Subunit of Protein Kinase A and Studies on the Expression of This Sequence in Rat Hepatomas and Regenerating Liver1

Jeffrey S. Roth, Ling-Ling Hsieh, Carl Peraino, and I. Bernard Weinstein2 Comprehensive Cancer Center and Institute of Cancer Research Â¡L-L.H., I. B. H'.J and Departments of Genetics and Development /J. S. R.Â¡and Medicine Â¡I.B. H'./, Columbia University, New York, New York 10032, and Division of Biological and Medical Research, Argonne National Laboratory, Arianne, Illinois 60439 [C. P.I

ABSTRACT subunit, which then phosphorylate specific intracellular targets (2, 3). A complementary DNA (cDNA) clone (B4) encoding the catalytic Notwithstanding suggestions that free regulatory subunits subunit of a cAMP-dependent protein kinase (PKAc) was isolated from have a biological function independent of the titration of the a XgtlO rat brain cDNA library, using a synthetic oligonucleotide probe catalytic subunit (4), the principal effect of cAMP on cells whose sequence was based on the known amino acid sequence of a bovine appears to be the activation of PKAc. It is therefore of interest cardiac PKAc. Sequence analysis of this clone revealed a region of 1002 to study PKAc directly as a means of approaching cAMP- nucleotides which encodes a protein that is 92% homologous to amino acids 17-350 of the bovine cardiac PKAc protein. This clone lacks coding dependent phenomena. Shoji et al. (5) deduced the amino acid sequences for amino acids 1-16 of the latter protein. Nevertheless, it sequence of this enzyme isolated from bovine heart, finding it provided a useful probe to analyze expression of the related gene in a to be composed of 350 residues; other characteristics of this variety of systems. Northern blot analyses using a <2P-labeled probe protein, such as phosphorylation sites (6) and A'-terminal tetra- prepared from a 0.6-kilobase /VI fragment of clone B4 revealed an decanoate capping (7), have been extensively studied. More abundant 4.6-kilobase band in rat brain RNA and lesser amounts of this recently, several groups have isolated cDNA clones encoding 4.6-kilobase RNA in rat heart and liver. A 4.6-kilobase RNA was also this protein from murine, bovine, and human cDNA libraries detected in RNA samples obtained from mouse fibroblasts. This probe (8-11). Significantly, these studies have revealed that, in par also detected homologous RNA in a variety of nonrodent species. In allel with recent work on PKC (for review see Refs. 12 and 13), subsequent experiments, this cDNA was used as a probe to elucidate the there exists a family of genes encoding cAMP-dependent pro role of PKAc in post-surgical hepatic regeneration and diethylnitrosamine-induced hepatomas in the rat. These experiments revealed that, tein kinases, which differ in their primary amino acid sequences, following partial hepatectomy, PKAc mRNA is decreased 3-fold by 12 their message size, and their tissue specificity. In this paper we li. returning to normal by 72 h; hepatomas showed no consistent pattern describe the isolation of a clone from a rat brain cDNA library of change in PKAc mRNA levels as compared to controls. Our results which encodes a PKAc similar to the fi-form previously isolated indicate that this cDNA encodes an isoform of PKAc which is distinct from murine and bovine sources and examine its expression in from PKAc-a isolated by Uhler et al. (Proc. Nati. Acad. Sci. USA, 83: various tissues and species. It was of particular interest to use 1300-1304,1986) but highly homologous to PKAc-0 isolated by Showers this cDNA as a probe to explore the role of PKAc gene and Maurer (J. Biol. Chem., 261: 16288-16291, 1986), that depression expression in cellular proliferation in hepatic regeneration and of cAMP-dependent protein phosphorylation may be an important mech hepatic neoplasia in the rat. Experimental evidence has impli anism in the regeneration of mature rat liver but is not a consistent cated cAMP, and by extension cAMP-dependent protein phos alteration in chemically induced hepatoma, and that this cDNA is useful phorylation, as an important negative regulator of cell growth. as a probe for the study of the role of PKAc gene expression in growth Early work (14) demonstrated that cAMP, when added to control, particularly in rodent species. transformed cells in culture, causes a partial reversion of the transformed phenotype. Subsequent studies by numerous inves INTRODUCTION tigators working in a variety of systems (15, 16) have explored this idea, with considerable variability of findings. In hepatoma The importance of protein phosphorylation in the regulation cells, cAMP-associated receptors (e.g., adrenergic, glucagon, of cellular growth and metabolism is widely recognized and has and prostaglandin) have been shown to be decreased, as were been a major focus of research efforts in recent years (for review overall cAMP levels as compared to controls (17, 18). In see Ref. l). Among the principal protein kinases which regulate contrast, in liver cells which have emerged from quiescence cellular processes is the cAMP-dependent protein kinase following partial hepatectomy, many studies have demonstrated (PKA3). The PKA holoenzyme is a heterotetramer composed transient increases in intracellular cAMP levels, but an overall of two regulatory subunits which bind to and inhibit two cata decrease in adenylate cyclase activity with time (19), and ele lytic subunits. PKA is activated when 4 molecules of cAMP, vated levels of/3-adrenergic receptors (20). formed in the cell in response to various tissue-specific extra In order to explore this issue at the level of expression of the cellular signals, bind to the regulatory subunit dimer with high gene encoding the effector molecule of cAMP, PKAc, we have affinity, causing it to release two active molecules of catalytic used the cDNA whose isolation is described here as a probe to measure levels of PKAc gene expression in regenerating rat Received 5/23/89; revised 12/4/89. liver following partial hepatectomy and in a series of primary The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in rat hepatomas. Our results suggest that cellular proliferation in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. hepatomas and in hepatic regeneration may be mechanistically 1This research was supported by National Cancer Institute Grant CA 26056 different, the latter being associated with a fall in PKAc levels. (to I. B. \V.), by an award from the National Foundation for Cancer Research (to I. B. W.). and by a Medical Scientist Training Program (to J. S. R.). 2To whom requests for reprints should be addressed, at College of Physicians and Surgeons. Columbia University. Comprehensive Cancer Center, 701 West MATERIALS AND METHODS 168th Street, New York. NY 10032. 3The abbreviations used are: PKA. protein kinase A; PKAc. catalytic subunit Preparation of the Probe. The 54-base oligonucleotide probe (Fig. I) of protein kinase A; cDNA. complementary DNA; SSC. standard saline citrate and a 19-base oligonucleotide primer which hybridized to the nonde [20 x SSC (3 M NaCI and 0.3 M trisodium citrate)]; PKC. protein kinase C. generate .V end of the probe Â»eresynthesized on an automated DNA 1675

