Expression of the Type I Regulatory Subunit of cAMP-dependent Protein Kinase in Escherichia coZi*

An expression vector has been constructed for the type I regulatory subunit of CAMP-dependent protein kinase. A cDNA clone for the bovine R'-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 photola~ling with 8-azidoadenosine 3':5'-m~no["~P]phosphate following transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Maximum a

An expression vector has been constructed for the type I regulatory subunit of CAMP-dependent protein kinase. A cDNA clone for the bovine R'-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 p h o t o l a~l i n g with 8-azidoadenosine 3 ' : 5 ' -m~no["~P]phosphate following transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of isopropyl-@-D-thiogalactopyranoside. R-subunit accumulated in large amounts only in the stationary phase of growth, and the addition of isopropyl-@-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 R'-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 R*-subunit was a soluble, dimeric protein, and no significant proteolysis was apparent in the cell extract. The purified R'-subunit (a) bound 2 mol of cAMP/mol of R monomer, (b) reassociated with Csubunit to form holoenzyme, and (c) 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 36,000. In all of these properties, the expressed protein was indistinguishable from R' 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 2 ' gene of the vector. This NH2-terminal sequence was confirmed by amino acid sequencing.
CAMP-dependent protein kinase, thought to be the primary mediator of cAMP action in eukaryotic cells, is composed of both regulatory (R) and catalytic (C) subunits (1). The inactive holoenzyme is an aggregate of both subunit types, R2C2, and activation by cAMP promotes dissociation into R,-(CAMP), and two monomeric C-subunits which are catalyti-* This work was supported by American Cancer Society Grants NP-419 and CD-255A. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ' '~U e T~~e m e n~" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Supported in part by United States Public Health Service Training Grant AM-07233. cally active. Different forms of the kinase have been identified and by convention are referred to as types I and I1 based on their elution from DEAE-cellulose (2). These forms can be distinguished on the basis of size and autophosphorylation and also are antigenically distinct (3, 4). In addition, there are soluble and rnembrane-associated forms of the kinase (5-7). Most evidence suggests that the catalytic subunits are very similar (8) and that those features which distinguish the various holoenzyme forms can be attributed exclusively to the R-subunits (9, 10). Bovine R' has been cloned recently and appears to be the product of a single gene (11). The type I1 regulatory subunits, on the other hand, represent a larger family of proteins. At least two unique gene products have been identified (12), and a qualitative comparison of various tissues suggests that this diversity may be widespread (13,14). The reason for this diversity is not yet apparent.
Despite differences, there are general features that are conserved in all R-subunits. These include cAMP binding, formation of a dimeric protein following dissociation by CAMP, and a recognition site for catalytic subunit. Furthermore, a well-defined domain structure appears to be retained in each R-subunit. In general, the protein can be divided into thirds based on the amino acid sequence. The NHz-terminal third of the molecule contains a hinge region that is highly susceptible to proteolytic cleavage (15-1'7), contains an essential recognition site for the C-subunit (18), and is the major site of interaction between the two protomers of the R d' lmer (19). Finally, it is the NH2-terminal segment, at least in the R"-subunit, that contains the primary antigenic determinants, both in the dissociated R-subunit and in the holoenzyme (12,1'7). The COOH-terminal two-thirds of the molecule contain two in tandem gene duplicated segments (20, 21) which represent two CAMP-binding sites. Our knowledge of the structure of the CAMP-binding sites has been greatly facilitated by their homology with the catabolite gene activator protein (CAP') in Escherichia coli (22). Homologies in amino acid sequences initially established the relatedness of the CAMP-binding domains in these two proteins (23). Comparison of photolabeling of R with the crystal structure of CAP has confirmed that cAMP binds in an analogous manner in both proteins and that the protein folding in each CAMPbinding domain of R is similar to that which is seen in CAP 124).
In order to understand this molecule at the molecular level, it will be necessary to solve the crystal structure of the protein.
In conjunction with this, the use of site-directed mutagenesis can provide an important tool for asking questions about functional sites and about the specific conformational changes associated with CAMP-dependent protein kinase. As a first  The R'-subunit is expressed at high levels, is soluble, and appears to be functionally equivalent to the protein purified from bovine tissue.

