Identification of Critical Determinants for Autoinhibition in the Pseudosubstrate Region of Type Ialpha cAMP-dependent Protein Kinase*

(Received for publication, May 30, 1996, and in revised form, September 12, 1996)

Celeste E. Poteet-Smith Dagger , John B. Shabb §, Sharron H. Francis Dagger and Jackie D. Corbin Dagger

From the Dagger  Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615 and the § Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine, Grand Forks, North Dakota 58202-9037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The consensus substrate site for cAMP-dependent protein kinase (PKA) is Arg-Arg-Xaa-Ser(P)-Xaa and the autoinhibitory domain of the PKA type Ialpha regulatory subunit (RI subunit) contains a similar sequence, Arg92-Arg-Arg-Arg-Gly-<UNL>Ala</UNL>-Ile-Ser-Ala-Glu. The italicized amino acids form a putative pseudosubstrate site (Ser is replaced with <UNL><IT>Ala</IT></UNL>), which together with adjacent residues could competitively inhibit substrate phosphorylation by the PKA catalytic subunit (C subunit). The present studies determine the contributions of Arg92-95, Ile98, and Glu101 to inhibitory potency. Amino-terminal truncation of RI subunit through Arg92 (Delta 1-92) or Arg93 (Delta 1-93) had no detectable effect on inhibition of C subunit. Truncation through Arg94 (Delta 1-94), or point mutation of Arg95 within truncated mutants (Delta 1-93.R95A or Delta 1-92.R95A), caused a dramatic reduction in inhibitory potency. Truncation through Arg95 (Delta 1-95) had a greater effect than did replacement or deletion of Arg94 or Arg95 alone. Using full-length RI subunit, the inhibitory potency was reduced by replacing Ile98 with Ala, Gly, or Gln, but not by replacing it with Val. The inhibitory potency of RI subunit was unchanged when Glu101 was replaced with Ala or Gln. It is concluded that Arg94, Arg95 and, to a lesser extent, Ile98 are vital constituents of PKA autoinhibition by type Ialpha R subunit.


INTRODUCTION

cAMP-dependent protein kinase (PKA)1 is a tetramer comprising a dimer of regulatory subunits (R subunit) and two catalytic subunits (C subunit). In the absence of cAMP, PKA is maintained as an inactive holoenzyme complex (R2C2). cAMP binding to the R subunit causes dissociation of the R and C subunits and subsequent activation of the kinase (1). There are two major classes of PKA holoenzyme, types I and II, denoted by the R subunit isoform associated with C subunit (2). Despite numerous differences in the R subunit isoforms, they share the same basic domain structure: a short amino-terminal dimerization domain, an autoinhibitory domain located in the amino-terminal segment of the protein, and two cAMP-binding domains toward the carboxyl-terminal end.

The autoinhibitory domain of the R subunit contains a sequence that mimics the consensus phosphorylation sequence of PKA substrates (Arg-Arg-Xaa-Ser(P)-Xaa). In the type II R subunit (RII subunit), this sequence (Arg-Arg-Val-Ser(P)-Val) is a substrate for C subunit, but in the RI subunit, the phosphorylatable Ser is substituted with a non-phosphorylatable Ala residue to form a pseudosubstrate site (Arg-Arg-Xaa-<UNL>Ala</UNL>-Xaa). The pseudosubstrate sequence is believed to interact with the C subunit catalytic site to competitively inhibit substrate phosphorylation (3).

The phosphorylation or pseudophosphorylation site is designated as the P residue, while residues located amino-terminal or carboxyl-terminal to P are designated as minus or plus residues, respectively (4). The P-3 and P-2 Arg residues are important determinants for phosphorylation of PKA substrates (5, 6). Replacement of either residue in peptide substrate analogs profoundly impairs the phosphorylation of the peptide. The P-3 and P-2 Arg residues are invariant in all known R subunit pseudosubstrate sequences and are required for potent inhibition of C subunit (3, 7, 8). They are also found in the pseudosubstrate sequence of the high affinity, heat-stable protein kinase inhibitor of PKA, PKI (Arg-Arg-Gln-<UNL>Ala</UNL>-Ile) (9). The importance of these two Arg residues in PKI has also been well documented using peptide analogs (10, 11), and more recently by co-crystallization of C subunit with the peptide derived from the inhibitory segment of PKI, PKI-(5-24) (4, 12).

Although many natural substrates for PKA conform to the consensus sequence of Arg-Arg-Xaa-Ser(P)-Xaa, many others contain only a single basic residue at either P-3 or P-2, and/or have additional basic residues more amino-terminal to P-3 (13). Basic residues are frequently found at the P-4, P-6, or both positions in PKA substrates (13), yet other substrates contain a cluster of four basic residues at the P-2 through P-5 positions (14, 15, 16). Both P-4 and P-6 basic residues are important recognition factors for phosphorylation of the peptide derived from rabbit skeletal muscle phosphorylase kinase (beta  subunit) (17). A similar pattern of basic amino acid residues is noted in the PKA regulatory proteins. A P-6 Arg residue is present in the RII subunit substrate site and in the PKI pseudosubstrate site, while the RI subunit has a cluster of four Arg residues at the P-2 through P-5 positions. The PKI P-6 Arg is crucial for potent inhibition of C subunit (4, 11). The contribution made toward C subunit inhibition by the RII subunit P-6 Arg residue, or the RI subunit P-4 and P-5 Arg residues, has not been investigated previously. Based on the evidence above, it is possible that each of the four Arg residues in the pseudosubstrate region of RI subunit interacts with an acidic residue(s) in the active site of C subunit.

The P+1 residue is frequently, but not exclusively, a large hydrophobic residue in PKA substrates (13). Studies with synthetic peptide substrates based on the phosphorylation sequence of pyruvate kinase (Arg-Arg-Ala-Ser(P)-Val) suggested that a large hydrophobic residue at the P+1 position (Phe, Leu, or Ile) is a positive determinant for substrate phosphorylation (13). However, small (e.g. Gly), charged (e.g. Lys, Arg, or Glu), or hydrophilic residues (e.g. Ser) at this position are negative determinants for phosphorylation of these peptide substrates by C subunit (13). The P+1 residue is conserved as Ile or Val in the autoinhibitory domains of all known sequences of PKA and PKG, and as Ile in PKI (10). PKI peptide analog studies (18) and the crystal structure of the C subunit·PKI-(5-24) complex (4) demonstrated the importance of a large P+1 hydrophobic residue for potent inhibition of C subunit (18). The contribution of the RI subunit P+1 hydrophobic residue toward C subunit inhibition has not been investigated to date.

The Glu at the P+4 position is invariant in the regulatory domain of all known sequences of PKA and PKG. Mutational analysis of Saccharomyces cerevisiae C subunit identified three Lys residues required for recognition of the yeast R subunit (19); the corresponding residues in mammalian C subunit (Lys189, Lys213, Lys217) lie distal to the carboxyl-terminal residue (P+3) of bound PKI-(5-24) (4, 12), and one of these Lys residues could interact with the P+4 Glu. The importance of the P+4 Glu in the autoinhibitory domain has not been previously addressed.

Using truncation and site-directed mutagenesis, the present report addresses the importance of the P-5,P-4, P-3, P-2, P+1 and P+4 residues for autoinhibition of type Ialpha PKA.