1Â« 20 if 40 60 â€¢¿ â€¢¿ â€¢¿ â€¢¿ CAA TTC CTA TCC AAA GCC AAA CAA CAC TTT CTC ACG AAA TGG GAG AAC CCT CCC CCC ACT Clu PhÂ« LÂ«u SÂ«r Lys AlÂ« LyÂ« Glu Asp PhÂ« LÂ«u Arg LyÂ»Trp Glu AÂ«n Pro Pro Pro SÂ«r 70 80 00 100 110 120 â€¢¿ AAT GCT GGG CTT GAG GAT TTT GAA AGG AAA AAA ACC TTC GGA ACG CGT TCC TTC GGA AGA Am Ali Cly LÂ«uGlu AÂ«p PhÂ« Glu Arg Lys Lyl Thr lÂ«u Gly Thr Gly SÂ«rPhÂ»Cly Arg

130 140 IBP 160 170 180 â€¢¿ CTC ATG CTC CTA AAC CAT AAC CCC ACT GAG CAC TAC H'is LyÂ« Al* Thr Clu Gin Tyr TAC CCC ATC AAG ATC TTA CAC AAC Vsl U.I LÂ«u VÂ«l LyÂ« Tyr Ali U.t LyÂ« IIÂ»LÂ«uAÂ»pLyÂ« 190 200 210 220 230 240 â€¢¿ â€¢¿ â€¢¿ CAG AAG GTC CTT AAC CTA AAG CAA ATA CAG CAC ACT CTG AAT GAG AAC AGA ATC CTC CAC Gin LyÂ«Val Vil Lyi LÂ«uLyÂ«Gin II* Clu Hij Thr lÂ«u Asn Clu LyÂ«Arg IIÂ«LÂ«uG ln

260 260 270 280 290 300 â€¢¿ â€¢¿ â€¢¿ GCA GTC GAG TTC CCG TTC CTT GTT GGG CTC CAG TAT TCT TTT AAC GAT AAT TCT AAT TTA AIÂ«VÂ«IGlu PhÂ«Pro PhÂ«LÂ«uVil Gly LÂ«uGlu Tyr SÂ«rPhÂ«LyÂ»AÂ«pAÂ«n5Â«rA*n LÂ«u

310 320 330 340 360 360 â€¢¿ â€¢¿ TAC ATC GTT ATC GAA TAC CTT CCT CCC CCC CAA ATC TTT TCA CAT CTA AGA AGA ATT CGA Tyr Met Vil MÂ«t Clu Tyr Vil Pro Cly Gly Clu M.t PhÂ« SÂ«r HiÂ»LÂ«uArg Arg IIÂ« Gly

370 360 390 400 410 420 â€¢¿ â€¢¿ ACG TTC ACT GAA CCC CAT GCT CGT TTC TAT GCA GCT CAG ATC GTC CTA ACA TTT GAG TAC Ara PhÂ»Sor Clu Pro HiÂ« Al* Arg PhÂ«Tyr AlÂ«AlÂ«Gin IIÂ«V.I LÂ«uThr PhÂ«Glu Tyr 430 440 460 460 470 480 Fig. 1. The open reading frame of the cDNA clone B4. Upper lines indicate the nu- CTCLÂ«uCATHI CCC GAA AAC CTC TTA ATT CAC CAC cleotide sequence; the corresponding amino Â»TCCSÂ«rCTCLÂ«uCACA.pCTCLÂ«uATCIIÂ«TACTyrACAArgGATA.pCTCLÂ«uAACLyÂ»Pro Glu AÂ»n LÂ«u LÂ«u I Iâ€¢AspH is acid sequence is shown below this. Differences between the translated nucleic acid sequence 490 600 BIB E20 630 640 of B4 and that of a bovine cardiac PKAc (5) CACGinCGT are underlined. Regions of homology of amino TTC GCC AAG ACA CTC AAG CGC AGG ACT TCC GlyTACTyrATCIIÂ«CACGinCTCVilACAThrCATAspTTTPhÂ«GGGCly PhÂ«AIÂ« LyÂ« Arg V.I Lys Gly Arg Thr Trp acid sequences to a protein kinase C (12) are in boxes. The regions of B4 corresponding to 660 660 670 680 690 600 the synthetic probe used to isolate it are dou ble-underlined. ACA TTC CGC CCA ATC AAG CGT TAC AAT PhÂ«TOTCy,Cly610ACCThrCCAProGACClu620TACTyrCIGLÂ«uGCCAlÂ«Pro630CACGluATCIIÂ«IIÂ«640CTCLÂ«uAGCSÂ«rLys Gly Tyr Asn

