From the § Howard Hughes Medical Institute,
Department of Chemistry and Biochemistry, School of
Medicine, University of California, San Diego,
La Jolla, California 92093-0654
Received for publication, July 19, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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Limited trypsin digestion of type I
cAMP-dependent protein kinase holoenzyme results in a
proteolytic-resistant The regulatory (R)1
subunits of cAMP-dependent protein kinase (PKA) are
multifunctional proteins that control in a variety of ways the
physiological functions of this ubiquitous protein kinase (1, 2). While
the R subunits have long been recognized as the primary receptor for
cAMP and the major physiological inhibitor for the catalytic (C)
subunit in eukaryotic cells, it is now apparent that these highly
modular proteins play other important roles as well (3). The R subunits
of PKA have a well defined domain structure that consists of a
dimerization domain at the NH2 terminus followed by an
autoinhibitor site and two-tandem cAMP-binding domains. While the
portion of the R subunit COOH-terminal to the inhibition site is
responsible for high affinity binding of the C subunit and cAMP, the
remaining NH2 terminus serves as an adaptor for binding to
kinase anchoring proteins (3) and other adaptor proteins, such as the
SH3-containing Grb2 (4). This segment or region of the R subunit is
responsible for in vivo subcellular localization and
targeting of PKA.
There are two general classes of PKA, designated as type I and type II,
due exclusively to differences in the R subunits, RI and RII (5-7).
Four different regulatory subunits, RI While both RI and RII contain two tandem and highly conserved
cAMP-binding domains at the carboxyl terminus (25), RI and RII differ
significantly at their amino terminus, especially at a proteolytically
sensitive hinge region that binds to the peptide recognition site of
the C subunit. The hinge region of RII subunits contains a serine at
the phosphorylation (P) site that can be autophosphorylated by the C
subunit (26); whereas RI subunits contain a pseudo phosphorylation site
(Arg-Arg-Gly-Ala-Ile) and type I holoenzyme has an essential high
affinity-binding site for MgATP (27, 28). When an autophosphorylation
site was introduced into the RI subunit by replacing the alanine with a
serine, the mutant RI subunit became a good substrate for the C
subunit. However, the formation of holoenzyme was no longer facilitated
by MgATP, and the affinity of the mutant holoenzyme for cAMP was
independent of MgATP (29). In contrast, elimination of the
autophosphorylation site by point mutation causes the RII subunit to
lose its ability to revert transformed fibroblasts (30).
The amino acid sequence flanking the phosphorylation site of protein
kinase substrates has been shown to be important for substrate
specificity. Most PKA phosphorylation sites contain Arg at the P-3 and
P-2 sites and a hydrophobic amino acid at the P+1 position (31). A
close examination of the primary sequence immediately
NH2-terminal to the inhibition site of the R subunits reveals that there is significant sequence diversity between RI and RII
in this region (Fig. 1). This suggests
that recognition of RI and RII by the C subunit at this region could be
different. We tested this hypothesis by introducing specific mutations
into the NH2-terminal proximal side, an adjacent region
that interacts with the sequence NH2-terminal to the
inhibition site of R subunit, of the active site cleft of the C
subunit. Interaction between mutant C subunits and RI Materials--
All chemicals used are either reagent grade or
molecular biology grade. Kemptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly, was
synthesized at the Peptide and Oligonucleotide Facility at the
University of California, San Diego, and further purified by
reverse-phase preparative high performance liquid chromatography
before use. The concentration of the peptide substrate was determined
by turnover with the catalytic subunit under the conditions of limiting
peptide. Oligonucleotides were synthesized with a DNA synthesizer
(Applied Biosystem Inc., model 380B). Pyruvate kinase from rabbit
muscle and lactate dehydrogenase from bovine heart were from Sigma.
Protein Preparation--
Mutations R133A and D328A were
introduced into the C subunit by the Kunkel method as described
previously (32). The mutant proteins C(R133A) and C(D328A) were
expressed in Escherichia coli BL21(DE3) using the pLWS-3
vector (33). The recombinant wild-type and mutant C subunits were
purified by phosphocellulose chromatography and FPLC Mono S column
(34). Isozyme II was pooled and used for all experiments.