EXPERIMENTAL PROCEDURES
Materials-Reagents were purchased from the following companies: histone IIA, phenylmethylsulfonyl fluoride, isopropyl-8-D-thiogalactopyranoside (IPTG), and bovine serum albumin, Sigma; Nu-ptosyl-L-lysine chloromethyl ketone and CAMP, United States Biochemical Corp. Plasmids and Bacterial Strains-Plasmid 62C12 which contains the full-length cDNA of bovine R' in pBR322 was kindly provided by G . Stanley McKnight from the University of Washington, Seattle (11). pUC7 (25) was used to subclone the cDNA for expression. The strains of E. coli that were used for transformation were JM105 (26) and 222 (27). Strain 222 was a generous gift from Alan Peterkofsky, National Institutes of Health.
Other Proteins-C-subunit was purified from porcine heart (28) and R' from porcine skeletal muscle (19).
Construction of the Expression Vector-Isolation of cDNA from clone 62C12 and construction of the expression vector are summarized in Fig. 1. DNA manipulations were carried out according to Maniatis et al. (29). Following transformation, cells were plated on LB plates containing ampicillin (50 pg/ml). Replicates of these plates were prepared on nitrocellulose filters and screened with a restriction fragment that was labeled with 32P at its 5'-end using T4 polynucleotide kinase and [y-32P]ATP. Positive colonies were further characterized by restriction analysis.
Detection of Regulatory Subunit-Since basal levels of cAMP are high in E. coli, we devised the following method for the accurate detection of R in crude extracts independent of exogenous CAMP. This method could be used for rapid screening. Protein samples were electrophoresed on 12.5% polyacrylamide gels (1.5 mm) containing sodium dodecyl sulfate according to the method of Laemmli (30). Proteins were then transferred from the gels to nitrocellulose filters using an electroblotting apparatus (Hoefer Scientific Instruments). Gels were electrotransferred for 4-6 h at 500 mA in 20 mM Tris, 154 mM glycine, 20% methanol (pH 8.3). The nitrocellulose was incubated for 1 h at room temperature with 0.05% Tween 20 in 150 mM sodium chloride and 10 mM Tris (pH 7.5) and then washed at room temperature with the same buffer except that Tween 20 was omitted. The filter was incubated with 8-N3[32P]cAMP (20 nM) in the same buffer for 30 min in the dark at room temperature. After washing with icecold buffer, the filter was irradiated with a UV s-11 lamp (254 nm) for 5 min, washed again, blotted dry, and subjected to autoradiography. The R-subunit also was detected by Millipore filtration using Purification of the Regulatory Subunit-L-broth (5 ml) containing ampicillin (50 pg/ml) was inoculated with pLST-1 and grown at 37 "C until the cell density reached ODmo = 0.1-0.5. The culture was then transferred to 1 liter of L-broth containing ampicillin and incubated (12-16 h). Unless indicated, all subsequent procedures were carried out at 4 "C. The cells were centrifuged at 5,000 X g for 30 min, and the resulting pellet was resuspended in buffer I (20 mM potassium phosphate, 5 mM EDTA, 5 mM 2-mercaptoethanol, 15 mg/liter N"p-tosyl-L-lysine chloromethyl ketone, and 15 mg/liter phenylmethylsulfonyl fluoride). This suspension was passed through a French pressure cell twice and then centrifuged at 5,000 X g for 20 min. The pellet was re-extracted with the same buffer. Ammonium sulfate was added slowly to the pooled supernatant fractions until a concentration of 70% was reached. After 1 h, the precipitated protein was recovered by centrifugation at 12,000 X g for 30 min, redissolved in buffer I, and dialyzed against the same buffer overnight. Fractions were assayed for [3H]cAMP binding and analyzed by polyacrylamide gel electrophoresis followed by photolabeling as described above. The dialyzed sample was then added to CAMP-agarose and gently rotated overnight. After removing the supernatant solution, the resin was washed with buffer I containing 2 M NaCl until the absorbance at 280 nm reached 0 and then washed again with buffer I. The R-subunit was eluted with 2 volumes of cAMP (50 mM) in buffer I at 30 "C for 1 h. The elution was repeated at least once.
The eluate from the CAMP-agarose resin frequently contained some proteolytic degradation products of R in addition to full-length R. The degradation products were removed by gel filtration on a 2 X 70-cm Sephadex G-150 column equilibrated with 25 D M potassium phosphate, 5 mM 2-mercaptoethanol, 5 mM EDTA, and 150 mM NaCl (pH 6.5). Fractions (2 ml) were collected at a flow rate of 7 ml/h. The R-subunit eluted close to the void volume and was well-resolved from the 35-kDa proteolytic fragment.