EXPERIMENTAL PROCEDURES

Materials

The pT7-7 vector was from S. Tabor (Harvard University, Cambridge, MA) (20, 21). Oligonucleotides were synthesized in the DNA core facility at Vanderbilt University. Escherichia coli strain BL21(DE3) is described in Ref. 22. E. coli strain CJ236 and helper phage M13K07 were from Invitrogen and the pBluescript KS(-) vector was from Stratagene. The Sequenase Version 2.0 DNA sequencing kit was from U. S. Biochemical Corp. Restriction endonucleases were from Promega, New England Biolabs, or Boehringer Mannheim. [alpha -32P]dATP and [2,8-3H]cAMP were from Amersham, [gamma -32P]ATP was from DuPont NEN, and 8-N3-[32P]cAMP was from ICN. Bovine serum albumin (essentially fatty acid-free, catalogue no. A-6003), cyclic nucleotides, and histone were from Sigma. Kemptide was from Peninsula Laboratories. Urea was from Boehringer Mannheim. Cyanogen bromide-activated Sepharose 4B was from Pharmacia Biotech Inc. P-81 phosphocellulose paper was from Whatman. All other reagents were purchased from Sigma. C subunit was prepared from bovine heart according to the method of Sugden et al. (23). N6-H2N(CH2)2-cAMP was synthesized according to Dills et al. (24) and was purified by Sephadex G-25 chromatography (4.5 × 16.5 cm) in 50 mM NH4HCO3, pH 7.8, according to Corbin et al. (25). N6-H2N(CH2)2-cAMP-Sepharose was prepared according to Dills et al. (24, 26, 27).

Construction of RI Subunit Deletion Mutants

A T7 RNA polymerase/promoter system, pT7-7, was used for bacterial overexpression of the type Ialpha R subunit. The RI subunit cDNA was subcloned into pT7-7 as described previously (28) to create pT7RNI and pT7R. pT7RNI, an intermediate vector, contained the entire RI subunit cDNA, which was out-of-frame. pT7R was the wild type (WT) RI subunit bacterial expression vector in which the RI subunit start codon was mutated to a NdeI site to allow direct in-frame fusion with the pT7-7 start codon, also encoded by a NdeI restriction site.

KS(-)R was created for use in oligonucleotide-directed mutagenesis by subcloning the 1118-base pair (bp) SacI-SalI fragment of pT7RNI (encoding all but 52 base pairs from the 5' end of the RI subunit cDNA) into the SacI-SalI fragment (2882 bp) of the M13-derived phagemid, pBluescript KS(-). Synthetic oligonucleotides 1-13 (Table I) were used to mutate KS(-)R by site-directed mutagenesis using the Kunkel method (29). Oligonucleotides 1-5 were used to mutate the KS(-)R sequence on the 5' side of the Arg92, Arg93, Arg94, Arg95, and Gly96 codons to a NdeI site (CATATG). Oligonucleotides 6-8 were used to create a NdeI site 5' to the Arg93 or Arg94 codons, and to mutate the codons for Arg94 (CGA) or Arg95 (GCG) to Ala (GCT) in KS(-)R. Introduction of a NdeI restriction site allowed for direct in-frame fusion with the NdeI restriction site encompassing the pT7R start codon, thus deleting the 5' segment of the RI subunit cDNA. Oligonucleotides 9-13 were used to mutate the Ile98 codon to Val (GTT), Ala (GCT), Gly (GGT), and Gln (CAG), and the Glu101 codon to Ala (GCT) in KS(-)R. A 700-bp PstI fragment, released from the previously described pUC13-based RI subunit expression vector (pUC13R) (30), was subcloned into the PstI site of M13mp8 to create M13.PstR. Oligonucleotide 14 was used to mutate the Glu101 codon (GAG) to Gln (CAA) in M13PstR using a commercially available mutagenesis kit (Oligonucleotide-directed in Vitro Mutagenesis, version 2, code RPN.1523; Amersham).

Table I.

Synthetic oligonucleotides for RI subunit mutagenesis

The oligonucleotides are complementary to the cDNA coding strand except for the bold and underlined nucleotides.
1) Delta 1-91 5'-GCGTCGCCGCCG<UNL><B>CATATG</B></UNL>CACCACCGGGTTAG-3'
2) Delta 1-92 5'-CCGCGTCGCCG<UNL><B>CATATG</B></UNL>CTTCACCACCGGGTTAG-3'
3) Delta 1-93 5'-GCCCCGCGTCG<UNL><B>CATATG</B></UNL>GCCCTTCACCACC-3'
4) Delta 1-94 5'-TGATGGCCCCGCG<UNL><B>CATATG</B></UNL>CCGGCCCTTCA-3'
5) Delta 1-95 5'-CGCTGATGGCCCC<UNL><B>CATATG</B></UNL>CCGCCGGCCCT-3'
6) Delta 1-93.R94A 5'-GATGGCCCCGCG<UNL><B>AGCCATATG</B></UNL>GCCCTTCACCACCG-3'
7) Delta 1-92.R95A 5'-CGCTGATGGCCCC<UNL><B>AGC</B></UNL>TCGCCG<UNL><B>CATATG</B></UNL>CTTCACCACCGGGTTAG-3'
8) Delta 1-93.R95A 5'-CGCTGATGGCCCC<UNL><B>AGC</B></UNL>TCG<UNL><B>CATAT</B>G</UNL>GCCCTTCACCACCG-3'
9) I98V 5'-GTAGACCTCAGCGCT<UNL><B>A</B></UNL>A<UNL><B>C</B></UNL>GGCCCCGCGTCG-3'
10) I98A 5'-GTAGACCTCAGCGCT<UNL><B>AGC</B></UNL>GGCCCCGCGTCG-3'
11) I98G 5'-GTAGACCTCAGCGCT<UNL><B>ACC</B></UNL>GGCCCCGCGTCG-3'
12) I98Q 5'-GTAGACCTCAGCGCT<UNL><B>CTG</B></UNL>GGCCCCGCGTCG-3'
13) E101A 5'-GGTGTAGAC<UNL><B>AGC</B></UNL>AGCGCTGAT-3'
14) E101Q 5'-GTGTAGAC<UNL><B>T</B></UNL>T<UNL><B>G</B></UNL>AGCGCTGAT-3'

Functional pT7R expression vectors encoding truncated RI subunit cDNA (Delta 1-91, Delta 1-92, Delta 1-93, Delta 1-94, Delta 1-95, Delta 1-93.R94A, Delta 1-92.R95A, and Delta 1-93.R95A) were created by subcloning the 585-596-bp NdeI-EcoRI fragments from KS(-)R mutants 1-8, into the large NdeI-EcoRI fragment of pT7R. The 760-bp PvuII-EcoRI fragments were isolated from KS(-)R mutants 9-13 and subcloned into the large PvuII-EcoRI fragment of pT7R to create functional mutant pT7R expression vectors encoding full-length RI subunits (I98V, I98A, I98G, I98Q, and E101A). The NaeI-XhoI fragment from M13.PstR mutant 14 (351 bp) was subcloned into the large NaeI-XhoI fragment of pUC13R. The 518-bp PvuII-XhoI fragment of pUC13R was subcloned into the large pT7R PvuII-XhoI fragment to create a functional mutant pT7R expression vector encoding the full-length E101Q RI subunit.

The integrity of the entire cDNA segment that was subjected to the mutagenesis procedure, including the cloning and mutation sites, was verified directly in the pT7R expression vectors by dideoxynucleotide sequencing of double-stranded DNA for mutants 1-12 and 14. DNA sequencing of mutants 1-8 and 14 was performed using a commercially available kit (Sequenase version 2.0, U. S. Biochemical Corp.) and mutants 9-12 were sequenced using an Automated Biosystems, Inc. model 373A DNA sequencer in the Cancer Center DNA Core facility of Vanderbilt University. The cloning sites for mutant 13 were verified by digestion with PvuII and EcoRI, and the mutation was verified by dideoxynucleotide sequencing using the aforementioned kit.

Bacterial Expression

E. coli strain BL21(DE3) cells were transformed with wild-type or mutant pT7R vectors the day prior to expression. Bacterial expression was performed as described in Ref. 28, except that the media contained 0.1 mg/ml ampicillin and the cells were pelleted at 5000 × g for 15 min each time. The bacterial pellets were stored at -20 °C until homogenization and were viable for more than 1 year when stored in this manner. Initially, the expression cultures yielded ~3-5 mg of RI subunit/liter of culture (5-h induction). Due to the necessity for greater quantities of protein, the expression time was later increased to 36 h, yielding 10-80 mg of RI subunit/liter culture.

