A cAMP-binding phosphoprotein in Drosophila heads is similar to the regulatory subunit of the mammalian type II cAMP-dependent protein kinase

Cyclic AMP (cAMP)-dependent regulation of in vitro phosphorylation of several proteins including a cAMP-binding protein was studied with crude membrane and cytosol fractions from Drosophila heads. Phosphorylation of at least seven distinct proteins was enhanced in the presence of cAMP. Interestingly, however, the phosphorylation of a 56 kDa protein was apparently reduced by cAMP in the membrane but not in the cytosol fraction. The following data strongly indicate that the 56 kDa phosphoprotein in both membrane and cytosol fractions is a cAMP-binding protein, very similar to the regulatory subunit (RII) of a mammalian cAMP-dependent protein kinase, and that its binding to cAMP makes this protein very susceptible to the action of phosphatases: (i) cAMP highly stimulated the dephosphorylation of the 56 kDa phosphoprotein by the endogenous phosphatase in the membrane fraction. (ii) The dephosphorylation of a similar 56 kDa phosphoprotein in the cytosol fraction by an exogenous, cAMP-independent, alkaline phosphatase was also highly stimulated by cAMP. (iii) The 56 kDa phosphoprotein was covalently bound to cAMP by u.v. irradiation. (iv) The alkaline-phosphatase treatment reversibly converted this phosphoprotein to a 53 kDa non-phosphorylated protein. (v) The 53 kDa protein was selectively bound to cAMP-agarose and subsequently eluted by cAMP and high salt. (vi) This protein served as a substrate for the catalytic subunit of a mammalian cAMP-dependent protein kinase.     

Retinoylation of the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases in HL60 cells.

Retinoylation (retinoic acid acylation) is a post-translational modification of proteins occurring in a variety of eukaryotic cell lines. There are at least 20 retinoylated proteins in the human myeloid leukemia cell line HL60 (N. Takahashi and T.R. Breitman (1990) J. Biol. Chem. 265, 19, 158-19, 162). Here we found that some retinoylated proteins may be cAMP-binding proteins. Five proteins, covalently labeled by 8-azido-[32P]cAMP which specifically reacts with the regulatory subunits of cAMP-dependent protein kinase, comigrated on two-dimensional polyacrylamide gel electrophoresis with retinoylated proteins of Mr 37,000 (p37RA), 47,000 (p47RA), and 51,000 (p51RA) labeled by [3H]retinoic acid treatment of intact cells. Furthermore, p47RA coeluted on Mono Q anion exchange chromatography with the type I cAMP-dependent protein kinase holoenzyme and p51RA coeluted on Mono Q anion exchange chromatography with the type II cAMP-dependent protein kinase holoenzyme. An antiserum specific to RI, the cAMP-binding regulatory subunit of type I cAMP-dependent protein kinase, immunoprecipitated p47RA. An antiserum specific to RII, the cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase, immunoprecipitated p51RA. These results indicate that both the RI and the RII regulatory subunits of cAMP-dependent protein kinase are retinoylated. Thus, an early event in RA-induced differentiation of HL60 cells may be the retinoylation of subpopulations of both RI and RII.     

Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase

The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.     

Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.     

Phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase in intact smooth muscle

A monoclonal antibody was used to quantitate changes in the extent of phosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase in intact bovine tracheal smooth muscle. The autophosphorylated and nonphosphorylated forms of the regulatory subunit (RII) were separated in sodium dodecyl sulfate-polyacrylamide gels and identified by immunoblot analysis. Addition of cAMP to tissue extracts resulted in rapid dephosphorylation of RII (t 1/2 = 20s at 4 degrees C) while addition of MgATP caused complete conversion to the phosphorylated form. Under basal conditions, 56% of RII in intact muscle was phosphorylated when the tissue was homogenized under conditions which fully inhibit protein kinase and phosphatase activities. Incubation with isoproterenol caused a dose-dependent decrease in the phosphorylation state of RII (EC50 = 5 X 10(-8) M). Incubation with high concentrations of isoproterenol, 1-methyl-3-isobutylxanthine, or forskolin caused maximal decreases in the phosphorylated form to 12-18% of the total RII. The effect of isoproterenol was rapid (t 1/2 = 15 s at 37 degrees C), reversible, and could be blocked with the antagonist propranolol. Contraction of the smooth muscle with K+ or low (less than 1 microM) concentrations of carbachol had no effect on the phosphorylation level. A decrease in the basal phosphorylation level to 41% was observed with 10 microM carbachol which was additive with the dephosphorylation produced by isoproterenol. The time course of isoproterenol-induced dephosphorylation of RII paralleled that of muscle relaxation, consistent with a role of cAMP-dependent protein kinase activation in relaxation of smooth muscle.     