650 660

AAG GCA GTG CAC TGC TGG GCA TTG GGT GTA CTC ATC TAC CAG ATG GCT GCT GGC TAC CCC Lys AIÂ« V.I Asp Trp Trp AlÂ«LÂ«uGly V.I LÂ«uIIÂ«Tyr Clu M.t. All All Gly Tyr Pro

670 680 690 700 710 720 CCG TTC TTT GCT GAC CAG CCA ATT CAG ATT TAT GAG AAG ATT err GCC CCA AAG CTC CGC Pro PhÂ« PhÂ« All Asp G ln Pro IIÂ« Gin IIÂ« Tyr Glu Lys IIÂ« Vil All Gly Lys V*l Arg 730 740 760 760 770 780 TTC CCG TCG CAC TTC ACT TCC CAT CTC AAC CAC CTT CTG CGG AAT CTC CTC CAG GTG CAT PhÂ« Pro SÂ«r HiÂ« PhÂ« SÂ«r SÂ«r Asp LÂ«u Lys Asp LÂ«u LÂ«u Arg Asn LÂ«u LÂ«u Gin V.I Asp

790 800 810 820 830 840

CTC ACC AAG CGC TTT GGA AAC CTT AAG CAC CGG GTT AAT GAC ATC AAA AAC CAC AAG TGG LÂ«u Thr Lys Arg PhÂ« Gly Asn LÂ«u Lys Asp Gly VÂ»I Asn Asp IIÂ« Lys AÂ»nHis Lys Trp 860 860 870 880 890 900 â€¢¿ TTT CCT ACT ACC GAA TGG ATC GCC ATT TAC CCC AGA AAG GTT GAC GCT CCT TTC ATA CCA PhÂ« AlÂ» Thr Thr Glu Trp IIÂ«Ala IIÂ«Tyr Pro Arg Lys Vil Glu All Pro PhÂ« IIÂ«Pro

910 920 eae 940 960 9Â«Â« â€¢¿ â€¢¿ AAA TTC GCCLys CACAÂ«p| CAAClu CAA GAG ATC CGA Lyi PhÂ«AAA ClyTTTPhÂ«GCCClyCATAspACAThrTCTSÂ«rAACAsnTTCPhÂ«CAT] AspTATTyrCAA Clu Glu Glu IIÂ« Arg

970 980 990 1000 . â€¢¿ GTC CGT ATA ACA GAA AAA TCT GCA AAG GAC TTT TCT GAA TTT V.I Arg IIÂ« Thr Glu Lys Cys Gly Lys Glu PhÂ« SÂ«r Glu PhÂ«

Synthesizer (Applied Biosystems 380A). The oligonucleotide probe was Three pmol of the probe in a volume of 3 u\ were hybridized to 15 designed, using probable codon usage tables (21), to hybridize to a pmol of the primer in a volume of 1.1 u\, together with 1.5 /Â¿'of lOx nucleic acid corresponding to amino acids 217-235 of a bovine cardiac reverse transcriptase buffer (22). at 55Â°Cfor2 h. This mixture was then PKAc whose amino acid sequence had been determined by Shoji et al. cooled briefly and combined with 2 M'each of "P-labeled and unlabeled (5). Yields of the synthesized oligonucleotide were analyzed using a dATP. dCTP, dGTP, and dTTP, 1 u\ of lOx reverse transcriptase trityl ion colorimetrie assay, as described by the manufacturer. The buffer, 0.5 ^1water, and 1 n\ avian myeloblastosis virus (AMV) reverse primer was purified on a 20% acrylamide-50% urea gel, and the probe transcriptase (Life Sciences). This mixture was incubated at 37"C for 1 was purified using a CIS reverse phase high performance liquid chro- h, and the resulting 12P-labeled probe was used to screen a cDNA matography column run with a continuous 0-40% acetonitrile gradient. library, as described below. 1676

