From the Department of Pharmacology, University of Illinois, Chicago, Illinois 60612
Received for publication, September 9, 2002, and in revised form, October 18, 2002
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ABSTRACT |
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The present studies mapped the protein kinase A
(PKA) phosphorylation site of G Protein phosphorylation is a well established and ubiquitous
mechanism for regulating protein function. Such regulation can impact
cellular signaling at multiple levels including enzymatic activity,
protein structure, protein translocation, and protein-protein interactions, among others. Regarding seven-transmembrane receptors, evidence has been provided that phosphorylation of specific sites can
serve to alter receptor-G protein coupling, initiate receptor internalization, and ultimately modulate cross-membrane receptor signaling (1). Whereas such effects of receptor phosphorylation have
been well documented, considerably less is known regarding the
prevalence or the possible signaling consequences of G protein phosphorylation (2-6). Nevertheless, several reports have provided evidence that G protein phosphorylation can alter heterotrimer complex
formation and downstream signaling events. Specifically, Kozasa and
Gilman (4) demonstrated that in vitro phosphorylation of
G Reagents--
G13SRIpep
(CLLARRPTKGIHEY) and G13SRIpepP
(CLLARRPpTKGIHEY (where pT represents phosphothreonine)) peptides were
synthesized by Multiple Peptide Systems (San Diego, CA). An N-terminal
cysteine was added to each peptide to aid in coupling to carrier
protein for future antibody production. Outdated human platelet units were obtained from Heartland Blood Centers (Aurora, IL). G418 was
obtained from Calbiochem; PKAcat (protein kinase A,
catalytic subunit, bovine heart), protein A-Sepharose, aprotinin,
leupeptin, coumeric acid, luminol, 8-Br-cAMP, NaF, and CHAPS were
obtained from Sigma; [ In Vitro Phosphorylation of Purified
G Cell Culture and Transfection--
COS-7 cells and CHO cells
were maintained in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. COS-7 cells were
transiently transfected when the cells were 80-90% confluent in
100-mm culture dishes. The medium was replaced with serum-free
Dulbecco's modified Eagle's medium, and the cells were transfected
using the LipofectAMINE Plus method (Invitrogen) according to the
manufacturer's protocol, using 10 µg of DNA and 20 µl of
LipofectAMINE per transfection. After 3 h at 37 °C, the medium
was supplemented with 1 volume of 20% fetal bovine serum, Dulbecco's
modified Eagle's medium. Experiments were performed 48 h after
transfection. Transient transfection of COS-7 cells with cDNAs for
G Immunoprecipitation and Phosphorylation of
G DNA Constructs and Site-directed
Mutagenesis--
G Ligand Affinity Chromatography--
12 units of outdated human
platelets were pooled and incubated with 3 mM aspirin for
45 min. Solubilized platelet membranes were then prepared as previously
described (8), yielding a typical protein concentration of 4.5 mg/ml.
Solubilized membranes were phosphorylated and purified by ligand
affinity chromatography, as described (8). Briefly, solubilized
proteins were incubated with 1 mM 8-Br-cAMP or vehicle
(H2O), 100 µl of ATP, 60 mM
CaCl2, and incubation medium (45 mM histidine
HCl, 50 mM KH2PO4, 20 mM NaF, 120 mM KCl, pH 7.4) for 30 min at
30 °C. The phosphorylated samples were supplemented with 500 mM KCl, 0.5 mg/ml asolectin, 20% glycerol, 0.2 mM EGTA, 10 mM CHAPS and were incubated on the ligand affinity columns overnight at 4 °C. Unbound proteins were washed with buffer D (20 mM Tris base, 10 mM
CHAPS, 20% glycerol, 500 mM KCl, 0.2 mM EGTA,
0.5 mg/ml asolectin, pH 7.4) supplemented with 20 mM NaF
and 2 mM orthovanadate. TXA2 receptor-G protein complexes were eluted from the column with the TXA2
antagonist BM13.177 in buffer D at a flow rate of 0.125 ml/min. A 3-ml
elution fraction was collected, dialyzed in 10 mM
NH4HCO3, and then lyophilized. Proteins were
reconstituted into 150 µl of H2O and were subjected to
SDS-PAGE.
Pull-down Assay for GTP-Rho--
Rho activation was determined
as previously described (20). CHO-G Statistical Analysis--
Data were analyzed according to the
analysis of variance using Dunnett's multiple comparison post-test or
Student's t test, as indicated, using GraphPad PRISM
statistical software (San Diego, CA). Statistical significance is
defined as p < 0.05, p < 0.01, or
p < 0.002, as indicated.
