From the Howard Hughes Medical Institute, the
Department of Chemistry and Biochemistry and the
§ Department of Pharmacology, University of California, San
Diego, La Jolla, California 92093-0654
Received for publication, October 22, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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For optimal activity the catalytic subunit
of cAMP-dependent protein kinase requires a phosphate on
Thr-197. This phosphate anchors the activation loop in the proper
conformation and contributes to catalytic efficiency by enhancing the
phosphoryl transfer rate and increasing the affinity for ATP (1). The
crystal structure of the catalytic subunit bound to ATP, and the
inhibitor peptide, IP20, highlights the contacts made by the Thr-197
phosphate as well as the role adjacent residues play in contacting the
substrate peptide. Glu-203 and Tyr-204 interact with arginines in the
consensus sequence of PKA substrates at the P-6 and P-2 positions,
respectively. To assess the contribution that each residue makes to
peptide recognition, the kinetic properties of three mutant proteins
(E203A, Y204A, and Y204F) were monitored using multiple peptide
substrates. The canonical peptide substrate, Kemptide, as well as a
longer 9-residue peptide and corresponding peptides with alanine
substitutions at the P-6 and P-2 positions were used. While the effect
of Glu-203 is more localized to the P-6 site, Tyr-204 contributes to
global peptide recognition. An aromatic hydrophobic residue is
essential for optimal peptide recognition and is conserved throughout
the protein kinase family.
The predominant regulatory mechanism used by eukaryotic cells to
convey a message from external stimuli is phosphorylation, mediated by
protein kinases. These messages control regulation of diverse pathways
in response to stress, antigen presentation, and development to name a
few. Members of the protein kinase family are related through a
structurally conserved catalytic core comprised of two lobes. The
smaller N-terminal lobe dominated by Several specific determinants contribute to recognition of substrates
and physiological inhibitors by the active C-subunit. Most prominent is
the positioning of Arg at the P-6, P-3, and P-2 positions in the
substrate. Most substrates have either a P-6 and P-3 Arg or a P-3 and
P-2 Arg combination (5, 6). Additionally, there is a preference for a
large hydrophobic residue at the P+1 position, with little constraint
placed at the P-1 position. Residues in the C-subunit that contribute
to peptide recognition are located primarily in the large lobe with
many of the residues located specifically within the activation segment of the enzyme.
The activation segment, broadly defined as residues 184-208, lies on
the surface of the large lobe and is essential for organizing the
entire enzyme's active site. The activation segment contains little
secondary structure, yet includes several distinct functional regions.
First is the magnesium-positioning loop, residues 184-187, which
positions the magnesium essential for coordinating the To further define the role of the P+1 loop in the overall organization
of peptide binding, in particular at sites other than the P+1 site,
Ala-scanning mutagenesis of the entire loop was carried out (17). We
focus here on Glu-203 and Tyr-204 for further kinetic analysis. Our
goals were to determine whether these residues contribute to localized
recognition of the P-2 and P-6 Arg, respectively, or whether they
contribute more globally to peptide recognition. An additional mutant,
Y204F, was engineered to assess the contributions of the aromatic ring
without the hydrogen bonding to Glu-230. The steady-state kinetic
parameters of the mutants were measured using several synthetic peptide
substrates. The traditional heptapeptide substrate, Kemptide (LRRASLG),
was assayed in addition to a longer 9-residue peptide (GRTGRRNSI). The
longer peptide was modified by substituting an Ala for Arg at the P-2
position or P-6 position. The results indicate that there is a greater
communication among the sites of substrate recognition than was
previously appreciated by simple examination of the crystal structure
and lead us to define this segment more globally as the
peptide-positioning loop.