Wild type RI Holoenzyme Formation--
PKA holoenzymes were reconstituted
from individually purified C and R subunits by mixing freshly isolated
R and C subunits in a molar ration of 1:1.2 and dialyzed against buffer
A (20 mM potassium phosphate, 100 mM KCl, 2 mM dithiothreitol, 0.1 mM ATP, 1 mM
MgCl2, and 5% glycerol, pH 6.5) extensively at 4 °C to
remove excess cAMP. The dialyzed sample was assayed for PKA activity first in the absence of cAMP and then in the presence of 100 µM cAMP to confirm the formation of the PKA holoenzyme.
PKA holoenzyme complex was further purified on a Superdex 200 FPLC gel
filtration column to remove excess C subunit.
Trypsin Digestion of the Holoenzyme--
A typical reaction
mixture (12 µl) contained 20 µg of RI Sequencing of Cleavage Products--
After electrophoresis,
proteins were transblotted onto a polyvinylidene difluoride (0.2 µm,
Bio-Rad) membrane. The electroblotting was performed in 10 mM Caps, 10% methanol (pH 11) buffer at 200 mA constant
current for 1 h. After rinsing with water (3 times) and methanol,
the membrane was stained with 0.1% Coomassie R-250 in 1% acetic
acid, 40% methanol for 2 min. The membrane was destained in
50% methanol and patted dry after rinsing with water. The protein bands of interest were excised from the membrane with a razor blade and
subjected to NH2-terminal protein sequencing.
Protein Kinase Activity Assay--
The enzymatic activity of the
C subunit was measured spectrophotometrically with a coupled enzyme
assay (38). In this assay, the formation of the ADP is coupled to the
pyruvate kinase and lactate dehydrogenase reactions. The reaction rate
is determined by following the decrease in absorbance at 340 nm.
Reactions were pre-equilibrated at room temperature and initiated by
adding the peptide substrate. The Michaelis-Menten parameters for ATP
and Kemptide were determined by fixing one substrate at near saturating concentration while varying the concentration of the other
substrate. Inhibition of the catalytic subunit by R or PKI was
assayed by mixing varying amounts of inhibitor with 20 nM C
subunit and subsequently determining the decrease of phosphotransfer activity.
A radioisotopic method (39) was also used to measure the activity of
the C subunit at low concentration, typically around 100 pM. With this assay, the inhibition constants of R and PKI to the catalytic subunit can be accurately determined. The kinase reaction mixture (50 µl) contained 50 mM Mops (pH 7.0),
10 mM MgCl2, 0.25 mg/ml bovine serum albumin,
0.1 mM peptide, and 0.1 mM ATP at 100 cpm/pmol,
100 pM catalytic subunit, and varying amount of inhibitor,
RI Surface Plasmon Resonance--
Surface plasmon resonance was
used to measure binding between wild-type/mutant C subunits and R
subunits, RI
Kinetic constants of binding were obtained using the BIAcore
pseudo-first order rate equation,
Data Analysis--
The Michaelis constant, Km
and maximal velocity, Vmax were determined from
plots of initial velocity, v versus substrate concentration, [S] according to,
The apparent inhibition constant,
Ki,app, was obtained directly
fitting the inhibition curves of C subunits by R subunits or PKI
according to,
Since both PKI and R subunits act as competitive inhibitors of the
catalytic subunit when Kemptide is used as a substrate, the inhibition
constant, Ka, can be further calculated from the
Ki,app using the Morrison equation,
Proteolytic Digestion of Type I Holoenzyme Complex--
Trypsin
digestion of type I holoenzyme resulted in two stable protein fragments
that corresponded to Isoform Specific Differences in the P-11 to P-4 Region of RI and
RII Subunits--
The proteolysis results led us to focus on potential
interactions between the first few residues (P-11 to P-4) that lie
NH2-terminal to the consensus peptide of R subunits at the
active site cleft of the C subunit. A close comparison of the structure
of C:PKI-(5-24) and the isoform-specific differences between the RI
and RII subunits (Fig. 1) suggested that there may be key differences
in the way that the R subunits in the P-11 to P-4 region
NH2-terminal to the consensus site complement the active
site of the C subunit. This led us to further probe the interactions
between the C subunit and R subunit isoforms by introducing two
mutations (R133A and D328A) into the C subunit around the active site cleft.