In Vitro PKA phosphorylation of Purified
G Inhibition of G PKA Does Not Phosphorylate G Binding of
In these studies, COS-7 cells were transfected with either wild type or
mutant G Ligand Affinity Co-purification of TXA2 Receptor-G
Protein Complexes--
The next series of experiments extended the
above finding and investigated possible consequences of
G
The ability of 8-Br-cAMP to cause such a selective effect on the
G13 heterotrimeric complex is consistent with previous
results, which have shown that PKA is capable of phosphorylating
G PKA-mediated Inhibition of Rho Activation--
Since
G Heterotrimeric G proteins are ubiquitously expressed across
different cell lines and serve as important mediators in the initiation and maintenance of cellular function. As such, these proteins are
particularly well poised to serve as targets for modulation of the
separate signaling pathways. One mechanism by which the functional
activity of G proteins might be regulated is through the process of
phosphorylation. For example, results from studies by Aragay and Quick
(5) suggested that thyrotropin-releasing hormone responses require PKC
phosphorylation of G In addition to PKC, evidence has been provided that PKA is also capable
of phosphorylating G On this basis, we next examined whether PKA phosphorylation of the
switch I region Thr203 residue of G Our final series of experiments investigated the effects of
G Based on the above considerations, we propose a model by which PKA may
regulate G13 signaling (Fig.
8). In the resting state, an equilibrium
exists between the intact receptor-G protein complex and the
dissociated complex. This dissociation-reassociation cycling process
defines basal GTPase activity, which is normally low because most
receptor-G protein exists in the complexed form. This basal activity
also defines the resting activation state of the downstream effectors.
On the other hand, the cycling rate can be markedly accelerated in the
presence of agonist. Thus, according to the classical model (31), the
agonist induces a shift in the equilibrium in favor of heterotrimer
dissociation, which is characterized by increased GDP-GTP exchange on
the G13 and studied the
consequences of its phosphorylation. Initial experiments using purified
human G
13 and the PKA catalytic subunit established that
PKA directly phosphorylates G
13. The location of this
phosphorylation site was next investigated with a new synthetic peptide
(G13SRIpep) containing the PKA consensus sequence (Arg-Arg-Pro-Thr203) within the switch I region of
G
13. G13SRIpep produced a
dose-dependent inhibition of PKA-mediated
G
13 phosphorylation. On the other hand, the
Thr-phosphorylated derivative of G13SRIpep
possessed no inhibitory activity, suggesting that G
13
Thr203 may represent the phosphorylation site. Confirmation
of this notion was obtained by showing that the
G
13-T203A mutant (in COS-7 cells) could not be
phosphorylated by PKA. Additional studies using co-elution affinity
chromatography and co-immunoprecipitation demonstrated that
G
13 phosphorylation stabilized coupling of G
13 with platelet thromboxane A2 receptors
but destabilized coupling of G
13 to its
subunits.
In order to determine the functional consequences of this
phosphorylation on G
13 signaling, activation of the Rho
pathway was investigated. Specifically, Chinese hamster ovary cells
overexpressing human G
13 wild type
(G
13-WT) or G
13-T203A mutant were
generated and assayed for Rho activation. It was found that
8-bromo-cyclic AMP caused a significant decrease (50%;
p < 0.002) of Rho activation in G
13
wild type cells but produced no change of basal Rho activation levels
in the mutant (p > 0.4). These results therefore
suggest that PKA blocks Rho activation by phosphorylation of
G
13 Thr203.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
12 and G
z by
PKC1 results in
conformational changes that inhibit interaction of the G
and G
subunits. A similar PKC-mediated phosphorylation effect on G
-G
interaction was observed by Fields and Casey (3) for G
z
and by Murthy et al. (6) for G
i1/2.
Offermanns et al. demonstrated that a
PKC-dependent phosphorylation of G
12 and
G
13 occurred when human platelets were activated by
thrombin, the TXA2 mimetic U46619, or the phorbol ester
phorbol 12-myristate 13-acetate (PMA) (7). Collectively, the
above results therefore suggest that different G
subunits can indeed
serve as substrates for PKC. Whereas the functional consequences of
such phosphorylation remain largely unknown, the strategic position of
G proteins in the signaling cascade suggests that
phosphorylation-mediated changes in G protein conformation or activity
could serve as a significant regulatory point in the signaling process.
In this connection, we recently provided evidence that TXA2
receptor-coupled G
13 is phosphorylated through a
PKA-mediated process (8). These results represented the first
demonstration that a platelet G
subunit is phosphorylated by
cAMP-dependent kinase and provided a possible explanation
for the high sensitivity of TXA2 receptor signaling to
increased cAMP levels. The present studies extend these findings by
characterizing the specific PKA phosphorylation site in
G
13 and identifying the molecular and functional
consequences of this phosphorylation. Our results demonstrate that PKA
phosphorylates G
13 at Thr203, which is
situated within the functionally important switch I region of the G
subunit (9-14). This phosphorylation results in stabilization of
G
13 coupling to TXA2 receptors and
destabilization of G
13 coupling to its
subunits.
