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INTRODUCTION |
The cAMP-dependent protein kinases
(cAPKs)1 play a key role in
many signal transduction processes, mediating the majority of the known
effects of cAMP in the eukaryotic cell. These multisubstrate enzymes
regulate the activity of proteins involved in signal transduction, energy metabolism, cell proliferation, or differentiation by
phosphorylation of Ser or Thr residues, which alters the biological
properties of the target proteins (1-3). The activation of a hormone
receptor that is linked by a heterotrimeric G-protein complex to
adenylate cyclase leads to an increase in the concentration of the
second messenger cAMP. Binding of cAMP activates the inactive cAPK
holoenzyme tetramer by inducing dissociation of two monomeric catalytic
(C) subunits from the regulatory (R) subunit dimer. The active C
subunits are now able to phosphorylate target proteins in the cytosol
or can translocate to the nucleus, activating cAMP-mediated gene expression (4). The presence of different isoforms of catalytic (C
, C
, and C
) and
regulatory (RI
, RI
, RII
,
and RII
) subunits suggests specific functions for these
isoforms (5). However, preferential coexpression of any of these C
subunits with either the type I or the type II R subunits has not been
reported. The
and
isoforms of the catalytic subunit have nearly
identical catalytic properties and a broad tissue distribution;
C
is generally the predominant form, whereas the
C
subunit is only found in testis (6). The two general isoforms of the R subunit, type I and type II, differ in molecular weight, tissue specificity, subcellular distribution, and expression pattern during development and cell cycle (2). The RI subunit is mainly
found in the cytoplasm, whereas the RII subunit, which can bind to
protein kinase A-anchoring proteins with high affinity, is localized in
discrete particulate fractions in association with either membrane
organelles or the cytoskeleton (7). The RII subunits contain a Ser at
their autoinhibitory site and are readily autophosphorylated upon
holoenzyme formation. The phosphorylation of this residue lowers the
reassociation rate of the phosphorylated RII subunit with the released
C subunit at least 5-fold (8). The decreased affinity prolongs the
activation of the C subunit unless dephosphorylation of the RII subunit
occurs (9). In contrast, the RI
subunit contains a
pseudophosphorylation site, where the Ser is replaced by Ala and binds
MgATP with high affinity (10, 11). Another class of specific cAPK
inhibitors are the heat-stable protein kinase inhibitors (PKIs) (12,
13). These relatively small proteins bind with high affinity to the C
subunit and favor the nuclear export, thereby blocking the cAMP response element-regulated gene expression (14).
The human protein kinase PrKX is related to the catalytic subunit of
cAMP-dependent protein kinases (15) but is distinct from
the isoforms C
, C
, and C
.
PrKX has 53.2% identity to the human C
subunit of cAPK
(PKA-C
) in the catalytic core region. This degree of
homology is much lower than the similarity of the two human isoforms
C
and C
(90.5% identity). PrKX shows the
highest sequence similarity to the DC2 protein kinase from
Drosophila melanogaster (62.4% identity). DC2 is a homologue of the major Drosophila catalytic subunit gene DC0
and can be inhibited by the heat-stable PKI and the regulatory subunit type I (16).
The catalytic core of PrKX is highly conserved, whereas the N terminus,
which is important for stability and orienting subdomains in
PKA-C
(17), is completely different. PrKX mRNA is
present in a variety of tissues, with the highest levels of expression in fetal and adult brain, kidney, and lung. Low levels of expression are also detected in adult placenta, heart, liver, skeletal muscle, and
pancreas and in fetal liver (15). The gene PRKX is located on the human chromosomal subregion Xp22.3 and has a homologue called
PRKY on the Y chromosome. An abnormal interchange between the X and the Y chromosomes that happens particularly frequently between PRKX and PRKY leads to the sex reversal
disorder of XX males and XY females (18). This report focuses on the
characterization of this novel protein kinase, PrKX, its interaction
with known inhibitors of cAPK, and its regulation by the second
messenger cAMP in vitro and in vivo.
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EXPERIMENTAL PROCEDURES |
Cloning and Construction of PrKX Expression Vectors--
The
cDNA for PrKX was isolated from a random primed HeLa cell library
(19). This cDNA differed from the original isolate described by
Klink et al. (15) by a Val to Ala substitution at position
42. To facilitate purification of the recombinant protein, an
N-terminal His6 tag (MGSSHHHHHHSSG) was added to the full-length construct, which was ligated into the StuI and
HindIII sites of the SV-Sport vector (Life Technologies,
Inc.). High efficiency electroporation of this construct into COS cells
resulted in overexpression of the PrKX protein. A GST-PrKX fusion
protein was produced using the ESP yeast expression system in
Schizosaccharomyces pombe (Stratagene). A
SmaI/HindIII fragment was isolated from SVPrKX
and cloned into the StuI/HindIII sites of the HTb
shuttle vector (Life Technologies, Inc.), a
BamHI/HindIII fragment was isolated, and the
HindIII site was filled in with Escherichia coli
DNA polymerase Klenow fragment. This fragment was cloned into the
BamHI/SmaI sites of ESP.