Characterization of Catalytic Mutants--
Mutants C(R133A) and
C(D328A) expressed in E. coli to levels that were similar to
the wild-type C and could be purified readily to homogeneity using
conventional or co-lysis purification procedures. The purified mutants
(isozyme II) phosphorylated at Ser338, Thr197,
and Ser10 have atomic masses of 40,597 ± 5 and
40,642 ± 4, respectively, as determined by electron spray mass
spectroscopy. These values are in good agreement with the calculated
values of 40,597 and 40,638 as expected for the Arg to Ala and Asp to
Ala mutations.
The steady-state kinetic properties of the mutants were determined and
summarized in Table I. Similar to that of
the double mutant C(R133A,R134A) (32), mutant C(R133A) showed a
2-3-fold increase in its Km for both Kemptide and
ATP and a 2-fold increase in kcat. Mutant
C(D328A) displayed a 2-fold increase in its Km for
Kemptide while its kcat and
Km for ATP remained unchanged as compared with the
wild-type C.
Inhibition of C(R133A), C(D328A), and Wild Type C Subunits by
RI
The radioisotopic method was also used to study the tight interaction
between catalytic subunits and R subunits and PKI so that the effects
of mutations C(R133A) and C(D328A) on interaction with RI
The inhibition constants for RI to wild-type C and C(R133A) and for RII
to wild-type C and C(D328A) were also determined (Fig. 4, B
and C). These inhibition constants, summarized in Table II, established clearly that mutation of 133 reduced affinity of the C
subunit for RII and PKI but not for RI. In contrast, mutation of 328 affected interaction between C and RI but not RII and PKI.
R-C Interaction Measured by Surface Plasma Resonance--
As
mentioned above, interaction between C(R133A) and RI
The association and dissociation constants for wild-type, C(R133A), and
C(D328A) C subunits obtained by surface plasmon resonance measurements
were summarized in Table III. Overall,
the association and dissociation constants for C subunit to RI Two major interaction sites between the R and C subunits were
defined previously (36, 41, 42). One is the autoinhibitory consensus
site in the linker region of R that fills the active site cleft of the
C subunit and competes for the substrate-binding site (41). The other
site, termed a peripheral recognition site, lies COOH-terminal to the
inhibitory consensus site and complements the surface of C that is
dominated by phosphothreonine 197 and the activation
loop (36, 42, 43). This peripheral recognition site is an essential
docking surface for cAMP-binding domain A in the holoenzyme complex.
The exact boundaries of R/C interaction at the active site cleft,
however, are not clear at present. Using limited trypsin digestion to
map the core of the type I holoenzyme complex, the apparent protection
of two tryptic cleavage sites, at residues 76 and 90, between residue
72 and the pseudo-substrate inhibition site in the R subunit clearly
suggests that the NH2-terminal boundary of the RI subunit
interaction site is between residues 72 and 76, indicating that this
region that lies NH2-terminal to the inhibition site may
also play an important role in R/C interaction. Although all R subunit
isoforms share extensive sequence similarity and contain two tandem
cAMP-binding domains that are homologous to the cAMP-binding domain of
the cAMP receptor protein in E. coli (25), RI and RII show
distinct sequence differences that are conserved within each subfamily
(44). These sequence differences are most striking in the linker region
that extends from the dimerization/docking domain through the consensus
inhibitor site. In RII the consensus inhibitor site contains the
sequence RRXS, an autophosphorylation site for C (26),
whereas the RI has the sequence RRXA that cannot be
autophosphorylated but is important for high affinity binding of MgATP
(28). However, the differences between RI and RII cannot be accounted
for exclusively by the presence or absence of the autophosphorylation
site. Given the proteolysis results, coupled with modeling based
on the C:PKI crystal structure, we reasoned that the residues that
immediately precede the consensus site arginines might be important for
holoenzyme formation. This analysis led us to test two specific
sites, Arg133 and Asp328, on the surface of the
C subunit as potential isoform-specific interaction sites for the P-11
to P-4 regions of RI and RII.
Arg133 and Asp328, located on the
NH2-terminal proximal side of the peptide-binding site in
the catalytic subunit, define two potential binding sides that lie on
opposite sites of the peptide-binding cleft. Arg133 reaches
out from the large lobe and is part of the hydrophobic binding pocket
where the P-11 Phe in PKI docks, while Asp328, is part of
the mobile acidic patch in the COOH-terminal tail that is functionally
a part of the small lobe (Fig. 5).