Separate experiments also provided evidence that a functional
consequence of this phosphorylation is decreased basal Rho activation
levels. Thus, PKA-mediated G
13 Thr203
phosphorylation appears to represent a novel mechanism for the regulation of signaling through the G
13 pathway.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (4,500 Ci/mmol) was
purchased from ICN Biochemicals, Inc.; G13-N, Myc,
G
12, and G
common IgG and Rho A polyclonal
antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA); rabbit polyclonal anti-peptide antibodies to
TXA2 receptor (P2 Ab) and G
q (QL
Ab) were prepared as previously described (15); the Wizard Plus
Midiprep DNA Purification kit was from Promega (Madison, WI);
LipofectAMINE Plus and LipofectAMINE 2000 were purchased from
Invitrogen; the QuikChange site-directed mutagenesis kit was
from Stratagene (La Jolla, CA); horseradish peroxidase-conjugated goat
anti-rabbit IgG (H + L) was from Bio-Rad; the BCA protein assay kit was
from Pierce; and the Rho activation assay kit was from Upstate
Biotechnology, Inc. (Lake Placid, NY). Primers (T203A-DS, 5'-GGA TGC
CTT TGG CGG GTC TTC TGG CAA GCA GAA TAT CTT G-3'; T203A-US, 5'-GCC AGA
AGA CCC GCC AAA GGC ATC CAT GAA TAC GAC TTT G-3') were from Integrated
DNA Technologies, Inc. (Coralville, IA).
13--
G
13 (16) was purified from
Sf9 cells, as previously described (17). For phosphorylation of
purified G
subunits, 5 pmol of G
subunit was added to 20 mM [
-32P]ATP (4,500 Ci/mmol),
PKAcat (protein kinase A, catalytic subunit (50 ng; 60 units) or PKA vehicle (100 mM NaCl, 20 mM MES,
pH 6.5, 30 mM
-mercaptoethanol, 100 mM EDTA,
50% ethylene glycol) in phosphorylation buffer (25 mM
Tris-HCl, pH 7.5, 5 mM MgCl2, 125 mM CaCl2, 1 mM dithiothreitol) in a
total reaction volume of 115 µl, and the mixture was incubated for 30 min at 30 °C. The reaction was terminated by adding Laemmli (18)
sample buffer (62.5 mM Tris-HCl, pH 6.5, 3% SDS, 10%
glycerol). Proteins were separated by SDS-PAGE and were visualized by
silver staining (19) followed by autoradiography.
13, G
1, and Myc-tagged G
2
subunits was performed using LipofectAMINE 2000 reagent, according to
the manufacturer's instructions. Transfections in CHO cells with human
G
13 wild type (G
13-WT) or
G
13-T203A mutant plasmids were performed as described
above. After 3 days of transfection, G418 (500 µg/ml) was added for
selection. After 3-5 weeks, the stable cell lines were established and
confirmed by Western blot.
13--
The transfected cells were washed three times
with cold PBS and were harvested with lysis buffer (50 mM
Hepes, pH 7.4, 10 mM MgCl2, 1 mM
EDTA, 100 mM NaCl, 1% Triton X-100, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µg/ml leupeptin). The lysates were centrifuged
to remove insoluble material, and the protein concentration of the
supernatant was measured using the BCA protein assay method. The
protein concentration of each lysate was adjusted to be equal within an
experiment and was typically 0.50-2.0 mg/ml. Aliquots of 0.6 ml were
incubated overnight at 4 °C with 10 µg/ml G
13
antibody and were subsequently incubated with protein A-Sepharose beads
(55 µl of a 10% (w/v) suspension) for 4 h at 4 °C. The
immune complexed beads were washed once with lysis buffer and were
supplemented with resuspension buffer (50 mM Hepes, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.014%
Tween 20, 1 mM dithiothreitol). 85 µl of protein-bound
beads were then incubated with 50 µM
[
-32P]ATP, 150 ng of PKA, 1 mM cAMP, and
the indicated concentration of blocking peptide in a total volume of
125 µl. The mixture was incubated for 30 min at 30 °C, after which
time the beads were pelleted by centrifugation and washed three times
with resuspension buffer. The beads were then suspended in Laemmli
sample buffer plus resuspension buffer, and immune complexes were
eluted by boiling. The eluted proteins were subjected to SDS-PAGE,
silver staining, and autoradiography. Phosphorylated proteins on
autoradiograms were quantified using a densitometer (Protein Databases,
Inc., Huntington Station, NY), and values were normalized for the
amount of silver-stained protein on the gel.