Purification of Recombinant RI Subunits

Bacteria were disrupted as described previously (31) except that the homogenization buffer was 10 mM potassium phosphate, pH 6.8, 1 mM EDTA, 2 mM beta -mercaptoethanol (KPEM) plus 50 mM benzamidine (KPEMB) and 2.3 mM 3',5'-cyclic inosine monophosphate (cIMP). cIMP was included during the homogenization process to promote exchange with the cAMP bound to RI subunits and thus increase the subsequent efficiency of RI subunit binding to N6-H2N-(CH2)2-cAMP Sepharose affinity resin during purification.

Free cyclic nucleotide was removed from the extract by fractionation on a Sephadex G-25 superfine column (2.75 × 26 cm) equilibrated in KPEMB. Protein-containing fractions (~70 ml) were collected and clarified by centrifugation at 12,000 × g for 30 min. The extract was chromatographed on a 1.5 × 3.5-cm N6-H2N(CH2)2-cAMP Sepharose column (equilibrated in KPEMB) at a flow rate of ~1 drop/12 s, or was divided and chromatographed over two columns (1.5 × 3.5 cm and 1.5 × 11.5 cm). In most instances, the flow-through was passed over the column(s) two or three times. RI subunits were isolated by the method of Dills (24) with the exceptions noted in Ref. 28. In addition, 30 mM cIMP was used to elute the affinity resin instead of 10 mM. Fractions containing protein (detected by Bio-Rad protein assay) were pooled and concentrated on a Centriprep-30 concentrator (Amicon) to a final volume of approximately 0.6 ml. Full-length and truncated RI subunits were resolved from proteolytic fragments and from free cyclic nucleotide on a Sephadex G-100 superfine column (0.9 × 58.5 cm) equilibrated in KPEM containing 0.2 M NaCl. Fractions were assayed for [3H]cAMP binding activity, and cIMP elution was measured by UV absorbance at 248 nm. The fractions containing RI subunit were pooled and concentrated on Centricon-30 devices (Amicon), followed by a desalting step with KPEM. The full-length and truncated RI subunits were >95% pure according to SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Purification of cAMP-saturated Native RI Subunit

Native type Ialpha R subunit was purified from a bovine lung homogenate that was primarily used for the purification of cGMP-binding, cGMP-specific phosphodiesterase (cG-BPDE) (32) and type Ialpha PKG (33) or it was purified from rabbit skeletal muscle according to Ref. 34. When using bovine lung homogenate, protein extract was adsorbed to DEAE-cellulose followed by batch elution with 0.1 M NaCl in 20 mM potassium phosphate, pH 6.8, 1 mM EDTA, and 25 mM 2-mercaptoethanol. After discarding the initial 0.5 liter of eluate, the next 1.5 liters of eluate was collected for cG-BPDE purification, and the following 1 liter was collected for purification of PKG and R subunit. This 1 liter of extract was slowly loaded onto N6-hexyl-cAMP-Sepharose (5 × 2.75 cm) equilibrated in KPEM containing 25 mM 2-mercaptoethanol (KPEM25) at 4 °C. The affinity resin was washed with 2 liters of KPEM25 containing 2 M NaCl, followed by 600 ml of KPEM25. The column was batch-eluted with 10 mM cGMP in KPEM25. Following elution of the bulk of PKG, monitored by SDS-PAGE of elutions, the affinity resin was batch-eluted with aliquots of KPEM25 containing 10 mM cAMP to elute types Ialpha and IIalpha R subunit. Fractions containing both types of R subunit were pooled and chromatographed on DEAE Sephacel (0.9 × 5.5 cm) equilibrated in KPEM containing 0.05 M NaCl at 4 °C. Types I and II R subunit were resolved by eluting the DEAE column with a linear salt gradient (0.05-0.35 M NaCl) in KPEM. The RI subunit was further purified by sucrose gradient (5-20%) centrifugation to separate it from PKG contaminants. The RI subunit fractions from five sucrose gradients were pooled and concentrated on a Centriprep-30 device. The protein was then chromatographed on a 0.9 × 58.5-cm Sephadex G-100 superfine column (equilibrated in KPEM containing 0.2 M NaCl) to separate full-length RI subunit from proteolytic fragments of RI subunit and PKG. RI subunit fractions were pooled and frozen at -20 °C in 50% KPEM and 50% glycerol. Native RI subunit from bovine lung contained saturating levels of cAMP as determined by assay of cAMP content (25), and contained ~5-10% PKG proteolytic fragments as detected by SDS-PAGE.

Preparation of cIMP-saturated Native RI Subunit

Native RI subunit (purified from bovine lung, above) was concentrated and the glycerol was removed by multiple centrifugations and dilutions on a Centricon-30 device. cIMP (at a final concentration of 1.1 mM) was added to the RI subunit solution (10:1 ratio of cIMP to cyclic nucleotide binding sites), and the solution was stored at 4 °C for several weeks to facilitate cAMP/cIMP exchange. RI subunit was then chromatographed on a Sephadex G-25 column (0.9 × 13 cm) equilibrated in KPEM to resolve protein from free cyclic nucleotide. cIMP-saturated RI subunit was pooled and concentrated using a Centricon-30 device.

Determination of Stoichiometry for cIMP Bound to RI Subunits

287 µg of WT or mutant RI subunit was denatured at 95 °C for 2 h in the presence of 13.5 mM HCl (final pH ~2-3) to release cyclic nucleotide. The solution was briefly centrifuged at 15-min intervals during incubation. Following neutralization, the sample was chromatographed at 23 °C on a Sephadex G-25 superfine column (0.9 × 13 cm) equilibrated in KPEM to purify cyclic nucleotide (25). cIMP elution was quantitated using the cIMP extinction coefficient (epsilon 1 mM = 12.3 absorbance units at 248 nm). The released cyclic nucleotide was verified to be cIMP by a UV absorbance scan at 220-320 nm.

Protein Sequence Analyses

The NH2-terminal sequence of various proteins was determined by sequential Edman degradation. The analyses were performed by the core sequencing facility at the University of Washington, Seattle, WA and by the Peptide Sequencing and Amino Acid Analysis Shared Resource at Vanderbilt University.

Cyclic Nucleotide Binding and Dissociation Assays

The cyclic nucleotide binding assay was used to identify RI subunit fractions eluted from gel filtration columns and to quantitate purified RI subunits. [3H]cAMP binding activity was measured as described previously (34) except that 1.1 µM [3H]cAMP (2800-5500 cpm/pmol cAMP) was used, and the reaction mixture was diluted with 2.5 ml of ice-cold 10 mM potassium phosphate, pH 6.8, 1 mM EDTA (KPE) and the tubes were each rinsed with 2.5 ml of KPE. RI subunit was quantitated based on a stoichiometry of 2 mol of cAMP/mol of RI subunit monomer.

[3H]cAMP dissociation assays were performed as described previously (35) using 0.2 µM [3H]cAMP (3 × 104 cpm/pmol) and 4 nM RI subunit in the equilibrium exchange reaction. A 500-fold molar excess of unlabeled cAMP was used in the dissociation reaction.

Inhibition of C Subunit by Native, WT, and Mutant RI Subunits

Immediately before use, C subunit was diluted in Dilution Buffer (50 mM potassium phosphate, pH 6.8, 0.1 mM dithiothreitol, and 1 mg/ml bovine serum albumin (BSA)). A final concentration of 21 pM C subunit was preincubated with varying concentrations of WT or mutant RI subunit (in KPEM) in Kinase Mix (20 mM Tris, pH 7.4, 20 mM magnesium acetate, 0.1 mM ATP, 0.5 mg/ml BSA, and 0.1 mM isobutylmethylxanthine) for 15-30 min at 30 °C. The protein kinase assay was initiated by the addition of a final concentration of 81 µM Kemptide, 0.071 mM ATP, and [gamma -32P]ATP (1000 cpm/pmol). The total assay volume was 35 µl. Following a 60-120-min incubation at 30 °C, the reaction was terminated by spotting 20 µl of reaction mixture onto P-81 phosphocellulose paper and immediately placing the papers into 75 mM phosphoric acid. The P-81 papers were washed a minimum of five times in 75 mM phosphoric acid, dried and counted in 10 ml of non-aqueous scintillant, or counted by Cerenkov radiation.