Screening of cDNA Libraries. XgtlO CHI rat brain cDNA libraries mentary 54-base oligonucleotide probe to detect a gene encod (library A, courtesy of R. Axel, Columbia University; library B. courtesy ing this peptide sequence (see "Materials and Methods"). This of J. Brosius, Mount Sinai School of Medicine) were plated at a density of 5 x IO4clones/15-cm plate, with a total number of plaques of 6 x synthetic oligonucleotide differed from the corresponding re IO5. These plates were blotted with Hybond-N nylon membranes gion of the cDNA which we subsequently isolated (see below) at only five positions other than those anticipated on the basis (Amersham Corp., Arlington Heights, IL) and processed according to the manufacturer's protocol. Filters from library A were prehybridized of codon preference tables. in a solution of 6x SSC, 5x Denhardt's solution, 50 ITIMNa2PO4,1% Two XgtlO cDNA libraries derived from female CHI rat sodium dodecyl sulfate, 20% formamide, and 2.5 Â¿igdenatured salmon brain were used in these studies. Library A (provided by R. sperm DNA, in a total volume of 125 ml, for 2 h at 42Â°C.The filters Axel) had an estimated complexity of 1 x IO6 clones, and were then hybridized in a solution of identical composition but with library B (provided by J. Brosius) had a complexity of 6 x IO6 the addition of the above-described 12P-labeled probe, at 42Â°Cfor 24 h, clones. Library A failed to yield a full length PKAc cDNA in a total volume of 50 ml. The filters were then washed to a stringency clone. It did, however, provide a clone that contained a 600- of 0.2x SSC. Positively hybridizing clones were plaque purified by base pair Pst\-Pst\ fragment encoding a polypcptide highly replating at lower density and screening as before. Single hybridizing homologous to a portion of bovine PKAc. This Pstl-Pstl frag plaques were stored in SM buffer containing 0.1% gelatin (22) and a trace of chloroform. Filters from library B were prepared and positive ment was used to screen library B for a full length cDNA clone. clones were picked and purified in a manner identical to that used with Fourteen recombinant plaques hybridized to this probe. Three library A. of these appeared to contain the same insert, which was ap DNA Sequencing and Vectors. DNA fragments obtained from the proximately 3 kilobases in length. The most abundant of these, above-described clones were subcloned into two Gemini series vectors clone B4, was subcloned into plasmid and phage vectors, and a (pGEMl or pGEM2) (Promega Biotech), for restriction mapping and detailed sequence analysis of clone B4 was performed (see chemical sequencing (23), or into bacteriophage ml3 vectors (mplS "Materials and Methods"). The sequence obtained revealed a and mpl9), for dideoxy chain-termination sequencing (24, 25). Plas- 1002-nucleotide open reading frame followed by a stop codon mids were prepared by the alkaline lysis method and banded in a CsCI gradient (22). Restriction mapping of DNA was performed with en (Fig. 1). Also indicated in Fig. 1 is the extensive homology of zymes from either New England Biolabs or Boeringer Mannheim the predicted amino acid sequence encoded by B4 to the previ Biochemicals. Sequencing gels were 0.4 mm and contained 6% acryl- ously reported amino acid sequence of bovine cardiac PKAc. amide:bis (19:1) and 8 M urea. Despite this remarkable homology (92%), the B4 cDNA se Liver Tissue Preparation. All livers were from Sprague-Dawley rats quence is slightly truncated at its 5' end, since it lacks the 5' (Charles River Laboratories, Wilmington. MA) and were from females terminal 51 nucleotides that would encode a 17-amino acid unless otherwise indicated. Liver tumors were initiated neonatally by a sequence present at the amino terminal end of the bovine PKAc single i.p. injection of diethylnitrosamine and, following weaning, rats were placed on a diet containing phÃ©nobarbital(0.025-0.1% of diet) to sequence. Southern blot analyses are consistent with a single promote the formation of tumors. The rats were killed by cervical copy of the corresponding gene in the rat genome (data not dislocation. The livers were removed and were immediately excised and shown). frozen in liquid nitrogen. Control livers were obtained from 200-day- We examined the expression of RNAs homologous to rat old female Sprague-Dawley rats obtained from Charles River Labora PKAc in various tissues, using B4 as a probe in Northern blot tories. The pathology of the samples was verified by routine histology. analysis. Fig. 2 indicates that we detected a 4.6-kilobase band Additional details have been previously described (26). in RNA samples isolated from various rat tissues (brain, heart, Liver regeneration experiments were carried out by placing female and liver), the K16 and K22 rat liver epithelial cell lines, the Sprague-Dawley rats on a diet containing either 30% casein (control) or 0.05% phÃ©nobarbitalplus casein for 16 days prior to partial hepa- Rat6 fibroblast cell line, human esophagus, the human GM6167 fibroblast cell line, two samples of mouse liver, and tectomy. Cervical dislocation was performed on three rats each at 12. the Chinese hamster ovary' cell lines CHO 10001 and CHO 24, 48, 72, and 168 h following surgery. Tissues were immediately frozen in liquid nitrogen and stored at -70Â°Cuntil processing. Control 10260 (a variant of CHO 10001, which is deficient in PKA livers for each animal were those tissues removed at the time of hepatectomy. 1 2 345 6 789 10 11 12 Northern Blot Analyses. Total RNA was isolated from various cell lines and tissues using a previously described extraction procedure (27). Equivalent amounts of RNA (10 Mg/lane) were loaded onto 1% agarose gels containing 6% formaldehyde, electrophoresed, and blotted onto nylon membranes, as described by the manufacturer (Amersham, Ar lington Heights. IL). The studies on regenerating rat liver and rat hepatomas (Figs. 4 and 5) employed polyadenylated RNA (4 Â¿ig/lane), -4.6kb which was isolated as previously described (26). A "P-labeled probe of -3.3kb the Pstl fragment of clone B4 was prepared by nick translation (28). -2.6kb hybridized at 42Â°Cto the nylon filters containing rat RNAs in buffer containing 50% formamide, and washed to a stringency of O.Ix SSC at 65Â°C,according to the manufacturer's protocol (Amersham). Filters containing RNAs from other species were hybridized to the same probe at 37Â°Cinbuffer containing 40% formamide and washed to a stringency of 2x SSC at 50Â°C.Molecular weights of the bands were based on the 18S and 28S ribosomal markers. All lanes contained equivalent amounts of RNA, based on ethidium bromide staining. Fig. 2. Northern blot analysis of RNA samples from various tissues using (he B4 sequence as a probe. Each lane contained 10 ^g of total RNA from rat brain RESULTS (lane /). heart (lane 2), and liver (lane 3): rat liver epithelial cell lines K16 (lane 4) and K22 (lane 5): ral6 fibroblasts (lane f>);human esophagus (lane 7); human An 18-amino acid peptide sequence (residues 217-235) pres GM6167 fibroblasts (lane H); mouse liver (lanes V and 10): and the Chinese hamster ovary cell lines 10001 and 10260 (lanes II and 12). Molecular weights ent in bovine cardiac PKAc (5) was used to design a comple are indicated at the n^ltt. For additional details see "Materials and Methods." 1677