13 was cloned from a human
adenocarcinoma LoVo cell line and was subcloned into a pcDNA3
vector. The G
13 mutation with the substitution of Ala
for Thr203 was performed using the Stratagene QuikChange
site-directed mutagenesis kit according to the manufacturer's
protocol. The final mutant was verified by DNA sequencing.
13-WT and
CHO-G
13-T203A mutant cells were seeded on six-well plates, grown to 80% confluence, and serum-starved for 24 h.
Following treatment with 8-Br-cAMP (1 mM) at 37 °C for
15 min, the cells were washed once with PBS and harvested with 300 µl
of lysis buffer (50 mM Tris-HCl, pH 7.5 containing 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, 1 mM
dithiothreitol, 50 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml
each of leupeptin and pepstatin, and 0.2% Nonidet P-40). The cell
lysates were clarified by centrifugation at 15,000 × g
for 1 min and incubated with 20 µg of glutathione
S-transferase-Rho binding domain fusion protein conjugated with glutathione beads at 4 °C for 1 h. The beads
were washed three times with lysis buffer and subjected to SDS-PAGE on
a 12% gel. Bound RhoA was detected by Western blot using a polyclonal
antibody against RhoA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
13--
Whereas we previously demonstrated that cAMP
induces phosphorylation of TXA2 receptor-coupled
G
13 in human platelets (8), these results did not
determine whether the observed phosphorylation is directly mediated by
PKA or whether additional kinases are involved. In order to address
this question, additional studies were performed using human
G
13 purified from Sf9 cells (16) with
[
-32P]ATP in the presence or absence of the PKA
catalytic subunit (PKAcat). Electrophoretic separation and
silver staining of these samples revealed the presence of a single
protein at 44 kDa corresponding to G
13 (Fig.
1a, lanes
1 and 2) but no such band in the presence of PKA
alone (Fig. 1a, lane 3). Analysis of
this gel by autoradiography revealed substantial phosphorylation of
G
13 (at 44 kDa) when PKA is added (Fig. 1b,
lane 1) but no phosphorylation (Fig.
1b, lane 2) in the absence of PKA.
These experiments therefore establish that PKA alone is sufficient to
phosphorylate G
13. The specificity of this
G
13 phosphorylation was examined in parallel experiments by assaying a separate platelet G protein, G
i (purified
from Sf9 cells as previously described) (17). Fig. 1a
illustrates a single protein band (lanes 4 and
5) corresponding to G
i. However, it can also
be seen (Fig. 1b, lanes 4 and
5) that no phosphorylation of G
i was observed
in either the presence or the absence of PKA. Collectively, these
results demonstrate that PKA directly phosphorylates G
13
and that this reaction does not require intermediate kinases or other
co-factors.
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Fig. 1.
In vitro PKA phosphorylation of
purified G 13. a,
silver-stained gel of purified G
13 and G
i
in the presence and absence of PKA. b, autoradiogram of
phosphorylated G
13 and G
i in the presence
or absence of PKA. Results are representative of three separate
experiments.
13 Phosphorylation by a
G
13 Peptide Containing a PKA Consensus Sequence--
A
14-amino acid synthetic peptide (G13SRIpep)
corresponding to the specific G
13 amino acid sequence
Leu197-Tyr209 was initially used to locate the
PKA phosphorylation site of G
13. This amino acid region
was selected because analysis of the primary structure of the
G
13 subunit revealed only one PKA consensus sequence
represented by Arg-Arg-Pro-Thr203. On this basis,
G13SRIpep, which encompasses this region, was used to probe the Leu197-Tyr209 region for
potential phosphorylation. Thus, it was reasoned that PKA-mediated
phosphorylation of G
13 would be blocked or reduced by a
competing peptide substrate (G13SRIpep)
containing an amino acid sequence that is identical to that contained
within G
13. In these experiments, human wild type
G
13 was immunoprecipitated from transiently transfected
COS-7 cell using protein A-Sepharose beads coupled to an anti-peptide
antibody directed against the N terminus of G
13
(G13-N IgG). The
subunit-antibody-bead complex was then
incubated with PKAcat and [
-32P]ATP in the
presence or absence of G13SRIpep. Following
incubation, the proteins were eluted from the beads and were subjected
to gel electrophoresis and autoradiography. It can be seen that the efficiency of immunoprecipitation was comparable for each
G
13-transfected COS-7 cell sample (Fig.
2a, lanes
1-6). It can also be seen that PKA induced a substantial
G
13 phosphorylation (Fig. 2b, lane 1). Furthermore, upon the addition of
G13SRIpep, there was a dramatic dose-dependent inhibition of this PKA-mediated effect (Fig.