Preparation of PKA Holoenzyme from WT RI Subunit and Native C Subunit

cIMP-saturated WT RI subunit (3.8 µM) and native C subunit (14.7 µM) were incubated for 30 min at 30 °C in Kinase Mix containing 0.23 mM ATP and 10 mg/ml BSA (final volume = 15 µl). 1 µl of reaction was diluted into 19 µl of Dilution Buffer and saved for subsequent assay of kinase activity (± cAMP). The remainder of the reaction was cooled on ice and then loaded onto a Sephadex CM50 column (0.9 × 0.5 cm) equilibrated in KPEM at 4 °C. Approximately 1 ml of KPEM was added once the sample had entered the resin, and 1-drop fractions (~23 µl) were collected. The fractions were assayed for phosphotransferase activity using the peptide substrate Kemptide (LRRASLG) (81 µM) in the presence of Kinase Mix containing 0.3 mM ATP and 10 mg/ml BSA plus 40 µM Kemptide, [gamma -32P]ATP (~33 cpm/pmol), with or without 2 µM cAMP. Fractions collected between 47 and 138 µl (~drops 3-6) contained ~70% of the total holoenzyme, and the kinase activity was 87% cAMP-dependent. Fractions collected between 139 and 230 µl (~drops 7-10) contained ~18% of the total holoenzyme, and the kinase activity was 73% cAMP-dependent.

Determination of cIMP Ka for Cyclic Nucleotide-depleted Holoenzyme (WT RI Subunit and Native C Subunit)

The activation constant (Ka) of cIMP for PKA holoenzyme was determined by measuring the transfer of phosphate to Kemptide in the presence of cIMP (0-20 µM). Holoenzyme was diluted to 24 nM in Dilution Buffer. 10 nM holoenzyme was combined with 0.1 mg/ml Kemptide (118 µM) and cIMP (0-20 µM) in Kinase Mix containing 0.2 mM ATP and 10 mg/ml BSA plus [gamma -32P]ATP (~30 cpm/pmol). The 24-µl reaction was incubated for 10 min at 30 °C before spotting 15 µl onto P-81 phosphocellulose paper. The papers were washed five times in 75 mM phosphoric acid, dried, and counted in scintillant. A 20% basal kinase activity was subtracted from each activity determination prior to calculating the Ka.

Preparation of Cyclic Nucleotide-free RI Subunits

Urea (0.67 mg of urea/µl of enzyme) was added to cIMP-saturated RI subunit (at 23 °C) for a final concentration of 8 M urea. After a 5-min maximum incubation at 23 °C, the urea/enzyme mixture was loaded onto a Sephadex G-25 superfine column (0.9 × 13 cm) equilibrated in KPEM containing 8 M urea. The column was developed at 23 °C with KPEM plus 8 M urea, and 1-ml fractions were collected. Protein-containing tubes (fractions 4-6) were separately dialyzed (4 °C, 24-48 h) against KPEM immediately as each fraction eluted from the column. Since cIMP eluted in fractions 8-10, it was easily separated from the RI subunits. Approximately 45 min elapsed from the time that urea was added to the protein until the time that the last fraction was placed into dialysate.


RESULTS AND DISCUSSION

Amino-terminal truncation and site-specific mutants (Table II) of the RI subunit were employed to investigate the contributions that conserved residues within and near the pseudosubstrate region make toward the potency with which RI subunit inhibits C subunit kinase activity. The individual roles of Arg92-95 were assessed by creating truncation mutants of RI subunit that were sequentially deleted through each of residues 91 through 95. Amino-terminal truncation mutants were also prepared in which Arg94 or Arg95 was replaced with Ala by site-directed mutagenesis. To examine a potential role for additional residues in the putative autoinhibitory domain, two other highly conserved residues (Ile98 and Glu101) were also investigated utilizing site-specific mutants of full-length RI subunit. Ile98 was mutated to Val, Ala, Gly, and Gln; Glu101 was changed to Ala and Gln. Finally, to provide a more comprehensive study, and to minimize certain pitfalls inherent to each form of RI subunit, inhibition of C subunit was examined using four forms of RI subunit: 1) cAMP-saturated, 2) cIMP-saturated, 3) cyclic nucleotide-saturated with excess cyclic nucleotide in the solution, and 4) cyclic nucleotide-free.

Table II.

Truncations and mutations in the pseudosubstrate region of the type Ialpha R subunit

The residue number is indicated above the sequences. The NH2-terminal residue is denoted by a superscript 1 in the truncated RI subunits, and the mutated residues are capitalized and underlined.
Regulatory subunit          P-5   P-4  P-3   P-2         P   P+1               P+4

      92  93  94  95  96  97  98  99  100 101
Wild type . . . Arg Arg Arg Arg Gly Ala Ile Ser Ala Glu . . .
 Delta 1-91  Met1 Arg Arg Arg Arg Gly Ala Ile Ser Ala Glu . . .
 Delta 1-92       Met1Arg Arg Arg Gly Ala Ile Ser Ala Glu . . .
 Delta 1-93           Met1Arg Arg Gly Ala Ile Ser Ala Glu . . .
 Delta 1-94               Met1Arg Gly Ala Ile Ser Ala Glu . . .
 Delta 1-94*                   Arg1Gly Ala Ile Ser Ala Glu . . .
 Delta 1-93.R95A           Met1Arg <UNL><B>ALA</B></UNL> Gly Ala Ile Ser Ala Glu . . .
 Delta 1-92.R95A       Met1Arg Arg <UNL><B>ALA</B></UNL> Gly Ala Ile Ser Ala Glu . . .
 Delta 1-95                       Gly1Ala Ile Ser Ala Glu . . .
I98V . . . Arg Arg Arg Arg Gly Ala <UNL><B>VAL</B></UNL> Ser Ala Glu . . .
I98A . . . Arg Arg Arg Arg Gly Ala <UNL><B>ALA</B></UNL> Ser Ala Glu . . .
I98G . . . Arg Arg Arg Arg Gly Ala <UNL><B>GLY</B></UNL> Ser Ala Glu . . .
I98Q . . . Arg Arg Arg Arg Gly Ala <UNL><B>GLN</B></UNL> Ser Ala Glu . . .
E101A . . . Arg Arg Arg Arg Gly Ala Ile Ser Ala <UNL><B>ALA</B></UNL> . . .
E101Q . . . Arg Arg Arg Arg Gly Ala Ile Ser Ala <UNL><B>GLN</B></UNL> . . .

Confirmation of the Structural Integrity of Recombinant RI Subunits

All segments of RI subunit cDNA subjected to a mutagenesis reaction were sequenced to verify the integrity of the mutation and subcloning sites before overexpression in bacteria. The recombinant RI subunits were purified to apparent homogeneity as shown in Fig. 1 (see "Experimental Procedures"). Consistent with previous results (36), the full-length RI subunit migrated at Mr ~ 49,000 when analyzed by SDS-PAGE, considerably larger than the molecular weight calculated by the amino acid sequence for bovine skeletal muscle RI subunit (42,804) (37). The truncated RI subunits migrated at Mr ~35,000 on SDS-PAGE, but were calculated to have molecular weights of ~31,790-32,414 by amino acid sequence. The purified WT, E101Q, and all truncated RI subunits were subjected to amino acid sequence analysis to verify the predicted primary structure at the amino termini. The initiator Met was present in proteins in which the penultimate residue was Arg, but it was absent from those in which this residue was Ala (WT, E101Q, Delta 1-94*) or Gly (Delta 1-95). These results were consistent with the previous finding that cleavage of the initiator Met from proteins during post-translational modification is dependent on the size of the adjoining residue (38). Protein expressed from the Delta 1-93.R94A cDNA (two preparations) did not have the expected amino-terminal sequence. This mutant lacked the initiator Met as well as the Ala that was intended to replace Arg94. Since the product of Delta 1-93.R94A differed from Delta 1-94 only in the absence of the initiator Met residue it was named Delta 1-94*. Presumably Ala94 was removed from the protein during cleavage of the initiator Met or during protein purification by an exoproteinase. The cAMP dissociation characteristics of the truncated and full-length mutants were compared to that of WT RI subunit to verify that the mutations did not impair the overall viability of the proteins. The cAMP dissociation rates for all mutant RI subunits were comparable to that of WT. Fig. 2 displays the characteristic biphasic cAMP dissociation (exchange) pattern of WT, and those of representative mutants: Delta 1-94, E101A, and I98A.


