THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 271, No. 17, Issue of April 26, pp. 10175–10182, 1996
Printed in U.S.A.
Functional Malleability of the Carboxyl-terminal Tail in Protein
Kinase A*
(Received for publication, August 2, 1995, and in revised form, January 24, 1996)
Anton Chestukhin‡, Larisa Litovchick‡, Dmitry Schourov‡, Sarah Cox§, Susan S. Taylor§, and
Shmuel Shaltiel‡¶
From the ‡Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel and the
§Department of Chemistry, University of California, San Diego, California 92093-0654
The catalytic (C) subunit of protein kinase A (PKA) is
regarded as a framework for the protein kinase family.
Its sequence is composed of a conserved core (residues
40 –300) between two segments at the amino and carboxyl termini of the protein. Since the various protein
kinases differ in their specificity, it seems reasonable to
assume that these nonhomologous segments may be involved in endowing each kinase with its individual specificity. Here we present data to show that the cluster of
acidic amino acids (328DDYEEEE334) at the carboxyl-terminal “tail” of the C subunit, specifically Tyr330, contributes to its substrate recognition. This is based on three
complementary lines of evidence: (i) on a conformationsensitive cleavage of the C subunit by a kinase-splitting
membranal proteinase that specifically recognizes this
cluster, to demonstrate the occurrence in solution of
“open” (cleavable) and “closed” (noncleavable) conformations of the C subunit; (ii) on analysis of the threedimensional structures of the open and closed conformations of the C subunit, showing an ;7-Å movement of
the phenolic hydroxyl of Tyr330 to reach (in the closed
conformation) an ;3-Å distance from the nitrogen atoms
of the Arg residue at position p-3 of the PKA consensus
sequence; and (iii) on single-site mutations of the C subunit (e.g. Y330A) that show a significant contribution of
Tyr330 to the Km of PKA for its substrates/inhibitors and
to its catalytic efficacy (Vmax/Km).
Discovered as a key enzyme in the cellular response to hormones that function via cAMP (1– 4), protein kinase A (PKA)1
is now regarded as a prototype for the large family of protein
kinases, which share a considerable homology with the sequence of the catalytic (C) subunit of PKA (5, 6). With the
elucidation of the three-dimensional structure of the C subunit
by x-ray crystallography (7, 8), these studies established the
basic architectural features of the C subunit, including its
* This work was supported by the Minerva Foundation (Munich,
Germany) and by the Ministry of Science (Israel) with the Gesellschaft
für strahlen und Umweltforschung (Nürenberg, Germany) and in part
by the Ben and Joyce Eisenberg Foundation, the Forchheimer Center
for Molecular Genetics, and the Simone Mallah Fund at the Weizmann
Institute of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
¶ Incumbent of the Kleeman Chair in Biochemistry at the Weizmann
Institute of Science. To whom correspondence should be addressed.
Tel.: 972-8-9343920/9342906; Fax: 972-8-9342804; E-mail: lishalt@
weizmann.weizmann.ac.il.
1
The abbreviations used are: PKA, protein kinase A; C subunit,
catalytic subunit; KSMP, kinase-splitting membranal proteinase; PKI,
PKA inhibitor; PKS, PKI substrate; ATPgS, adenosine 59-O-(thiotriphosphate); App(NH)p, adenyl-59-yl b,g-imidodiphosphate; App(CH2)p,
adenyl-59-yl b,g-methylenediphosphate.
unique nucleotide fold, the spatial relationship of the conserved
amino acid residues present at its active site (7–11), the occurrence of a helix motif (12) that complements the conserved
catalytic core (residues 40 –300) (7, 9, 10), and the scaffolding
common to the family of the protein kinases, upon which the
individual features of each kinase are built (13).
Already in the early eighties, it was shown that the C subunit of PKA has a very malleable structure in solution, undergoing distinct conformational changes around physiological pH
and ionic strength (14 –16). Furthermore, these changes were
shown to be fully reversible and to be triggered by substrates
and inhibitors of the kinase. The first indications of this conformational malleability were obtained by measuring the relative chemical reactivity of the two SH groups of the kinase,
located at its core (Cys199) and at its carboxyl-terminal tail
(Cys343) (14 –17). Subsequently, the specific malleability of the
carboxyl-terminal tail was illustrated by the intriguing conformation dependence of a specific proteolytic cleavage of this tail
by a kinase-splitting membranal proteinase (KSMP), which
was found to be quite specific for the kinase (18 –25). These
findings, regarding the functional relevance of the malleability
of the C subunit, were supported by studies that monitored the
structural consequences of binding inhibitors and substrates
on the C subunit, using circular dichroism (26, 27), differential
labeling of carboxyl groups in the carboxyl-terminal tail of the
C subunit with a water-soluble carbodiimide (28), and small
angle x-ray scattering (29) of this kinase.