Asp328 was identified as a putative recognition site for a
basic residue at the P-4 position of peptide substrates when an
oriented peptide library was screened to determine the optimal peptide
substrate (45). Substitution of Arg177 and
Lys178 (the Arg133/Arg134
equivalents) of the yeast C subunit with Ala caused a 4.2-fold decrease
in binding affinity for the yeast R subunit, which is more closely
related to the mammalian type II R subunit, without impairing the
catalytic activity (43). The equivalent mutations in murine C subunit
(R133A/R134A) led to a defect in PKI interaction without significantly
affecting the interaction with the RI subunit (32). Arg133,
together with Tyr235, Pro236, and
Phe239 form the hydrophobic P-11 binding pocket that is
necessary for tight binding of PKI (32). Based on these observations
and on the isoform-specific differences in RI and RII in the P-6 to P-4 region, mutations of R133A and D328A were introduced to dissect interaction between the catalytic subunit and specific regulatory subunit isoforms.
(1-72) regulatory subunit core, indicating
that interaction between the regulatory and catalytic subunits extends
beyond the autoinhibitory site in the R subunit at the
NH2 terminus. Sequence alignment of the two R subunit
isoforms, RI and RII, reveals a significantly sequence diversity at
this specific region. To determine whether this sequence diversity is
functionally important for interaction with the catalytic subunit,
specific mutations, R133A and D328A, are introduced into sites adjacent
to the active site cleft in the catalytic subunit. While replacing
Arg133 with Ala decreases binding affinity for RII,
interaction between the catalytic subunit and RI is not affected. In
contrast, mutant C(D328A) showed a decrease in affinity for binding RI
while maintaining similar affinities for RII as compared with the
wild-type catalytic subunit. These results suggest that sequence
immediately NH2-terminal to the consensus inhibition site
in RI and RII interacts with different sites at the proximal region of
the active site cleft in the catalytic subunit. These isoform-specific
differences would dictate a significantly different domain organization
in the type I and type II holoenzymes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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(8), RI
(9), RII
(10),
and RII
(11) have been identified. Regulatory isoforms are
differentially expressed in tissues (12-14), and their subcellular
distribution also appears to be distinct (8-11, 15-18). The existence
of a family of protein kinase A anchoring proteins (AKAPs) that tether
the type II regulatory subunits to specific subcellular structures has
been well documented (3), and AKAPs for both RI and RII (19, 20) as
well as an RI-specific AKAP protein (21) have been identified recently.
At present the exact physiological functions of RI and RII are not
clear. However, growing evidence suggests that they are functionally different. This is supported by the fact that although the ratio of the
total R subunits/C subunits in normal tissue was found to be relatively
constant around 1:1, the relative amount of RI and RII varies and
depends highly on physiological conditions and the hormonal status of
the tissue (13, 14, 22, 23). One recent study shows that in RII
gene
knockout mice, an increase level of RI
compensates the loss of
RII
in brown fat cells. The switching of PKA isoform from type II to
type I resulted in an increased basal level of PKA activity and
increased energy expenditure. The RII
knockout mice are leaner and
protected against diet-induced obesity (24). These results demonstrate
clearly that RI and RII are functionally distinct.
and RII
was
then quantitatively examined by kinetic and surface plasmon resonance
studies.
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Fig. 1.
Sequence alignment of the hinge of different
R subunit isoforms. Amino acid sequences immediately
NH2-terminal to the autoinhibitor sites
(underlined) in R subunits are compared with that of PKI.
P (the phosphorylation site), P-3, P-4, P-5, P-6, P-11, and
P+1 sites are marked by arrows.
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ABSTRACT
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was purified using DE52 anion exchange chromatography
followed by FPLC gel filtration on Superdex 200 (Amersham Pharmacia
Biotech) as described previously (35). H6-RI
was purified on nickel resin (Qiagen) and RII
was isolated by
co-purification with H6-C using a protocol similar to that
of Hemmer et al. (36). Briefly, equal volumes of E. coli cultures that overexpressed RII
and H6-C were
mixed and co-lysed in the presence of 5 mM MgATP. After
batch binding the holoenzyme onto Ni2+ resin, the free RII
was eluted with 5 mM cAMP. Pooled fractions from the cAMP
elution were precipitated with ammonium sulfate, and further purified
by FPLC gel filtration on a Superdex 200 column. To obtain cAMP-free R
subunits, RI
and RII
were dialyzed against 8 M urea
to completely remove cAMP. The chemically denatured R subunits were
then refolded by dialysis to remove the urea (37), and further purified
by passing through a Superdex 200 gel filtration column. All proteins
were at least 95% pure judged by SDS-polyacrylamide gel electrophoresis.