Fig. 1. SDS-polyacrylamide gel electrophoresis of recombinant RI subunits purified from E. coli. WT and mutant RI subunits were purified from BL21(DE3) expression cultures by the procedure described under "Experimental Procedures." The purified samples were analyzed on 15% SDS-polyacrylamide gels stained with Coomassie Blue. WT and various purified RI subunit mutants are represented in this figure. Lane 1, WT; lane 2, I98V; lane 3, I98A; lane 4, I98G; lane 5, I98Q; lane 6, Delta 1-91; lane 7, Delta 1-92; lane 8, Delta 1-94; lane 9, Delta 1-95. M represents low molecular weight standards from Pharmacia.
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Fig. 2. cAMP dissociation from WT and mutant RI subunits. [3H] cAMP dissociation was performed as described under "Experimental Procedures." In short, the cyclic nucleotide binding sites of WT (open circle ), Delta 1-94 (square ), E101A (triangle ), and I98A (diamond ) RI subunits were saturated with [3H]cAMP, followed by the addition of a 500-fold molar excess of unlabeled cAMP. The quantity of [3H]cAMP bound to RI subunits at various time points (B) was determined by Millipore filtration of aliquots diluted in KPE buffer. Be represents [3H]cAMP bound to RI subunits at time 0. Each curve represents the mean of the following number of assays: WT = 29, Delta 1-94 = 2, E101A = 3, and I98A = 2.
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Inhibition of C Subunit by cAMP-saturated RI Subunits

In the absence of cAMP, PKA is maintained as an inactive holoenzyme complex R2C2 with an affinity of ~0.2 nM between R and C subunit (39). cAMP binding to the R2 in the R2C2 complex decreases its affinity for C subunit by 4-5 orders of magnitude (40, 41, 42), resulting in dissociation of the R and C subunits and subsequent activation of the kinase (1) (Reaction R1). A ternary complex involving the R and C subunits and cAMP (exemplified as R2cAMP4C2) is formed as an intermediate during both activation and inactivation of PKA (40, 43, 44, 45). Other types of ternary complexes (e.g. R2cAMP2C) are also formed, but are not shown in this simplified Reaction R1 (46, 47).
<UP>R</UP><SUB>2</SUB><UP>C</UP><SUB>2</SUB>+4<UP>cAMP</UP> &rlhar2; <UP>R</UP><SUB>2</SUB><UP>cAMP</UP><SUB>4</SUB><UP>C</UP><SUB>2</SUB> &rlhar2; <UP>R</UP><SUB>2</SUB><UP>cAMP</UP><SUB>4</SUB>+2<UP>C</UP>
(<UP>inactive</UP>)<UP>                            </UP>(<UP>active</UP>)
<UP>R<SC>eaction</SC></UP> 1
The forward reaction is exemplified in Fig. 3A, which shows that holoenzyme was activated by cAMP in a concentration-dependent manner. Activation was evident when the cAMP concentration exceeded 2 nM, and holoenzyme was fully activated at ~75-100 nM cAMP. Half-maximal activation of WT holoenzyme (Ka) was 21 nM as determined by a Hill plot.


Fig. 3.

Effect of cAMP and cIMP on the interaction of RI subunit and C subunit. A, holoenzyme was prepared from cIMP-saturated WT RI subunit and native C subunit as described under "Experimental Procedures." 1 nM WT holoenzyme was incubated with varying concentrations of cAMP in the presence of 20 mM Tris, pH 7.4, 20 mM magnesium acetate, 0.1 mM ATP, 0.5 mg/ml BSA, 0.1 mM isobutylmethylxanthine, and 81 µM Kemptide substrate for 30 min at 30 °C. The reaction was terminated and the amount of Kemptide phosphorylation was determined. Ka was calculated by a Hill plot. B, C subunit (21 pM) was preincubated with increasing concentrations of cAMP-saturated native type Ialpha R subunit in the presence of 20 mM Tris, pH 7.4, 20 mM magnesium acetate, 0.1 mM ATP, 0.5 mg/ml BSA, 0.1 mM isobutylmethylxanthine for 19 min at 30 °C. The reactions were initiated by the addition of 81 µM Kemptide substrate, 0.071 mM ATP, and [gamma -32P]ATP (1000 cpm/pmol), and the incubation proceeded for an additional 105 min at 30 °C. The reactions were terminated, and the amount of Kemptide phosphorylation was determined. The same results were obtained using two independent preparations of native RI subunit from bovine lung and rabbit skeletal muscle. C, C subunit (21 pM) was preincubated with varying concentrations of cIMP-saturated WT (bullet ) or native (square ) RI subunit for 22 min at 30 °C under the same conditions as described above. The reactions were initiated by the addition of Kemptide substrate, and the incubation proceeded for an additional 140 min at 30 °C. The reactions were terminated, and the amount of Kemptide phosphorylation was determined. IC50 values were calculated from Hill plots.


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The reverse of Reaction R1, inhibition of C subunit by formation of the R2C2 complex, is facilitated in vivo by the action of cyclic nucleotide phosphodiesterases which degrade cAMP (48, 49). Because of the high affinity with which cAMP is bound to R subunit in vitro, and because of its dissociation-reassociation characteristics, cAMP remains associated with R subunit throughout extensive molecular sieve chromatography, ion exchange chromatography2 and dialysis (3). In fact, a significant portion (~20%) of intracellular R subunit contains bound cAMP even in the basal state (50). Because much of the in vitro characterization of native R subunit has utilized the cAMP-bound form of the protein, the reverse reaction was initially examined by incubating cAMP-saturated native RI subunit (R2cAMP4) with the purified C subunit (Fig. 3B). C subunit was inhibited by RI subunit in a concentration-dependent manner until the concentration of bound cAMP added to the reaction exceeded 2 nM, at which point the inhibition by RI subunit was reversed, presumably because the equilibrium of Reaction R1 shifted back toward the right. These results are consistent with the results presented in Fig. 3A, which showed that the inactive holoenzyme complex predominated until the added cAMP reached 2 nM, whereupon the equilibrium shifted to the right, toward cAMP saturation of the R subunit and dissociation of the holoenzyme complex. The concentration of cAMP in the reaction at the point at which the equilibrium shifted to the right (2 nM) correlated well with the mean equilibrium binding constant (KD) of cAMP for RI subunit (~1.4 nM) (51). When the concentration of cAMP in the reaction exceeded the KD (Fig. 3, A and B), the equilibrium of cAMP binding to RI subunit shifted toward formation of the R2cAMP4 complex. These considerations demonstrate the technical difficulties involved in quantitating the potency with which R subunit inhibits C subunit kinase activity when using R subunit maintained in the physiological, cAMP-bound form. Therefore, attempts were made to remove cAMP from RI subunit either by exchanging it for a lower affinity cyclic nucleotide or by urea denaturation as described in the following sections.

