From the Laboratory of Allergic Diseases, NIAID,
National Institutes of Health, Rockville, Maryland 20852, the
§ Department of Cellular and Molecular Medicine, University
of California School of Medicine, San Diego,
La Jolla, California 92093, the ¶ Department of Physiology,
Cornell University, Weill Medical College,
New York, New York 10021, and
Molecular Pharmacology Group,
Division of Biochemistry and Molecular Biology, Institute of Biomedical
and Life Sciences, University of Glasgow,
Glasgow G12 8QQ, Scotland, United Kingdom
Received for publication, October 9, 2002, and in revised form, January 17, 2003
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ABSTRACT |
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The amplitude of signaling evoked by stimulation
of G protein-coupled receptors may be controlled in part by the GTPase
accelerating activity of the regulator of G protein signaling (RGS)
proteins. In turn, subcellular targeting, protein-protein interactions, or post-translational modifications such as phosphorylation may shape
RGS activity and specificity. We found previously that RGS16 undergoes
tyrosine phosphorylation on conserved tyrosine residues in the RGS box.
Phosphorylation on Tyr168 was mediated by the
epidermal growth factor receptor (EGFR). We show here that endogenous
RGS16 is phosphorylated after epidermal growth factor stimulation of
MCF-7 cells. In addition, p60-Src or Lyn kinase phosphorylated
recombinant RGS16 in vitro, and RGS16 underwent
phosphorylation in the presence of constitutively active Src (Y529F) in
EGFR G protein-coupled receptors
(GPCRs),1 the largest family
of proteins in the human genome (1), mediate extracellular signals that
control such diverse processes as sensation, cognition, cell growth and
proliferation, cell migration, and hormone secretion. GPCRs signal
through a common element, the heterotrimeric G protein, whose One means by which RGS proteins may be modified is phosphorylation
(6-17). Sst2 undergoes signal-dependent phosphorylation by
MAP kinase, which slows Sst2 degradation (6). 14-3-3 proteins interact
with phosphorylated RGS7, which reduces RGS7 GAP activity (7). Protein
kinase C phosphorylates RGS2 on a serine residue in the RGS box,
resulting in decreased GAP activity toward G In addition to direct effects on RGS enzymatic activity, Ser
phosphorylation may alter intracellular localization of certain RGS
proteins. Protein kinase A phosphorylates RGS10, and mutation of the
protein kinase A target Ser residue prevents RGS10 translocation from
the plasma membrane and cytosol to the nucleus (12). Upon illumination
of rod outer segments, RGS9-1 translocates to lipid rafts (13), where
it undergoes Ser phosphorylation by an endogenous kinase (14).
RGS proteins also contain conserved tyrosine residues in the RGS box
that appear to affect their function. Phosphorylation of the RGS
domain-containing protein PDZ-RhoGEF by focal adhesion kinase enhances
Rho activation after stimulation of G In this study we investigated further mechanisms of RGS16 tyrosine
phosphorylation and their possible effects on RGS16 function. We found
that RGS16 was a substrate for Src family kinases in vitro
and in mammalian cells. Src-mediated RGS16 phosphorylation did not
affect RGS16 intracellular localization or GAP activity, but instead
appeared to slow RGS16 protein degradation. Moreover, sustained
phosphorylation correlated with an expanded pool of RGS16 and increased
GAP activity in cell membranes. These results demonstrate that RGS16
function is modulated by tyrosine phosphorylation in mammalian cells,
suggesting a novel form of feedback regulation of G protein signaling
by tyrosine kinases.
Reagents--
ATP, sodium orthovanadate, hydrogen peroxide
(H2O2), doxorubicin, EGF, and carbachol were
purchased from Sigma. PP2, PP3, and the proteasome inhibitor MG132 were
obtained from Calbiochem. Pervanadate consisted of a mixture of 0.1 mM sodium orthovanadate and 10 mM
H2O2.
Cells, Proteins, and Plasmids--
HEK 293T and CHO-K1 cells
were maintained in supplemented Dulbecco's modified Eagle's medium
(Invitrogen) containing 10% fetal bovine serum, 2 mM
L-glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, and 20 ng/ml gentamicin, in a humidified 5%
CO2 incubator at 37 °C. BaF3 cells stably expressing
mouse HA-RGS16, the gift of Cheryl Miller (Millennium Pharmaceuticals),
were grown in RPMI + 10% fetal bovine serum and interleukin-3 (10 ng/ml, PeproTech). For transfections, 2 × 106 cells
were plated in 100-mm tissue culture dishes and transfected with
appropriate plasmids for 18 h using LipofectAMINE reagent (Invitrogen) or for 5 h using Superfect (Qiagen) according to the
manufacturer's instructions. After removal of the DNA mixture, the
cells were incubated in serum-free medium until harvest 24-48 h after transfection.
Plasmids encoding pcDNA3-HA-RGS16 (human) (the kind gift of Drs.