Expression and Purification of PrKX--
PrKX expressed in COS
cells was purified using Ni-nitrilotriacetic acid affinity
chromatography as described previously (19). Fractions containing the
highest concentration of PrKX as determined by Coomassie staining of
SDS-polyacrylamide gels were pooled, dialyzed against storage buffer
(20 mM Tris, pH 8.0, 150 mM NaCl, 0.25%
Nonidet P-40, 20% glycerol, 1 mM dithiothreitol), and
stored at
20 °C. GST-PrKX was overexpressed in S. pombe, purified using glutathione-agarose, and eluted with 10 mM glutathione (21).
Purification of PKA-C
, RI
,
RII
, and GST-PKI--
Wild-type recombinant
PKA-C
was overexpressed in E. coli BL21.DE3
and purified by phosphocellulose chromatography as described previously
(22). The fractions were further purified using Mono S chromatography
(Amersham Pharmacia Biotech) (23). Recombinant RI
and
RII
subunits were overexpressed in E. coli
222, purified as described previously (24), and stored at
20 °C.
To obtain cAMP-free R subunit, the R subunit was unfolded with 8 M urea and refolded as described by Buechler et
al. (25). Protein purity was checked by SDS-PAGE (26). GST-PKI was
overexpressed in E. coli BL21.DE3, purified using
glutathione-agarose, and eluted with 10 mM glutathione
(21).
Kinetic Assays--
The specific activity was determined using
the spectrophotometric assay of Cook et al. (27) and the
heptapeptide Kemptide (LRRASLG, Bachem) as substrate. The standard
assay was performed at 22 °C in Buffer C (100 mM MOPS,
pH 7.0, 1 mM ATP, 10 mM MgCl2, 1 mM dithiothreitol) with 250 µM Kemptide.
Alternatively, activity was measured by a modified radioactive assay
according to Roskoski (28) using Buffer D (20 mM MOPS, pH
7.0, 50 mM KCl, 1 mM ATP, 10 mM
MgCl2, 1 mM dithiothreitol) at 30 °C. The
Km values for ATP and Kemptide were determined based
on Michaelis-Menten kinetics, and data evaluation was performed using
the software GraphPad Prism. The effect of the free Mg2+
concentration on PrKX activity was examined using varying
concentrations of Mg2+ and constant amounts of ATP and
substrate. The free Mg2+ concentrations were calculated
using the software Bound and Determined based on equations provided by
Brooks and Storey (29). The heat denaturation studies were performed
according to Yonemoto et al. (30).
Autophosphorylation--
1.2 µM PrKX was incubated
in Buffer D with [
-32P]ATP at 30 °C for 60 min. The
inhibition of autophosphorylation was tested by adding a 2-fold molar
excess of cAMP-free RI
or RII
subunit in
either the absence or presence of 20 µM cAMP; adding a
2-fold molar excess of GST-PKI or 20 mM EDTA; or heating
PrKX at 60 °C for 5 min prior to the incubation. The
autophosphorylation was stopped by adding SDS sample buffer. The
samples were analyzed by SDS-PAGE and autoradiography.
Inhibition Studies with GST-PKI, RI
, and
RII
--
60 nM PrKX was incubated with
increasing amounts of GST-PKI and tested for activity with the
spectrophotometric assay in Buffer C at 22 °C. The
Ki values were determined using the equation of
Cheng and Prusoff (31) based on KD values determined by surface plasmon resonance (SPR). For inhibition studies with the
regulatory subunits, 100 nM PrKX was incubated with
increasing amounts of either cAMP-free RI
subunit or
RII
subunit in Buffer C at 22 °C and tested for
Kemptide phosphorylation activity with the spectrophotometric assay.
Determination of Activation Constants (Ka) for
cAMP--
Holoenzyme at a concentration of 5 nM formed by
mixing PKA-C
or PrKX and cAMP-free RI
subunit in a 1:1.2 molar ratio in Buffer D was incubated for 2 min at
room temperature with varying concentrations of cAMP between 1 nM and 2.5 µM. The reaction was started by
adding 1 mM Kemptide, and the resulting phosphotransferase activity was measured using the radioactive assay.
SPR--
SPR experiments were performed using a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden). Here, the interaction between an immobilized component, referred to as the ligand, and a molecule in
the mobile phase, the analyte, was determined. Changes in surface concentration are proportional to changes in the refractive index on
the surface resulting in changes in the SPR signal, plotted as response
units (RU). 1000 RU correspond to a surface concentration of 1 ng/mm2 (32). The association and dissociation of cAMP-free
RI
was determined on a surface containing 580 RU of
GST-PrKX immobilized via an
GST antibody at a flow rate of 30 µl/min in Buffer H. To determine the interaction between PKI and
PrKX, 160 RU of GST-PKI were immobilized using the
GST antibody
surface, whereas for the interaction with PKA-C
the
immobilization level was reduced to 60-75 RU. A lower immobilization
level of the ligand avoids rebinding effects in the dissociation phase
that can arise due to fast on-rates. The regeneration of the antibody
surface, i.e. the removal of the complete protein complex
bound to the antibody, was performed using 10 mM glycine
(pH 2.2) or 0.05% SDS and resulted in a completely regenerated and
functional antibody surface. For the measurement of the interaction
between PrKX or PKA-C
and the RII
subunit, the C subunits were covalently coupled to a CM5 chip via
primary amines (300 RU of His6-PrKX and 475 RU of
PKA-C
) as described (33), and the cAMP free
RII
subunit was injected at a flow rate of 10 µl/min
in Buffer G or Buffer H. All buffers used contained 0.005% surfactant
P20. The regeneration of the surface was performed in Buffer G with 100 µM cAMP and 2.5 mM EDTA. To determine
unspecific binding, blank runs were performed on a control surface
without ligand, and these values were subtracted. Kinetic constants
were calculated by nonlinear regression of data using the Biaevaluation
software, version 2.1 or 3.0 (Biacore). The association rate constant
was calculated according to the equation,
|
(Eq. 1)
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where R is the SPR signal in response units,
ka is the association rate constant,
kd is the dissociation rate constant, and
C is the concentration of the injected analyte at any time
t during association. This equation describes the response
at any time during association and can be used for nonlinear regression
analysis of single curves. The dissociation rate constant was
calculated according to the equation,
|
(Eq. 2)
|
where Rt is the response at time
t, and R0 is the time at an arbitrary
starting point t0. With the rate constants determined and known analyte concentrations, the equilibrium binding constants were calculated according to the following equation.