Inhibition of C Subunit by cIMP-Saturated RI Subunits

In theory, a quantitative measure of C subunit inhibition would be possible if the RI subunit preparation were bound with a low affinity cyclic nucleotide instead of with cAMP. Thus, cAMP was replaced with cIMP, which exhibits an affinity that is ~20-fold lower than that of cAMP for the type Ialpha RI subunit cyclic nucleotide binding sites (52). Since expression of RI subunit in E. coli produces the cAMP-saturated form of the enzyme, the exchange of cIMP for cAMP was performed during the preparation of bacterial extracts (see "Experimental Procedures"). This facilitated subsequent purification of RI subunits using N6-H2N-(CH2)2-cAMP Sepharose affinity resin. RI subunit was eluted from the affinity resin with cIMP, and the excess cIMP was separated from RI subunit by molecular sieve chromatography, producing cIMP-saturated recombinant RI subunits (~2 mol of cIMP/mol of RI subunit monomer) (see "Experimental Procedures"). The cIMP-saturated form of type Ialpha native RI subunit (from bovine lung) was also prepared. In this case, the purified preparation of cAMP-saturated native RI subunit was incubated with cIMP to allow exchange, followed by molecular sieve chromatography (see "Experimental Procedures"). The retention of cIMP by the RI subunits was unexpected since removing bound cAMP by exchanging for a lower affinity cyclic nucleotide (e.g. cGMP), followed by a procedure such as ion exchange chromatography (30), molecular sieve chromatography (41) or dialysis (53) to remove the lower affinity cyclic nucleotide, is a strategy commonly employed by investigators, including our own laboratory (30).

Unlike the cAMP-saturated RI subunit, the cIMP-saturated WT and native RI subunits completely inhibited C subunit in a concentration-dependent manner; both WT and native proteins exhibited similar IC50 values, 0.36 nM and 0.40 nM, respectively (Fig. 3C). The IC50 values were determined by Hill plots and represent the concentration of RI subunit required to inhibit C subunit kinase activity by 50%. The cIMP concentrations introduced into these assays via the R2cIMP4 complex (50 nM) were calculated to be well below the Ka of cIMP for WT holoenzyme, which was ~750 nM as determined by a Hill plot (data not shown). The inhibition curves for selected cIMP-saturated RI subunit mutants are illustrated in Fig. 4; complete or nearly complete inhibition of C subunit was achieved with each of the RI subunit mutants.


Fig. 4. Inhibition of C subunit activity by cIMP-saturated WT and mutant RI subunits. C subunit (21 pM) was preincubated for 15-30 min at 30 °C with varying concentrations of either WT (bullet ), Delta 1-94* (triangle ), Delta 1-93.R95A (square ), Delta 1-95 (black-down-triangle ), I98Q (open circle ), I98A (down-triangle), or I98G (black-square) RI subunits as described in Fig. 3B. The reactions were initiated by the addition of Kemptide substrate, and the incubation proceeded for an additional 60-120 min at 30 °C. The reactions were terminated, and the amount of Kemptide phosphorylation was determined. 100% catalytic activity was determined to be ~6 pmol/min/ng of C in the absence of RI subunit and cAMP. Each curve represents the mean of >= 4 assays for the Arg92-95 mutants, 2 assays for the Ile98 mutants, and 31 assays for WT RI subunit.
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Residues 1-91, Arg92-95 Tetrad

Table III summarizes the IC50 values calculated for the cIMP-saturated truncated RI subunit mutants (RcIMP2) that were used to examine the contribution of the amino-terminal segment of RI subunit, and each residue of the Arg92-95 tetrad. The cIMP-saturated truncated RI subunits are represented as RcIMP2 instead of R2cIMP4 since the dimerization domain has been eliminated by truncating the protein. Truncation of the amino-terminal segment of RI subunit through Gly91 (Delta 1-91) did not impair the inhibitory potency of this mutant compared to that of WT. In the crystal structure of the C subunit·PKI-(5-24) complex, the PKI P-6 Arg forms a hydrogen bond with Glu203, the P-3 Arg forms hydrogen bonds with Glu127 and Glu331 and the P-2 Arg hydrogen bonds with Glu230 and Glu170 (4). Since the RI subunit pseudosubstrate region contains four arginine residues (P-5,P-4,P-3,P-2), it is possible that each Arg could interact with one or more of these Glu residues in C subunit, and thus contribute to the high affinity interaction with C subunit. However, truncation through Arg92 (P-5) or Arg93 (P-4) did not detectably impair the inhibitory potency of either of these mutants (Delta 1-92 and Delta 1-93). The importance of the P-3 residue (Arg94) was investigated using two RI subunit mutants (Delta 1-94 and Delta 1-94*) which were truncated through Arg94. The inhibitory potency of Delta 1-94 was ~17000-fold lower than that of WT, while the Delta 1-94* mutant, which lacks the initiator Met, was ~21000-fold less potent than WT. Thus, the initiator Met was not detrimental to the interaction of Delta 1-94 with C subunit. The requirement for a P-2 Arg (Arg95) was examined using the truncated Delta 1-93.R95A mutant. Substitution of Arg95 with Ala within Delta 1-93.R95A profoundly reduced the inhibitory potency of this truncated mutant (~31000-fold). Removal of all of the amino-terminal Arg residues, Delta 1-95, reduced the inhibitory potency of RI subunit for C subunit by ~54000-fold. This was an additive decrease in effectiveness compared to that observed when Arg94 or Arg95 were deleted or mutated individually.

Table III.

Contribution of the amino-terminal segment and each residue of the Arg92-95 tetrad of the RI subunit autoinhibitory domain

IC50 is defined as the concentration of RI subunit required to inhibit 50% of Kemptide phosphorylation by C subunit. The concentration of C subunit was 0.02 nM.
Regulatory subunit IC50
cIMP-saturated Cyclic nucleotide-free

nM nM
    -5-4-3-2     P
     92 93 94 95
Wild type . . . R  R  R  R  G  A  I  S . . . 0.36 0.72
 Delta 1-91    M  R  R  R  R  G  A  I  S . . . 0.27 0.25
 Delta 1-92       M  R  R  R  G  A  I  S . . . 0.15 NDa
 Delta 1-93          M  R  R  G  A  I  S . . . 0.14 ND a
 Delta 1-94             M  R  G  A  I  S . . . 6100 31
 Delta 1-94*                R  G  A  I  S . . . 7400 7
 Delta 1-93.R95A          M  R  A  G  A  I  S . . . 11,200 44
 Delta 1-95                   G  A  I  S . . . 19,500 537
 Delta 1-92.R95A       M  R  R  A  G  A  I  S . . . 9000 37

a  ND, not determined.

These results demonstrated that both Arg94 and Arg95 are critical determinants for potent inhibition of C subunit, but that neither Arg92 nor Arg93 is detectably involved. The results also indicated that the amino-terminal 91 residues are not involved in autoinhibition, despite the established role of the PKI residues which correspond to residues 79-87 in RI subunit (4, 18). The results are in agreement with similar studies in full-length RI (8) and RII subunits (7), and with PKI peptide analog studies (10, 11), in which the P-3 and P-2 Arg residues were shown to be required for C subunit inhibition. The data presented here suggested that the requirement for a basic residue is approximately the same for the P-3 or P-2 position, with perhaps a slightly greater requirement at the P-2 position. This conclusion is consistent with findings showing that a basic residue is of slightly greater significance at the P-2 position than at the P-3 position of the peptide substrate Kemptide (6), but contrary to findings using PKI peptide analogs (11).

Although neither Arg92 (P-5) nor Arg93 (P-4) is required for potent inhibition of C subunit, it is possible that these highly conserved residues make minor contacts with C subunit that would be more evident in the absence of the P-3 or P-2 inhibitory residues (Arg94 or Arg95). It is also plausible that some flexibility exists in the interaction between pseudosubstrate residues and C subunit that would permit a shift in the spatial location so that the P-4 and P-3 Arg residues could serve the same role as the P-3 and P-2 Arg residues. The P-4-P-3 arrangement of basic residues is seen in naturally occurring PKA substrates such as phosphorylase kinase (beta  subunit) (54) and glycogen synthase (site 1b) (55). These substrates have a P-4 Lys and a P-3 Arg residue, but do not possess an Arg residue at the P-2 position. The Delta 1-92.R95A mutant, in which an Ala residue was substituted for the P-2 Arg, was used to examine the interaction between the RI subunit P-4-P-3 Arg residues and the active site of C subunit. The Delta 1-92.R95A mutant was an extremely poor inhibitor of C subunit, comparable to Delta 1-93.R95A (Table III), clearly demonstrating that the RI and C subunit interaction is not flexible enough to permit spatial repositioning of the Arg residues by even a single residue.