The cleavage of the C subunit by KSMP yields a specific
clipped product devoid of kinase activity (18). This cleavage
was found to occur in the native structure of the C subunit, but
not in its heat-denatured form (19, 20), suggesting that KSMP
recognizes specifically the three-dimensional structure of the C
subunit. The KSMP cleavage site in the native C subunit was
recently identified2 as Glu332–Glu333, within the cluster of
acidic amino acids at the carboxyl-terminal tail (327FDDYEEEEI335). This cluster, which accommodates a tyrosine residue (Tyr330) and is located between two hydrophobic amino
acids (Phe327 and Ile335), constitutes the major biorecognition
element for KSMP, as indicated by studies with monoclonal
antibodies that recognize clusters of Glu and Tyr and the antiidiotypes of these antibodies, and by studies with synthetic
peptides based on the sequence around the above-mentioned
cluster of acidic amino acids (23).
Interestingly, a conformation-dependent cleavage by KSMP
was shown to occur also in two other kinases that contain a
cluster of acidic amino acids at their carboxyl-terminal tails: in
the epidermal growth factor receptor kinase and in the insulin
receptor kinase (21, 22). These findings led us to propose that
2
A. Chestukhin, K. Muradov, L. Litovchick, and S. Shaltiel, manuscript in preparation.
10175
10176
Functional Malleability of the Carboxyl-terminal Tail in PKA
the malleable tail at the carboxyl terminus of each of these
kinases may well play “an important role in creating the preferential affinity of these kinases for their in vivo substrates”
(22, 23), i.e. this malleability may have a functional role in their
substrate recognition.
X-ray studies have recently shown that in the crystalline
state, the C subunit assumes two different conformations referred to as “open” and “closed” (10, 30). These two conformations represent different relative orientations of the small and
large lobes of the C subunit and are associated with the opening and closing of the cleft between the two lobes, which forms
the ATP-binding pocket at the active site. This domain movement (31) is accompanied by a movement of the carboxylterminal tail of the C subunit (10), as expected from the abovementioned studies in solution.
Here we show that the conversion of the open conformation
of the C subunit to its closed conformation in the ternary
complex with its substrates and inhibitory analogs involves an
approach of the cluster of acidic amino acids (especially Tyr330)
toward a key biorecognition element in the peptide substrates
of the C subunit. The importance of this interaction is highlighted by the fact that a single-site mutation of this tyrosine
(Y330A) results in a significant increase in the Km of the
enzyme for its peptide substrate, Leu-Arg-Arg-Ala-Ser-Leu-Gly
(Kemptide); a reduction in its catalytic efficacy as reflected in
the Vmax/Km value for this peptide; and a reduction in the
specific activity of the mutant enzyme for a protein substrate
(histone type II-A).
MATERIALS AND METHODS
Preparation and Assay of the C Subunit—The C subunit of bovine
PKA was purified as described by Reimann and Beham (32). This
purification involves chromatography on DE52, affinity elution of the C
subunit by cAMP, and chromatography on hydroxylapatite. To these we
added a purification step on a Mono S column (HR 5/5, Pharmacia,
Uppsala). The fractions with kinase activity obtained from the hydroxylapatite column were pooled, dialyzed against the equilibration buffer,
and applied to the Mono S column pre-equilibrated with 20 mM NaPi
buffer (pH 6.8) containing 1 mM EDTA and 1 mM dithiothreitol. The
loaded column was washed with the equilibration buffer and then
eluted with a linear gradient of 20 –200 mM NaPi (pH 6.8). This column
yields two isoforms of the C subunit: CA and CB (33, 34). The CB isoform
was used routinely in this study since it was previously shown that it
constitutes a slightly better substrate for KSMP (34). The enzyme was
assayed as described previously (35) with the following modification.
The phosphoprotein pellet formed by precipitation with trichloroacetic
acid was washed on Whatman GF/C glass filters.
Purification of KSMP—Brush-border membranes obtained from kidneys of 12-week-old Wistar rats were prepared as described by Evers et
al. (36) and used in the preparation of KSMP. Starting with 60 rat
kidneys (total weight of ;80 g), we usually obtained a membrane
preparation that contained ;130 mg of protein. This preparation was
subjected to solubilization (30 min at 4 °C) by a solution (100 ml) of 0.8%
(w/v) b-octyl glucoside and 20 mM Tris-HCl (pH 7.1), ascertaining that
the detergent/protein ratio (w/w) was ;6:1. The insoluble components
were removed by centrifugation at 100,000 3 g (30 min at 4 °C). The
supernatant containing essentially all of the KSMP activity was subjected to purification on a DEAE-Sephacel column (2 3 10 cm) preequilibrated with a buffer (pH 7.1) composed of Tris-HCl (20 mM),
MgCl2 (1.5 mM), NaCl (50 mM), and b-octyl glucoside (0.5%, w/v). The
concentration of NaCl in the b-octyl glucoside extract was adjusted to 50
mM, and the extract was applied to the column. After loading, the
column was washed with 10 volumes of the pre-equilibration buffer,
and then KSMP was eluted by raising the concentration of NaCl in this
buffer to 175 mM. The eluted material was diluted with 20 mM Tris-HCl
(pH 7.1) to bring the concentration of b-octyl glucoside to 0.3% (w/v).