Protein Sequencing-Sequencing was carried out on an Applied Biosystems Gas-phase Sequencer. Two nanomoles of R-subunit were sequenced, and the phenylthiohydantoin derivatives were analyzed on a Beckman HPLC system using a cyano column (IBM) according to the procedure of Hunkapillar and Hood (32).

RESULTS
Construction of the Expression Vector-In order to construct an expression vector for the cloned R' gene, a fulllength cDNA insert was isolated from a derivative of a pBR322 clone, 62C12, which contains the complete coding sequence of bovine R' (11). The procedure for isolation of t h e restriction fragment containing R' is summarized in Fig. 1. The R' restriction fragments indicated were ligated into pUC7, and following transformation, colonies were screened to determine which ones contained the full-length inserts. Twenty colonies were selected, and t h e DNA from each was prepared and digested with BglI in order to establish the orientation of the insert. The DNA from three clones gave a restriction pattern which indicated that t h e R' coding segment was inserted in the correct orientation with regard to the lac 2' gene ( Fig. 1). These clones were further characterized for expression of the protein.
Expression of Regulatory Subunit-The initial transformation was done with E. coli JM105. Expression of the protein could be monitored in several ways. Because this protein has a high affinity for CAMP, we chose to use photolabeling with 8-N3[32P]cAMP as a method for detecting functional protein.
Since the intracellular concentration of cAMP in E. coli is high, it was not feasible to photolabel directly in total cell extracts. Instead, photolabeling was carried out following SDS-polyacrylamide gels electrophoresis of total extracts as described previously. As indicated in Fig. 2, one clone expressed a CAMP-binding protein in significant amounts. The clones which had the R' insert in the opposite orientation did not produce detectable amounts of protein, and no photolabeled protein was visualized in cells transformed with the parent pUC7 plasmid. Surprisingly, two other clones which, based on the BglI restriction mapping, also contained plasmid with the insert in the correct orientation did not express detectable amounts of protein.
The photolabeled protein had a molecular weight that was slightly larger than R' purified from bovine tissue. This is consistent with the pUC construct where the AUG start codon is supplied by the lac 2' gene in the plasmid, yielding a fusion protein which contains 10 additional amino acids at the NH2 terminus. The expressed protein was not degraded significantly in crude extracts.
Although pUC7 contains the lac promoter, addition of IPTG during the log phase of growth blocked the production of Rsubunit (Fig. 3). In the absence of IPTG, R-subunit accumulated in large amounts only in the stationary phase of growth. Very low levels of R-subunit were detected during the first 10 h of growth (data not shown). Quantitation of photolabeled The pBR322 plasmid containing the R' insert (62C.12) was digested with NarI, and the 3.6-kilobase (kb) fragment containing the cDNA was isolated. Following partial digestion with NcoI, the two large fragments, the full-length cDNA fragment ,(1155 base pairs (bp)), and a truncated fragment (1020 base pairs) were isolated as a mixture, filled-in with Klenow fragment, and ligated into pUC7 that had been linearized with HincII. The 135-base pair fragment also was isolated, radiolabeled with [y-'*P]ATP, and used to identify those clones which contained the full-length insert. Sites for restriction enzymes EcoRI (E) and BglI (B) are indicated. The three restriction fragments resulting from digestion with BglI are indicated by dashed arrows and were used to identify the orientation of the insert. The orientation of the lac Z' gene, ampicillin-resistance gene (Amp), tetracycline-resistance gene (tet), and origin of replication (ori) are indicated by arrows.
protein using purified R' as a standard indicated that up to 4 mg of R-subunit were being produced per liter of culture.
Characterization and Purification of the Expressed Protein-Having demonstrated that R-subunit was being expressed, we next compared the behavior of the expressed fusion protein with that of the native protein purified from bovine tissue. Selective photolabeling with 8-N3cAMP indicated already that the high affinity CAMP-binding site was functional. Another feature that is observed with purified R' is that the protomers of the dimer are cross-linked by interchain disulfide bonds. When the expressed protein was subjected to electrophoresis in the absence of 2-mercaptoethanol, it also migrated as a dimer (Fig. 4).