Effects of Substitutions at Ile98

The P+1 position is highly conserved as Ile or Val in the autoinhibitory domains of all species of PKA, PKG and in PKI. Substitution of Ile22 (P+1) with Gly in PKI peptide analogs reduced the inhibitory potency of the peptide 150-fold (18). In the crystal structure of the C subunit·PKI-(5-24) complex, Ile22 (P+1) is situated in a hydrophobic pocket on C subunit comprising Leu198, Pro202, and Leu205, indicating that a P+1 hydrophobic residue is essential to a high affinity interaction between PKI and C subunit (4). Since a role in C subunit inhibition has not been previously demonstrated for the R subunit P+1 residue, mutants of the RI subunit P+1 residue (I98V, I98A, I98G, and I98Q) were used to investigate the importance of aliphatic side chain length and steric constraints on the ability of RI subunit to potently inhibit C subunit. The IC50 values for the cIMP-saturated, full-length RI subunits (R2cIMP4) mutated at the P+1 position (Ile98) are presented in Table IV. Conservative replacement of Ile98 with Val (I98V) in full-length RI subunit did not impair the IC50 value of I98V compared to that of WT. When the aliphatic side chain length at the P+1 position was reduced the IC50 values were increased by ~5300-fold and 14000-fold for I98A and I98G, respectively, compared to that of WT. When the side chain length was increased, as in the I98Q mutant, the IC50 value was ~4200-fold greater than that of WT. The loss of inhibitory potency was greatest for I98G, but substitution of Gly at the P+1 position could cause a conformational change that alters the position of other critical residues in the pseudosubstrate region. The increased length of the Gln side chain and its hydrophilic nature are the most probable factors causing the reduced inhibitory potency for the I98Q mutant. The results suggested that the RI subunit P+1 hydrophobic residue is quite important for a potent interaction with C subunit, and that the length of the aliphatic side chain is crucial. Strict steric constraints at the P+1 site are also indicated by Kemptide analog studies. Substitution of the P+1 residue in Kemptide with Pro (LRRASPG) resulted in a significant reduction in substrate capacity (56), but introduction of Nalpha -methyl Leu in place of Leu at the P+1 position resulted in only a moderate loss of substrate capacity (57).

Table IV.

Contribution of Ile98 and Glu101 of the RI subunit autoinhibitory domain

IC50 is defined as the concentration of RI subunit required to inhibit 50% of Kemptide phosphorylation by C subunit. The concentration of C subunit was 0.02 nM.
Regulatory subunit IC50
cIMP-saturated Cyclic nucleotide-free

nM nM
Wild type 0.36 0.72
I98V 0.16
I98A 1900 2.9
I98G 5000
I98Q 1500
E101Q 0.18
E101A 0.59

Effects of Substitutions at Glu101

Glu101, at the P+4 position in RI subunit, is invariant in the regulatory domain of all known sequences of PKA and PKG. To examine the putative electrostatic interaction between Glu101 and Lys residues of C subunit (see Introduction), the acidic charge of Glu101 was eliminated by substitution of Glu101 with Gln (E101Q) or Ala (E101A) in full-length RI subunit. The IC50 values for the cIMP-saturated Glu101 mutants (Table IV) were similar to that of WT RI subunit. Despite the highly conserved nature of the P+4 Glu and its proximity to the pseudosubstrate site, Glu101 does not appear to play an important role in C subunit inhibition, although it could serve some other function.

Influence of cIMP on Inhibition of C Subunit by RI Subunits

Although complete or nearly complete inhibition of C subunit was achieved using each of the cIMP-saturated RI subunit mutants, concentrations as high as 20-50 µM were required by most RI subunit mutants to inhibit C subunit completely, compared to 0.01 µM for WT (Fig. 4). C subunit was inhibited in those reactions containing 50 µM mutant RI subunit (100 µM cIMP), despite the fact that the cIMP concentration in the reaction was calculated to exceed the Ka by >100-fold. The results of studies with cAMP-saturated RI subunit (Fig. 3B) would have predicted less C subunit inhibition as the concentration of the R2cIMP4 or RcIMP2 complex approached the Ka of cIMP for holoenzyme (750 nM). When using cIMP-saturated WT RI subunit, cIMP would not be expected to interfere with holoenzyme formation since the assays are performed using very dilute RI subunit (25 nM) where the concentration of cIMP (50 nM) would be far below the Ka (750 nM). It is suggested that the large rightward shift exhibited in the inhibition curves by certain RI subunit mutants must reflect the following considerations. 1) The decreased affinity between the pseudosubstrate site of the mutant RI subunit and the catalytic site of C subunit causes a rightward shift in the equilibrium of Reaction R1 (toward R2cIMP4 (or RcIMP2) and active C subunit); 2) the necessity of using mutant R2cIMP4 (or RcIMP2) at concentrations that vastly exceed the mean equilibrium binding constant of cIMP for RI subunit (~28 nM) (51, 52) causes a dramatic shift in the equilibrium of cIMP binding to RI subunit (toward formation of the R2cIMP4 (or RcIMP2) complex). The second consideration causes a shift of the equilibrium of Reaction R1 even further to the right than that caused by mutation of RI subunit alone, thereby resulting in the high IC50 values determined for the cIMP-saturated RI subunit mutants. This is supported by the observation that the equilibrium of Reaction R1 was progressively shifted to the right when cIMP-saturated Delta 1-94* (9.8 µM RI subunit:19.6 µM cIMP) and C subunit (21 pM) were incubated in the presence of increasing concentrations of exogenous cIMP (up to 20 µM) (data not shown).

Inhibition of C subunit by R2cAMP4 or R2cIMP4 in the Presence of Excess Cyclic Nucleotide

Under physiological conditions the PKA holoenzyme complex (R2C2) is inactive (see Reaction R1). The PKA ternary complex (R2cAMP4C2) is also largely inactive (IC50 ~ 15 µM for type I enzyme) when the reverse reaction of Reaction R1 is measured using pharmacological concentrations of R2cAMP4 and a vast excess of cAMP, such that the RI subunit remains in the R2cAMP4 form (40). Since the IC50 values obtained for several cIMP-saturated RI subunit mutants approximate the IC50 value obtained for this native ternary complex, it was necessary to determine if these values might reflect the inhibited mutant ternary complexes instead of the R2C2 (full-length RI subunit mutants) or RC (truncated RI subunit mutants) holoenzyme complexes.