This material was applied to a hydroxylapatite column (1.5 3 4 cm)
pre-equilibrated with Tris-HCl (pH 7.1) containing b-octyl glucoside
(0.3%, w/v). After loading, the column was washed and then eluted with
the following series of eluents: 100, 150, 275, and 500 mM NaPi, all at
pH 7.1 and containing b-octyl glucoside (0.5%, w/v). The protein fraction
eluted by the buffer containing 150 mM NaPi was found to possess the
highest KSMP specific activity. This fraction was subjected to further
purification on a Mono Q column (HR 5/5, Pharmacia) after dialysis
against a buffer composed of Tris-HCl (pH 7.1), MgCl2 (1.5 mM), and
b-octyl glucoside (0.5%, w/v). Prior to loading the column, the fraction to
be subjected to chromatography was dialyzed overnight at 4 °C (two
buffer changes) against the buffer used for the column pre-equilibration. After loading, the column was washed with 10 volumes of the
equilibration buffer and then eluted with a 0 – 400 mM NaCl gradient.
The fractions that eluted from the column at ;200 mM NaCl (which had
a maximal KSMP specific activity) were pooled and used.
Assay of KSMP—KSMP was assayed as described previously (18, 19)
with the following modifications. The reaction mixture (usually a total
volume of 20 ml) contained 1 mg of the C subunit in a buffer (pH 7.1)
composed of Tris-HCl (20 mM), MgCl2 (1.5 mM), and b-octyl glucoside
(0.2%, w/v). The reaction was allowed to proceed at 23 °C for 2–10 min
(depending on the activity of the KSMP preparation used) and arrested
by adding the sample buffer used for polyacrylamide gel electrophoresis
in the presence of SDS, as described by Laemmli (37), and then boiling
(5 min at 95 °C). The gels used usually had a polyacrylamide gradient
of 5–20%. The sample buffer used was composed of SDS (1%, w/v),
dithiothreitol (40 mM), EDTA (5 mM), and Tris-HCl (20 mM) at pH 6.8.
After electrophoresis, the gels were stained with Coomassie Blue
(0.25%, w/v) in 50% (v/v) methanol and 7% (v/v) acetic acid, and the
extent of cleavage was determined by a computing densitometer with
ImageQuant software. Initial rates of cleavage were calculated by determining the formation of the clipped C subunit with time and extrapolation to time 0. A control cleavage of the C subunit was run alongside
for reference.
Substrates and Inhibitors of PKA—The ATP analogs and the histone
(type II-A) used were purchased from Sigma. Synthetic peptide fragments of the PKA inhibitor known as PKI (residues 6 –22 or 5–24, with
a carboxyl terminus in an amide or free carboxyl form) (38), the phosphorylatable substrate analog of PKI-(5–24) denoted PKS (for structures, see Scheme I), and the PKA peptide substrate known as Kemptide (39) were either purchased from Sigma or synthesized in our
laboratory.
Preparation of C Subunit Mutants—The wild-type murine Ca subunit gene was cloned into the pRSET-B vector (Invitrogen) as described
elsewhere.3 Site-directed mutations were introduced by oligonucleotidedirected mutagenesis of a uracil-containing single-stranded Kunkel
template (40). The wild-type or mutant enzyme-carrying vector was
used for transformation of the Escherichia coli BL21(DE3) strain. The
conditions used for growing, induction, and expression were as described elsewhere (41, 42). Expression was allowed to proceed for 4 h
after induction, and then the bacteria were collected by centrifugation
and lysed in an ultrasound disintegrator using 20 mM Tris-HCl (pH 7.5)
containing 2 mM MgCl2, 1 mM dithiothreitol, and 0.1% (w/v) b-octyl
glucoside. The insoluble particles were removed by centrifugation, the
supernatants were then collected, and the level of expression was measured by SDS-polyacrylamide gel electrophoresis. The supernatants
were then purified further by chromatography on a Whatman phosphocellulose P-11 column as described elsewhere (43). The enzyme content
was measured by quantitative immunoblotting using an ECL detection
system (Amersham International, Buckinghamshire, United Kingdom)
for developing. X-ray films were exposed to the ECL-developed blots,
and the former were quantitated by computing densitometer scanning.
The C subunit purified from bovine heart was routinely included as a
standard in these immunoblots. The rabbit primary antibodies used
were raised against a peptide (6AKKGSEQESVKEFLAKAK23) whose
sequence is identical to that of an amino-terminal segment in the C
subunit.
Assay of the Kinase Activity of Expressed C Subunit Mutants—Kemptide was used as a peptide substrate for measuring the kinase activity
of expressed C subunit mutants. The assay was performed as described
elsewhere (44). Phosphorylation was carried out at 30 °C in a final
volume of 50 ml containing 20 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 1 mM
dithiothreitol, a 1– 40 ng/ml concentration of the kinase (depending on
the specific activity of the tested C subunit mutant), 0.1 mCi of
[g-32P]ATP (specific activity of 3000 Ci/mmol) per assay, and varying
concentrations of ATP and Kemptide. The reaction was allowed to
proceed for 3 min and arrested by a stopping solution containing 100
mM ATP, 100 mM EDTA, and 3% (w/v) SDS in 20 mM Tris-HCl (pH 7.5).