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. PKAc IN RAT HEPATOMAS AND REGENERATING RAT LIVER activity) (29). The relative abundance of this RNA species in Fold Induction of mRNA the rat brain, heart, and liver samples (which contain equivalent amounts of RNA/lane) corresponds approximately to the re ported relative tissue abundance of PKAc protein in these tissues (30). It was also of interest to compare our rat B4 clone to the murine PKAc-Â«and PKAc-0 clones isolated by other investi gators (8, 9, 11). Fig. 3 indicates a Northern blot of mouse liver RNA hybridized to nick-translated probes prepared from the B4 clone (Fig. 3/1), the murine PKAc-a (8) (Fig. 3Ã„),or the murine PKAc-/Ã(11) (Fig. 3C). As is apparent in the figure, both the rat B4 and the murine PKAc-0 probes detected a prominent 4.6-kilobase band, whereas the murine PKAc-a probe detected a fainter lower molecular weight band of ap 30 40 50 proximately 2.3 kilobases. The presence of low molecular Tant (hr.) weight RNA bands in tissues from mouse liver, CHO 10001 cells, and human brain, using B4 as a probe (Fig. 2), and in mouse liver, using murine PKAc-0 as a probe (Fig. 3C), may 1 23456 7 891011 12 represent cross-hybridization to other isoforms of PKAc; alter natively, they may indicate RNA degradation products of the intact higher molecular weight form. This aspect requires fur ther study. Fig. 4A depicts the densitometric data obtained from North -4.6kb ern blot analysis of the time course of expression of PKAc in regenerating rat liver. RNA was collected from triplicate ani mals at time points of 12, 24, 48, 72, and 168 h after partial hepatectomy from phenobarbital-fed and casein control rats, as described in "Materials and Methods." Error bars represent 1 SE. The level of message expression at the 168-h time point is B unchanged from 72 h and is not shown. It is apparent from the Fig. 4. Levels of PKAc RNA expression following partial hepatectomy in the figure that liver regeneration is associated with a 3-fold reduc rat. A, densitometric data of Northern blot analysis of rat liver mRNA probed tion in the abundance of PKAc mRNA at 12 h following partial with clone B4 at 12, 24, 48. 72, and 168 h following partial hepatectomy (168-h time points not shown). Error bars represent 1 SE. Diets were as described in hepatectomy. This reduction is statistically significant based on "Materials and Methods."â€”, phÃ©nobarbitaldiet; , casein diet. B, example calculations of the SE of the three densitometric data points. of Northern blot analysis densitometrically depicted in A. Data shown are from 12-h post-hepatectomy livers (lanes 1-6) along with their internal controls (taÃ±es These data also suggest that the fall in PKAc message levels 7-12). Lanes 1-3 and 7-9, from rats fed phÃ©nobarbitaldiet; lanes 4-6 and 10- returns to baseline by 72 h. Furthermore, we found that these 12, from rats fed the casein control diet. All lanes contained equivalent amounts changes were not affected by the administration of phÃ©nobar (4 /jg) of rat liver polyadenylated mRNA. bital, a known promoter of liver tumor induction in rats (26). Fig. 48 is an example of the Northern blot analysis from which 1 234567 891011121314151617181920 the densitometric data were obtained. Shown are the 12-h time point RNA samples obtained from the partially hepatectomized and control animals. Since the rats used in this study were