Materials--
Reagents were obtained as follows: pRSETB
expression vector (Invitrogen, Carlsbad, CA);
[ Site-directed Mutagenesis of the PKA Catalytic
Subunit--
cDNA for the murine PKA C Expression of Murine PKA Catalytic Subunit--
Wild type and
mutant C-subunits were expressed in the E. coli strain
BL21(DE3). Cells were grown in YT medium containing 100 µg/ml
ampicillin at 37 °C to an optical density at 600 nm of 0.5-0.8, induced with 0.5 mM
isopropyl- Purification of Catalytic Subunit--
Wild type and mutant
proteins were purified using phosphocellulose chromatography and Mono S
fast protein liquid chromatography. Briefly, cell pellets were
resuspended in lysis buffer (30 mM MES, pH 6.5, 1 mM EDTA, 50 mM KCl, 5 mM
Catalytic Activity Assays--
The kinetic values for the
proteins were obtained by a direct phosphorylation filter-binding assay
using [
Kinetic data were fitted to the equation Phosphorylation of C-subunit Mutant Proteins--
Experiments
were performed as described previously (17). Briefly, wild type, G200A,
and T201A were expressed in E. coli with the addition of the
potent inhibitor, H-89, added at induction for the wild type to prevent
autophosphorylation. Bacterial cell pellets were lysed, and the soluble
fraction was used as substrate for phosphorylation by PDK-1. PDK-1 was
obtained by transfecting 293 cells and immunoprecipitating from the
soluble cell lysate using an antibody to its N-terminal Myc tag.
Phosphorylation was assessed using antibodies specific for the
phosphorylated Thr-197. The activity of these PDK-1-phosphorylated
proteins was assayed using the PepTag PKA activity assay. This assay
uses a fluorescent-tagged Kemptide substrate, where a change in net
charge occurs upon phosphorylation. This change is detected by a shift
in its direction of mobility when run on an agarose gel.
Prior Ala scanning mutagenesis studies were performed to assess
how the residues in the activation segment contribute to
phosphorylation on Thr-197 (17). A particularly interesting mutation,
highlighted in these experiments (Y204A), yielded an enzyme that did
not phosphorylate Kemptide, but was able to autophosphorylate when
expressed in E. coli. Since autophosphorylation requires
similar local recognition factors as those for exogenous peptide
substrates, further exploration of this phenomenon was warranted.
Mutation of the adjacent residue, Glu-203, generated a mutant enzyme
active toward Kemptide and capable of autophosphorylation.
Surprisingly, Kemptide does not possess a P-6 residue capable of
interacting with this charged side chain. The question of whether
Glu-203 would discriminate among substrates as Y204A if there were a
P-6 Arg could be determined by using a longer peptide substrate. Based
on these properties, E203A and Y204 were selected for a more rigorous
kinetic analysis using various peptide substrates.
Steady-state Kinetic Parameters for the Activation Loop
Mutants--
Since the two mutant enzymes, E203A and Y204A, were both
able to autophosphorylate in E. coli but showed differences
in activity using a qualitative assay, the kinetic parameters of both
were analyzed more quantitatively. Direct phosphorylation of the
peptide substrate LRRASLG, Kemptide (25), measured by the incorporation of 32P, was used to determine the kinetic properties.
Alanine substitution at Glu-203 and Tyr-204 increased the
Km for Kemptide by ~10-fold (Table
I). Although both mutations equally
affected Km, kcat was
decreased only for the Y204A mutant. This decrease in
kcat from 33 s Steady-state Kinetic Parameters for a Longer Peptide
Substrate--
While much kinetic data are available for Kemptide
phosphorylation, this short peptide does not exploit all the subsites
in the binding pocket. Since the crystallographic molecular model shows
that Glu-203 interacts with the P-6 Arg of the substrate (Fig.
1A), but there is no P-6
position in the Kemptide substrate, it was necessary to utilize longer
peptides to more fully appreciate the role of Glu-203. A 9-residue
peptide with a P-6 Arg, as well as altered peptides with alanines
replacing the two arginines involved in substrate recognition at the
P-2 and P-6 position, were thus used as comparative substrates. As
expected, the added contacts offered by the larger substrate
results in a 30-fold lower Km compared with
Kemptide (Table II). Alanine substitution at positions 203 and 204 results in 30-fold increases in
Km compared with wild type C. These effects are
similar to those observed for Kemptide and the alanine mutants.
Finally, both alanine mutants have nominal effects on
kcat using GRTGRRNSI as a substrate.