Determination of the Specific Activity of the Wild-type C Subunit and
of Its Mutants Using a Protein Substrate—These specific activities were
determined with histone type II-A. The reaction mixtures (200 ml)
contained a 1–25 ng/ml concentration of the mutant kinase preparation
3
W. M. Yonemoto and S. S. Taylor, manuscript in preparation.
Functional Malleability of the Carboxyl-terminal Tail in PKA
SCHEME 1. Structure of PKA peptide inhibitors and substrates.
(depending on the specific activity of the tested C subunit mutant), 6
mM MgCl2, 1 mg/ml histone, 1 mg/ml bovine serum albumin, 1025 M
ATP, 0.5 mCi/ml [g-32P]ATP, and 0.1% b-octyl glucoside in 20 mM
Tris-HCl (pH 7.1). Phosphorylation was allowed to proceed for 3 min at
30 °C (securing a linear dependence of the incorporation of phosphate
with time and with enzyme concentration) and then arrested by adding
0.5 ml of 10% (w/v) trichloroacetic acid. A 4-ml volume of 1% (w/v)
trichloroacetic acid in phosphate-buffered saline was then added, and
the mixture was poured on GF/C glass fiber filters placed on a vacuum
manifold. The filters were then washed four times with 4 ml of 1% (w/v)
trichloroacetic acid in phosphate-buffered saline and transferred to
scintillation vials, and their radioactivity was measured in a Packard
Tri-Carb scintillation counter. Determinations were performed in
triplicates.
Determination of the Kinetic Parameters of the C Subunit Mutants
and Comparison with Those of the Wild-type C Subunit—The kinetic
parameters for the phosphotransferase reaction of the C subunit mutants and those of the wild-type C subunit were calculated using the
rate equation given by Cleland (45) for bireactant enzyme-catalyzed
reactions that follow a sequential pathway (as is the case for PKA) (16,
46 –50): v 5 VAB/(KiaKb 1 KbA 1 KaB 1 AB). In this equation, v is the
initial velocity; A and B are the substrate concentrations; V is the
limiting rate at high substrate concentrations; Ka and Kb are the Km
values for substrates A and B, respectively; and Kia is the dissociation
constant for substrate A.
In practice, we measured the activity of the various mutant kinases
(as well as the wild-type enzyme) at seven different concentrations of
ATP (from 5 to 200 mM), each at seven different concentrations of
Kemptide (from 50 to 400 mM). The data obtained were linearized by
least-square analysis, and the results were plotted in double-reciprocal
plots of 1/v versus 1/A or 1/B. The numerical values of slopes and y axis
intercepts were extracted from the equations describing these lines.
The intercepts and slopes obtained were replotted versus the reciprocal
concentration of ATP and Kemptide, and the kinetic constants (e.g. for
substrate B) were calculated from the following equations: slope 5
(KiaKb/V)1/B 1 Ka/V and y axis intercept 5 (Kb/V)1/B 1 1/V. It should be
noted that, under the conditions used in these determinations, the
phosphate incorporation was linear with time and with enzyme concentration. The values given in Table II were obtained from three experiments, each including all mutants side by side with the wild-type
enzyme.
Comparison of the X-ray Structures of the Open and Closed Conformations of the C Subunit—This comparison was based on the three
following structures: (a) a closed conformation obtained from the ternary complex of the murine recombinant Ca subunit with the inhibitor
peptide PKI-(5–24) and MnATP, a structure refined to 2.2 Å (51)
(Brookhaven National Laboratory Protein Data Bank code 1ATP,
R1ATPSF); (b) a closed conformation obtained from the binary complex
of the murine recombinant Ca subunit with the inhibitor peptide PKI(5–24) and with detergent, a structure refined to 2.0 Å (52) (Brookhaven
National Laboratory Protein Data Bank code 2CPK); and (c) an open
conformation obtained from the binary complex of the porcine heart C
subunit with the diiodinated inhibitor peptide PKI-(5–24), a structure
refined to 2.9 Å (30) (Brookhaven National Laboratory Protein Data
Bank code 1CTP, R1CTPSF). Distances between individual atoms in
the above-mentioned structures were measured using a Silicon Graphics Indigo computer and an Insight II program (Version 2.3) from
Biosym Technologies (San Diego, CA).
RESULTS AND DISCUSSION
The elucidation of the functional role of the cluster of acidic
amino acids at the carboxyl-terminal tail of the C subunit of
PKA is based on three complementary lines of evidence: (a) on
10177
FIG. 1. Effect of PKA ligands on the cleavage of the C subunit
of PKA by KSMP. The cleavage was carried out under the conditions
described under “Materials and Methods”: with no addition (control;
panel I), with ATP (an excess of 4 mol/mol of C subunit; panel II), with
PKI-(6 –22) (an excess of 4 mol/mol of C subunit; panel III), and with an
equimolar mixture of ATP and PKI-(6 –22) (C subunit/ATP/PKI fragment 5 1:4:4; panel IV). The reaction mixtures (24 ml) contained 1 mM
C subunit and 6.5 mg/ml KSMP. The cleavage was allowed to proceed at
23 °C for the time periods indicated and then arrested as described
under “Materials and Methods.” C9 is KSMP-clipped C.
studies in solution, using the conformation-sensitive cleavage
of the C subunit by KSMP and monitoring the effect of PKA
substrates and inhibitors on this cleavage; (b) on an analysis of
the x-ray structures of the C subunit in two different conformations (open or closed), focusing on the different positioning
of the cluster of acidic amino acids; and (c) on single-site mutations of Tyr330 and a quantitative assessment of the contribution of this residue to the interaction of the C subunit with
its substrates and inhibitors.
