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Isolation of a Complementary DNA Encoding the Catalytic Subunit Of (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 ¡L-L.H., I. B. H'.J and Departments of Genetics and Development /J. S. R.¡and Medicine ¡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 diethylnitrosa- mine-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 »eresynthesized on an automated DNA 1675 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. PKAc IN RAT HEPATOMAS AND REGENERATING RAT LIVER 1« 20 if 40 60 •¿ •¿ •¿ •¿ CAA TTC CTA TCC AAA GCC AAA CAA CAC TTT CTC ACG AAA TGG GAG AAC CCT CCC CCC ACT Clu Ph« L«u S«r Lys Al« Ly« Glu Asp Ph« L«u Arg Ly»Trp Glu A«n Pro Pro Pro S«r 70 80 00 100 110 120 •¿ AAT GCT GGG CTT GAG GAT TTT GAA AGG AAA AAA ACC TTC GGA ACG CGT TCC TTC GGA AGA Am Ali Cly L«uGlu A«p Ph« Glu Arg Lys Lyl Thr l«u Gly Thr Gly S«rPh»Cly Arg 130 140 IBP 160 170 180 •¿ CTC ATG CTC CTA AAC CAT AAC CCC ACT GAG CAC TAC H'is Ly« Al* Thr Clu Gin Tyr TAC CCC ATC AAG ATC TTA CAC AAC Vsl U.I L«u V«l Ly« Tyr Ali U.t Ly« II»L«uA»pLy« 190 200 210 220 230 240 •¿ •¿ •¿ CAG AAG GTC CTT AAC CTA AAG CAA ATA CAG CAC ACT CTG AAT GAG AAC AGA ATC CTC CAC Gin Ly«Val Vil Lyi L«uLy«Gin II* Clu Hij Thr l«u Asn Clu Ly«Arg II«L«uG ln 260 260 270 280 290 300 •¿ •¿ •¿ GCA GTC GAG TTC CCG TTC CTT GTT GGG CTC CAG TAT TCT TTT AAC GAT AAT TCT AAT TTA AI«V«IGlu Ph«Pro Ph«L«uVil Gly L«uGlu Tyr S«rPh«Ly»A«pA«n5«rA*n L«u 310 320 330 340 360 360 •¿ •¿ TAC ATC GTT ATC GAA TAC CTT CCT CCC CCC CAA ATC TTT TCA CAT CTA AGA AGA ATT CGA Tyr Met Vil M«t Clu Tyr Vil Pro Cly Gly Clu M.t Ph« S«r Hi»L«uArg Arg II« Gly 370 360 390 400 410 420 •¿ •¿ ACG TTC ACT GAA CCC CAT GCT CGT TTC TAT GCA GCT CAG ATC GTC CTA ACA TTT GAG TAC Ara Ph»Sor Clu Pro Hi« Al* Arg Ph«Tyr Al«Al«Gin II«V.I L«uThr Ph«Glu Tyr 430 440 460 460 470 480 Fig.
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