The P-2-substituted peptide, GRTGRANSI, displays the highest
Km with the wild type enzyme compared with the other peptides (Table II). The Km values are also elevated for both mutant proteins using this peptide, although the largest effect occurs with Y204A. The Km value for GRTGRANSI is 2- and 30-fold larger for E203A and Y204A, respectively, than for
wild type (Table II). Although the Km values for the
P-2-substituted peptide to wild type and the mutants are higher than
those for GRTGRRNSI, the kcat values are
similar. The P-6-substituted peptide, GATGRRNSI, shows a similar trend
with E203A displaying higher affinity than Y204A. The
Km for GATGRRNSI is 6- and 30-fold larger for E203A
and Y204A, respectively, than for wild type (Table II). The
kcat values for the P-6-substituted peptide are
close in value to those for GRTGRRNSI. Overall, for all peptides
studied, substitution at Tyr-204 has the most profound effects on
apparent substrate affinity. This is surprising in light of the crystal
structure, which demonstrates that this residue makes no direct
contact with the peptide.
Kinetic Parameters for Y204F--
The results from the above
kinetic experiments suggest that Tyr-204 may be contributing more to
peptide recognition than the electrostatic interaction between the P-2
Arg and Glu-230. The Y204F mutant was engineered to assess the
contributions of the aromatic ring to this phenomenon. Any differences
between this mutant and the wild type would be a result of the loss of
the Tyr-204 hydroxyl group. Consistent with the other mutant proteins, Y204F did not display a change in the Km for ATP
(Table I). The Ki value for Ala-Kemptide was similar
to the other mutant proteins. Using the Kemptide substrate the
kcat was found to be similar to that for wild
type. While the Km is ~2-fold higher than that for
wild type, this change is much smaller than those for the alanine
mutants. These data suggest that the aromatic ring is, indeed, making a
significant contribution to peptide recognition. The GRTGRRNSI kinetics
display similar trends in Km values as those for the
Kemptide substrate. While the Km for GRTGRRNSI is
30-fold higher for Y204A than that for wild type, this
Km is only 3-fold higher for Y204F compared with
wild type. The P-2-substituted peptide, GRTGRANSI, did have a
Km larger than the Glu-203 mutant protein, but it is
still about 5-fold less than the Ala mutant at 204.
Activity of Phosphorylated G200A and T201A--
The P+1 loop
contains other residues necessary for function but not for direct
recognition of substrate side chains. These include Gly-200 and
Thr-201, both conserved in Ser/Thr kinases. When alanine mutations are
engineered at these sites and expressed in Escherichia coli,
the resulting proteins are unphosphorylated on Thr-197 and inactive
(17). The mutant proteins analyzed thus far, E203A, Y204A, and Y204F,
have all been phosphorylated on Thr-197, and perhaps the inactivity of
G200A and T201A is the result of under phosphorylation. To test this,
G200A and T201A, were expressed in E. coli along with a wild
type control. Since unphosphorylated C-subunit is the best control, the
wild type protein was induced in the presence of H-89, a potent PKA
inhibitor that disrupts autophosphorylation. The soluble fraction of
these bacterial cell lysates was used as substrate material for
phosphorylation by the Thr-197 kinase,
3-phosphoinositide-dependent protein kinase-1 (PDK-1). The
success of the phosphorylation reaction was tested using antibodies
specific for the phosphorylated form of Thr-197. Fig.
2A depicts immunoblots of the
material in the PDK-1 reaction. The C-subunit antibody shows that each
reaction contained the same amount of C-subunit, and the
phospho-Thr-197 antibody indicated that each is phosphorylated with
similar efficiency. Any lack of activity relative to wild type will not
be due to low expression or poor phosphorylation by PDK-1. Aliquots
from the phosphorylation reactions were tested for activity toward
Kemptide using a qualitative assay. Fig. 2B indicates that
even when phosphorylated on Thr-197, the perturbation of Gly-200 and
Thr-201 abolishes activity.
After carrying out a qualitative screen of alanine mutants made in
the activation loop residues (17), two C-subunit mutants were selected
for kinetic analysis based on their unusual kinetic parameters. The
Y204A mutant showed reduced Kemptide activity in the qualitative PepTag
assay, but was able to autophosphorylate when expressed in E. coli. This apparent contradiction led to a closer examination of
its kinetic parameters. Replacement of Glu-203 with Ala led to a
protein that was active and able to autophosphorylate. The
structure suggests that the latter residue is involved in recognition
of the P-6 arginine (16), a residue not found in the Kemptide
substrate. Kinetic analysis of these two mutants revealed that the P+1
loop, as well as these two specific residues, plays a global role in
organizing the binding of peptide substrates. Each residue not only
contributes to a local site, but also shows more long range effects.