2b, lanes 2-5). Quantitation of this
peptide inhibition is illustrated in Fig. 2c, where it can
be seen that G
13 phosphorylation was significantly
blocked at a peptide concentration of as little as 50 µM,
and was completely inhibited at a peptide concentration of 500 µM. Fig. 2a, lanes 7 and
8, demonstrate that the vector-transfected COS-7 cells do
not reveal detectable amounts of G
13 under these conditions, and consistent with this finding, there was no observed PKA-induced phosphorylation in the 44-kDa region of the
immunoprecipitated protein samples (Fig. 2b,
lanes 7 and 8). These findings using transfected COS-7 cells therefore confirm our previous results in
platelets showing PKA-mediated phosphorylation of G
13
and suggest that the PKA phosphorylation site of G
13 is
contained within the Leu197-Tyr209 sequence.
However, since it is possible that the ability of
G13SRIpep to block phosphorylation was due to a
nonspecific peptide effect, a control peptide was evaluated in
subsequent experiments. Specifically, it was reasoned that the most
appropriate control would employ a peptide with an amino acid sequence
identical to that of G13SRIpep, the only
difference being substitution of a phosphorylated Thr in the peptide
sequence. Thus, the phosphorylated peptide should serve as a poor
substrate for PKA. On this basis, G13SRIpepP
was synthesized and tested for its ability to affect PKA-mediated phosphorylation under the same experimental conditions as those used
for G13SRIpep. As can be seen in Fig
3a, lanes
1-4, equal amounts of G
13 protein were
immunoprecipitated under each experimental condition. It can also be
seen (Fig. 3b, lane 1) that PKA
addition again resulted in substantial G
13
phosphorylation. However, in contrast to the effects of
G13SRIpep, the addition of
G13SRIpepP was completely ineffective in
blocking this PKA-mediated phosphorylation (Fig. 3b,
lanes 2 and 3), even at a
concentration (500 µM) that resulted in complete
inhibition by G13SRIpep. The quantitative analysis of these phosphorylation profiles is illustrated in Fig. 3c. Since G
13 contains only a single PKA
consensus sequence within Leu197-Tyr209, these
findings therefore suggest 1) that G13SRIpep
specifically acts to block PKA-induced phosphorylation of
G
13; 2) that the location of the PKA phosphorylation
site is contained within the Leu197-Tyr209
sequence of G
13; and 3) that Thr203 serves
as the phosphorylation site within this sequence.
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Fig. 2.
Inhibition of
G 13 phosphorylation by a
G
13 peptide sequence.
a, silver stain of immunoprecipitated G
13
protein. b, autoradiogram of phosphorylated
G
13. c, dose-dependent inhibition
of PKA-induced phosphorylation of G
13 by
G13SRI peptide; n = 6. Statistical analysis
was performed by analysis of variance. *, p < 0.05;
**, p < 0.01.
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Fig. 3.
No Inhibition of PKA-induced phosphorylation
of G 13 by G13SRI-P
peptide. a, silver stain of immunoprecipitated
G
13 protein. b, autoradiogram of
phosphorylated G
13; c, quantitative analysis
of PKA-induced phosphorylation; n = 4. Statistical
analysis was performed by analysis of variance. *, p < 0.05; **, p < 0.01.
13-T203A Mutant
Protein--
In order to provide independent confirmation that
Thr203 is in fact the residue phosphorylated by PKA, a
mutant G
13 (G
13-T203A) was produced in
which Thr203 was changed to Ala by site-directed
mutagenesis. In these experiments, the G
13-T203A mutant
(or wild type G
13) was immunoprecipitated from
transiently transfected COS-7 cells using the G13-N
antibody, and the
subunit-antibody-bead complex was subjected to
phosphorylation, as described earlier. It can be seen (Fig.
4a, lanes
1-4) that the efficiency of immunoprecipitation was
comparable for both wild type and mutant protein. However, a comparison
of the phosphorylation profiles reveals that substitution of
Thr203 with Ala resulted in a complete loss of PKA-mediated
G
13 phosphorylation (Fig. 4b). In this
regard, some experiments revealed a minor phosphorylation in the mutant
with or without PKA. However, this apparent phosphorylation was not
found to be significantly different from vector control, and no
detectable phosphorylation was observed in the mutant autoradiogram (Fig. 4b, lanes 3 and 4).
The complete absence of PKA-induced phosphorylation in the T203A
mutant, in combination with the previous results, therefore provides
evidence that PKA phosphorylates a single site at Thr203
contained within switch I region of G
13.
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Fig. 4.