Does KSMP Distinguish between the Open and Closed Conformations of the C Subunit?—In an attempt to find out
whether there is a correlation between the open and closed
conformations of the C subunit (recently identified by x-ray
crystallography (9, 10)) and the cleavability of the kinase by
KSMP, we monitored the cleavage of the C subunit by KSMP
under conditions in which the kinase forms binary or ternary
complexes with its substrates or inhibitors, i.e. in molecular
species equivalent to those prevailing in the various crystalline
structures of the C subunit.
The closed conformation of the C subunit was established in
x-ray studies (8 –10) using the ternary complex of the kinase
with ATP and a synthetic peptide derived from the aminoterminal portion of the heat-stable PKA inhibitor (PKI-(5–24))
(Scheme I) (38). To compare the structural evidence obtained in
the crystalline state with the structural evidence obtained in
solution, we carried out a systematic study of the effect of ATP
and of the synthetic PKI-derived peptide on the KSMP cleavage
of the C subunit. As shown in Fig. 1, this cleavage of the C
subunit is essentially unaffected by ATP when present in a 4:1
molar ratio and is somewhat slowed down in the presence of a
4:1 molar excess of a synthetic peptide derived from the aminoterminal portion (residues 6 –22) of PKI. However, when these
ligands are added together to the C subunit, there is a complete
inhibition of the KSMP cleavage (Fig. 1).
By monitoring the initial rates of cleavage by densitometric
scanning of such gels, we obtained a quantitative assessment of
the effect of various ligands on the KSMP cleavage of the C
subunit. As shown in Fig. 2A, ATP alone affords very little
protection from cleavage when added in an excess of up to 10
mol/mol of C subunit. Under the same conditions, the PKI
fragment alone affords a considerable protection, although not
a complete one. However, full protection from cleavage is
achieved under conditions that allow the formation of a ternary
complex (i.e. with a combination of the PKI fragment, ATP, and
the C subunit in a 1:1:1 molar ratio). This is in agreement with
the fact that ATP enhances the affinity of PKA for PKI-(5–24)
by ;10,000-fold (from ;23 mM to 2.3 nM) (33, 49).
The results presented above seem to indicate that open and
closed conformations of the C subunit similar to those observed
10178
Functional Malleability of the Carboxyl-terminal Tail in PKA
FIG. 3. Effect of ATP and its analogs on the cleavage of the C
subunit by KSMP. The experiment was carried out as described in the
legend to Fig. 2.
FIG. 2. Quantitative determination of the cleavage of the C
subunit of PKA in the presence of different molar excesses of
PKI-(6 –22), ATP, ADP, and App(NH)p as well as equimolar combinations of PKI-(6 –22) with ATP or ATP analogs. The cleavage of
the C subunit was carried out as described under “Materials and Methods” and in the legend to Fig. 1. The percentage of cleavage was
determined in each case on the basis of relative initial rates, calculated
from the amount of clipped C subunit formed with time. These values
were extrapolated to time 0 to obtain the given initial rates. Each value
represents the mean 6 S.E. of three separate determinations. The
initial rate of cleavage in the absence of any addition was taken as
100%.
in the crystalline state (9, 10) probably exist also in solution.
Furthermore, they indicate that KSMP can distinguish between such structures, cleaving the C subunit in its open
(loose) conformation(s), but not in its closed (tight) conformation(s). Interestingly, the binary complex of the C subunit with
PKI-(6 –22), which is partially protected from cleavage (Fig.
2A), was found to occur in the crystalline state in both a closed
and an open conformation (9, 10, 30, 52). An explanation for
this finding could be that in the presence of the PKI fragment,
there is an equilibrium between the open and closed conformations and that this equilibrium is shifted to one or the other
direction upon crystallization. However, we cannot exclude the
possibility that the iodinated analog favors a different conformation since the two different conformations observed with the
binary complex were actually obtained with the PKI fragment
itself and with its diiodinated analog, respectively.
In this context, it should be mentioned that when KSMP was
originally identified, we observed that the regulatory subunits
of PKA (without ATP) protect the C subunit from cleavage by
KSMP (18, 19). This is in agreement with the partial protection
afforded by PKI-(6 –22) alone. However, since the regulatory
subunits are large proteins, we could not exclude the possibility
that the protection from cleavage observed with these subunits
is merely due to a steric hindrance, i.e. to a reduced accessibil-
ity of the KSMP cleavage site in the C subunit. In addition, it
was previously shown that although the substrate/pseudosubstrate sites of the regulatory subunits of PKA and those of the
PKI fragment both interact with the C subunit at the catalytic
site, the regulatory subunit-binding site is partially distinct
from the PKI-binding site (53).