This work redefines the P+1 loop and suggests that it should more
appropriately be described as the peptide-positioning loop, as its
contributions go well beyond recognition of only the P+1 residue. In
addition, the loop contributes either directly or indirectly to
recognition of the P-site, the P-2 site, and the P-6 site.
The steady-state kinetic parameters presented in this study explain why
Y204A displays lower activity compared with wild type and E203A using
the Pep-Tag assay. Although both mutants bind Kemptide with equivalent,
poor affinity compared with wild type, the larger decrease in
kcat/Km, the catalytic
efficiency term, for Y204A compared with E203A is due to a lower
turnover number, kcat (Table I). Indeed, the
Ki values for Ala-Kemptide are much higher than the
Km values for Kemptide confirming this point.
By studying the longer peptide substrate, GRTGRRNSI, and its
derivatives we were able to better evaluate substrate recognition determinants. This peptide contains P-2 and P-6 arginines that can be
used to assess the roles of Tyr-204 and Glu-203. While the latter
interaction is direct, the former is mediated indirectly via Glu-230
(Fig. 1A). In general, the Km values for GRTGRRNSI are lower for all the enzymes studied, consistent with improved affinities of the longer peptides. Furthermore, the relative changes in Km values for the mutants follow those
for the Kemptide Km values. Alanine substitution at
positions 203 and 204 lead to Km increases of
10-fold for Kemptide (Table I), whereas these substitutions lead in
Km increases of between 2- and 30-fold for GRTGRRNSI
and its derivatives (Table II). When either mutant protein was assayed
with the substrate peptides lacking the corresponding arginine residue
(i.e. P-2 Ala substitution, Y204A; P-6 Ala substitution,
E203A), elevations in Km are obtained. For example,
the Km value for GATGRRNSI is 7-fold larger than
that for GRTGRRNSI with wild type (Table II). Furthermore, the
Km for GRTGRANSI with Y204A is ~30-fold
larger than that for GRTGRRNSI and wild type (Table II). These results
demonstrate that both Glu-203 and Tyr-204 contribute to peptide binding
to an extent not predicted by the x-ray structure. For instance, the
crystal structure clearly illustrates that the hydroxyl group of
Tyr-204 interacts with Glu-230, which in turn is in hydrogen bonding
distance from the substrate P-2 arginine. In light of the kinetic
parameters for the Y204A mutant, it seemed likely that the aromatic
ring of the tyrosine might also be contributing to peptide recognition
perhaps through an interaction between the P-2 Arg and the Glu-203 and Tyr-204 are involved in peptide recognition of a specific
substrate residue but also contribute to overall substrate recognition
indirectly. The direct interactions are clearly shown in the crystal
structure by the proximity of Glu-203 to the P-6 Arg and the network of
interactions involving P-2 Arg recognition, termed the P-2 nodule (Fig.
1B, Table III). In the P-2
nodule Tyr-204 with Arg-133 is aiding the positioning of Glu-230, which
interacts with the P-2 Arg. Additionally, Glu-170 in the catalytic loop helps coordinate the P-2 arginine. When the Glu-203 mutant is assayed
with Kemptide the results show an increase in the Km value that is quite larger than expected for a mutant whose substrate determinant was not present. This observation suggests that Glu-203 influences peptide binding beyond the P-6 interaction, perhaps through
the P+1 loop. The same holds true for Y204A. This mutation disrupts P-2
Arg binding on the substrate, but a peptide with an alanine substituted
at this position increased the Km value even more.
Perhaps the P-2 residue itself is contributing to the stability of the
enzyme by coordinating those residues that are involved in its binding.