1 2 1 2 1 2

Fig. 5. Levels of PKAc RNA expression in diethylnitrosamine-induced rat hepatomas. Northern blot analysis of rat liver mRNAs probed with clone B4. Lane 1-5, and 8, hepatic adenomas (males); lanes 6, 7, 9 and 10, hepatocellular carcinomas (males); lanes 11-15. hepatic adenomas (female); lanes 16 and 17, controls (males); lanes 18-20, controls (females). All lanes contained 4 ng poly -4.6kb adenylated mRNA/lane. Ethidium bromide staining of gels indicated that all lanes contained equal amounts of RNA. Diagnoses were made by routine histo- -3.3kb pathology. as previously described (8). -2.2kb outbred, it was necessary to use the liver resected at the time of hepatectomy as an internal control for each regenerating liver removed at different times following hepatectomy. The mark edly and consistently decreased PKAc message expression is readily apparent in the figure. Fig. 5 shows the Northern blot hybridization analysis of the Fig. 3. Northern blot analysis of mouse liver RNA. All of the lanes contained expression of PKAc mRNA in rat liver tumors, which were 10 >igof total RNA from mouse Â¡Â¡verRNA.which was hybridized to the following induced as described in "Materials and Methods." As is appar probes: the rat B4 probe (A), the murine PKAc-Â«probe (B). and the murine PKAc-ji probe (C). Lanes I and 2 are duplicates. Molecular weights are indicated ent from the figure, there was no consistent pattern of the mean at the right. For additional details see "Materials and Methods." induction or reduction of expression of this gene in liver tumors. 1678

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. PKAc IN RAT HEPATOMAS AND REGENERATING RAT LIVER when compared to the normal livers of adult control rats, nor liver regeneration studies revealed a significant decrease in the were there significant differences in the expression of this gene abundance of PKAc mRNA levels in rat liver 12 h following in hepatocellular carcinoma versus adenomas. Adenomas in partial hepatectomy. This change was independent of whether many male rats showed increased levels of PKAc gene expres the rats were fed the liver tumor promoter phÃ©nobarbital.These sion, as compared to controls, and several of the adenomas in results suggest that decreased expression of this gene may play female rats displayed minor decreases in PKAc mRNA levels, a role in liver regeneration by modulating the level of cAMP- these alterations, however, were inconsistent within these dependent protein phosphorylation in these cells; furthermore, groups. These conclusions were confirmed by densitometric they imply that the mechanism of hepatocyte promotion by scanning of the autoradiograms. Ethidium bromide staining of phÃ©nobarbitalis not by alteration in PKAc gene expression. ribosomal markers indicated that equal amounts of RNA were This putative role of a reduction in PKAc expression in liver loaded in each lane. regeneration is emphasized by the finding (36) that overall levels of protein synthesis and RNA synthesis are increased at 7 and 16 h following partial hepatectomy, despite our finding DISCUSSION of a decrease in the level of a specific PKAc mRNA during that The present studies describe the isolation from a rat brain time period. These decreases in PKAc may be mechanistically cDNA library of a cDNA clone, designated B4, that contains important in enabling mature hepatocytes to emerge from an open reading frame of 1002 nucleotides which encodes a quiescence and restore hepatic organ mass to normal levels. In protein that is 94% homologous to the amino acid sequence of contrast to the findings in regenerating liver, we did not find a a bovine cardiac PKAc (5). The predicted amino acid sequence consistent change in the mean level of PKAc mRNA in a series contains regions which are conserved among several different of carcinogen-induced rat hepatomas, although increases in protein kinases (31), including the catalytic regions of PKC PKAc message levels in several individual adenomas in male (13) and the cGMP-dependent protein kinase (32). A compar rats and, to a lesser extent, decreases in levels in some adenomas ison with the amino acid sequence of bovine cardiac PKC in female rats were seen. The significance of this heterogeneity strongly suggests that the B4 clone is truncated by at least 51 is not known. nucleotides at its 5' end. Presumably this occurred during In summary, we have isolated a cDNA which encodes the construction of the cDNA library, since the 5' end of the open catalytic subunit of PKAc but which is slightly truncated at the 5' end. Attempts to isolate this fragment