Effects of cAMP-binding site mutations on intradomain cross-communication in the regulatory subunit of cAMP-dependent protein kinase I

Each protomer of the regulatory subunit dimer of cAMP-dependent protein kinase contains two tandem and homologous cAMP-binding domains, A and B, and cooperative cAMP binding to these two sites promotes holoenzyme dissociation. Several amino acid residues in the type I regulatory subunit, predicted to lie in close proximity to each bound cyclic nucleotide based on affinity labeling and model building, were replaced using recombinant techniques. The mutations included replacement of 1) Glu-200, predicted to hydrogen bond to the 2'-OH of cAMP bound to site A, with Asp, 2) Tyr-371, the site of affinity labeling with 8-N3-cAMP in site B, with Trp, and 3) Phe-247, the position in site A that is homologous to Tyr-371 in site B, with Tyr. Each mutation caused an approximate 2-fold increase in both the Ka(cAMP) and Kd(cAMP); however, the off-rates for cAMP and the characteristic pattern of affinity labeling with 8-N3-cAMP differed markedly for each mutant protein. Furthermore, these mutations affect the cAMP binding properties not only of the site containing the mutation, but of the adjacent nonmutated site as well, thus confirming that extensive cross-communication occurs between the two cAMP-binding domains. Photoaffinity labeling of the native R-subunit results in the covalent modification of two residues, Trp-260 and Tyr-371, by 8-N3-cAMP bound to sites A and B, respectively, with a stoichiometry of 1 mol of 8-N3-cAMP incorporated per mol of R-monomer (Bubis, J., and Taylor, S. S. (1987) Biochemistry 26, 3478-3486). A stoichiometry of 1 mol of 8-N3-cAMP incorporated per R-monomer was observed for each mutant regulatory subunit as well, even when 2 mol of 8-N3-cAMP were bound per R-monomer; however, the major sites of covalent modification were altered as follows: R(Y371/W), Trp-371; R(E200/D), Tyr-371, and R(F247/Y), Tyr-371.     

Molecular basis for regulatory subunit diversity in cAMP-dependent protein kinase: crystal structure of the type II beta regulatory subunit

BACKGROUND: Cyclic AMP binding domains possess common structural features yet are diversely coupled to different signaling modules. Each cAMP binding domain receives and transmits a cAMP signal; however, the signaling networks differ even within the same family of regulatory proteins as evidenced by the long-standing biochemical and physiological differences between type I and type II regulatory subunits of cAMP-dependent protein kinase. RESULTS: We report the first type II regulatory subunit crystal structure, which we determined to 2.45 A resolution and refined to an R factor of 0.176 with a free R factor of 0.198. This new structure of the type II beta regulatory subunit of cAMP-dependent protein kinase demonstrates that the relative orientations of the two tandem cAMP binding domains are very different in the type II beta as compared to the type I alpha regulatory subunit. Each structural unit for binding cAMP contains the highly conserved phosphate binding cassette that can be considered the "signature" motif of cAMP binding domains. This motif is coupled to nonconserved regions that link the cAMP signal to diverse structural and functional modules. CONCLUSIONS: Both the diversity and similarity of cAMP binding sites are demonstrated by this new type II regulatory subunit structure. The structure represents an intramolecular paradigm for the cooperative triad that links two cAMP binding sites through a domain interface to the catalytic subunit of cAMP-dependent protein kinase. The domain interface surface is created by the binding of only one cAMP molecule and is enabled by amino acid sequence variability within the peptide chain that tethers the two domains together.     