In order to more fully characterize the protein, the expressed R'-subunit was purified to homogeneity according to the procedure described under "Experimental Procedures." Following disruption in a French pressure cell, the R-subunit was found primarily in the supernatant fraction (Fig. 5). Determination of cAMP binding after dialysis of the redissolved ammonium sulfate pellet indicated a yield of 2-4 mg R'-subunit/liter of original culture, which was consistent with the initial estimate based on photolabeling. The R-subunit was purified further by affinity chromatography. Following elution from the affinity resin a t 30 "C, both the native protein and a significant amount of proteolytic fragment were observed (Fig. 6). The R-subunit was purified to homogeneity by gel filtration on Sephadex G-150 with the intact R-subunit eluting in a position that corresponded to a dimeric protein.
The intact R-subunit had a molecular weight of 48,000 on SDS-polyacrylamide gels. The amino-terminal sequence of

this protein was Thr-Met-Ile-Thr-Asn-Ser-Pro-Pro-Asp-Ser-
Met-Ala-Ser-Gly-Thr-Thr-Ala. This sequence confirmed that the expressed protein represents a fusion of 10 amino acids contributed by the lac 2' gene plus the entire coding region of bovine R'. The amino acids contributed by the lac 2' gene are italicized. Methionine a t position 11 is the beginning of the coding region for R'.
The cAMP binding properties of the purified protein were Finally, the purified R-subunit was incubated with a slight excess of C-subunit under conditions that should lead to the formation of holoenzyme. When excess C-subunit was removed by ion-exchange chromatography, the remaining protein was totally dependent on added cAMP for enzymatic activity (data not shown). In addition, analysis on SDSpolyacrylamide gels demonstrated equivalent amounts of Rand C-subunits, confirming the reassociation of stoichiometric amounts of both subunits to form holoenzyme (Fig. 6).
Expression in Other Strains-In order to facilitate purifi-  35,000) corresponds to a proteolytic fragment of the R-subunit and was removed subsequently by gel filtration). Right, purified R-subunit was incubated with bovine heart C-subunit. Following removal of excess C-subunit, formation of holoenzyme was assessed for dependence of activity on cAMP and by gel electrophoresis. Free C-subunit (C) and R-subunit ( R ) are indicated in the first and third lanes, respectively. The reconstituted holoenzyme is in the center lane. Proteins were visualized with Coomassie Blue .   FIG. 7. Expression of R-subunit in E. coli 222. E. coli 222 was transformed with pUC7 and with pUC7 containing the R' insert (pLST-1). Cells were pelleted from overnight cultures, dissolved in gel loading buffer, and subjected to electrophoresis. Protein was photolabeled as described in "Experimental Procedures." E. coli JM105 transformed with the same plasmid was included as a control (left). R' standard is indicated on the right. Only the portion of the gel showing radioactivity is shown. cation by affinity chromatography and to potentially improve expression of the R-subunit, a strain of E. coli, 222, was used which lacks adenylate cyclase (vu-) and which also contains a variant of CAP that binds to DNA in the absence of cAMP (crp*) (27). When E. coli 222 was transformed with pLST-1, the expression of R-subunit during the late stationary phase of growth was increased to 15-20 mg/liter based on cAMP binding and estimation of photolabeling (Fig. 7). In this strain, cAMP binding could be measured directly in the cell extract because it lacked endogeneous CAMP. The expressed R-subunit was a totally soluble dimeric protein which was purified to homogeneity by affinity chromatography followed by gel filtration.

DISCUSSION
The cDNA corresponding to the complete coding region of the bovine type I regulatory subunit of CAMP-dependent Expression of the Type I Regulatory Subunit 11095 protein kinase was inserted into the HincII site of pUC7.
When the repressor overproducing strain of E. coli, JM105, was transformed with a plasmid that contained the R' insert in the same orientation as the lac 2' gene, a clone was isolated which expressed up to 4 mg of R-subunit/liter of culture. Two other transformants were isolated from the same ligation mixture which, on the basis of DNA analysis, were identical to the above clone. Nevertheless, these two clones did not produce large amounts of R-subunit. Further experimentation is now underway to ascertain what percentage of transformants produces R-subunit and to determine whether the lack of expression in these clones is due to a host mutation. The plasmid that has consistently yielded transformants that express R-subunit was stored as a 20% glycerol stock from cells harvested in the mid-log phase of growth, and this plasmid is subsequently referred to as pLST-1.