No PKA-induced phosphorylation of
G 13-T203A mutant protein.
a, phosphorylation of G
13 WT and
G
13-T203A mutant protein by PKA; n = 6. b, autoradiogram of phosphorylated G
13
protein. c, silver stain of immunoprecipitated
G
13 protein.
to Wild Type G
13 and
G
13-T203A Mutant Protein--
The next series of
experiments investigated the molecular consequences of this
PKA-mediated Thr203 phosphorylation. Because
phosphorylation is known to alter protein conformation and
function, it is possible that Thr203 phosphorylation may
change the conformational or chemical characteristics of
G
13 and thereby alter its normal function in the
receptor-coupled signaling process. Indeed, this possibility might be
anticipated for two reasons: 1) Thr203 resides within
switch I region of the G
13 subunit, and 2) the switch I
region is considered important for modulating activation of the
subunit and for regulating its affinity for the
subunits (16).
Based on these considerations, we examined whether PKA phosphorylation
of the switch I region Thr203 residue affects the stability
of the G
13 heterotrimeric complex.
13 (G
13-T203A) as well as with
and Myc-tagged
2. The
subunits were then
immunoprecipitated with anti-Myc antibody. It can be seen that
co-transfection of
and Myc-tagged
2 cDNAs alone
(without G
13) showed no G
13 protein in
either the lysate or the immunoprecipitate (Fig.
5, row a,
lane 3, and row b,
lane 3). As expected, G
13 was
immunoprecipitated (Fig. 5 row a, lane
2) and present in the lysate (Fig. 5, row
b, lane 2) when the cells were
co-transfected with
, Myc-tagged
2 cDNAs, and
wild type G
13. On the other hand, no such
G
13 immunoprecipitation was observed (Fig. 5,
row a, lane 1) when the
co-transfection was done with G
13-T203A mutant.
Furthermore, Western blotting of total cell lysates demonstrated that
both constructs of G
13 were expressed at the same levels
(Fig. 5, row b, lanes 1 and 2), suggesting that the inability of mutated
G
13 to complex with
1
2 was
not due to insufficient expression. These findings therefore suggest
that mutation of G
13 at Thr203 disrupts
heterotrimer stability.
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Fig. 5.
binding to wild
type G
13 and
G
13-T203A mutant protein. The
figure illustrates an immunoblot of G
13 that
co-precipitated with Myc-tagged
from transfected COS-7 cells.
Wild type G
13 co-immunoprecipitated with
subunits
(lane 2), whereas the mutant G
13
protein did not (lane 1).
13 Thr203 phosphorylation on stability of
the receptor-G
13 complex. To this end, affinity
purification of the receptor-G protein complex was employed. This
technique has previously been used to co-purify native TXA2
receptors with their associated G
proteins (21, 22) and was used to
demonstrate PKA phosphorylation of TXA2 receptor-coupled
G
13 (8). In the present experiments, solubilized platelet membranes were prepared and incubated in the presence or
absence of 1 mM 8-Br-cAMP plus [
-32P]ATP.
The TXA2 receptor-G protein complexes were then purified by
affinity chromatography (8), and the column eluate was assayed for
TXA2 receptor, G
13, G
q, and
subunits by immunoblotting. It can be seen that in the absence of
8-Br-cAMP (Fig. 6a,
lane 1) TXA2 receptors co-purified
with their associated G protein heterotrimeric complexes. Thus, the
TXA2 receptor elute fractions yielded positive
immunoreactivity for the G
13, G
q, and
G
subunits. It can also be seen, however, that 8-Br-cAMP treatment
produced a significant shift in this elution profile (i.e. a
substantial increase in co-eluted G
13 and a substantial
decrease in co-eluted G
) (Fig. 6a, lane
2). On the other hand, 8-Br-cAMP treatment produced no
change in the amounts of either G
q or TXA2
receptor (Fig. 6a, lanes 1 and
2). Quantitation of these results is illustrated in Fig.
6b.
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Fig. 6.
Effect of PKA phosphorylation on
TXA2 receptor-G protein complex formation.
a, immunoblot of TXA2 receptor and G proteins
eluted from the ligand affinity column in the absence (lane
1) or presence (lane 2) of 1 mM 8-Br-cAMP. The illustrated blot represents an experiment
that yielded one of the largest differences between experimental and
control conditions. b, quantitation of average protein
elution levels from four different experiments (determined by
densitometric scan); *, p < 0.05.
13 (8) but incapable of phosphorylating either
G
q (23) or platelet TXA2
receptors.2 Furthermore,
since it is known that co-elution of complexed proteins is proportional
to the affinity between these proteins, the observed shifts in the
elution profiles for both G
13 and
subunits suggest that PKA-mediated Thr203 phosphorylation of
G
13 has two separate effects on G
13.
First, this phosphorylation appears to destabilize its association with G
subunits, resulting in
dissociation. This finding is
consistent with the results in Fig. 5 using Myc-tagged
2. Second, Thr203 phosphorylation appears to
stabilize the association between TXA2 receptors and
G
13. Thus, PKA-mediated phosphorylation within the
switch I region of G
13 appears to impact the interaction of this G
subunit with each of its protein partners.