The regulatory subunit of the type II cAMP-dependent protein kinase in rabbit ovaries is the RII beta isoform.

Based on RII autophosphorylation, photoaffinity labeling with 8-N3[32P]cAMP, and Western blot analysis we have identified the RII isoform found in rabbit corpora lutea as RII beta. The RII beta subunit found in rabbit corpora lutea differs from the RII beta found in rat follicles and corpora lutea in that it migrates at Mr 52,500 on SDS-PAGE and shifts to Mr 53,000 when phosphorylated.     

Conformational changes of type II regulatory subunit of cAMP-dependent protein kinase on cAMP binding

The effect of cAMP on the conformation of the regulatory subunit of type II cAMP-dependent protein kinase (RII) from bovine heart was investigated by UV-difference and circular dichroism (CD) spectroscopy. The UV-difference spectrum of RII with and without cAMP showed a positive band around 278 nm and a negative band at 256 nm. Similarly, cAMP enhanced the ellipticity of RII in the region between 280 and 300 nm and decreased that between 250 and 280 nm. In addition, cAMP transformed the far-UV CD spectrum of RII from that of a negative band minimally at 209 nm with a shoulder at 223 nm to one with two minima at 222 and 211 nm. These data show that cAMP induces conformational changes of RII upon binding. Such structural changes may be the basis of activation of cAMP-dependent protein kinases by cAMP.     

Expression cloning of a cDNA encoding the type II regulatory subunit of the cAMP-dependent protein kinase

We report here the isolation and sequence of a cDNA for the type II regulatory subunit of the cAMP-dependent protein kinase (cAMP-PK) from a lambda gt-11 cDNA library derived from a porcine epithelial cell line (LLC-PK1). The cDNA was detected by immunological screening using an affinity purified polyclonal antibody for bovine RII. DNA sequence analysis of the 467 bp EcoRI insert confirmed the identity of the clone, because the deduced amino acid sequence corresponded to the published sequence for the bovine RII protein. Northern analysis of total RNA from the LLC-PK1 cells indicated a single mRNA species of about 6.0 kb, probably derived from a single copy gene.     

PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 micromol/(min.mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RIalpha) of cAMP-dependent protein kinase (KD = 10 nM) and the heat-stable protein kinase inhibitor (KD = 15 nM), but not with the type II regulatory subunit (RIIalpha, KD = 2.3 microM) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RIalpha subunit and by protein kinase inhibitor in vitro, but only weakly by the RIIalpha subunit. The inhibition by the RIalpha subunit is reversed by addition of nanomolar concentrations of cAMP (Ka = 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the Calpha subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RIIalpha subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RIalpha subunit and by phosphorylating the RIIalpha subunit in the absence of cAMP.     

Immunochemical properties of the regulatory subunit of cAMP-dependent protein kinase II]

Immunochemical analysis of the cAMP-dependent protein kinase regulatory subunit type II was performed with the use of two rabbit antisera elicited to a free R-subunit from pig brain and to a RcAMP complex. Quantitative precipitation of the homogeneous antigen revealed six determinants on the R-molecule. Of these at least one is localized in the R-fragment (37 kD), the others--in the N-terminal part of the R-molecule. The antigenic determinants seem to be remoted from the cAMP-binding centers, since the attachment of the affinity purified antibody Fab-fragments to the R-subunit did not influence the cAMP-binding activity of the latter. The antibodies to RcAMP caused dissociation of the holoenzyme. The antibody Fab-fragment binding to the R-subunit prevented its association with the catalytic subunit. The results of immunochemical analysis suggest that the R-subunit adopts different conformations when bound to cAMP or to the catalytic subunit.     

