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J. Biol. Chem., Vol. 275, Issue 36, 28167-28172, September 8, 2000
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,
,,,,, and§§
From the Department of Medicine, University Hospital
of Freiburg, Hugstetterstr. 55, 79106 Freiburg, Germany, the
§ Department of Medicine, ¶ Division of Signal
Transduction and Department of Surgery, and
Department of Psychiatry, Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
02215, and the ** Department of Physiology, University of Freiburg,
79106 Freiburg, Germany
Received for publication, April 6, 2000, and in revised form, June 7, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Regulator of G protein signaling (RGS) proteins
function as GTPase-activating proteins (GAPs) that stimulate the
inactivation of heterotrimeric G proteins. We have recently shown that
RGS proteins may be regulated on a post-translational level (Benzing, T., Brandes, R., Sellin, L., Schermer, B., Lecker, S., Walz, G., and
Kim, E. (1999) Nat. Med. 5, 913-918). However, mechanisms controlling the GAP activity of RGS proteins are poorly understood. Here we show that 14-3-3 proteins associate with RGS7 and RGS3. Binding
of 14-3-3 is mediated by a conserved phosphoserine located in the
G Regulator of G protein signaling
(RGS)1 proteins share a
conserved RGS domain that binds Ubiquitously expressed in all eukaryotic cells (10, 11) 14-3-3 proteins
include nine distinct isotypes ( Here we show that RGS3 and RGS7 contain a functional 14-3-3-binding
site within their RGS domains and that a significant fraction of both
proteins normally exists bound to endogenous 14-3-3. The binding of
RGS3 and RGS7 to 14-3-3 is phosphorylation-dependent; the
primary 14-3-3-binding site in RGS7 involves serine 434, a region
implicated in interactions with G Plasmids--
Flag-tagged versions of human RGS7 (7, 18) and
human RGS3 (a kind gift of Drs. K. M. Druey and J. H. Kehrl)
were generated by polymerase chain reaction and standard cloning
techniques. Site-directed mutagenesis was used to insert mutations in
RGS7. Point mutations were verified by sequence analysis. A
GST.14-3-3 Co-Immunoprecipitation--
Co-immunoprecipitation experiments
were performed as described (18). Briefly, HEK 293T cells were
transiently transfected by the calcium phosphate method. After
incubation for 24 h, cells were washed twice and lysed in a 1%
Triton X-100 lysis buffer. After centrifugation (15,000 × g, 15 min, 4 °C) cell lysates containing equal amounts of
total protein were incubated for 1 h at 4 °C with the
appropriate antibody followed by incubation with 40 µl of protein
G-Sepharose beads (mouse monoclonal antibodies) or protein
A-Sepharose (rabbit polyclonal antisera) (Amersham Pharmacia Biotech) for approximately 3 h. The beads were washed
extensively with lysis buffer and bound proteins were resolved by 10%
SDS-PAGE.
Metabolic Labeling and Immunoprecipitation--
HEK 293T cells
were transiently transfected with plasmid DNA as indicated. After
24 h methionine/cysteine-free Dulbecco's modified Eagle's medium
containing 1% dialyzed fetal bovine serum was applied for 30 min, then
1 mCi of Tran35S-Label (ICN) containing
[35S]methionine/cysteine was added for 6 h. Cells
were harvested, lysates immunoprecipitated, and precipitates subjected
to SDS-PAGE. Densitometric analysis of nonsaturating autoradiographs
utilizing NIH image software was corrected for the number of
incorporated methionines and cysteines of the respective proteins.