13 is known to signal through the Rho pathway (16,
24-27), the final series of experiments investigated the effects of
G
13 Thr203 phosphorylation on the basal Rho
activation state. In these studies, CHO cell lines overexpressing human
G
13-wild type (G
13-WT) or G
13-T203A mutant (which cannot undergo PKA
phosphorylation; Fig. 4) were assayed for G
13-Rho
activation. Fig. 7 illustrates the fraction of activated Rho A relative to total Rho A in the cell homogenate. Using G
13-WT cells, it was found that the
addition of 1 mM 8-Br-cAMP caused a substantial decrease in
basal Rho activation levels (Fig. 7, lanes 1 and
2). On the other hand, 8-Br-cAMP did not significantly
decrease Rho activation in the G
13-T203A mutant (Fig. 7,
lanes 3 and 4). The average of seven
separate experiments revealed that 8-Br-cAMP caused a 50 ± 14%
inhibition (p < 0.002) in the G
13-WT
cells but had no significant effect (5 ± 13%; p > 0.4) in the mutant cells. These results therefore demonstrate that
G
13 T203A mutation decreases the ability of 8-Br-cAMP to block Rho activation, suggesting that G
13
phosphorylation at Thr203 interferes with the
G
13 signaling process.
View larger version (38K):
[in a new window]
Fig. 7.
PKA-mediated inhibition of Rho
activation. The figure illustrates Rho activation in
G 13-WT-transfected CHO cells (lanes
1 and 2) and G
13-T203A mutant
(lanes 3 and 4). Effects of 8-Br-cAMP
on basal Rho activation levels are shown (lanes 2 and 4).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
16. This suggestion was based on the
finding that mutation of the G
16 PKC phosphorylation sites blocked the thyrotropin-releasing hormone-induced responses (5).
Other studies have indicated that PKC-mediated phosphorylation can
down-regulate G protein activity. Specifically, Glick and Casey (28)
reported that phosphorylation of G
z by PKC substantially reduced its activation. Furthermore, Murthy et al. reported
that PKC activation leads to phosphorylation of
G
i1/G
i2, and blocks downstream
signaling events mediated through both G
i1- and
G
i2-coupled receptors. Moreover, they suggested that
phosphorylation of G
subunits could also indirectly affect responses
mediated by
subunits. Thus, it appears that PKC-mediated G
subunit phosphorylation may play a significant role in regulating G
protein function and hence its downstream signaling processes.
subunits in vitro (29, 30). Furthermore, we recently demonstrated that TXA2
receptor-coupled G
13 is phosphorylated by PKA in
vivo (8). The present studies extended these findings by
characterizing the specific PKA phosphorylation site in
G
13 and identifying the molecular and functional
consequences of this phosphorylation. Our initial results showed that
PKA phosphorylation of G
13 does not require intermediate
kinases or other co-factors. Our subsequent studies using site-specific
peptides and site-directed mutagenesis provided evidence that PKA
phosphorylates G
13 at a single site (i.e.
Thr203 contained within the switch I region of the G
subunit). Because this phosphorylation occurs at a conformationally
sensitive site of the of the G
subunit, it is possible that such
phosphorylation could potentially change the structural or chemical
characteristics of G
13 and thereby alter its interaction
with receptor-G13
partners or its downstream
signaling capacity. In this connection, switch I and II regions are
structurally and functionally analogous to those first described in the
small GTPase Ras (9), whereas the switch III region is unique to
heterotrimeric G proteins. These switch regions are domains of the
subunit that have been found to undergo conformational changes during G
protein activation induced by GDP-GTP exchange (9-14). Furthermore,
crystallization studies have revealed that the switch I region contains
critical sites for not only binding GTP (13, 22) but also for binding the
subunits (13). Consequently, this region is thought to be
important for modulating activation of the
subunit as well as for
regulating the affinity of the
subunit for the
subunits (13).
13
affects the stability of the G
13 heterotrimeric complex or the interaction of this complex with TXA2
receptor protein. Using co-elution affinity chromatography and
co-immunoprecipitation, it was found that G
13
phosphorylation stabilized the coupling of G
13 to
platelet TXA2 receptors but destabilized the coupling of
G
13 to its
subunits. Thus, phosphorylation of
G
13 at Thr203 appeared to produce structural
effects on the receptor-G protein heterotrimer complex.