2C2
holoenzyme, 35 ng of trypsin, and with or without 8 mM
cAMP. The digestion reaction was started by adding the protease, and 5 µl of reaction mixture was withdrawn and mixed with 5 µl of 2 × SDS sample buffer after incubating at room temperature for 10 or 30 min. The samples were boiled for 2 min and loaded onto a 12%
SDS-polyacrylamide electrophoresis gel for analysis of the digestion products.
, RII
, or PKI. The reaction was pre-equilibrated at room
temperature for 10 min and initiated by adding the peptide substrate.
Following a 10-min incubation, aliquots (45 µl) were withdrawn,
spotted onto discs of Whatman P81 paper, and immediately immersed in 75 mM phosphoric acid (10-20 ml/sample) to terminate the
reaction. After washing three times in phosphoric acid and once with
ethanol, the discs were dried under a heating lamp. Radioactivity was
measured by liquid scintillation spectrometry.
and RII
. C(R133A), C(D328A), and wild-type C
subunits were immobilized to a sensor chip (CM dextran surface) by
direct amine coupling as described previously (40). All surface plasmon
resonance experiments were performed in running buffer containing 20 mM Mops (pH 7.0), 150 mM KCl, 0.1 mM ATP, 1 mM MgCl2, 0.5 mM dithiothreitol, and 0.005% polysorbate 20, a nonionic
detergent surfactant, at room temperature. For each run, five
injections at different R concentrations between 30 and 500 nM were performed and sensorgrams were collected for each
injection. The C subunit surface was regenerated by injection of 10 µl of 10 µM cAMP and 4 mM EDTA in running
buffer following each injection of R subunit.
Where kasso is the association rate,
kdiss is the dissociation rate, C is
the concentration of injected analyte, and R is the
response. The dissociation rate, kdiss, was
calculated by integrating the rate equation when C = 0, yielding ln(Rt1/Rtn) = kdiss(tn
(Eq. 1)
t1). Plots of dR/dt versus
Rt have a slope of kdiss. Plotting
kdiss against C gives a slope that is equal to
the kasso. Affinity constants were calculated
from the equation Kd = kdiss/kasso.
The maximal velocity was then converted to
kcat by dividing Vmax by
the total enzyme concentration.
(Eq. 2)
where V and V0 are the
measured kinetic rate of the catalytic subunit in the presence or
absence of inhibitor, respectively, [I] is the total inhibitor concentration.
(Eq. 3)
where [S] is the Kemptide concentration and
Km is the Michaelis constant of catalytic subunit
for Kemptide.
(Eq. 4)
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(1-72)R (band A, Fig. 2) and
(1-8)C (band B, Fig. 2) based
on NH2-terminal protein sequencing. Cleavage of the
holoenzyme in the presence of cAMP led to further degradation of the
regulatory subunit. Between residues 72 and 92 (P-4 Arg) there are two
potential trypsin cleavage sites at residues 76 and 90. The apparent
protection of these two cleavage sites in the holoenzyme complex
indicated that this region of the R subunit (between residues 73 and
91) was also involved in holoenzyme formation.
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Fig. 2.
Trypsin digestion of the type I holoenzyme in
the absence and presence of cAMP. Type I holoenzyme was digested
by trypsin in the presence and absence of cAMP as described under
"Experimental Procedures." Arrows indicate the original
position of the R and C subunit, and cleavage products A and B.
Kinetic parameters for mutant and wild-type C subunit
, RII
, and PKI--
Having demonstrated that the kinetic
properties of the mutant catalytic subunits were not significantly
perturbed, the ability of these mutant C subunits to be inhibited by
RI
, RII
, and PKI was tested using the spectrophotometric assay.
When 20 nM of the wild-type and mutant catalytic subunits
were used in the assay, cAMP-free RI
titrated the C(R133A) and
wild-type C subunits stoichiometrically, indicating that the affinity
of RI
for these C subunits is much lower than 20 nM.