RGS3 Pull-down Assay--
HEK 293T cells were transiently
transfected with F.RGS3. After incubation with staurosporine cells were
lysed in 1% Triton X-100, 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 50 mM NaF, 15 mM
Na4P2O7, 2 mM
Na3VO4, 1 mM EDTA, and protease
inhibitors for 15 min on ice. Following centrifugation the supernatant
was incubated for 1 h at 4 °C with 6 µg of recombinant
purified GST.14-3-3 In Vivo Co-Immunoprecipitation from Brain--
For preparation
of brain protein extracts whole brains of female BALB/c mice (20 g in
body weight, Charles River) were removed and homogenized in 4 ml of
brain lysis buffer (20 mM Tris, pH 7.5, 0.1% Triton X-100,
40 mM NaCl, 50 mM NaF, 15 mM
Na4P2O7, 2 mM
Na3VO4, 1 mM EDTA, containing
protease inhibitor mixture and 44 µg/ml phenylmethylsulfonyl
fluoride) for 15 min on ice. Following centrifugation and
ultracentrifugation (100,000 × g, 4 °C, 30 min), the supernatant was divided into 2 fractions and immunoprecipitated with specific anti-RGS7 antiserum and control antibody followed by incubation with protein G-Sepharose. Resulting precipitates were subjected to immunoblot analysis with
anti-14-3-3 mAb (Santa Cruz) followed by incubation with horseradish
peroxidase-coupled secondary antiserum and enhanced chemiluminescence.
Pull-down of Native RGS7 from Brain--
GST and GST.14-3-3 In Vitro Phosphorylation and Interaction--
In
vitro phosphorylation of GST.RGS7315-469 and
MBP.RGS7315-469 was performed for 30 min at 37 °C in a
100-µl reaction in a buffer containing 20 mM HEPES, pH
7.4, 10 mM MgCl2, 0.1 mM
CaCl2, 100 µM ATP, 20 µg/ml diacylglycerol,
100 µg/ml phosphatidylserine, 0.03% Triton X-100, and the indicated
amount of recombinant RGS7 protein. The phosphorylation was initiated
by the addition of 0.5 units of recombinant protein kinase C- Fluorescence Polarization Studies--
For fluorescence
polarization assays GST.14-3-3 GTP Hydrolysis Assays--
Single turnover GTPase activity
measurements were carried out as described (22-24). Recombinant
myristoylated G ERK1/2 Phosphorylation--
For the determination of ERK1/2
phosphorylation HEK293T cells were transfected with the plasmid DNA as
indicated. After transfection cells were serum-starved overnight and
incubated in the absence/presence of carbachol. After 15 min the
stimulation was stopped by placing the cells on ice and exchange of the
media with ice-cold phosphate-buffered saline. Cells were harvested,
lysed in a 1% Triton X-100 lysis buffer containing 20 mM
Tris, pH 7.5, 50 mM NaCl, 50 mM NaF, 15 mM Na4P2O7, 2 mM Na3VO4, 1 mM EDTA,
protease inhibitors. The lysate was cleared by centrifugation and equal
amounts of protein were separated by 12% SDS-PAGE. Dually
phosphorylated ERK1/2 was visualized with phosphospecific antisera (New
England Biolabs) that detects ERK1/2 only when phosphorylated at
threonine 202 and tyrosine 204 (Thr(P)-E-Tyr(P) motif). Equal loading
was confirmed by reprobing the membrane with To test whether 14-3-3 binds and regulates RGS proteins we
used RGS3, RGS7, 14-3-3
-interacting portion of the RGS domain; interaction with 14-3-3 inhibits the GAP activity of RGS7, depends upon phosphorylation of a
conserved residue within the RGS domain, and results in inhibition of
GAP function. Collectively, these data indicate that
phosphorylation-dependent binding of 14-3-3 may act as
molecular switch that controls the GAP activity keeping a substantial
fraction of RGS proteins in a dormant state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
subunits of heterotrimeric G
proteins and stimulates their intrinsic GTPase activity. By
accelerating the inactivation of GTP-bound G subunits, RGS proteins
serve as negative regulators of G protein-mediated signaling pathways and inhibit and redirect G protein-stimulated cellular responses (1-4). Heterotrimeric G proteins transduce a wide variety of receptor-mediated signals across the plasma membrane (5). The ability
of RGS proteins to diminish the magnitude and duration of G
protein-dependent signaling mandates tight regulation of their GAP activity. RGS proteins are subject to both transcriptional and post-translational regulation (6-9). However, mechanisms directly
controlling GAP activity of RGS proteins are poorly understood.