Interaction of the regulatory subunit of a type II cAMP-dependent protein kinase with mammalian sperm flagellum

We have reported previously (Horowitz, J. A., Toeg, H., and Orr, G. A. (1984) J. Biol. Chem. 259, 832-838) that most of the type II cAMP-dependent protein kinases in rat sperm are associated with the flagellum. We have now identified flagellar polypeptides which are capable of forming tight complexes with the regulatory subunit of type II cAMP-dependent protein kinase (RII). Flagellar RII-binding polypeptides were identified using an RII overlay/immunoblot procedure and had apparent subunit Mr of 120,000, 80,000, and 57,000 in rat and 120,000 and 57,000 in bovine flagella. RII is released from the flagellum by disulfide reducing agents, e.g. 1 mM dithiothreitol (DTT). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie Blue staining of the DTT-released material shows that a limited subpopulation of flagellar polypeptides are solubilized by disulfide-reducing agents. Neither tubulin, the dynein ATPase, or any of the RII-binding proteins are released by 1 mM DTT, and thin section electron microscopy revealed that the morphology of the flagellum is unaltered by reducing conditions. Our data established that RII is not linked to the flagellum via a direct disulfide bridge. We propose that RII is released from the flagellum, a highly disulfide cross-linked structure, due to structural changes in the flagellum which disrupts the interaction between RII and its binding proteins.     

Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts

Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.     

The neural type II regulatory subunit of cAMP-dependent protein kinase is present and regulated by hormones in the rat ovary

In this study purified isoforms of rat ovarian regulatory subunit of type II cAMP-dependent protein kinase (R-II) were compared with R-II purified from rat brain. A special neural form of R-II has been previously described in bovine brain. Analysis by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved three isoforms of rat ovarian R-II (R-II54, Mr = 54,000; R-II52, Mr = 52,000; and R-II51, Mr = 51,000) compared to two R-II isoforms in rat brain (R-II54 and R-II52). Polychromatic silver-stained peptide maps of purified R-II subunits indicated that peptides generated from both rat ovarian R-II52 and R-II51 were similar (if not identical) to the peptides of the neural form, R-II52, purified from rat brain. These peptides differed markedly from those generated from R-II54 of either rat ovary, brain, or heart. Ovarian R-II52/51 photoaffinity labeled with 8-N3-[32P]cAMP and analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was shown to consist of three (rather than two) isoelectric variants, which were similar to three variants resolved from rat brain R-II and clearly distinct from that of rat heart R-II54. An antibody which recognized both the R-II54 and R-II52/51 isoforms of rat ovarian extracts also recognized both forms of rat brain R-II (R-II54 and R-II52) and similar forms in extracts of rat adrenal and parotid glands. These results strongly suggest that the R-II52 isoform previously designated as a neural specific form of R-II is present in high concentrations in a nonneural tissue, the rat ovary.     

Deletion of cAMP-binding site B in the regulatory subunit of cAMP-dependent protein kinase alters the photoaffinity labeling of site A

Photoaffinity labeling with 8-azidoadenosine 3':5'-monophosphate is a highly selective method for probing the cAMP-binding sites of the regulatory subunits of cAMP-dependent protein kinase and for identifying specific residues that are in close proximity to the cAMP-binding sites. The cAMP-binding site of a mutant RI-subunit has been characterized here and contrasted to the native RI-subunit. This mutant RI-subunit was generated by oligonucleotide-directed muta-genesis and lacks the entire second cAMP-binding domain which includes both of the residues, Trp260 and Tyr371, that are photolabeled in the native RI-subunit. The mutant RI-subunit, nevertheless, is photoaffinity-labeled with high efficiency, and the residue covalently modified was identified as Tyr244. The position of Tyr244 based on a computer graphic model of cAMP-binding site A is proposed and correlated with the presumed locations of Tyr371 and Trp260 in the native R-subunit. Photoaffinity labeling also can be used to detect functional cAMP-binding sites following electrophoretic transfer of the denatured protein to nitrocellulose. Labeling of the immobilized protein on nitrocellulose required a functional cAMP-binding site A that can be photoaffinity-labeled in solution based on the following criteria. 1) The type I R-subunit is photolabeled, whereas the type II R-subunit is not. A primary feature which distinguishes these two R-subunits is that the RI-subunit is photolabeled at both sites A and B, whereas covalent modification of the RII-subunit occurs only at site B. 2) The truncated mutant of the RI-subunit which lacks the entire second cAMP-binding domain can be photolabeled on nitrocellulose. 3) A mutant RI-subunit which can no longer be photolabeled in site B is still photolabeled on nitrocellulose. 4) A mutation which abolished cAMP binding to site A also abolished photoaffinity labeling after transfer to nitrocellulose.     