However, mutant C(D328A) required higher concentrations of RI
to be
inhibited to the same extent as the wild-type C subunit. Quantitative
analysis of the inhibition curve gave an apparent inhibition constant
of 28 nM (Fig. 3A)
and a calculated intrinsic inhibition constant of 2.2 nM
(Table II). The opposite was observed when stripped RII
was used. RII
, while it inhibited the wild-type C and C(D328A) readily, showed a decreased affinity toward C(R133A) (Fig. 3B). The measured
Ki,app and Ka of
RII
to C(R133A) were 21 and 1.9 nM, respectively (Table
II). Interaction between PKI and mutant catalytic subunits were also
measured. As shown in Fig. 3C, linear inhibition curves were
obtained when the wild-type and C(D328A) were titrated with PKI. In
contrast, the inhibition constants of PKI to C(R133A) were 888 and 83 nM, for Ki,app and
Ka, respectively. These results were in good
agreement with the early observation (32).
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Fig. 3.
Inhibition of the activity of wild-type and
mutants catalytic subunits by RI, RII, and PKI. The activity of 20 nM wild-type ( ), C(R133A) (
), and C(D328A) (
) in
the presence of varying amounts of RI (A), RII
(B), and PKI (C) was measured by the
spectrophotometric assay.
Intrinsic inhibition constants for mutant and wild-type C subunit
, RII
,
and PKI could be quantitatively assessed and compared. Since it was
possible to measure the PKA activity at very low concentration
(pM) with the radioisotopic method, subnanomolar affinity
could be easily measured. Inhibition of 100 pM wild-type and C(D328A) catalytic subunits by PKI was measured by
32P-phosphorylation of Kemptide, and the result was
summarized in Fig. 4A. It was
clear that unlike C(R133A), mutant C(D328A) interacted with PKI with
high affinity similar to the wild-type C-subunit. The inhibition
constant, Ki, for C(D328A) is 0.49 nM (Table II).
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Fig. 4.
Inhibition of the activity of wild-type and
mutants catalytic subunits by RI, RII, and PKI. The activity of
100 pM wild-type ( ), C(R133A) (
), and C(D328A) (
)
in the presence of varying amounts of PKI (A), RI
(B), and RII (C) was measured by the radioactive
assay.
and between
C(D328A) and RII
, was measured indirectly by the ability of these
mutants to be inhibited by the R subunits. Surface plasmon resonance
was used to further confirm these observations and to obtain a direct
estimation of the binding affinities of these mutant C subunits to
RI
and RII
. After immobilizing C, C(R133A), and C(D328A) to a
sensor chip by amine coupling, binding of RI
and RII
was measured.
were
8 × 105 M
1 s
1
and 2 × 10
4 s
1, respectively.
However, C(D328A) showed a slower association constant (4.5 × 105 M
1 s
1) and a
faster dissociation constant (5.9 × 10
4
s
1). This led to an apparent Kd of 1.3 nM, a 5-fold increase as compared with that of the
wild-type and C(R133A) C subunits. Although the C subunit interacted
with the RII
with similar affinity as with RI
, with
Kd values around 0.2 nM, both
association and dissociation constants were six times faster for
interaction between C subunits and RII
than for C subunit and RI
.
In contrast to RI
, C(R133A) displayed a Kd of 1 nM RII
, a 5-fold increase as compared with that of the
wild-type C subunit, interaction between the C(D328A) and RII
was
indistinguishable from wild-type C. This was exactly the opposite of
the observation made for interaction between the C subunits and RI
,
and was consistent with the inhibition data.
Apparent binding constants between R and C subunits measured by surface
plasmon resonance
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DISCUSSION
REFERENCES
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Fig. 5.
GRASP representation of the crystal structure
of the catalytic subunit of cAMP-dependent protein kinase
showing the locations of Arg133 and
Asp328. The surface of the C subunit that
precedes the active site cleft is shown with positions of
Asp328 and Arg133 highlighted. Also shown are
the residues that contribute to the P-2 site (Glu230 and
Glu170) and P-11 site (Tyr236,
Pro236, and Phe239). The P-3 to P+1 portion of
consensus site peptide from the MnATP·PKI-(5-24)·C subunit ternary
complex (1ATP) is also shown. The boxes indicate the two
surfaces thought to be complemented by RI and RII, respectively. The
figure was generated from the ternary complex where PKI-(5-24) was
deleted using the GRASP program.