, 
, 
, 
, 
, 
, 
, 
,
and 
) that modulate signaling events by binding to serine or
threonine-phosphorylated target proteins. 14-3-3 proteins have been
implicated in the activation of protein kinases, cell cycle control,
and regulation of apoptosis (12-17).
subunits and displaying sequence
conservation with other RGS family members. Furthermore, phosphorylation and subsequent interaction with 14-3-3 results in a
progressive decline in the GAP activity of RGS proteins. Our data
suggest that regulated phosphorylation/14-3-3 binding and
dephosphorylation within the RGS domain could function as a molecular
switch to turn off and on the GAP function of RGS proteins in
vivo.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
, kindly provided by Dr. Y.-C. Liu, was utilized to
generate Myc-, Flag-, and MBP-tagged versions. In some
experiments, an extended Myc-tagged 14-3-3 version was used to
differentiate transfected from endogenous 14-3-3 protein; this
construct contained eight additional amino acids (ERDSRGSL) at the COOH
terminus. m2 muscarinic receptor (OB-m2) was a kind gift of Dr. Silvio Gutkind.
prebound to glutathione-Sepharose beads
(Amersham Pharmacia Biotech). Bound proteins were separated by 10%
SDS-PAGE and RGS3 was visualized with anti-Flag antibody. Equal loading
of GST.14-3-3 was confirmed by Coomassie Blue staining of the gels.
fusion protein was immobilized on affinity chromatography minicolumns
(Bio-Rad) using glutathione-Sepharose beads; columns were washed
extensively and pre-equilibrated by three washes with lysis buffer (1%
Triton X-100, 20 mM Tris-HCl, pH 7.5, 50 mM
NaCl, 50 mM NaF, 15 mM
Na4P2O7, 2 mM
Na3VO4, 1 mM EDTA, and protease
inhibitors) at 4 °C. Whole brains of healthy adult WKY rats were
frozen in liquid nitrogen, homogenized in 4 ml of lysis buffer,
incubated on ice for 20 min, and centrifuged at 20,000 × g for 20 min. Supernatants were loaded on a GST or a
GST.14-3-3 column. Flow-through (identical volumes) was subjected SDS-PAGE and immunoblot analysis with anti-RGS7 antiserum, PKC, and
-catenin antibodies.
(1850 units/mg, Panvera) in enzyme dilution buffer or enzyme dilution buffer
alone (control). To monitor the incorporation of phosphate, the
unlabeled ATP was supplemented with 10 µCi of
[-32P]ATP, and radiolabeled
MBP.RGS7315-469 or GST.RGS7315-469 was
visualized by SDS-PAGE and autoradiography or spotted on nitrocellulose
filter and counted in a scintillation counter. For in vitro
interaction studies, purified recombinant protein (1 µg of
phosphorylated or unphosphorylated GST.RGS7315-469 or GST
alone) was immobilized to glutathione-Sepharose beads and incubated
with bacterial lysates containing 2.5 µg/ml recombinant MBP.14-3-3
for 90 min in 450 µl of binding buffer containing 50 mM
potassium phosphate, pH 7.5, 150 mM KCl, 1 mM
MgCl2, 10% (v/v) glycerol, 1% Triton X-100, and protease
inhibitors. The washed precipitate was separated on a 10% SDS
acrylamide gel. Bound MBP.14-3-3 was detected by immunoblotting
using an anti-MBP rabbit antiserum (New England Biolabs).
fusion proteins were expressed in
bacteria and purified on glutathione-Sepharose beads as described
previously (19, 20). Fluorescent peptides were synthesized using
N--Fmoc-protected amino acids and standard BOP/HOBt
coupling chemistry on an ABI 431A Peptide BioSynthesizer, with
fluorescein isothiocyanate connected to the peptide amino terminus via
a -alanine linker. Fluorescence polarization anisotropy was measured
using a Panvera Beacon 2000 Variable Temperature Fluorescence
Polarization System. Low fluorescence buffers and reagents (Panvera
Corp.) were used throughout. Binding curves were measured independently
in three separate experiments. 14-3-3 proteins were serially diluted
(0-223 µM) to a final volume of 150 µl in
phosphate-buffered saline in 6 × 50-mm borosilicate glass tubes.