13 Thr203 phosphorylation on downstream
signaling through Rho. It was found that 8-Br-cAMP caused a substantial
reduction of the basal Rho activation state in CHO cells overexpressing
wild type G
13. On the other hand, mutation of
G
13 Thr203 to Ala significantly blocked the
cAMP-mediated effects. Since separate experiments demonstrated that the
T203A mutant is incapable of PKA-mediated phosphorylation, these
results suggest that Thr203 phosphorylation serves as a
mechanism for PKA modulation of G
13 downstream
signaling. This finding is consistent with previous results that
demonstrated cAMP-dependent inhibition of Rho activation (27).
subunit (17). This process in turn causes a rapid dissociation
of G
from the
subunits, enabling both the
and the
to increase their activation of downstream effectors. Upon hydrolysis
of GTP to GDP, the receptor-G
complex reforms and is thereby
poised to undergo additional cycles of activation. On the other hand,
if this cycling process is interrupted (e.g. by G
subunit
phosphorylation), downstream effector activation could be significantly
altered. Specifically, our results have shown that PKA-mediated
phosphorylation of G
13 at Thr203 has two
structural effects: 1) it stabilizes the G
13-G
protein-coupled receptor complex, and 2) it inhibits
G
13 association. Whereas the mechanisms of these
effects are unknown, they may involve steric considerations or
phosphorylation-induced conformational changes. For example,
Thr203 is situated within the
interacting surface of
G
13. Consequently, phosphorylation of this site could
prevent binding between the
and
subunits and thereby inhibit
formation of the heterotrimeric complex. At the same time,
Thr203 phosphorylation may lead to conformational changes
that favor stabilization of the G
13-G protein-coupled
receptor binding interaction.
View larger version (21K):
[in a new window]
Fig. 8.
Proposed model of the mechanism by which
cAMP/PKA modulate G 13
signaling. a, cAMP activates PKA, which mediates
Thr203 phosphorylation of G
13 within the
switch I region. b, Thr203 phosphorylation of
G
13 stabilizes coupling of G
13 to
G protein-coupled receptor and/or prevents binding of switch I region
to effector(s). c, Thr203 phosphorylation of
G
13 decreases the stability of the G
13
heterotrimer complex, leading to
subunits release and inhibition
of heterotrimer complex reassociation.
release may transiently
influence downstream effectors.
Regarding functional effects, stabilization of the G-receptor
complex by phosphorylation of G
13 Thr203
would presumably lead to direct inhibition, since the G
subunit would be unavailable for downstream signaling. Alternatively, steric or
charge-related effects of phosphorylation may also decrease binding of
switch 1 region to the downstream effector (32), again leading to
inhibition. Our results showing decreased Rho activation by
G
13 Thr203 phosphorylation are consistent
with either of these possibilities. On the other hand, the consequences
of inhibited
reassociation by phosphorylation are less clear.
For example, one might think that initial dissociation of
would
lead to downstream activation. However, this activation phase may be
brief and self-limiting due to inhibition of cycling and G protein
adaptation, which has recently been shown to occur in the face of
persistent heterotrimeric dissociation (31). On this basis, the
downstream signaling response may be biphasic, and indeed there is
precedent for a transient platelet activation phase (33) caused by
protein kinase G (which presumably would also phosphorylate
Thr203).
In summary, the present findings identify a novel mechanism for
modulating G protein-coupled receptor signaling. Our demonstration of
PKA-mediated G13 Thr203 phosphorylation
provides evidence that a conformationally sensitive switch region of
G
13 can serve as a potential target for cAMP modulation.
This phosphorylation appears to have both structural and functional
effects in that it causes increased stability of G
13
coupling to G protein-coupled receptors, decreased stability of the
G
13 heterotrimer complex, and decreased basal Rho
activation state. Whereas the relative importance of this regulatory
process is presently unknown, it is noteworthy that phosphorylated
G
13 exists even at basal cAMP levels (8). This finding
raises the possibility that cAMP may exhibit tonic influences on
G
13 signaling even at low cellular cAMP levels. Finally,
sequence analysis reveals that G
12 also contains a PKA
consensus sequence in switch I region, whereas other G proteins
(e.g. G
q, G
i,
G
z, or G
s) do not. Thus, the proposed
ability of cAMP to modulate G
conformation/function may represent a
regulatory mechanism that is unique to the ubiquitously expressed
G
12/13 family. Clearly, additional studies will be required to investigate this interesting possibility.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL-24530 (to G. L.), GM 54159 and GM65160 (to T. V-Y.), and GM59427 (to T. K.).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.
Present address: Millennium Pharmaceuticals, Inc., 256 E. Grand
Ave., South San Francisco, CA 94080.
§ These two authors contributed equally to this work.
¶ To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave. (M/C 868), Chicago, IL, 60612. Tel.: 312-996-4929; Fax: 312-996-4929; E-mail: gcl@uic.edu.
Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M209219200
2 G. C. Le Breton, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; TXA2, thromboxane A2; PKA, protein kinase A; 8-Br-cAMP, 8-bromo-cyclic AMP; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; CHO, Chinese hamster ovary; WT, wild type.
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