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(Eq. 3)
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A 1:1 binding model assuming Langmuir conditions was applied to
the data.
Phosphorylation of RII
Subunit--
The
phosphorylation of RII
(2 µM) by catalytic
amounts of PrKX or PKA-C
(2.4 nM) was
performed with the radioactive assay at 30 °C. Aliquots of the
phosphorylated RII
subunit were spotted onto
phosphocellulose filters, washed three times in 0.5%
o-phosphoric acid for 10 min, and counted using a liquid scintillation counter. In parallel, aliquots were analyzed by SDS-PAGE
and autoradiography. Prephosphorylation of RII
(2 µM) was performed with catalytic amounts of
either PKA-C
or PrKX (2.4 nM) in Buffer D
using unlabeled ATP (1 mM). At certain time points,
[
-32P]ATP and the other kinase, PrKX or
PKA-C
, in the same concentration were added to the
sample. The effect of prephosphorylation on 32P
incorporation was demonstrated by SDS-PAGE and autoradiography.
Microinjection Experiments--
10T1/2 mouse embryo fibroblasts
were propagated in Dulbecco's modified Eagle's medium with 10% fetal
bovine serum to 50% confluence and then microinjected with plasmids
encoding HA-tagged PrKX and the mutant regulatory subunits RI(R209K)
and RII(R213K), which are deficient in their response to cAMP. To show
the effect of cAMP, cells were treated for 15 min with 1 mM
dibutyryl-cAMP before fixing the cells. Cells were fixed after 6 h
with 4% paraformaldehyde in PBS for 10 min, washed twice with PBS, and
permeabilized with 0.2% Triton X-100 in PBS for 15 min. The reaction
with the primary antibody was performed for 1 h at 37 °C with
monoclonal antibody against HA (BABCO) at a dilution of 1:200 in PBS
containing 0.5% Nonidet P-40 and 5 mg/ml bovine serum albumin. The
cells were then washed twice with PBS for 5 min at room temperature
before the secondary antibody reaction with a rhodamine-labeled donkey anti-mouse antibody (Jackson ImmunoResearch) at 1:200 dilution for 45 min at 37 °C. Injected cells were photographed with a Zeiss Axiophot
fluorescence microscope under a × 63 oil immersion lens.
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RESULTS |
Expression, Purification, and Biochemical Characterization of
PrKX--
The expression of PrKX in eukaryotic cells (COS cells,
S. pombe) yielded catalytically active protein kinase, which
was used for the characterization. Initial preparations of PrKX
overexpressed in bacteria were inactive and copurified with the
bacterial chaperon GroEl. Inactivity and association with the chaperon
most likely indicates folding problems, possibly due to a lack of
posttranslational modifications, i.e. phosphorylation (34).
Purification of PrKX was simplified by using fusion constructs with
either an N-terminal His6 tag that can easily be purified
with Ni-NTA affinity chromatography or a GST fusion tag (Fig.
1A). A wash step with cAMP (1 mM) was included in the purification strategy due to
cross-contamination with endogenous PKA-C
from COS
cells, which was initially detected by Western blot analysis with a
specific antibody against PKA-C
and by using mass
spectrometry.2 After this
wash step, no contamination with endogenous PKA-C
could
be detected by Western blot analysis (Fig. 1B). The specific activity of PrKX determined either by the coupled spectrophotometric assay (27) or the more sensitive radioactive assay using the cAPK
standard substrate Kemptide was 1.5 ± 0.7 µmol/(min·mg), 20-fold less than the specific activity for PKA-C
. The
Km values for ATP (127 ± 9 µM)
and Kemptide (58 ± 7 µM) were significantly higher
compared with the wild-type or the His6-tagged
PKA-C
(Table I). PrKX
phosphotransferase activity had an absolute requirement for
Mg2+. PrKX displayed the highest activity at micromolar
concentrations of free Mg2+, whereas higher concentrations
of free Mg2+ had inhibitory effects on PrKX activity (Fig.
2A). PrKX was also autophosphorylated. Autophosphorylation was inhibited by the addition of EDTA (see Fig. 4), chelating the Mg2+. This result
further confirmed the absolute requirement for Mg2+. Heat
denaturation experiments were performed to determine the thermal
stability of PrKX and yielded Tm values of 41.8 ± 0.2 °C for PrKX and 45.3 ± 0.2 °C for PKA-C
(Fig. 2B). Thus, the thermostability of PrKX is only
slightly reduced and comparable to PKA-C
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Fig. 1.