Fluorescein-tagged peptide was added (59 nM final concentration), mixed, and fluorescence polarization measured at
22 °C after a 120-s delay with a 16-s integration. Background fluorescence was measured for each sample prior to peptide addition. Binding data was analyzed by assuming that fluorescence polarization was a linear function of ligand binding (21), and that each 14-3-3 monomer contained a single peptide-binding site (19). Curves were fit
to the equation: LB/Ltot = Lf/(kD + Lf),
where LB is bound ligand,
Ltot is total ligand, Lf is
free ligand and kD is the dissociation constant, in
closed form using nonlinear regression analysis (Kaleidograph).
i1 subunits (Calbiochem, 250 nM) were loaded with [-32P]GTP (1.0 µM) for 20 min at 30 °C in 500 µl of buffer
containing 50 mM HEPES, pH 8.0, 5 mM EDTA, 2 mM DTT, and 0.1% Lubrol. The stoichiometry of GTP binding
of Gi1 subunits was 25-40%. For zero time point a
12.5-µl aliquot was removed and added to 375 µl of 5% (w/v) Norit
in 50 mM NaH2PO4. GTP hydrolysis
was initiated at 4 °C by adding 150 µl of the loaded
Gi1 subunits on ice to MgCl2 (15 mM final concentration) and unlabeled GTP (150 µM final concentration), with or without purified,
phosphorylated or unphosphorylated MPB.RGS7315-469 (1.0 µM final concentration), that was preincubated with
GST or GST.14-3-3 (final concentration 5 µM, 30 min on
ice) as indicated. Aliquots of 25 µl were removed from the hydrolysis
reaction, mixed with 375 µl of 5% (w/v) Norit in 50 mM
NaH2PO4 on ice, centrifuged at 10,000 rpm for 5 min, and counted by liquid scintillation spectrometry. Zero time values
were subtracted from all experimental points. Statistical analysis was
performed using the statistical and curve fitting functions of
SigmaPlot 4.01 (Jandel Scientific). Hydrolysis rate constants were
calculated according to Wang et al. (27). To demonstrate
statistical significance of differences in GTP hydrolysis, hydrolysis
rate constants were normalized, expressed as fold increase of basal
hydrolysis rate constant, and averages of these constants were depicted
in a table.
-actin and Amido Black
staining. The degree of dual phosphorylation of ERK1/2 was quantitated
by densitometric analysis of non-saturated radiographs with the NIH
Image software.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
, and 14-3-3 as model proteins. Both RGS3 and RGS7 specifically interacted with 14-3-3 proteins in transfected HEK 293T cells (Fig. 1). Epitope-tagged
14-3-3 co-precipitated with RGS3 and RGS7 but not with control proteins
and vice versa. Truncations of RGS3 and RGS7 were generated to localize
the site of interaction with 14-3-3 to the RGS domains (Fig. 1,
a and e). To more quantitatively assess the
amount of RGS3 complexed with 14-3-3 we labeled transiently transfected
HEK 293T cells with [35S]methionine/cysteine.
Immunoprecipitation from labeled cells revealed that approximately 70%
of the immunoprecipitated RGS3 was complexed with 14-3-3 (Fig.
2a). Since immunoprecipitation of Myc-tagged 14-3-3 immobilized only 15% of the RGS3, it appears that most of the RGS3 is bound to 14-3-3, whereas the majority of
14-3-3 is complexed with other cellular proteins. 14-3-3 proteins generally recognize their ligands only following serine/threonine phosphorylation (19, 20, 25). We therefore examined whether treatment
of HEK 293T cells with staurosporine, a broad spectrum inhibitor of
protein kinases, would prevent the phosphorylation of RGS proteins and
their subsequent interaction with 14-3-3. Staurosporine nearly
abrogated the interaction between RGS3 and 14-3-3 in vivo
and in vitro. Only trace amounts of 14-3-3 were co-immunoprecipitated in staurosporine-treated HEK 293T cells (Fig.
2b). Similarly, treatment of RGS3-expressing HEK 293T cells with staurosporine dramatically reduced binding of RGS3 to immobilized 14-3-3 in vitro (Fig. 2c). Note that the
staurosporine treatment did not cause a nonspecific reduction in the
cellular amounts of either RGS3 or 14-3-3 (Fig. 2 b and
c, lower panels). Similar experiments with RGS7
were precluded by the destabilization of RGS7 by staurosporine
treatment. Both RGS7 and 14-3-3 are highly abundant in mouse brain.
When we examined their endogenous interaction by co-immunoprecipitation
of mouse brain lysates (Fig.