are in progress. reading frame of B4 contains an EcoRl site, but this requires further study. Nevertheless, this cDNA has been shown to be useful as a probe During the course of our studies, other laboratories have in the study of the expression of the PKAc gene in two distinct reported the isolation from various sources of cDNAs for PKAc circumstances of altered growth, controlled hepatic regenera (8, 9, 11). It is of interest, therefore, to compare our results tion and dysregulated growth of liver tumors. In addition, the with theirs. Our Northern blot analyses, using a Pstl-Pstl frag present studies provide further evidence that PKAc, like PKC ment of clone B4 as a probe, detected a 4.6-kilobase RNA (13), represents a multigene family, which may help to explain species in both rat and mouse cells (Figs. 2 and 3). Ulher et al. the multiple roles these protein kinases play in the regulation (8, 11) have isolated two PKAc cDNAs (designated c-n and c- of gene expression and growth control. ÃŸ)and found that the c-o clone hybridizes to a 2.4-kilobase species and the c-/3clone hybridizes to a 4.3-kilobase species of ACKNOWLEDGMENTS murine RNA. The homology of our B4 clone to their c-a and c-ÃŸclones, at the nucleic acid level, is about 76 and 92%, The authors are indebted to Gerard Housey for valuable advice and respectively; the homology of B4 to the bovine c-/3 of Showers assistance, to Michael Gottesman for generously providing the Chinese and Maurer is 86%. Using probes generously supplied to us by hamster ovary cell lines, to Richard Axel and JÃ¼rgenBrosius for Dr. G. Stanley McKnight, University of Washington, we have providing the rat brain cDNA libraries, and to G. Stanley McKnight found that our rat clone, B4, detects a 4.6-kilobase RNA species for generously providing probes for the murine c-a and c-ÃŸforms of that appears to be the same as that detected by murine PKAc- PKAc. 0, whereas murine PKAc-a detects a 2.3-kilobase RNA (Fig. 3). These results, together with the above-described sequence REFERENCES homologies, suggest that our rat B4 clone and the murine PKAc-0 are species variants of the same isotype of PKAc. The 1. Hunter. T. A thousand and one protein kinases. Cell, 50: 823-829. 1987. 2. Beavo, J. A., Bechtel. P. J.. and E. G. Krebs. Mechanism of control for rodent genome, therefore, encodes at least two distinct PKAc cAMP-dependent protein kinase from skeletal muscle. Adv. Cyclic Nucleo- sequences. A human form of PKAc-a (10) and two isoforms of tide Res., 5:241-260, 1975. porcine PKAc (33) have also recently been cloned. The exist 3. Connelly, P. A.. Hastings, T. G., and Teimann, E. M. Identification of a ternary complex between cAMP and a trimeric form of a cAMP-dependent ence of multiple forms of PKAc, coupled with the existence of protein kinase. J. Biol. Chem., 261: 2325-2330, 1986. multiple forms of the regulatory subunit of PKA (34, 35), may 4. Nagamine, Y., and Reich, E. Gene expression and cAMP. Proc. Nati. Acad. provide mechanisms by which cAMP can exert complex and Sci. USA, Â«2:4606-4610, 1985. 5. Shoji. S., Titani. K., DÃ©maille.J. G., and Fischer, E. H. Complete amino different effects in different tissues. It remains to be determined acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP- whether the individual forms of PKAc differ with respect to dependent protein kinase. Proc. Nati. Acad. Sci. USA, 78: 848-851, 1981. their tissue-specific expression and regulation and the protein 6. Shoji. S.. Parmelee, D. C., Wade, R. D., Kumar. S.. Ericsson. L. H.. Walsh, K. A., Neurath, H., Long, G. L., DÃ©maille.J.G.. Fischer, E. H., and Titani. substrates that they phosphorylate. It is of interest that another K. Sequence of two phosphorylated sites in the catalytic subunit of bovine protein kinase, PKC, also belongs to a multigene family and cardiac muscle adenosine 3':5'-monophosphate-dependent protein kinase. J. Biol. Chem.. 254: 6211-6214. 1979. that there is differential expression of the individual genes in 7. Carr, S. A., Biemann, K., Shoji. S., Parmelee. D. C.. and Titani, K. n- different tissues (12, 13). Tetradecanoyl is the NH:-terminal blocking group of the catalytic subunit of The usefulness of our clone as a molecular probe to study cyclic AMP-dependent protein kinase from bovine cardiac muscle. Proc. Nati. Acad. Sci. USA, 79: 6128-6131, 1982. levels of expression of the gene encoding PKAc is well illus 8. Uhler. M. D., Carmichael. D. F., Lee, D. C.. Chrivia. J. C., Krebs, E. G., trated by the liver regeneration and hepatoma experiments. The and McKnight, G. S. Isolation of cDNA clones coding for the catalytic 1679