Certainly the absence of the P-2 Arg would disrupt the network of the
P-2 nodule.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheets is responsible for
nucleotide binding, while the larger C-terminal lobe made up primarily
of
-helices, relays substrate specificity (2).
cAMP-dependent protein kinase
(PKA)1 is one of the simplest
and best understood members of the protein kinase superfamily. It
exists as an inactive holoenzyme complex consisting of a regulatory (R)
subunit homodimer and two catalytic (C) subunits. Upon increased levels
of intracellular cAMP, each regulatory subunit cooperatively binds two
molecules of cAMP inducing a conformational change resulting in the
unleashing of the active catalytic subunits (3). The C-subunit is a
350-amino acid (4), 41-kD protein with the conserved kinase core
represented by residues 40-300. The core is flanked at the N terminus
by a 39-amino acid helical region and a 50-amino acid C-terminal tail,
with each flanking region undergoing co- or posttranslational
modifications. The simplicity of this molecule and its ability to
define the conserved and active kinase core allow it to serve as a
model for other enzymes in the family.
phosphate of
ATP. Residues 188-192 comprise
-sheet 9, the only element of
regular secondary structure. This segment interacts with the
A-helix outside the core as well as the essential phosphate on
Thr-197 in the activation loop. The activation loop follows with
residues 194-197. The activation loop is also a site of regulation for
most members of the protein kinase family, where phosphorylation on one
or two key Tyr, Thr, or Ser residues is required for optimal activity
(7). Based on structural comparisons of several active and inactive
protein kinases such as cdk (8-10), src (11), and hck (11-13),
phosphorylation at these positions appears to change the conformation
of the loop and arrange it in a position necessary for optimal activity
(14, 15). In the C-subunit of PKA the kcat is
reduced and the Km for ATP is increased when Thr-197
is not phosphorylated (1). This leads to a 50-fold decrease in
catalytic efficiency (Kcat/Km). The
activation loop of the kinase resides in the large lobe, and the
phosphorylated residue in the activation loop of the C-subunit,
Thr-197, makes several contacts within the large lobe (Arg-165 and
Arg-189), as well as one of the few interactions between the large lobe and the small lobe in the closed conformation (His-87). The next region
is the P+1 loop, 198-205. The properly positioned P+1 loop contains
regions that interact with the P+1 hydrophobic residue, as well as the
P-2 and P-6 arginines of peptide substrates. Examination of a
crystallographic molecular model consisting of the C-subunit bound to
MgATP and the inhibitor peptide IP20, residues 5-24 of the protein
kinase inhibitor, lends insight into the molecular nature of these
interactions (16). The model demonstrates that a hydrophobic pocket is
formed, where the side chains of Leu-198, Pro-202, and Leu-205 make the
largest contribution to the pocket (Fig. 1A). Also in the
P+1 loop Glu-203 forms a hydrogen bond with the P-6 Arg, and Try-204
forms a hydrogen bond with Glu-230, which directly interacts with the
P-2 Arg. These residues contribute to recognition nodules where distal
parts of the molecule come together. The residues that facilitate
recognition of the P-3 Arg, Glu-127, and Try-330 lie outside the
activation loop. Finally there are the conserved APE residues,
206-208. These residues serve as an anchor to the large lobe via
interaction with Arg-280, another conserved residue in the large lobe.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (PerkinElmer Life Sciences);
Escherichia coli strains BL21(DE3) (Novagen, Madison, WI);
P81 filter paper and P-11 phosphocellulose resin (Whatman Inc.,
Clifton, NJ); Mono S HR 10/10 (Amersham Biosciences); Muta-Gene
site-directed mutagenesis kit (BioRad, Hercules, CA); horseradish
peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences); H-89 (LC
Laboratories, Woburn, MA); SuperSignal West Pico chemiluminescent substrate detection kit (Pierce); oligonucleotides (Genosis-Sigma); the
PepTag PKA activity assay kit (Promega, Madison, WI); mouse monoclonal
anti-Myc and anti-hemagglutinin antibodies (Covance, Princeton,
NJ). The C-subunit antibodies were generated as described (19).
Antibodies to the phosphorylated Thr-197 were originally generated to
the phosphorylated Thr-500 of PKC and were a gift from A. Newton
(University of California, San Diego) (20). All peptide substrates were
synthesized at the Peptide and Oligonucleotide Facility at the
University of California, San Diego on a Milligen 9050 PepSyn peptide
synthesizer using standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) methodology activator
and purified by high-performance liquid chromatography. All DNA
sequencing was performed with the ABI Prism 310 Genetic Analyzer
from PE Applied Biosystems.