3a), 14-3-3 specifically
co-precipitated with RGS7 indicating an endogenous in vivo
interaction (Fig. 3b). To quantitatively assess the capacity of endogenous RGS7 to interact with 14-3-3, we determined the fraction
of endogenous RGS7 retained by a recombinant glutathione S-transferase-14-3-3 fusion protein immobilized to a
glutathione-Sepharose column. Unbound RGS7 was measured in the
flow-through by immunoblotting; PKC and -catenin levels were used
to correct for unspecific binding and equal loading (Fig.
3c). More than 50% of the RGS7 contained in brain lysates
was retained on a GST.14-3-3 column (Fig. 3d).
Collectively, these data indicate that RGS3 and RGS7 interact with
14-3-3 in a phosphorylation-dependent manner. This interaction
does not only occur in transfected cells but can also be demonstrated
with endogenous proteins. Furthermore, the data suggest that a
substantial fraction of RGS proteins is bound to 14-3-3 in
vivo.
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Fig. 1.
The RGS domains of RGS3 and RGS7
specifically interact with 14-3-3. Lysates were prepared from HEK
293T cells, transfected with epitope-tagged constructs as indicated,
and immunoprecipitated with Flag- or Myc-specific antibodies, and
resolved by SDS-PAGE. a, 14-3-3 co-precipitates with RGS3
as well as with truncations RGS3314-520 and
RGS3421-520, but not with RGS31-389 or the
control protein GFP (upper left panel); HC
denotes IgG heavy chain and LC light chain. Expression levels of
Myc-tagged 14-3-3 in cell lysates are shown in the lower
panel, the expression levels of Flag-tagged RGS3 truncations are
shown in the right panel. b, RGS3 co-precipitates with
14-3-3, but not with TRAF2 (upper panel). Expression
levels of Flag-tagged RGS3 in cell lysate are shown in the
lower panel; expression levels of myc.14-3-3 and
myc.TRAF2 are shown in the right panel. c,
14-3-3 co-precipitates with RGS3, but not with Tau protein
(upper panel). Expression levels of Flag-tagged RGS3, Tau,
and a HA-tagged 14-3-3 in the lysate are shown in the lower
panel. d and e, 14-3-3 co-precipitates
with RGS7 and the RGS7315-469 truncation that contains the
RGS domain of RGS7 (amino acid 333 to 448). Serine to alanine
substitution at position 434 of RGS7 (F.RGS7S434A,
RGS7315-469 S434A) abrogates the interaction with
14-3-3 (upper panels). Expression levels of myc.14-3-3
in cell lysates are shown in the lower panel (d)
and middle panel (e), respectively. Expression
levels of RGS7315-469 and RGS7315-469 S434A
are shown in the lower panel e.
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Fig. 2.
The interaction of RGS proteins with 14-3-3 is phosphorylation-dependent. a, autoradiograph
after in vivo labeling with
[35S]methionine/cysteine, and immunoprecipitation of
transiently transfected HEK 293T cells with anti-Flag or anti-Myc
antibodies. An extended myc.14-3-3 construct was used that is easily
distinguished from endogenous 14-3-3, labeled "14-3-3."
b, co-precipitation of 14-3-3 with RGS3 is decreased
after staurosporine pretreatment (0.5 µM, 2 h)
(upper panel). Expression levels of transiently transfected
Flag-tagged RGS3, Tau, and Myc-tagged 14-3-3 in HEK 293T cellular
lysates prior to IP are shown in the lower panel.
c, pull-down of RGS3 with GST.14-3-3 prebound to
glutathione-Sepharose beads is diminished after staurosporine
pretreatment. Precipitated RGS3 was detected with anti-Flag antibody
(upper panel). Expression of Flag-tagged RGS3 and Tau in
lysates of transiently transfected HEK 293T cells are shown in the
middle panel. Coomassie staining of GST.14-3-3, used to
pull down RGS protein, is shown in the lower panel.
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Fig. 3.