Expression and purification of PrKX.
A, SDS-PAGE analysis of His6-PrKX overexpressed
in COS cells, GST-PrKX overexpressed in S. pombe, and
PKA-C overexpressed in E. coli. The lanes are
labeled as follows: M, molecular mass standard; lane
1, His6-PrKX; lane 2, GST-PrKX; lane
3, PKA-C . The proteins were prepared as described
under "Experimental Procedures," and the gel was stained with
Coomassie Brilliant Blue. B shows an immunoblot with an
PKA-C antibody using different concentrations of
PKA-C as indicated on the blot and 2 µg of
His6-PrKX from COS-cells after a wash with cAMP. A
contamination with PKA-C in the preparation of PrKX
could not be detected.
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Table I
Kinetic properties of His6-tagged PrKX, wild-type and
His6-tagged PKA-C
S.E. values are given using at least three different preparations. Data
were fit assuming Michaelis-Menten conditions.
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Fig. 2.
Biochemical characterization of PrKX.
A shows the influence of Mg2+ on PrKX
phosphotransferase activity determined with the spectrophotometric
assay. ATP was used in a concentration of 1 mM in all
assays, and the free Mg2+ concentrations, calculated
according to Brooks and Storey (29), were varied as indicated.
B, thermostability of His6-PrKX ( ) and
wild-type PKA-C ( ). Thermal denaturation was carried
out in 20 mM MOPS, 150 mM KCl, 1 mM
dithiothreitol, pH 7.0, for 3 min. The remaining phosphotransferase
activity was determined in comparison to an untreated sample using the
spectrophotometric assay. Error bars denote sample S.D.
values.
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Regulation of PrKX Activity--
The effects of physiological
inhibitors of PKA-C
(the RI
subunit, the
RII
subunit, and PKI) on PrKX were investigated. Both
binding of these inhibitors and inhibition of phosphotransferase activity were quantified using different methods. The
autophosphorylation of PrKX (Fig. 3) was
significantly but not completely inhibited by a 2-fold molar excess of
cAMP-free RI
subunit or GST-PKI. In contrast, the
addition of a 2-fold molar excess of cAMP-free RII
subunit did not have any effect on PrKX autophosphorylation whether
cAMP was present or not. Phosphotransferase activity of PrKX using
Kemptide as substrate was significantly inhibited by both GST-PKI and
the RI
subunit (Fig. 4,
A and B). A 2-fold molar excess
of PKI or RI
subunit inhibited 80%
(Ki = 7 nM) or 95%
(Ki = 1 nM) of this activity,
respectively. Initial slopes of the inhibition curves indicated 1:1
stoichiometry. However, a 2-fold molar excess of RII
subunit did not lead to a detectable decrease of phosphotransferase
activity (Fig. 4B). Addition of a 25-fold molar excess of
RII
subunit was necessary to cause a 40% inhibition,
suggesting a much weaker inhibition by the RII
subunit
in the presence of ATP. This partial inhibition by the
RII
subunit could also be reversed by cAMP (Fig.
4C).

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Fig. 3.
Inhibition of autophosphorylation of
His6-PrKX. Autophosphorylation of PrKX (lane
1) in the presence of [32P]ATP and Mg2+,
detected by SDS-PAGE and autoradiography, was inhibited by the addition
of a 2-fold molar excess of RI subunit (lane
2) and GST-PKI (lane 6). The inhibition by the
RI subunit was reversed by cAMP (100 µM)
(lane 3). The RII subunit in the same molar
excess did not inhibit autophosphorylation of PrKX; however, the
RII subunit itself is phosphorylated by PrKX (lane
4). Addition of cAMP (100 µM) (lane 5)
did not change the autophosphorylation of PrKX in the presence of the
RII subunit. Addition of 20 mM EDTA
chelating the Mg2+ or heating of PrKX for 5 min at 60 °C
prior to the incubation completely abolished autophosphorylation
(lanes 7 and 8). The lower bands in
lanes 4 and 5 are proteolytic fragments of the
RII subunit.
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Fig. 4.
Regulation of PrKX phosphotransferase
activity. A, inhibition of His6-PrKX (60 nM) by increasing concentrations of GST-PKI; B,
inhibition of His6-PrKX (100 nM) by
RI ( ) and by RII subunit ( ) in
molar ratios as indicated on the plot. Clearly, no inhibition of PrKX
activity could be observed with RII at concentrations at
which RI completely abolished activity. C,
inhibition of 100 nM His6-PrKX with
RII subunit at higher concentrations. Even with a
25-fold molar excess, only 40% inhibition was achieved. However, this
partial inhibition could be reversed in the presence of cAMP.
D, activation constant (Ka) for cAMP
using holoenzymes of PrKX ( ) and PKA-C ( ) with the
RI subunit. The complexes were formed in the presence of
ATP/Mg2+ and activity was determined using the kinetic
assays as described under "Experimental Procedures." Error
bars denote sample S.D.
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Activation by cAMP--
The inhibition of PrKX activity by the
RI
subunit of cAPK could be reversed by the addition of
cAMP, as demonstrated in the autophosphorylation experiment (Fig. 3).