The IC50 of the WT ternary complex was measured by incubating C subunit with either WT-R2cIMP4 or WT-R2cAMP4 in the presence of a large excess (500 µM) of cIMP or cAMP (Fig. 5). The WT-R2cIMP4C2 and WT-R2cAMP4C2 ternary complexes exhibited IC50 values similar to each other (IC50 ~10-12 µM), and to that published for the native ternary complex (40). These values were ~4-5 orders of magnitude higher than the IC50 value measured for WT holoenzyme (R2C2) (0.36 nM; Table III). Inhibition of C subunit by a truncated mutant, cIMP-saturated Delta 1-93.R95A RI subunit, was also examined in the presence of excess cIMP to measure inhibition of the Delta 1-93.R95A ternary complex (RcIMP2C). Only 20% of C subunit activity was inhibited by 50 µM cIMP-saturated Delta 1-93.R95A when assayed in the presence of 500 µM cIMP (Fig. 5), whereas cIMP-saturated Delta 1-93.R95A exhibited an IC50 value of 11.2 µM when assayed in the absence of excess cIMP (Table III). Similarly, only 16% of C subunit activity was inhibited by 5 µM cIMP-saturated I98A when assayed in the presence of 500 µM cIMP (data not shown), whereas cIMP-saturated I98A exhibited an IC50 value of 1.9 µM when assayed in the absence of excess cIMP (Table IV). Assuming that there is no RC or R2C2 complex present when 500 µM cIMP is included in the reaction, these results suggest that the IC50 values obtained for the cIMP-saturated mutant RI subunits in the absence of excess cyclic nucleotide are indeed a measure of the inhibited RC or R2C2 holoenzyme complexes, and do not reflect the inhibited ternary complex. The results also indicate that the interactions between C subunit and WT- or mutant-R2cIMP4 or mutant-RcIMP2 are specific interactions and not due simply to the addition of a high concentration of RI subunit; the interaction of C subunit with either Delta 1-93.R95A-RcIMP2 or I98A-R2cIMP4 was profoundly reduced compared to that interaction with WT-R2cIMP4 when measured in the absence or presence of a large excess of cIMP. This also suggests that the same crucial residues interact with C subunit regardless of whether the measured inhibition is that of the ternary complex or of the holoenzyme complex. It follows from the above considerations that since a significant portion of R subunit is bound with cAMP under physiological conditions (50), a natural biological mutation of one of the pseudosubstrate Arg residues or the Ile residue would form constitutively active PKA.


Fig. 5. Effect of excess cyclic nucleotide on the inhibition of C subunit by RI subunit. C subunit (21 pM) was preincubated with cIMP-saturated WT RI subunit in the presence of 500 µM cIMP (open circle ) or 500 µM cAMP (down-triangle), or C subunit was preincubated with Delta 1-93.R95A (square ) in the presence of 500 µM cIMP at 30 °C for 19 min under the same conditions as described in Fig. 3B. The reactions were initiated by the addition of Kemptide substrate, and the incubations proceeded for an additional 120 min at 30 °C. The reactions were terminated, and the amount of Kemptide phosphorylation was determined.
[View Larger Version of this Image (23K GIF file)]


Inhibition of C Subunit by Cyclic Nucleotide-free RI Subunit Mutants

For the sake of comparison, it seemed prudent to examine the effect of RI subunit mutation(s) on inhibitory potency using the cyclic nucleotide-free form of RI subunit, whereby the interaction of R and C subunit is measured directly without the interference of cAMP or cIMP (Reaction R2).
<UP>R<SUB>2</SUB>C<SUB>2</SUB> &rlhar2; R<SUB>2</SUB> + 2C</UP>
(<UP>inactive</UP>)<UP>  </UP>(<UP>active</UP>)
<SC><UP>Reaction R2</UP></SC>
Investigators have previously used R subunit that was made cyclic nucleotide-free by urea denaturation (followed by renaturation) to examine the effect of R subunit mutation(s) on the inhibition of kinase activity (8, 58). Since the renatured R subunit has been shown to have the same affinity for C subunit as that of non-denatured R subunit (59), this method was selected for the preparation of cyclic nucleotide-free RI subunit in the present study. R subunits that have been treated with urea, however, have an increased rate of cyclic nucleotide-dissociation from the binding sites (i.e. decreased affinity for cyclic nucleotide) (60). Since the effect on cyclic nucleotide dissociation was found to be directly related to the length of time that the R subunits were exposed to urea (60), the exposure of the RI subunits to urea was temporally minimized as described under "Experimental Procedures."

The IC50 values for the cyclic nucleotide-free RI subunits were determined by Hill plots and are summarized in Tables III and IV. Urea treatment of WT RI subunit had minimal effect on the potency of inhibition of C subunit activity compared to that of untreated WT RI subunit (0.72 nM and 0.36 nM, respectively). The IC50 values obtained for the cyclic nucleotide-free RI subunit mutants were considerably lower than those for the cIMP-saturated mutant RI subunits, but within the same range as those previously reported for urea-treated R subunit mutants (8, 58). Deletion of the first 91 amino acid residues (Delta 1-91) of RI subunit did not impair the inhibitory potency of this mutant toward C subunit kinase activity. Compared to WT, the IC50 values for the Delta 1-94 and Delta 1-94* mutants were 43- and 10-fold greater, respectively, while those for Delta 1-93.R95A and Delta 1-92.R95A were 61- and 51-fold greater. When the P-3 and P-2 Arg residues were both deleted (Delta 1-95), the IC50 value increased by 746-fold over that of WT, which was considerably more than additive when compared with the IC50 of mutants in which these residues were deleted or mutated individually. These results suggested that Arg94 and Arg95 interact in a synergistic manner with the active site on C subunit. The inhibitory potency of a single cyclic nucleotide-free Ile98 mutant, I98A, was also measured and was 4-fold less potent toward C subunit than was WT.

Four different forms of RI subunit (cAMP-bound, cIMP-bound, cIMP-bound plus excess cyclic nucleotide, and cyclic nucleotide-free) have been used in the present study. Each form provides useful and sometimes distinct information about R subunit/C subunit interactions. The cAMP-saturated form of R subunit provides insights into cellular interactions, but presents a technical impediment in quantitatively assessing the interaction between R and C subunit. However, since a significant portion of R subunit is bound with cAMP under physiological conditions (50), the results of mutating the RI subunit reveal that a natural mutation of one of the pseudosubstrate Arg residues or the Ile residue would form constitutively active PKA in body tissues. The RI subunit that is saturated with the lower affinity cyclic nucleotide, cIMP, can be better used to determine the relative contribution of particular residues toward the inhibition of C subunit. However, a true quantitative measurement of this interaction is hindered even with this RI subunit since mutation of critical pseudosubstrate site residues reduces the R-C subunit affinity and necessitates the use of RcIMP2 or R2cIMP4 concentrations that far exceed the cIMP KD for RI subunit. This causes an exaggerated increase in the IC50 value. However, because the IC50 values are exaggerated, this form of R subunit is ideal for detecting small differences in the contribution of particular residues toward C subunit inhibition that might otherwise have been discounted. When assayed in the presence of excess cyclic nucleotide, the cIMP-saturated RI subunits are specific, but extremely weak inhibitors of C subunit. It is suggested that a more quantitative assessment of the R-C interaction is obtained using RI subunit that is made cyclic nucleotide-free by urea denaturation. Furthermore, considerably less of this RI subunit is required for inhibition than is required when using cIMP-saturated RI subunit, thus facilitating most experimental protocols. Although evidence suggests that the R and C subunit interaction is not impaired as a result of urea treatment of the RI subunit, this possibility cannot be ruled out since urea denaturation alters the affinity of R subunit for cyclic nucleotide (60).

In summary, the results of this study establish that both the P-2 and P-3 Arg, and, to a lesser extent, the P+1 Ile residues are critical for potent inhibition of C subunit by RI subunit. Neither the P-4 Arg, P-5 Arg, nor the P+4 Glu residue has a detectable effect. The results indicate that the interactions between the pseudosubstrate site Arg residues and the catalytic site acidic residues are not flexible enough to tolerate spatial re-positioning of the two required Arg residues from the P-2-P-3 to the P-3-P-4 positions.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant DK 40029. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: 702 Light Hall, Dept. of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615. Tel.: 615-322-4384; Fax: 615-343-0490.
1    The abbreviations used are: PKA, cAMP-dependent protein kinase; C subunit, catalytic subunit; R subunit, regulatory subunit; RI subunit, type Ialpha regulatory subunit; RII subunit, type II regulatory subunit; PKG, cGMP-dependent protein kinase; R2C2, holoenzyme complex of cAMP-dependent protein kinase; PKI, protein kinase inhibitor; cG-BPDE, cGMP-binding, cGMP-specific phosphodiesterase; WT, wild type; bp, base pair(s); BSA, bovine serum albumin; cIMP, 3',5' cyclic inosine monophosphate; PAGE, polyacrylamide gel electrophoresis.
2    C. E. Poteet-Smith, J. B. Shabb, S. H. Francis, and J. D. Corbin, unpublished observations.

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