Endogenous interaction of RGS7 with
14-3-3. a, Western blot analysis reveals that
RGS7 and 14-3-3 are highly abundant in mouse brain. b,
co-immunoprecipitation of endogenous RGS7 and 14-3-3 from mouse
brain. Mouse brain lysates were immunoprecipitated with control or
anti-RGS7 antiserum, and resolved by 15% SDS-PAGE. Co-precipitated
14-3-3 protein was visualized with an anti-14-3-3 antibody. The
first lane is a positive control (myc.14-3-3 ); the
second lane represents brain lysates before
immunoprecipitation; the third lane represents
immunoprecipitates from mouse brain with control antiserum; and the
fourth lane demonstrates the co-immunoprecipitation of
14-3-3 with RGS7 from mouse brain using a specific anti-RGS7 polyclonal
antiserum. LC denotes the position of the light chain.
c, precleared brain lysates containing native RGS7 were
incubated with either GST (control) or GST.14-3-3, immobilized on
glutathione-Sepharose beads. Equal amounts of the flow-through were
separated by SDS-PAGE and sequentially probed with anti-RGS7,
anti-PKC, and anti--catenin. d, densitometric
analysis, performed on non-saturating autoradiographs using the NIH
image software and corrected for levels of the control proteins PKC
and -catenin.
Mutational analysis revealed that serine 434 of RGS7 was critical for
binding to 14-3-3. Replacement of the serine residue at position 434 of
RGS7 by alanine completely abolished binding of 14-3-3 to both
full-length RGS7 and to the isolated RGS domain (RGS7315-469) (Fig. 1, d and e). To
demonstrate that phosphorylation of serine 434 is required for the
interaction of RGS7 and 14-3-3, the interaction of 14-3-3 with
unphosphorylated and phosphorylated RGS7 was tested in
vitro. As expected, no interaction was detectable between
bacterially expressed, unphosphorylated RGS7315-469 fused
to GST (GST.RGS7315-469) and 14-3-3 fused to MBP
(MBP.14-3-3) (Fig. 4a),
whereas phosphorylation by PKC enabled GST.RGS7315-469
to bind MBP.14-3-3. Replacement of serine 434 in RGS7 by aspartate to mimic serine phosphorylation facilitated a constitutive association between RGS7 and 14-3-3 that remained unaffected by subsequent phosphorylation with PKC (data not shown), suggesting that serine 434 is the relevant PKC phosphorylation site.
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Fluorescence polarization measurements confirmed that 14-3-3 rapidly
binds to a 14-mer phosphopeptide containing the serine 434 14-3-3-binding site of RGS7 with a kD of 15.9 µM (Fig. 4 b and c). Most published
affinities for 14-3-3 interacting peptide sequences range from 0.1 to 2 µM, using surface plasmon resonance (Biacore); however,
the interpretation of these affinities is complicated by an avidity
effect since dimeric 14-3-3 may simultaneously bind to two
phosphopeptides immobilized on the Biacore chip (19), while the
fluorescence polarization experiments were performed with solubilized
molecules. Given the strong interaction of 14-3-3 with RGS7 and RGS3 in
the co-immunoprecipitation and pull-down experiments, this moderate
kD suggests that additional factors such as
dimerization of RGS proteins or tandem binding of 14-3-3 may contribute
to the interaction of RGS proteins with 14-3-3 in vivo.
Indeed, both RGS3 and RGS7 contain at least two additional potential
14-3-3-binding sites in close proximity within the RGS domain. Although
our data clearly implicate serine 434 as a critical 14-3-3 binding
residue in RGS7, it is conceivable that simultaneous binding to
additional sites increases the affinity and stability of this
interaction. Several 14-3-3 binding proteins such as c-Raf-1, Cbl, and
BAD contain two 14-3-3 binding sequences separated by polypeptide
segments of various length, and tandem binding to adjacent 14-3-3 sites
has been shown to facilitate the formation of a high-affinity,
bidentate complex (19).
Based on the resolution crystal structure of RGS4 complexed with
activated Gi subunits (26) the 14-3-3 binding site at serine 434 in RGS7 aligns with one of the three putative domains required for Gi1 interaction and stimulation of GTPase
activity of Gi1 (Fig.