Carol Beadling and Kendall Smith (Cornell University Medical College)),
pcDNA3.1-RGS16-V5/His, and tyrosine mutants thereof have been
described previously (16) as have plasmids encoding His6RGS16, His6RGS2, and the purification of
recombinant RGS proteins from Escherichia coli. The plasmid
pUSEamp-Src (Y529F) (constitutively active) was purchased from Upstate
Biotechnology, Inc., as were the recombinant kinases Src, Lyn, Csk, and
Abl. In some kinase assays, Src or Btk kinases purified from Sf9
cells were used, as described elsewhere (17). The plasmid encoding the
m2 muscarinic receptor was the gift of J. Silvio Gutkind (NICDR,
National Institutes of Health).
Immunoprecipitation and Immunoblotting--
After stimulation,
we rinsed cells twice in ice-cold phosphate-buffered saline before
lysis in buffer P, containing 20 mM Tris, pH 7.5, 250 mM NaCl, 1 mM NaF, 1% Triton X-100, 1 mM Na3VO4, and a Complete tablet of
protease inhibitors (Roche Molecular Biochemicals). We
immunoprecipitated equal protein amounts (500-600 µg) with either
rabbit polyclonal anti-HA (2 µg ml In Vitro Phosphorylation--
We determined RGS phosphorylation
by incubating 10-25 pmol of His6RGS16 for 1 h at
30 °C with recombinant Lyn, Csk, or Abl kinases (1 µl) in buffer
containing 50 mM HEPES, pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 1 mg
ml Proteolytic Cleavage--
Protease digestion with endoproteinase
Lys-C (500 ng, Roche Molecular Biochemicals) was carried out after
phosphorylation in 30 µl of incubation buffer (25 mM
Tris, pH 8.5, 1 mM EDTA) for 2 h at 37 °C.
His-tagged RGS16 was phosphorylated as described for 1 h before
affinity purification by nickel-agarose chromatography. Beads were
washed twice in detergent-containing buffer P-Ni and once in protease
incubation buffer before the addition of protease. Digests were
separated on 16% Tricine gels and run alongside Mark 12 protein
ladders (Invitrogen).
Single Turnover GAP Assays--
We performed single turnover
GTPase assays by loading recombinant, myristoylated G Metabolic Labeling and Pulse-Chase Analysis--
BaF3/RGS16
cells (5 × 106 cells/ml) were washed once in
methionine-free RPMI (Biofluids) and then incubated for an additional 30 min in this medium to deplete intracellular methionine pools. [35S]Methionine (Amersham Biosciences, >1000 Ci/mmol, 10 mCi/ml) was added at a final concentration of 150 µCi/ml for 30 min
at 37 °C. Cells were then pelleted, washed in chase medium
(serum-free RPMI supplemented with cold methionine (Sigma, 15 mg/liter)), and incubated at 37 °C in chase medium for the indicated
times in the presence or absence of the appropriate agonist or
inhibitor. Cells were lysed in buffer P and immunoprecipitated with
either anti-RGS16 antibodies or HA-coupled agarose (Roche Molecular
Biochemicals) as indicated. Immunoprecipitates were resolved by
SDS-PAGE and exposed to autoradiography at Preparation of Membranes--
Generation of HEK 293 cells stably
expressing a 5-hydroxytryptamine 1A receptor
(5-HT1A)/G High Affinity GTPase Assays--
Steady-state GTPase activity
was determined in membranes expressing the fusion protein. Membrane
preparations (10 µg of total protein) were stimulated with the
indicated concentrations of 5-HT in an ATP-regenerating buffer system
(20 mM creatine phosphate, 0.1 unit/µl creatine kinase,
200 µM AMP-PNP, 500 µM ATP, 1 µM GTP, 2 mM ouabain (all from Sigma), 200 mM NaCl, 10 mM MgCl2, 4 mM dithiothreitol, 200 mM EDTA, pH 7.5, 80 mM Tris, pH 7.5), spiked with 50,000 cpm
[ Densitometry--
Immunoblots and autoradiographs were
quantitated using Scion Image software (Scion Corp.).
Phosphorylation of Endogenous RGS16 in MCF-7 Cells--
Our
previous studies demonstrated EGF-mediated RGS16 tyrosine
phosphorylation in transfected cells. To explore RGS16 phosphorylation further, we first investigated whether endogenous RGS16 underwent tyrosine phosphorylation in response to EGF stimulation in MCF-7 breast
carcinoma cells. Rgs16 was discovered as a target of the p53
tumor suppressor gene and shown to be up-regulated by genotoxic stress
in these cells (23). By using specific anti-RGS16 antibodies to
immunoblot MCF-7 cell lysates, we identified a single band of the
predicted molecular weight of RGS16 (23 kDa) (Fig.