Overexpression of the type II regulatory subunit of the cAMP-dependent protein kinase eliminates the type I holoenzyme in mouse cells

Mammalian tissues and cell lines express two major types of cAMP-dependent protein kinase, PKA-I and PKA-II, which can be distinguished at the molecular level by the presence of either type I or type II regulatory subunits in the holoenzyme. An expression vector for the mouse type II regulatory subunit (RII alpha) was transfected into ras-transformed NIH3T3 (R3T3) cells, which contain approximately equal amounts of both holoenzymes, PKA-I and PKA-II. In RII alpha-overexpressing R3T3 cells, PKA-II levels were increased, and the level of PKA-I declined. The decrease in PKA-I was dependent on the amount of RII alpha expressed, and at high levels of RII alpha expression, PKA-I was completely eliminated. In contrast, overexpression of the type I regulatory subunit (RI alpha) did not alter PKA isozyme levels. We propose that competition between RII alpha and RI alpha for a limited pool of catalytic subunit results in preferential assembly of PKA-II and that significant amounts of PKA-I are formed only if catalytic subunit is present in excess of the RII alpha subunit. The PKA-I isozyme, which is absent in untransformed 3T3 cells, is not essential for the transformed phenotype of R3T3 cells. RII alpha-overexpressing R3T3 cells that are devoid of PKA-I continued to exhibit a transformed phenotype including anchorage-independent growth. Overexpression of RII alpha provides a genetic approach that may prove useful in demonstrating specific functions for the two PKA isozymes in cAMP-dependent signal transduction pathways.     

Major 56,000-dalton, soluble phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase

It has been shown that cAMP-dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000-dalton phosphoprotein present in both dog sperm and in other cells containing axokinin-like activity. Since the regulatory subunit of a type II cAMP-dependent protein kinase is a ubiquitous cAMP-dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000-dalton cAMP-dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP-dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non-neural form of the enzyme and undergoes a phosphorylation-dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulatory subunit (RII) is quantitatively removed from these extracts using either immobilized cAMP or an anti-RII monoclonal affinity column, the ability to phosphorylate the 56,000- but not 40,000-dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP-dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase and not the motility initiator protein, axokinin.     

Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

An expression vector has been constructed for the type I regulatory subunit of cAMP-dependent protein kinase. A cDNA clone for the bovine RI-subunit has been inserted into pUC7. When Escherichia coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-azidoadenosine 3':5'-mono[32P]phosphate following transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of isopropyl-beta-D-thiogalactopyranoside. R-subunit accumulated in large amounts only in the stationary phase of growth, and the addition of isopropyl-beta-D-thiogalactopyranoside during the log phase of growth actually blocked the accumulation of R-subunit. Maximum expression (20 mg/liter) was achieved when E. coli 222 was transformed with the RI-containing plasmid. E. coli 222 is a strain that contains two mutations; it is cya- and also has a mutation in the catabolite gene activator protein (crp) that enables the protein to bind to DNA in the absence of cAMP. The expressed RI-subunit was a soluble, dimeric protein, and no significant proteolysis was apparent in the cell extract. The purified RI-subunit bound 2 mol of cAMP/mol of R monomer, reassociated with C-subunit to form holoenzyme, and migrated as a dimer on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis, yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties, the expressed protein was indistinguishable from RI purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z' gene of the vector. This NH2-terminal sequence was confirmed by amino acid sequencing.