5c). To test whether binding
of 14-3-3 interferes with the GTPase accelerating activity of RGS7, we
measured the GAP activity of RGS7 in single-turnover GTP hydrolysis
assays (22). GTP hydrolysis follows a single exponential time course equivalent to a first-order reaction which can be expressed by means of
the rate constant of a first-order reaction (27). Addition of 14-3-3 reduced the hydrolysis rate constant of phosphorylated RGS7 from 10- to
3.5-fold (Fig. 5a), while addition of 14-3-3 to
unphosphorylated RGS7 or Gi1 had no effect on the
hydrolysis rate constant (data not shown). In order to more
quantitatively assess the effect of phosphorylation and/or 14-3-3 interaction on GAP activity of RGS7 hydrolysis, rate constants of
several experiments were averaged and expressed as fold increase of
basal hydrolysis rate (Table I). Addition
of 14-3-3 to phosphorylated RGS7 almost completely abrogated the GTPase
stimulatory effect, suggesting that phosphorylation of serine 434 and
the subsequent interaction with 14-3-3 dramatically interferes with
binding of activated Gi1. These findings suggest that a
phosphorylation-dependent interaction between 14-3-3 and
the RGS domain may regulate the GAP activity of RGS proteins; however,
the kinase responsible for this phosphorylation in vivo has
yet to be determined. RGS3 impairs MAP kinase activation by mammalian G
protein-linked receptors in human embryonic kidney (HEK) cells (28). To
illustrate the functional consequences of the interaction between
14-3-3 and RGS proteins, we co-transfected the Gi-linked
m2 cholinergic receptor with and without RGS3 and 14-3-3 into HEK cells
expressing Rap1 and Rap1GAPII. Stimulation with carbachol (30 µM, 15 min) resulted in a strong dual phosphorylation of
ERK1 and ERK2 on threonine 202 and tyrosine 204 (Fig. 5b).
Dual phosphorylation of ERK1/2 within the T-E-Y motif leads to
the activation of the kinase and represents a sensitive measure
of ERK1/2 activity. RGS3 inhibited the carbachol-mediated MAP kinase
phosphorylation. Co-expression of 14-3-3 rescued the
Gi-induced MAP kinase phosphorylation from this
inhibitory effect of RGS3 (Fig. 5b), but did not affect MAP
kinase activation in the absence of RGS3 (data not shown). Equal
protein loading was ensured by reprobing the blot against actin and by
Amido Black staining. These data provide further evidence that binding
of 14-3-3 counteracts the inhibitory effect of RGS proteins on G
protein-initiated signaling.
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Alignment of the sequence bordering serine 434 in RGS7 with other RGS
members reveals a putative 14-3-3 binding motif: (K/E)-(K/R)-D-pS-Y-P (Fig. 5c) (19, 20). Since the 14-3-3-binding site in RGS7 is
conserved in other RGS members, we speculate that 14-3-3 binding may
similarly regulate the GAP activity of other RGS proteins. It is
unclear whether the reduction of RGS GAP activity depends upon a
conformation change induced by 14-3-3 binding or on the physical
impedance of the association of RGS and G. Our data suggest that
phosphorylation-dependent interaction of RGS proteins with
regulatory proteins such as 14-3-3 may rapidly and dynamically control
RGS GAP activity without altering their expression.
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ACKNOWLEDGEMENTS |
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We thank Dr. Lewis Cantley and Dr. Matthew J. F. Waterman for helpful discussions.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft (DFG) Grants BE 2212/2-1 (to T. B.) and WA 597/3-1 (to G. W.) and National Institutes of Health Grant HL03601 (to M. B. Y.).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: Unité de Biologie et Biochimie
Cellulaire, Facultés Universitaires Notre-Dame de la Paix, 61, rue de Bruxelles, 5000 Namur, Belgium.
§§ To whom correspondence should be addressed. Tel.: 49-761-270-3250; Fax: 49-761-270-3245; E-mail: walz@med1.ukl.uni-freiburg.de.
Published, JBC Papers in Press, June 21, 2000, DOI 10.1074/jbc.M002905200
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ABBREVIATIONS |
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The abbreviations used are:
RGS, regulator of G
protein signaling;
GST, glutathione S-transferase;
GAP, GTPase-activating protein;
PAGE, polyacrylamide gel electrophoresis;
PKC, protein kinase C-;
HEK, human embryonic kidney;
MAP, mitogen-activated protein.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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).
Phosphorylated MBP.RGS7315-469 (upper curve;
) enhances the rate of GTP hydrolysis by purified
G
).
b, HEK 293T cells were co-transfected with the m2 muscarinic
acetylcholine receptor, F.RGS3, and myc.14-3-3