To further examine the activation by cAMP, the PrKX/RI
holoenzyme was incubated with varying concentrations of this second
messenger, and phosphorylation of Kemptide was determined. The
activation constant (Ka) for the
PrKX/RI
holoenzyme is 43 nM (Fig.
4D), which is 2.5-fold lower compared with the
Ka for the PKA-C
/RI
holoenzyme (109 nM). Hill coefficients of the activation
constants constants are 2 and 2.4 for
PKA-C
/RI
and PrKX/RI
, respectively, suggesting a cooperative activation of both holoenzymes.
Interaction Studies Using SPR--
To demonstrate the interaction
between PrKX and its physiological inhibitors, SPR was employed. With
this method, association and dissociation rate constants were directly
determined, and the binding behavior of the inhibitors of cAPK, the
RI
subunit, the RII
subunit, and PKI was
further defined. The interaction between PrKX and the RI
subunit was examined using amine-coupled His6-tagged PrKX
or GST-PrKX, immobilized on an
GST antibody surface. As an analyte,
the cAMP-free RI
subunit was injected in the presence of
1 mM ATP/10 mM MgCl2 (Fig.
5). Regeneration of the surface was
performed with cAMP. The equilibrium binding constants
(KD) for the His6-tagged and GST-PrKX
were 5.4 and 4.9 nM, respectively, which suggests that the
different fusion tags do not influence the affinity to the
RI
subunit. However, the association and the
dissociation rate constants were affected slightly (Table
II). Comparable rate constants for the interaction between RI
subunit and either amine-coupled
His6-tagged or wild-type PKA-C
indicated
that the N-terminal His6 tag also did not change the
affinity to the RI
subunit (Table II). Only
GST-PKA-C
showed a slight difference, with a 2-fold lower dissociation rate constant.

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Fig. 5.
High affinity binding of GST-PrKX to the
RI subunit determined by SPR.
GST-PrKX was immobilized to an GST-antibody surface as described
under "Experimental Procedures," and cAMP-free RI
subunit in the concentrations indicated on the plot was injected
in the presence of ATP/Mg2+. The association and
dissociation phases were monitored for 300 s by following the
change in SPR signal, given in RU. Association and dissociation phases
are indicated on the plot.
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Table II
Rate and affinity constants for the interaction of the RI
subunit with PrKX and PKA-C using SPR
Association rate constants (ka), dissociation rate
constants (kd), and equilibrium binding constants
(KD) were determined for the interaction of
different fusion proteins of PrKX and PKA-C as immobilized
ligands with cAMP-free RI subunit as analyte in the presence
of ATP/Mg2+. His6-PrKX, His6-tagged
PKA-C , and wild-type PKA-C were immobilized via
amine-coupling to a CM5 surface, GST fusion-tagged PKA-C and
PrKX were immobilized via an GST antibody. Although there are
differences in the apparent rate constants, the KD
values of the interactions between the same species agree very well.
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PKI was immobilized site-directed via the GST fusion tag on an
GST
antibody surface. Because no physiological dissociation mechanism is
known, the protein complex was completely removed from the antibody
surface after each interaction analysis and subsequently reimmobilized
with GST-PKI at the same concentration level. KD
values of 15 and 0.5 nM were determined for PrKX and
PKA-C
, respectively, in the presence of 1 mM
ATP/10 mM MgCl2 (Fig.
6). The association rate constant for
PrKX was 4-fold lower, and the dissociation rate constant was 7.5-fold faster than for PKA-C
(Table
III).

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Fig. 6.
PKI binds with high affinity to
His6-PrKX and
PKA-C . GST-PKI was
immobilized to an GST antibody surface as described under
"Experimental Procedures." The association and dissociation phases
of His6-PrKX (A) and PKA-C
(B) injected in the presence of ATP/Mg2+ were
monitored by following the change in SPR signal, given in RU. The
concentrations of the analytes are given on the plots.
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Table III
Rate and affinity constants for the interaction of PrKX and
PKA-C with PKI using SPR
Association and dissociation rate constants and equilibrium binding
constants were determined from the interactions of His6-tagged
PrKX and PKA-C as analytes with GST-PKI immobilized via an
GST-antibody in the presence of ATP/Mg2+ as described under
"Experimental Procedures."
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Initial data suggested that the PrKX/RII
subunit
interaction is dependent on the absence of MgATP. This interaction was measured both in the absence and in the presence of MgATP using again a
variety of immobilization strategies and fusion tags. Similar binding
constants were obtained.2 In the absence of MgATP, the
KD value for the PrKX/RII
interaction
was 43 nM. However, in the
presence of MgATP, the KD value
increased dramatically to 2.3 µM (Fig. 7,
C and D; Table IV). In
comparison, the KD
values for the PKA-C
/RII
interaction were
0.1 nM in the absence and 0.7 nM in the
presence of MgATP (Fig. 7, A and B; Table
IV).

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Fig. 7.
Binding of
RII to
PKA-C and His6-PrKX in
the absence and presence of MgATP using SPR. PKA-C
(A and B) or PrKX (C and D)
was covalently immobilized via primary amines on a CM5 surface as
described under "Experimental Procedures." cAMP-free
RII subunit was injected in the concentrations indicated
on the plots. Measurements were performed in the absence (A
and C) or in the presence (B and D) of
1 mM ATP/10 mM MgCl2.
