(Received for publication, May 30, 1996, and in revised form, September 12, 1996)
From the 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
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
I regulatory subunit (RI subunit) contains a similar sequence,
Arg92-Arg-Arg-Arg-Gly-
-Ile-Ser-Ala-Glu.
The italicized amino acids form a putative pseudosubstrate site
(Ser is replaced with
), 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 (
1-92) or Arg93
(
1-93) had no detectable effect on inhibition of C subunit. Truncation through Arg94 (
1-94), or point mutation of
Arg95 within truncated mutants (
1-93.R95A or
1-92.R95A), caused a dramatic reduction in inhibitory potency.
Truncation through Arg95 (
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 I
R subunit.
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--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
P3 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-
-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 P3 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 (
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 P5,P
4,
P
3, P
2, P+1 and P+4
residues for autoinhibition of type I
PKA.
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. [
-32P]dATP and [2,8-3H]cAMP
were from Amersham, [
-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).
A T7 RNA
polymerase/promoter system, pT7-7, was used for bacterial
overexpression of the type I 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).
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Functional pT7R expression vectors encoding truncated RI subunit
cDNA (1-91,
1-92,
1-93,
1-94,
1-95,
1-93.R94A,
1-92.R95A, and
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 ExpressionE. 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.
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 -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 SubunitNative
type I 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 I
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 I
and II
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.
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 Subunits287 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 (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.
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 AssaysThe 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 SubunitsImmediately 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
[-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.
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,
[-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.
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 [-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.
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.
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.
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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, 1-94*) or Gly (
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
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
1-93.R94A differed from
1-94 only in the
absence of the initiator Met residue it was named
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:
1-94, E101A, and I98A.
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).
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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 I 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
[
-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 (
) or native (
) 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.
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 I 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 I
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.
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 (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 (
1-92 and
1-93). The importance of the
P
3 residue (Arg94) was investigated using two
RI subunit mutants (
1-94 and
1-94*) which were truncated
through Arg94. The inhibitory potency of
1-94 was
~17000-fold lower than that of WT, while the
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
1-94
with C subunit. The requirement for a P
2 Arg
(Arg95) was examined using the truncated
1-93.R95A
mutant. Substitution of Arg95 with Ala within
1-93.R95A
profoundly reduced the inhibitory potency of this truncated mutant
(~31000-fold). Removal of all of the amino-terminal Arg residues,
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.
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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 P3 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 (P5) 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 (
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
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
1-92.R95A mutant was an extremely poor inhibitor of C
subunit, comparable to
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.
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
N-methyl Leu in place of Leu at the
P+1 position resulted in only a moderate loss of substrate
capacity (57).
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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 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
1-93.R95A RI subunit, was also examined in the presence of excess
cIMP to measure inhibition of the
1-93.R95A ternary complex
(RcIMP2C). Only 20% of C subunit activity was inhibited by
50 µM cIMP-saturated
1-93.R95A when assayed in the presence of 500 µM cIMP (Fig. 5), whereas cIMP-saturated
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
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.
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).
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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 (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
1-94 and
1-94* mutants were 43- and 10-fold greater,
respectively, while those for
1-93.R95A and
1-92.R95A were 61- and 51-fold greater. When the P
3 and P
2 Arg
residues were both deleted (
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
P2 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.