C(R133A) and C(D328A) exhibited similar kinetic parameters both for
Kemptide and ATP as the wild-type C indicating interaction between the
mutant C subunits and this substrate is not significantly perturbed by
these mutations. On the other hand, a 5-fold decrease in binding
affinity between C(D328A) and RI was observed while interaction of
C(D328A) with RII
and PKI remained unchanged. Hence, it can be
concluded that Arg328 is a RI-specific interaction site.
This is consistent with the mutational study showing that equivalent
mutation in yeast C subunit does not affect its interaction with the
yeast R subunit, which is more closely related to the type II R
subunits (43). Based on sequence alignment of R subunits and PKI, only
RI subunits contain a conserved Arg at the P-4 position. The likely
reason for such selectivity is that a basic residue at P-4 position can interact favorably with Asp328 (45). Manual substitution of
the P-4 Gly of PKI with Arg in C:PKI crystal structure model confirms
that Asp328 is within hydrogen-bonding distance with the
guanidine group of the P-4 Arg.
Mutation R133A significantly impairs the interaction of C with PKI. The
measured inhibition constant, Ki, for the mutant C
was increased more than 300-fold, in a good agreement with an early
study (32). Interaction between C(R133A) and RII was also affected,
but to much less extent with a 5-fold decrease in binding affinity,
whereas C(R133A) mutant was identical to the wild-type C subunit in
terms of forming holoenzyme with the RI
subunit. The interaction of
R subunits with C at the NH2-terminal proximal side of the
peptide-binding site is thus isoform specific. Furthermore, RII is more
like PKI than RI in terms of interacting with C at the
NH2-terminal proximal side of the peptide-binding site.
This is in contrast to the consensus site peptide itself where PKI more
closely resembles RI having: 1) an Ala at the P site and 2) an ATP
requirement for tight binding to C. These differences can be attributed
to the diversity of the amino acid sequence immediately
NH2-terminal to the autoinhibition site in the RI and RII
subunits. In this region, while RI and RII differ significantly, sequence similarity between RII and PKI is apparent. Both RII and PKI
contain an Arg at the P-6 position and this Arg is important for PKI
binding to C (46). Although RII lacks the P-11 Phe, another important
amino acid in PKI that is critical for catalytic subunit recognition,
modeling of the C:PKI crystal structure by substituting the P-5
threonine with phenylalanine, a conserved amino acid residue in RII,
suggests that the aromatic side chain of this P-5 phenylalanine can
reach down to occupy the space that is filled by the P-11 Phe in PKI.
Thus, the highly conserved P-5 Phe in RII can potentially function in a
similar way as the P-11 Phe in PKI. Both side chains could fill the
same hydrophobic pocket formed by Arg133,
Tyr235, Pro236, and Phe239. The P-6
Arg of RII would then fill the same position as it does in PKI
interacting with Glu203.
The fact that RI and RII interact differentially with the C subunit at
P-6 to P-4 proximal to the active cleft could also have significant
consequences for the overall hydrodynamic features of the type I and
type II holoenzymes. If P-6 to P-4 residues direct R subunits in
different directions, then the NH2-terminal dimerization
domain which is also the docking site for AKAPs could be oriented
differently relative to the rest of the protein for RI and RII. This
difference in orientation of the NH2 termini of R subunits
would eventually lead to different overall structures between type I
and type II PKA holoenzymes (Fig. 6). The
Stokes radius of the type I holoenzyme (52 Å) versus the
type II holoenzyme (57 Å) is indicative of significant differences in
overall shape and, therefore, supports our interpretation. Furthermore,
the major parameters for determining these differences in Stokes radii are associated with the region that links the dimerization domain to
the consensus site peptide in R subunits based on the Stokes radii of
RI/RII chimeras where the dimerization domain of RI and RII
are
switched (47). These results are consistent with our observations that
the P-11 to P-4 linker regions in different R subunit isoforms
differentially interact with the C subunit in PKA holoenzyme complexes.