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Table IV
Interaction studies with RII subunit using SPR
Rate and affinity constants were determined for the interaction of
His6-tagged PrKX and wild-type PKA-C immobilized via
amine coupling with cAMP-free RII subunit as the analyte in
the absence or presence of ATP/Mg2+ as described under
"Experimental Procedures."
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Phosphorylation of RII
Subunit--
The results of
the autophosphorylation assay (Fig. 3) demonstrated that the
RII
subunit is an excellent substrate for PrKX. To
determine the rate of phosphorylation of the RII
subunit
2 µM RII
subunit were phosphorylated with
catalytic amounts (2.4 nM) of either PrKX or
PKA-C
in the absence or in the presence of 50 µM cAMP. In the presence of cAMP, there was a
32P incorporation of 0.95 mol/mol and 0.5 mol/mol after 20 min using PKA-C
and PrKX, respectively (Fig.
8A). Using higher
concentrations of both C subunits, a stoichiometry of about 1 mol/mol
was achieved, suggesting that a single residue is phosphorylated by
both C subunits.2 The initial slope of the phosphorylation
kinetic was used to determine the specific activity when using
RII
subunit as a substrate (Fig. 8A). In the
absence of cAMP, there was only low phosphorylation of
RII
by PKA-C
(0.07 ± 0.01 µmol/(min·mg)). However, upon the addition of cAMP, there was an
18-fold increase in activity (1.25 ± 0.08 µmol/(min·mg)). In
contrast, the RII
subunit was phosphorylated by PrKX
with a specific activity of 0.36 ± 0.06 µmol/(min·mg) in a
cAMP-independent manner, which is 3.5-fold lower than the
phosphorylation by PKA-C
in the presence of cAMP but
5-fold higher than the phosphorylation by PKA-C
in the
absence of cAMP. To determine whether PKA-C
and PrKX
phosphorylate the same residue on the RII
subunit, the
RII
subunit was prephosphorylated by either
PKA-C
and PrKX using unlabeled ATP. The phosphorylated
RII
was then used as a substrate in a second
phosphorylation experiment containing radiolabeled ATP and the other
kinase. In both cases, prephosphorylation led to a significant decrease
of 32P incorporation, indicating that PKA-C
and PrKX phosphorylate the same residue (Fig. 8B).

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Fig. 8.
Phosphorylation of
RII subunit by
His6-PrKX and PKA-C in
the absence and presence of cAMP. A, the
phosphorylation of RII subunit by PrKX ( , ) and
PKA-C ( , ) measured for 20 min using the assay
according to Roskoski (28). The open symbols indicate
phosphorylation in the absence of cAMP, and the closed
symbols indicate phosphorylation in the presence of cAMP.
Error bars denote sample S.D. B, phosphorylation
of RII by PrKX (lanes 1 and 2) or
by PKA-C (lanes 5 and 6),
demonstrated by SDS-PAGE and autoradiography, was performed in the
absence (lanes 1 and 5) and in the presence
(lanes 2 and 6) of 100 µM cAMP. In
a separate experiment, RII was first prephosphorylated
using PKA-C and unlabeled ATP for 5 min (lane
3) and 10 min (lane 4) and then incubated with
[ -32P]ATP and PrKX .The opposite experiment was
performed by prephosphorylating with PrKX and unlabeled ATP for 10 min
(lane 7) and 20 min (lane 8) and then incubating
with [ -32P]ATP and PKA-C . The
lower protein bands (lower molecular weight) are proteolytic
fragments of RII .
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In Vivo Interaction in Microinjected Cells--
Plasmids encoding
HA-tagged PrKX were microinjected in mouse embryo 10T1/2 fibroblasts.
PrKX expressed in vivo localized to the cytoplasm, but
addition of the membrane permeable dibutyryl-cAMP to the microinjected
cells induced a translocation of PrKX into the nucleus within about 15 min (Fig. 9). This suggests that PrKX interacts with endogenous regulatory subunit of
cAMP-dependent protein kinase present in the cytoplasm and
dissociates from the regulatory subunits in response to cAMP. To
clarify whether the cytoplasmic localization is due to interaction with
the regulatory subunit type I or type II, coinjections with plasmids
encoding mutant R subunits deficient in their response to cAMP were
performed. The coexpression of PrKX with RI
(R209K) shows
a cytoplasmic localization of PrKX in the absence and in the presence
of cAMP, whereas the coexpression with the corresponding mutant
RII
(R213K) shows the same localization pattern as for
PrKX alone (Fig. 9, A-C). These in vivo
experiments clearly demonstrate that the regulatory subunit type I is
the interacting regulatory protein for PrKX, whereas the mutant
RII
subunit is not able to keep PrKX in the cytosol.

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Fig. 9.
Microinjection of plasmids encoding HA-tagged
PrKX in mouse embryo 10T1/2 fibroblasts. A-C show an
immunostain of cells microinjected with PrKX after 6 h. Cells were
photographed either before (A) or after (B and
C) addition of dibutyryl-cAMP, as indicated at the
top. After 15 min, a clear translocation to the nucleus was
visible (C). D-G show cells in which PrKX was
coinjected with plasmids containing mutant forms of the RI subunit
(D and E) and the RII subunit (E and
F) in the absence (D and F) and
presence (E and G) of dibutyryl-cAMP. Both mutant
proteins are deficient in their ability to bind cAMP. Only in case of
the RI(R209K) subunit was PrKX kept in the cytosol, whereas RII(R213K)
had no effect on the subcellular localization.