Effect of ATP Analogs on the Cleavage of the C Subunit by
KSMP—In an attempt to find out whether the closed conformation of the C subunit observed with the PKI fragment and
ATP prevails not only with a peptide inhibitor but also with a
peptide substrate of the C subunit, we studied the effect of
nonhydrolyzable analogs of ATP on the KSMP cleavage, with
the aim of identifying an analog that can replace ATP in a
ternary complex, without allowing a phospho-transfer reaction
to take place in the course of the experiment.
As shown in Fig. 3, when the effect of ATP is monitored over
a wide range of nucleotide/C subunit molar ratios, protection of
the C subunit from cleavage can be achieved. However, this
occurs at an excess of 200 –300 mol of ATP/mol of kinase, i.e. at
an ATP concentration of 200 –300 mM, well above its Kd. Under
these conditions, GTP does not afford significant protection,
even if its excess is raised to 1000 mol/mol of C subunit (Fig. 3).
A few other nonhydrolyzable analogs of ATP, such as ATPgS,
App(NH)p, and App(CH2)p, or ADP (which has no g-phosphate)
afford some protection from cleavage, although a larger excess
of the analog is required for achieving complete protection of
the C subunit from cleavage (Fig. 3). This raises the possibility
that the closed conformation of the C subunit might be formed
in solution with ATP alone. However, at high concentrations of
nucleotides, the inhibition of the KSMP cleavage may, at least
in part, be due not to its effect on the C subunit, but to an
inhibition of the proteinase itself. This is due to the fact that
KSMP is a metalloenzyme (20), and such nucleotides can bind
the metal ions essential for its activity. In spite of this, the
effect of ATP analogs can be evaluated at low concentrations by
measuring their contribution in conjunction with a peptide
substrate or inhibitor of the C subunit. For example, when
either ADP or App(NH)p is added to the C subunit in addition
to PKI-(6 –22), it enhances the protection of the C subunit from
KSMP cleavage at an essentially stoichiometric molar ratio of
C subunit/PKI fragment/ATP analog (Fig. 2B), i.e. at a low
nucleotide concentration.
The Ternary Complex of the C Subunit with PKS (a Substrate
Analog of PKI) Is Also in the Noncleavable (Closed) Conformation—In view of the possibility of replacing ATP by a nonhydrolyzable analog, we could now assess the protection from
cleavage afforded to the C subunit by PKS (Scheme I) when
alone (binary complex) and when present with a nonhydrolyz-
Functional Malleability of the Carboxyl-terminal Tail in PKA
FIG. 4. A, effects of PKS (Scheme I), of the nonhydrolyzable ATP
analog App(NH)p, and of an equimolar combination of the two on the
cleavage of the C subunit by KSMP; B, effects of Kemptide and of ADP
as well as an equimolar combination of the two on the cleavage of the C
subunit by KSMP. The experiments were carried out under the conditions described in the legend to Fig. 2, except for the differences indicated in the figure itself.
able analog of ATP (ternary complex). It was found that PKS by
itself affords significant protection from cleavage and that
it provides almost complete protection in the presence of
App(NH)p at essentially stoichiometric ratios with the C subunit (Fig. 4A), which is similar to the PKI inhibition (Fig. 2A).
This result indicates that not only an inhibitor, but also a high
affinity phosphorylatable substrate of the C subunit provides
the kinase with considerable protection from cleavage. It
should be noted, however, that the commonly used peptide
substrate of PKA, Kemptide (39) (Scheme I), which does not
contain some of the affinity-contributing side chains in the PKI
fragment (Phe10, which interacts with subsite p-11, and Arg15,
which interacts with subsite p-6 (3, 8, 54, 55)), is not an inhibitor of the KSMP cleavage, even when added in a molar excess
of 400 mol of peptide/mol of C subunit (Fig. 4B). Furthermore,
even when ADP is added together with Kemptide, this peptide
substrate does not protect the C subunit from cleavage: the
partial protection observed (Fig. 4B, å) can be fully accounted
for by ADP alone (Fig. 4B, Ç). The failure of Kemptide (in
contrast to PKS) to provide protection to the C subunit from
KSMP cleavage could be associated with the significantly
higher affinity of the C subunit for PKS (3, 50, 54).
The Malleability of the Carboxyl-terminal Tail of the C Subunit Involves a Distinct Movement of Its Cluster of Acidic Amino
Acids—On the basis of a few crystal structures of the C subunit
that assume open or closed conformations (8, 10, 30), it was
recently concluded (10) that the apoenzyme of the kinase (no
10179
substrate or inhibitor bound) assumes an open conformation,
the binary complex of PKA with PKI-(5–24) alone can assume
an open or a closed conformation, while a ternary complex with
both ATP and PKI assumes a closed conformation (8 –11).
Small angle x-ray scattering and Fourier transform infrared
spectroscopy studies of the C subunit in solution have shown
that this kinase contracts upon complex formation with the
inhibitor peptide PKI-(5–22)-amide or with this peptide and
ATP (29).