The expression of R-subunit did not follow the typical pattern of IPTG induction that might be anticipated for a gene linked to the lac promoter. The molecular basis for this expression is not apparent. The R-subunit obviously binds cAMP with a high affinity and thus will titrate any cAMP that is produced. However, this should not lead to activation of the lac promoter. Although there are some reports that the R-subunit may interact with DNA (36), there is no evidence as yet that suggests that any R-subunit binds specifically and with a high affinity to DNA. The homologies of the R-subunit with CAP make this an intriguing speculation (37); however, based on the structural data available, there are no clear homologies between any R-subunit and the DNA-binding domain of CAP. Nagamine and Reich (37) cite a specific sequence in the R-subunit that may be analogous to a DNA recognition sequence in C A P however, this sequence lies in the middle of the CAMP-binding domains of R. When these domains are compared in more detail by building the Rsubunit sequences into the crystal structure of CAP, it is highly unlikely that this region would assume a structure analogous to the DNA-binding sequence in CAP.2 If the Rsubunit does interact with DNA directly, the sites of interaction almost certainly must be confined to the amino-terminal region of the protein.
Although further experimentation will be required in order to understand the mechanism being utilized for the expression of R-subunit, the protein can nevertheless be expressed reproducibly in large amounts. Maximum induction was observed when media was inoculated with cultures grown to OD,,, = 0.1-0.5 and then allowed to grow into late stationary phase in the absence of IPTG. Very little R was visualized in the log phase of growth, and large amounts accumulated only in the stationary phase. Addition of IPTG during the log phase of growth blocked the expression of R-subunit. Maximum expression of R-subunit was seen in E. coli strain 222. This is a strain which contains a double mutation. It lacks adenylate cyclase (cyu-) and also contains a variant of CAP (crp*) that binds DNA in a CAMP-independent manner (27). This strain expressed nearly 10-fold more R-subunit than did E. coli JM105, which is a strain that constitutively overexpresses the lac repressor.
Visualizing the R-subunit by photolabeling with 8-N,[32P] CAMP following electrotransfer to nitrocellulose provided a convenient method for detection which obviated the need to remove basal cAMP from crude extracts. Apparently, the protein refolds sufficiently following electrotransfer to nitrocellulose to restore its high affinity binding for CAMP. The high selectivity is apparent since no other proteins are labeled to any significant extent in the total cellular extract. This procedure thus appears to be a very specific method for detecting R-subunits following denaturation and separation on the basis of size.
The CAMP-binding protein that was expressed had a molecular weight that was slightly larger than native R' based on gel mobilities and was consistent with the design of the construct which should yield a fusion protein. The sequence of the construct was confirmed by amino acid sequencing. Proteolysis did not appear to be a major problem in the initial extraction even though this protein is particularly susceptible to limited proteolysis. The proteolytic fragment that was observed later in the purification was identical, based on gel mobilities, to the endogenously generated fragment of the native protein, and this also is consistent with the predicted construct. This susceptibility to limited proteolysis also confirmed that the expressed protein is folding in a manner that is analogous to the native protein.
Although the expressed protein contained 10 additional residues at the amino-terminal end, it behaved like the protein purified from mammalian tissues. The expressed R-subunit was a soluble dimer, and the specificity of interaction between the protomers was confirmed by the appearance of interchain disulfide bonds. This is similar to R' from porcine skeletal muscle (19), and although it has not been ascertained that this disulfide bonding is physiological, it nevertheless does indicate that the cysteine residues from adjacent chains are in close proximity in the dimer both in the mammalian protein and in the expressed R-subunit.
Another property that is associated with the amino-terminal region of the protein is the interaction with the C-subunit, and this function also appears to be intact. When purified Rsubunit was incubated with C-subunit, holoenzyme formed that was fully dependent on added cAMP for activity. Activation of the holoenzyme by cAMP indicated that the CAMPbinding sites were functional, and this was confirmed by photoaffinity labeling with 8-N3cAMP and by successful purification with a cAMP affinity resin. Quantitation of the CAMP-binding sites established that both classes of binding sites were functional in that the expressed protein and that the expressed protein binds maximally 2 mol of cAMP/Rmonomer.
By ail the above criteria, the expressed regulatory subunit appeared to be functioning in a manner totally analogous to its mammalian counterpart. This construct thus provides an efficient mechanism for further probing this molecule by directed mutagenesis. In addition to the functional sites described here, the molecule is known to have other functions such as specific interaction with microtubule-associated protein I1 (381, calcineurin (39), and p75 in brain (40). Other functions include a role as a phosphatase inhibitor (41) and a potential role as a topoisomerase I (36). These reports provide new and relatively unexplored insights into the role that this molecule may play in CAMP-mediated regulation in addition to its well-established role as an inhibitor of the catalytic subunit.
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