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DISCUSSION |
Based on sequence alignments with the catalytic subunits of cAPKs
from several organisms, the novel human protein kinase PrKX is
predicted to belong to the family of cAMP-dependent protein kinases. Comparison of primary sequences showed conservation of many
residues that have been demonstrated to be important for substrate
recognition and the interaction with the regulatory subunits type I and
type II and PKI in PKA-C
. This includes residues that
are important for binding the substrate consensus sequence,
RRX(S/T)Y, i.e. the binding site for
the P-2 Arg (Glu-170 and Glu-230), the P-3 Arg (Glu-127 and Asp-328)
and the P+1 (Leu-198, Pro-202, and Leu-205) (35), but are not conserved
throughout the protein kinase family (36). There is only one
conservative change, from Asp-328 to Glu in PrKX. The P-site and
Y are referred to as the phosphorylation site and a
hydrophobic residue, respectively. Therefore, it is likely that the
substrate recognition sequences of PrKX and PKA-C
are
similar, and Kemptide, the standard cAPK peptide substrate (LRRASLG),
can be used as substrate for PrKX. Despite higher Km
values and a 20-fold lower specific activity compared with
PKA-C
, the kinetic properties of PrKX from COS cells
still indicate that PrKX is a PKA-like kinase. The phosphotransferase
activity of PrKX has an absolute requirement for Mg2+ but
is reduced in the presence of millimolar concentrations of free
Mg2+, suggesting that PrKX also has two metal binding
sites, one activating and one inhibiting site, similar to
PKA-C
(37).
Several lines of evidence demonstrate that PrKX is a novel catalytic
subunit of the cAMP-dependent protein kinase: 1)
autophosphorylation and Kemptide phosphorylation of PrKX could strongly
be inhibited by the RI
subunit. The inactive holoenzyme
consisting of the RI
subunit and PrKX is activated at
2.5-fold lower concentrations of cAMP compared with the holoenzyme
consisting of RI
and PKA-C
. This
increased sensitivity to cAMP was also found for the holoenzyme
containing the neural form of the RI subunit, RI
(38)
and suggests an earlier response to cAMP. 2) Binding of both R
subunits, determined by SPR as described under "Results," can be
reversed completely by the addition of cAMP. 3) Microinjection with
plasmids encoding PrKX showed a nuclear translocation in response to a
membrane permeable analog of cAMP, dibutyryl-cAMP, indicating that PrKX
expressed in mouse embryo 10T1/2 fibroblasts is inhibited by endogenous
R subunit in the absence of cAMP. In the presence of cAMP, PrKX is
released and can translocate to the nucleus.
A major difference from PKA-C
is the regulation by the R
subunits. Autophosphorylation of PrKX was significantly inhibited by
the RI
subunit but was not affected by the
RII
subunit in the presence of ATP. Kemptide
phosphorylation by PrKX was inhibited stoichiometrically by the
RI
subunit but only by a large molar excess of the
RII
subunit. This suggests a much lower affinity to the
RII
subunit in the presence of physiological concentrations of MgATP. Using SPR, these data were confirmed and
compared with the interaction of PKA-C
. The
KD value for PrKX and the RI
subunit
determined in the presence of ATP is 5 nM, whereas the
corresponding value for PKA-C
is in the subnanomolar
range and corresponds well with the value reported by Hofmann (10). The
affinity of PrKX for the RII
subunit is strongly
dependent on the MgATP. In the absence of MgATP, the affinity to
RII
(KD = 43 nM) was
430-fold less than for PKA-C
. In the presence of
physiological concentrations of MgATP, the affinity decreased to a
KD of 2.3 µM, which is almost 2 orders
of magnitude lower. In contrast, the decrease in affinity was only
7-fold in the case of PKA-C
(Table IV). Comparing now
the KD values between PKA-C
and PrKX in the presence of MgATP, a 3300-fold difference was observed. These
data imply that under physiological conditions the RII
subunit is a potent inhibitor for PKA-C
but not for
PrKX.
Coinjection of DNA encoding PrKX with DNA encoding mutant forms of the
RI and the RII subunit further demonstrated that only the RI subunit,
not the RII subunit, is able to keep PrKX in the cytosol. Both mutant
proteins, RI(R209K) and RII(R213K), are deficient in cAMP binding to
the cAMP binding site A (39). RI(R209K) mutant protein does not respond
to dibutyryl-cAMP at the concentrations used, preventing the release of
PrKX as shown in Fig. 9E. If the cells are injected with a
plasmid encoding the RII(R213K) mutant protein, the same concentration
of dibutyryl-cAMP causes a translocation of PrKX to the nucleus,
indicating that PrKX is kept in the cytosol, most likely by the
endogenous RI subunit, and is not anchored by the cAMP-insensitive
mutant RII subunit.