The conformational change involved in the conversion of an
open to a closed conformation of the C subunit was described as
a “rotation of the small lobe of the enzyme and a displacement
of the carboxyl-terminal 30 residues” (10). As shown in Fig. 5,
these two conformations distinctly differ in the location of the
cluster of acidic amino acids (327FDDYEEEEI335; highlighted
in yellow), which moves closer to the peptide inhibitor. This
movement is quite pronounced for Tyr330, whose phenolic hydroxyl approaches the nitrogen atoms of the Arg18 side chain
(yellow) stemming from the backbone of the peptide inhibitor
(red). The movement of Tyr330 of PKA toward the Arg18 side
chain of the inhibitor is noteworthy since this arginine residue
is located in a position equivalent to subsite p-3 in PKA substrates and since this subsite constitutes an important specificity element in the consensus sequence of physiological PKA
substrates (3, 39, 54 – 60). This movement of Tyr330 supports
our suggestion that the cluster of acidic amino acids at the tail
of PKA is involved “in creating the preferential affinity of the
kinase for its in vivo substrates” (22, 23).
Table I illustrates the movement of the individual amino acid
side chains of the cluster upon converting the open conformation of the C subunit to its closed conformation. It is clear, for
example, that Phe327, Glu332, Glu333, and Glu334 move 3– 4 Å
closer to distinct atoms in the protein core. It is also clear that
the phenolic hydroxyl of Tyr330 (Fig. 5 and Table I) moves ;7 Å
closer to the charged nitrogen atoms of Arg18, to reach a distance of ;3 Å (Table I).
In an attempt to assess the involvement of Tyr330 in the
substrate recognition process, we prepared three single-site
mutations of the Tyr residue at position 330 and measured the
effect of each of these mutations on the Km and Vmax of the
kinase. As shown in Table II, replacing Tyr330 by Ala results in
an ;6-fold increase in Km for ATP and almost a 30-fold increase
in the Km for the peptide substrate Kemptide. In parallel, there
is a 40-fold decrease in Vmax. Therefore, this single-site mutation of Tyr330 causes a very significant disruption of the catalytic efficacy of the kinase, as evident from an ;1100-fold
reduction in the Vmax/Km ratio for Kemptide (Table II).
A major factor in this disruption is the reduction in the
affinity of the kinase for its peptide substrate, raising the
possibility that Tyr330, and especially the phenolic ring absent
in the Y330A mutant, may contribute directly to the substrate
recognition of the kinase. To further analyze this contribution,
we prepared two other mutants: a Y330F mutant, which does
not possess the phenolic hydroxyl yet still has the phenyl ring,
and a Y330S mutant, which lacks the phenyl ring yet still has
a hydroxyl group (even though it is at a shorter distance from
the peptide backbone). The results depicted in Table II indicate
that both the phenyl ring and the phenolic hydroxyl contribute
to the Km of the kinase for its peptide substrate: the phenyl ring
presumably contributes to maintaining the preferred conformation for peptide and ATP binding by providing packing or
van der Waals interactions with residues in the conserved core,
and the phenolic hydroxyl contributes to an interaction with
the basic amino acid at position p-3, i.e. to the substrate recognition of the enzyme. It should be emphasized that this interaction (possibly a hydrogen bond) is only one out of several
10180
Functional Malleability of the Carboxyl-terminal Tail in PKA
FIG. 5. Upper panel, a-carbon backbone
structures of the open and closed conformations of the C subunit of PKA (10, 30).
The cluster of acidic amino acids with
their side chains (yellow) and the location
of the PKI fragment (red) are highlighted.
Note the approach of Tyr330 (within the
cluster of acidic amino acids) to the Arg
side chain (yellow) stemming from the
peptide inhibitor backbone (red) upon the
conversion of the C subunit from its open
to its closed conformation. Lower panel,
space-filling model of the open and closed
conformations of the C subunit. Note the
change in location of the phenolic ring of
Tyr330 (white arrow) in the open and
closed conformations relative to the nitrogen atoms (dark blue) in the Arg side
chain (yellow) of the peptide inhibitor
(red).
interactions involved in the recognition of the consensus sequence by PKA. This is evident from the fact that both the
Y330F and Y330A mutants are still active enzymes (although
catalytically less efficient).
In search of additional evidence illustrating the importance
of the phenyl ring and the phenolic hydroxyl of Tyr330 in the
catalytic function of the kinase, we tested each of the abovementioned Tyr330 mutants on a protein substrate of the C
subunit. As shown in Table III, the replacement of Tyr330 by
either Ala or Ser significantly lowers the specific activity of the
enzyme even on a protein substrate. A more limited yet still
significant effect was observed with the Y330F mutant. The
difference in the relative effect of the various mutations on the
catalytic function of the enzyme is not clear. However, it might
be associated with the fact that the binding of protein substrates to the C subunit most likely involves multiple interac-
tions, and consequently, a smaller relative contribution is left
to each atom in an individual side chain. In fact, this may be
the reason behind the observation that PKA, which optimally
acts on peptide substrates containing an RRXS/T sequence (39,
58, 60), also acts on protein substrates containing an XRXS/T
sequence, lacking an Arg residue at position p-3 (59).