subunii of mouse cAMP-dcpcndcnt protein kinase. Proc. Nad. Acad. Sci. NY: Cold Spring Harbor Press. 1983. USA. 83: 1300-1304. 1986. 23. Maxam. A. M.. and Gilbert. W. Sequencing end labelled DNA with base- 9. Showers. M. O.. and Maurer. R. A. A cloned bovine cDNA encodes an specific chemical cleavages. Methods Enzymol.. 65:499-560. 1977. alternale form of the catalytic subunit of cAMP-dependcnt protein kinase. J. 24. Messing. J.. and Viera. J. A new pair of m 13 vectors for selecting cither Biol. Chem.. 261: 16288-16291. 1986. DNA strand of double-digest restriction fragments. Gene. 19:269-276,1982. 10. Maldonado. F., and Hanks. S. K. A cDNA clone encoding human cAMP- 25. Sanger. F. Determination of nucleotide sequences in DNA. Science (Wash. dependent protein kinase catalytic subunit c-alpha. Nucleic Acids Res.. 16: DC). 214: 1205-1210. 1981. 8189-8190. 1988. 26. Hsieh. L. L.. Hsiao. W-L.. Peraino. C.. Maronpot. R. R., and Weinstein, I. 11. Uhler. M. D.. Chrivia, J. C., and McKnight. G. S. Evidence for a second B. Expression of retroviral sequences and oncogenes in rat liver tumors isoform of the catalytic subunit of cAMP-dependent protein kinase. J. Biol. induced by diethylnitrosaminc. Cancer Res.. 47: 3421-3424, 1987. Chem.. 261: 15360-15363. 1986. 27. Karin. M.. Martial, J. A., and Baxter. J. D. A method for isolation of intact, 12. Housey, G. M.. O'Brian. C. A.. Johnson, M. D.. Kirschmeier. P.. and translationally active ribonucleic acid. DNA. 2: 329-335. 1983. Weinstein. I. B. Isolation of cDNA clones encoding protein kinase C: 28. Rigby. P. W.. Dieckmann. M.. Rhodes. C., and Berg. P. Labelling of evidence for a protein kinase C-related gene family. Proc. Nati. Acad. Sci. deoxyribonucleic acid to high specific activity in vitro by nick translation USA. 84: 1065-1069. 1987. with DNA polymerase I. J. Mol. Biol.. 13: 237-251. 1978. 13. Nishizuka, Y. The molecular heterogeneity of protein kinase C and its 29. LeCam. A., Nicolas. J. C.. Singh. T. J.. Cabrai. F., and Pastan. I. Cyclic implications for cellular regulation. Nature (Lond.). 334: 661-665. 1988. AMP-dependent phosphorylation in intact cells and in cell-free extracts from 14. Sheppard. J. R. Restoration of contact-inhibited growth to transformed cells Chinese hamster ovary cells: studies with wild type and cyclic AMP-resistant by dibutyryl adenosine 3'5'-cyclic monophosphate. Proc. Nati. Acad. Sci. mutants. J. Biol. Chem.. 256: 933-941. 1981. USA. 6Â«.-1316-1320. 1971. 30. Minakuchi. R.. Takai. Y.. Yu. B.. and Nishizuka. Y. Widespread occurrence 15. Puck. T. T. Cyclic AMP, the microtubule-microfilament system, and cancer. of calcium-activated, phospholipid dependent protein kinase in mammalian Proc. Nati. Acad. Sci. USA. 74: 4491-4495. 1977. tissues. J. Biochem., 89: 1651-1654. 1981. 16. Ro/.cngurt, E. A.. Legg, A., Strang, G., and Courtenay-Luck, N. Cyclic AMP: 31. Hanks, S. K.. Quinn. A. M.. and Hunter. T. The protein kinase family: a mitogenic signal for Swiss 3T3 cells. Proc. Nati. Acad. Sci. USA, 78:4392- conserved features and deduced phylogeny of the catalytic domains. Science 439620. 1982. (Wash. DC). 241: 42-52. 1988. 17. Leichtling. B. H.. Su. V.. Wimalesena. J.. Harden. T. K., Wolfe. B. B., and 32. Takio. K.. Wade. R. D.. Smith. S. B.. Krebs, E. G.. Walsh, K. A., and Titani. Wicks. W. D. Studies of cAMP metabolism in cultured hepatoma cells: K. Guanosine cyclic 3'.5'-phosphate-dependent protein kinase. a chimeric absence of functional adenylate cyclase despite low cAMP content and lack protein homologous with two separate protein families. Biochemistry. 23: of hormonal responsiveness. J. Cell. Physiol.. 96: 215-223. 1978. 4207-4218. 1984. 18. Wicks. W. D.. Leichtling. B. H.. Wimalesena, J., Roper. M. D., Su, J. L., 33. Adavani. S. R.. Schwartz. M.. Showers, M. O., Maurer. R. A., and Hcm- Su, Y. F., Howell, S.. Harden. T. K., and Wolfe, B. B. Regulation of cAMP mings. B. A. Multiple mRNA species code for the catalytic subunii of the metabolism, protein kinase activation, and specific en/ymc synthesis in cAMP-dependenl protein kinase from LLC-PK1 cells. Eur. J. Biochem.. cultured hepatoma cells. Adv. Cyclic Nucleotide Res.. 9: 411-424. 1978. 167: 221-226, 1988. 19. Mahler, S. M., and Wilce, P. A. Dcsensitization of adenylate cyclase and 34. Eppler, C. M., Bayley. H.. Greenberg. S. M., and Schwartz. J. H. Structural cyclic AMP flux during the early stages of liver regeneration. J. Cell. Physiol.. studies on a family of cAMP-binding proteins in the nervous system of 136: 88-94. 1988. Aplysia. J. Cell BioL. 102: 320-331. 1986. 20. Sandnes, D., Sand, T. E.. Sager, G.. Brnstad. G. O., Refsnes. M. R., 35. Titani, K., Sasagawa. T.. Ericsson. L. H., Kumar. S.. and Smith. S. B. Amino Gladhang. 1. P.. Jacobsen. S., and Christoffersen, T. Elevated level of beta- acid sequence of the regulatory subunit of bovine type I adenosine cyclic 3'- adrenergic receptors in hepatocytes from regenerating rat liver. Exp. Cell 5'-phosphate dependent protein kinase. Biochemistry. 2^:4193-4199. 1984. Res., 165: 117-126, 1986. 36. Laks. M. S.. Harrison. J. J.. Schwoch. G., and Jungmann. R. A. Modulation 21. Lathe. R. Synthetic oligonucleotide probes deduced from usage data: theo of nuclear protein kinase activity and phosphorylation of histone H l subspe retical and practical considerations. J. Mol. Biol., 1H3: 1-12. 1985. cies during the prereplicative phase of rat liver regeneration. J. Biol. Chem.. 22. Maniatis. T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor. 256:8775-8785. 1981.

1680

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. Isolation of a Complementary DNA Encoding the Catalytic Subunit of Protein Kinase A and Studies on the Expression of This Sequence in Rat Hepatomas and Regenerating Liver

Jeffrey S. Roth, Ling-Ling Hsieh, Carl Peraino, et al.

Cancer Res 1990;50:1675-1680.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/50/6/1675

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/50/6/1675. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.