-subunit in the bacterial
expression vector pRESTB was used as a template for Kunkel-based
site-directed mutagenesis as described previously (21). All mutations
were made using the Muta-Gene kit as per the manufacturer's
recommendations. DNA sequencing analysis confirmed the presence of the
correct mutation.
-D-thiogalactopyranoside, incubated for an
additional 6 h at 24 °C, collected by centrifugation, and stored frozen. Cells were lysed with a French pressure cell (American Instruments) at pressures between 1000 and 1500 p.s.i. using 15 ml
of lysis buffer/liter culture. Insoluble material was removed by
centrifugation at 25,000 × g at 4 °C for 45 min.
-mercaptoethanol), lysed, pelleted, diluted with cold water, and
batch-bound to P11 resin (1 g of resin/liter of culture) overnight at
4 °C. Resin was batch-washed in running buffer (30 mM
MES, pH6.5, 1 mM EDTA, 5 mM
-mercaptoethanol), and eluted with running buffer containing
potassium phosphate at 0, 50, 90, 250, and 500 mM. Wild
type C-subunit eluted at 90 mM, while the mutant proteins
typically eluted at 250 mM. Elutions were diluted with 3 volumes of cold water and bound to the Mono S 10/10 column. The
proteins were eluted in 20 mM potassium phosphate, pH 6.5, 5 mM
-mercaptoethanol with a 0-500 mM KCl gradient.
-32P]ATP (22). The assays were performed as
described (23). Briefly, the C-subunit (0.25-1.0 nM) was
incubated in 50 mM MOPS (pH 7.0), 0.1 M KCl, 10 mM MgCl2, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 2.5 µCi of [
-32P]ATP,
1 mM unlabeled ATP, and peptide substrate. To determine the
Km (ATP), peptide concentrations were
held constant and the total ATP was varied from 1.0 µM to
2.0 mM. To determine the Km values for
peptide substrate, the ATP concentration was fixed and the peptide
substrate varied. Reactions were initiated with the addition of peptide
substrates and incubated at 30 °C in a final volume of 50 µl.
Reactions were terminated with 20 µl of 50% acetic acid. Aliquots
were spotted on P81 filter disks and washed together in 0.5%
phosphoric acid (four times, 500 ml, 10 min). Filter disks were rinsed
once with acetone, air-dried, and counted in 5 ml of EcoLume.
Background reactions containing no peptide substrate were subtracted
from all data. All reactions were performed in triplicate.
= Vmax[S]/([S]Km)
where
is the reaction rate, Vmax in the
maximum rate, [S] is the concentration of the
variable substrate, and the Km is the Michaelis
constant. Inhibitor constants were determined by assaying at various
concentrations of the inhibitor at fixed substrate concentration. Data
were fitted to a single-site binding model using GraphPad Prizm version
3.02, and Ki values were extrapolated from
IC50 values using the relationship of Cheng and Prusoff:
Ki = IC50/(1 + [S]/Km) (24).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 to 0.9 s
1 results in a decrease in catalytic efficiency
(kcat/Km) of over 400-fold
for the Y204A mutant as compared with a 15-fold reduction for the E203A
mutant enzyme. The Km for ATP was similar for all of
the enzymes assayed, indicating that these mutations are not
influencing the nucleotide pocket. Since the Km for
peptide binding does not reflect a true Kd for
binding peptide (26), the Ki for each protein was also determined using Ala-Kemptide (LRRAALG), an active site-directed inhibitor (27). These values were increased ~40-fold for both mutant
enzymes relative to the wild type enzyme.
Steady-state kinetic parameters for P + 1 mutant proteins using
kemptide as substrate
View larger version (36K):
[in a new window]
Fig. 1.
Interactions made by residues of the
peptide-positioning loop and the P-2 nodule. As observed in
1atp.pdb (A) the P+1 loop, white, forms a
hydrophobic pocket that aids in the binding of the substrate,
tan, P+1 hydrophobic residue. Other residues within this
sequence are involved in recognition of other substrate determinants:
Glu203 with the P-6 position and Tyr-204 through Glu-230 in the
recognition of the P-2 Arg. B, the crystal structure of the
C-subunit demonstrates that recognition of the P-2 Arg involves
residues from different parts of the molecule: Glu270, Arg133, Glu230,
and Tyr204.