As indicated in Fig. 6, the P-5 and P-4 Arg's in RI would direct the
peptide chain toward the carboxylate-rich COOH-terminal tail of the C
subunit, most likely locking the COOH-terminal tail firmly into a
closed conformation. This is totally consistent with the fact that
MgATP is required for the formation of type I holoenzyme, and MgATP is
essential for the closed conformation of the C subunit (28, 48). It is
also consistent with the early observations that the type I holoenzyme
is sensitive to dissociation with high salt or histones (28) which both
would disrupt the acid-rich tail and lead to the release of ATP and activation of the holoenzyme. In contrast, the same region in RII could
interact with the NH2-terminal proximal side of the inhibitor recognition site of C in a similar way as PKI, making contact
with C(R133A) and the P-11 hydrophobic pocket. These interactions are
exclusively with the large lobe and do not require a closed conformation. In addition, they are hydrophobic and thus would not be
as sensitive to salt. The presence of a phosphor-Ser at the P site of
the RII subunits further dictates that the conformation of C when it is
bound to RII will assume a more open conformation. These interactions
will orient the NH2-terminal dimerization domain of RII
toward the large lobe (Fig. 6). This diversity in peptide recognition
in close proximity to the active site also has implications for peptide
substrate specificity and may explain the lack of single consensus
sequence for substrates of PKA (31).
|
Of course, differential binding of RI and RII
to the C subunit at
the region just NH2-terminal to active site cleft would not
necessarily leads to different orientations of the entire NH2-terminal domain of the R subunit in type I and type II
holoenzymes. The model presented in Fig. 6 is only the most likely
possibility. Additional experimental data are required to validate our
proposed model as well as the specific location of the
NH2-terminal domain. However, this model is supported by a
number of previous findings. In a recent study, Gangal et
al. (50) showed that interaction of the RII subunit, but not the
RI subunit, with the myristylated C subunit led to an increase in both
NH2-terminal backbone flexibility and exposure of the
NH2-myristate. In an earlier report, Herberg et
al. (51) demonstrated that deletion of the
NH2-terminal A helix of the C subunit significantly
affected the interaction between the C subunit and RII subunit, but not
the RI subunit. Collectively, these data and our current study strongly
support a model that the orientation of the NH2 terminus of
the R subunit is isoform-specific and that differential interaction
between the R subunit isoforms and the C subunit at the region
NH2-terminal to the pseudo substrate site maybe responsible
for this difference.
The effects of the R133A mutation on the C-PKI interaction versus RI confirm that the region responsible for the high affinity binding of C resides primarily on the NH2-terminal side of the inhibition site (46, 49). On the other hand, perturbation on R-C interaction caused by both R133A and D328A mutations is much milder, indicating that residues important for tight binding of C are mainly located at the COOH-terminal side of the autoinhibitor site in R subunits (46, 49). However, results from this study suggest that although amino acid sequences NH2-terminal to the autoinhibitor site in R does not contribute significantly to the affinity of R-C interaction, this region may be functionally extremely important for distinguishing RI and RII isoforms.
Clearly, PKA isoforms I and II possess different physiological
functions in vivo, and RI and RII subunits exert their
distinct functions by assuming specific cellular localization and
different cAMP sensitivity. The ability of the catalytic subunit to
interact differentially with RI and RII subunits provides another
mechanism for fine turning the regulation of PKA within cells although
the precise nature of such regulation remains to be elucidated.
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ACKNOWLEDGEMENTS |
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We thank Siv Garrod for excellent technical support on protein sequencing, Teresa Clifford for assistance in protein purification, and Dr. Elzbieta Radzio-Andzelm for preparation of Fig. 5.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM34921 (to S. S. T.).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.
¶ Jeane B. Kempner Scholar and supported by American Cancer Society postdoctoral fellowship PF-4315. Present address: Dept. of Pharmacology and Toxicology, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031.
To whom correspondence should be addressed. Tel.:
858-534-3677; Fax: 858-534-8193; E-mail: staylor@ucsd.edu.
Published, JBC Papers in Press, November 10, 2000, DOI 10.1074.jbc.M006447200
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ABBREVIATIONS |
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The abbreviations used are: R, cAMP-dependent protein kinase regulatory subunit; AKAP, protein kinase A anchoring protein; C, cAMP-dependent protein kinase catalytic subunit; Caps, 3-(cyclohexylamino)-1-propanesulfonic acid; Grb2, growth factor receptor-bound protein 2; Mops, 3-(N-morpholino)propanesulfonic acid; PKA, cAMP dependent protein kinase; PKI, heat stable protein kinase inhibitor; SH3, Src homology domain 3; FPLC, fast protein liquid chromatography.
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