Inhibitors of cAPK, such as PKI and the RI subunit, contain
pseudo-substrate sites, whereas the RII subunit is a substrate of
PKA-C
that inhibits with a subnanomolar binding constant (10). The phosphorylation of the RII
subunit in the
presence of cAMP leads to an at least 5-fold reduction in the affinity for PKA-C
(8), which is in excellent agreement with the SPR studies presented here. PrKX also phosphorylates the
RII
subunit, but neither autophosphorylation nor
Kemptide phosphorylation is significantly inhibited by
RII
subunit. Thus, PrKX phosphorylates the
RII
subunit in a cAMP-independent manner, whereas
PKA-C
in catalytic amounts phosphorylates the
RII
subunit significantly only in the presence of this
second messenger. The phosphorylation of the RII
subunit
occurs at the autoinhibitory site, and PrKX most likely phosphorylates
the same residue as PKA-C
, as demonstrated by the
prephosphorylation assay. Because the phosphorylation of the RII
subunit lowers the reassociation rate of the type II holoenzyme, PrKX
could modulate the activation state of PKA when its catalytic subunit
associates with the RII subunit.
Another important role of the regulatory subunits type I and II is to
target the C subunits to distinct subcellular compartments. The RI
subunit serves as an cytoplasmic anchor for the C subunit, as well as
for PrKX, as demonstrated here, whereas the RII subunit keeps the C
subunit at specific organelles via specific protein kinase A-anchoring
proteins (7). A dual-specificity protein kinase A-anchoring protein
that binds both RI and RII subunits has been recently described (40).
In this way, the RI and the RII subunits may serve to localize the C
subunit close to relevant substrates or local pools of cAMP, providing
distinct functions for these isoforms. Indeed, it has been suggested
that the type I isoform is a positive effector of growth, and the type
II isoform relates more to tissue differentiation (2). Besides the
possibility that PrKX as a type I cAPK might have a specific function
in this pathway itself, the alteration of the affinity for one of the regulatory subunits, i.e. by phosphorylation of the RII
subunit by PrKX, might change the balance between both isoforms or the subcellular localization of PKA-C
. Therefore, PrKX may
act to modulate the distinct functions of these isoforms.
Although the crystal structure of the holoenzyme has not been solved,
some important features for the interaction between the C and R
subunits have been determined. In addition to the pseudosubstrate site,
two basic residues (Lys-213 and Lys-217) have been demonstrated to be
essential for holoenzyme formation with the RI
subunit
(41). Both are conserved in PrKX, with only a conservative change from
Lys-217 to Arg. The N terminus of PKA-C
contains a
myristylation motif and a long amphipathic helix (A-helix), which is
important for the structural integrity and the correct orientation of
subdomains at the cleft interface. Mutations in the N terminus strongly
affect the activation of the RII
but not the
RI
holoenzyme (17). The N terminus of PrKX is completely
different from that of PKA-C
and lacks both the
myristylation motif and the long amphipathic helix. However, PrKX has a
proline-rich motif that might be a putative binding site for SH3 or WW
domains. Previous work has demonstrated that SH3 domains recognize
sequences containing proline and hydrophobic residues (42), and it is
already clear that the protein-protein interactions mediated by these
domains play an important role in the control of different signaling
pathways (43). The WW domain is a protein module present in a number of
signaling and regulatory proteins and is known to be a binding site for
proline-rich peptides (44). The differences in the N terminus, the
preferential binding of PrKX to one of the regulatory subunits, and the
phosphorylation of the RII
subunit are important
features that distinguish PrKX from PKA-C
not only in
structure but also in subcellular localization and involvement in other
signal transduction pathways in the eukaryotic cell.
PKI, a specific inhibitor of PKA-C
(12), also interacts
with PrKX with high affinity, as demonstrated by SPR. This interaction
significantly inhibits Kemptide phosphorylation and autophosphorylation
activity of PrKX. The KD value for PKA-C
and GST-PKI is about 0.5 nM,
determined for the first time by SPR, and corresponds well with the
value determined by Whitehouse and Walsh (45), whereas the
KD for PrKX and GST-PKI (KD = 15 nM) is 30-fold higher compared with PKA-C
.
Although the physiological function of PKI remains unclear, one role
may be as a transport protein for the nuclear export of
PKA-C
(14, 46), thus regulating the transcriptional activation of gene expression induced by cAMP response element-binding proteins (CREBs). A number of eukaryotic genes are regulated by the
PKA-C
-mediated phosphorylation of CREBs at Ser-133, and this transcriptional activation is rate-limited by the nuclear entry of
PKA-C
(47). PrKX has a putative nuclear localization signal (48) in the C terminus (RRX11KHHR) and
thus might also be involved in the regulation of gene expression, but
the subcellular localization and in vivo function of PrKX
remains to be elucidated.
A novel class of inhibitors for the cAMP-dependent protein
kinases PKA-C
and PrKX are the viral nonstructural
proteins of adeno-associated virus type 2 Rep78/52. Infection of
primary human cells with adeno-associated virus type 2 and
overexpression of the Rep proteins lead to a decrease in cellular
proliferation and to growth arrest (49). In infected cells, the viral
Rep proteins might affect the activity of both kinases or its
regulation by cAMP. In addition, the subcellular localization mediated
by the interaction with the regulatory subunits, RI and RII, and the
transcriptional regulation by CREB phosphorylation might also be
influenced. The mechanism of Rep-mediated inhibition of cell growth has
not been established yet, but the inhibition of PKA-C
and PrKX by Rep78/52, as demonstrated by Chiorini et al.
(19) may explain some of the effects of Rep expression.