Concluding Remarks—The results presented above, taken
together with earlier data in the literature, make it reasonable
to conclude that the structural malleability of the carboxylterminal “tail” of the C subunit and specifically the mobility of
its cluster of acidic amino acids (328DDYEEEE334) may have a
functional role in creating the specific affinity of the kinase for
its physiological substrates. This is based on the following
observations. (a) The structural malleability of the C subunit
occurs under physiological conditions, i.e. in solution, at neutral pH, and at physiological values of ionic strength. The
Functional Malleability of the Carboxyl-terminal Tail in PKA
10181
TABLE I
Movement of the cluster of acidic amino acids at the tail of the C subunit
300
305
310
315
320
325
330
335
340
345
350
Tail sequence: TDWIAIYQRKVEAPFIPKFKGPGDTSNFDDYEEEEIRVSINEKCGKEFSEF
cluster of acidics
Distancesa
Residue
Atom
Residue
Movementa
Atom
Open
Closed
(Å)
Phe327
Asp328
Asp329
Tyr330
Glu331
Glu332
Glu333
Glu334
Ile335
Leu173
CE2
CD2
8
(Å)
4
4
5
3
NSMc
7
3
3
4
4
3
3
4
NSM
b
OD2
OH
Lys47
Arg18 d
OE2
OE1
OE1
CG1
Thr48
Arg56
Arg45
Met58
NZ
5
NE
10
Side chain facing protein exterior
OG1
6
NH1
6
NH1
8
CE
4
a
Distances and movements should be taken as approximate values in view of the high temperature factors (B factors) of the amino acid side
chains in this region. Therefore, the values given are rounded to whole Ångstroms. It should be noted, however, that the high B factors are actually
a reflection of the structural malleability in this region of the molecule.
b
In the binary complex structure of the murine recombinant C subunit, the side chain of Arg18 in PKI has two conformations (52). In one of these,
the atom OD2 of Asp328 in the C subunit approaches the atom NE of Arg18 in PKI to reach a hydrogen-bonding distance (;2.5 Å). This interaction
is not seen in the ternary complex of the murine recombinant C subunit.
c
No significant movement (,0.5 Å).
d
This Arg residue is located at position 18 in PKI-(5–24) (p-3 in the consensus sequence).
Km and Vmax
experiments.
TABLE II
Effect of the Tyr330 mutation on the Km and Vmax values of the C subunit as measured with a peptide substrate
values were determined as described under “Materials and Methods.” Each value represents the result (6S.E.) of three different
Km
Mutant
ATP
Kemptide
mM
40 6 5
51 6 5
62 6 7
230 6 15
Wild-type
Y330F
Y330S
Y330A
Specific activitya
10
Wild-type
Y330F
Y330S
Y330A
a
28
Vmax (3106)/Km
ATP
Kemptide
2500
800
500
11
16,700
1300
700
15
nmol/min/
mg
662
31 6 4
45 6 3
170 6 21
TABLE III
Effect of the Tyr330 mutation on the specific activity of the C subunit
as measured with a protein substrate
The specific activity of the wild-type enzyme and of each of the
mutants was determined as described under “Materials and Methods.”
Each value represents the result (6S.E.) of three different determinations.
Mutant
Vmax
% of wild-type
mol P/min/mg
84.2 6 2.8
54.9 6 4.8
6.3 6 0.7
13.7 6 3.1
100.0
65.2
7.5
16.3
The protein used as substrate was histone type II-A.
structural changes associated with this malleability are fully
reversible in solution, and the conformations involved are interconvertible by means of substrates and inhibitors of the
kinase (14 –17, 23–27). (b) Removal of the tail by KSMP (which
recognizes the Asp328–Glu334 stretch) inactivates the kinase
(18 –20, 23). (c) The carboxyl groups in this stretch are accessible to modification in the free C subunit using a water-soluble
carbodiimide, but are protected when the C subunit is complexed with ATP and PKI-(5–24) (28). (d) The catalytic subunit
of the kinase significantly contracts in solution upon forming a
complex with PKI-(5–22)-amide (29). (e) The conditions (C subunit/substrate or inhibitor ratios) favoring or preventing the
KSMP cleavage of the C subunit in solution are equivalent to
those needed to form an open or a closed conformation, respectively, of the C subunit in the crystalline state (this paper). (f)
A detailed analysis of the three-dimensional structures of the C
100 6 4
41 6 6
30 6 4
2.5 6 0.8
subunit in the open and closed conformations (10, 30) shows a
movement (;7 Å) of the phenolic hydroxyl of Tyr330 to reach (in
the closed conformation) a distance of ;3 Å from the nitrogen
atoms of the Arg residue at position p-3 of the PKA consensus
sequence (this paper). (g) Point mutations of the C subunit (e.g.
Y330A) show a significant contribution of Tyr330 to the affinity
of PKA for its substrates/inhibitors and to its catalytic efficacy
(this paper). The carboxyl-terminal tail of the C subunit and
presumably of other protein kinases may therefore constitute
an important biorecognition element for the purpose of specific
catalysis and regulation.
Acknowledgments—We thank Misha Batkin and Lorraine Pelosof for
help in the initial stages of the preparation of PKA mutants and Siv
Garrod for purification of the iodinated peptide PKI-(5–24).
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