Kinetic values for P + 1 loop mutant proteins using variations of
a longer peptide substrate
View larger version (52K):
[in a new window]
Fig. 2.
Activity of phosphorylated G200A and
T201A. The Thr-197 kinase, PDK-1, was used to phosphorylate the
indicated proteins in an in vitro reaction. The success of
the reactions was assessed by immunoblotting with antibodies specific
for the phosphorylated Thr-197 and for total protein with a C-subunit
antibody (A). The blots indicate that there are similar
amounts of protein in each reaction and that they are all are
phosphorylated with similar efficiency. B indicates the
results of the PepTag activity assay. When the fluorescent-tagged
Kemptide substrate gets phosphorylated, it undergoes a change in net
charge of +1 to 1, altering its direction of migration on an agarose
gel. Here phosphorylated peptide migrates toward the top of the page,
indicating activity. Only the wild type protein demonstrates
activity.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
electrons of the aromatic ring. The Y204F mutant protein does indeed
demonstrate that the aromatic ring contributes to substrate
recognition. The consequence of losing this side chain is seen in the
crystal structure of the Y204A mutant
protein.2
P-2 nodule residues and interactions
Both Glu-203 and Tyr-204 lie within a sequence that forms a hydrophobic pocket that binds the substrate's P+1 hydrophobic residue. The positioning of this loop appears to be of critical importance. Hydrophobic residues are contributing to the pocket, and the other non-hydrophobic residues contribute to the proper positioning of the loop and of the substrate. Table IV lists the residues that make up the P+1 loop as well as the interactions in which they are involved. The Glu-203 and the Tyr-204 residues on this loop do not contribute to its hydrophobicity with their side chains; instead they are directed away from the hydrophobic pocket. The increased Km value for mutant proteins with peptide substrates lacking their corresponding arginine may be due to disruption of the P+1 loop and other interactions it makes. Sequence alignments for members of the kinase family highlight the importance of this P+1 loop in substrate recognition (18). There are distinct sequence differences between Ser/Thr kinases and Tyr kinases in this loop. These changes reflect the need to accommodate the larger substrate tyrosine. The Thr-201 position is conserved as either a Ser or Thr in Ser/Thr kinases but is almost exclusively Pro in the Tyr kinases. The Glu-203 position is conserved in the AGC superfamily of kinases, but not in other Ser/Thr kinases, whereas this position is basic in the Tyr kinases. The Tyr at 204 is somewhat conserved in the Ser/Thr kinases with some substitutions of Phe or Trp but is always aromatic in the Tyr kinases. The inactivity of G200A and T201A, even when phosphorylated, further demonstrates the importance of these individual residues as well as the global nature the contributions this loop makes.
|
The C-subunit is an enzyme that is poised to phosphorylate its
substrates. Crystallographic molecular models show the
peptide-positioning loop on the surface of the enzyme, prepared to bind
substrate. This is evident in its exhaustive list of substrates, which
are involved in wide-ranging cellular functions. This characteristic works for the C-subunit because it is also unique as a kinase in its
mechanism of regulation. Although there are key sites of phosphorylation that are required for activity, regulation does not
occur through the dynamic transfer of a phosphate on/off at the
activation loop. Instead regulation occurs through the R-subunits and
further through the A-kinase-anchoring-proteins or AKAPs that target
the PKA signal to the various parts of the cell. It will be at this
level where substrate specificity will next be described.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ronald Aimes for helpful discussions and Dr. Kenneth Humphries for manuscript review.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM19301 (to S. S. T.) and Training Grant DK07233 from the National Institutes of Health (to M. J. M.).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: Howard Hughes Medical Institute, Dept. of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0654. Tel.: 858-534-8190; Fax: 858-534-8193; E-mail: staylor@ucsd.edu.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M210807200
2 J. Yang, C. Juliano, X. Nguyen-hu, L. F. Ten Eyck, and S. S. Taylor, manuscript submitted.
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ABBREVIATIONS |
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The abbreviations used are: PKA, protein kinase; C, catalytic; R, regulatory; YT, yeast-tryptone; MES, 4-morpholineethanesulfonic acid.
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