1A, top). The intensity of the
band increased after treatment of the cells with doxorubicin,
suggesting an increase in RGS16 protein expression similar to the rise
in RNA levels observed in the previous study (23). To confirm the
identity of this band as RGS16, we immunoprecipitated MCF-7 cell
lysates or recombinant RGS16 with anti-RGS16 antibodies or anti-RGS4 as
a control. Immunoprecipitates were then immunodetected with the same
anti-RGS16 antibody used for immunoprecipitation. We visualized the
23-kDa band only in anti-RGS16 immunoprecipitates (Fig. 1A,
bottom). Finally, to determine whether endogenous RGS16
underwent tyrosine phosphorylation, we treated the cells with either
medium alone or EGF, and we immunoblotted cell lysates with either
anti-phosphotyrosine (Fig. 1B, top) or anti-RGS16
(Fig. 1B, bottom). RGS16 was not phosphorylated under basal
conditions but was significantly phosphorylated after EGF treatment.
These results suggest that RGS16 tyrosine phosphorylation occurs in
response to a physiological stimulus in MCF-7 cells.
Src Phosphorylates RGS16 in Vitro--
To determine which, if any,
additional kinase(s) mediate RGS16 tyrosine phosphorylation, we tested
the capacity of purified RGS16 to act as a substrate for various
kinases in vitro. We incubated RGS2 or RGS16 with
recombinant kinase and [ In Vivo RGS16 Phosphorylation in the Presence of Constitutively
Active Src--
We next determined whether Src plays a role in RGS16
phosphorylation in mammalian cells. This hypothesis is difficult to
test directly in cells that express both Src and EGFR because EGFR stimulates Src kinase activity (24), and in turn Src enhances EGFR
activity (25). To minimize a possible effect of EGFR kinase, we
examined RGS16 phosphorylation in CHO-K1 cells, which do not express
functional EGFR but do express a closely related EGFR family member
ErbB2 (26, 27). To determine whether these cells respond to EGF, we
measured MAP kinase activation after EGF or carbachol stimulation in
cells transfected with m2R by immunoblotting with anti-phospho-ERK and
ERK1 antibodies. Similar to previous studies, EGF treatment of CHO-K1
cells did not induce ERK activation, whereas increased ERK activity was
observed after carbachol treatment (28) (Fig.
3A). To evaluate RGS16
phosphorylation in the presence of Src activation, we expressed
HA-RGS16 with control or constitutively active Src (Y529F) plasmids,
and we assessed RGS16 phosphorylation by immunoprecipitation with
anti-HA and detection with anti-phosphotyrosine antibodies. RGS16
underwent pronounced tyrosine phosphorylation in the presence of active
Src (Fig. 3B). To determine whether endogenous Src kinase(s)
in CHO-K1 cells mediated RGS16 phosphorylation, we expressed
RGS16-V5/His and treated cells with pervanadate, an inhibitor of
tyrosine phosphatases. We affinity-purified recombinant His-tagged
proteins with Ni2+/nitriloacetic acid-coupled agarose and
immunoblotted with anti-phosphotyrosine and anti-V5. There were no
tyrosyl-phosphorylated proteins in affinity precipitations from
vector-transfected or RGS16-transfected cells without pervanadate
stimulation. In contrast, we observed strong RGS16 phosphorylation in
pervanadate-treated cells, which was blocked by the Src-specific
inhibitor PP2 (Fig. 3C). This result indicates that
pervanadate-induced RGS16 phosphorylation in CHO-K1 cells is
attributable to activity of endogenous Src kinase(s).
Receptor-induced RGS16 Tyrosine Phosphorylation Is Sensitive to Src
Kinase Blockade--
We have shown previously (16) that GPCR
stimulation (m2R) induces RGS16 tyrosine phosphorylation. To examine
whether Src plays a role in GPCR-induced RGS16 tyrosine
phosphorylation, we utilized BaF3 B-lymphoblastoid cells lacking
endogenous RGS16 that stably overexpress HA-RGS16. We stimulated cells
with CXCL12, which activates endogenous CXCR4 chemokine receptors
coupled to G Tyr177 Is the Putative Site of Src-induced
Phosphorylation--
To determine which residue might be
phosphorylated by Src, we incubated purified Src and ATP with
recombinant WT RGS16 or mutants and detected phosphorylation by
immunoblotting with anti-phosphotyrosine. We observed Src-induced
phosphorylation of RGS16 WT and reduced phosphorylation of Y168F but no
phosphorylation of Y177F or the double mutant. These results suggest
that Src-mediated phosphorylation requires both residues (Fig.
5A). However, we hypothesize
that the diminished phosphorylation of the Y168F mutant may be because of altered protein folding or conformation, as this mutation reduces GAP activity of the recombinant protein in vitro,
independently of phosphorylation (16). To map the phosphorylation site
in the WT protein, we digested phosphorylated RGS16 with endoproteinase Lys-C (EndoLys-C). This protease cleaves at the C terminus of lysine
residues, and RGS16 contains 15 potential cleavage sites. Of the
tyrosine-containing peptides, EndoLys-C is predicted to generate a
C-terminal fragment consisting of residues 174-202 (containing
Tyr177) plus an additional 13 linker residues contained in
the plasmid used to generate recombinant RGS16. Together, this
polypeptide has an expected mass of 4.2 kDa. After incubation with Src
kinase plus [
To determine the site of phosphorylation of RGS16 in cells, we
expressed HA-RGS16 WT or mutants with active Src (Y529F) in CHO-K1
cells. We immunoprecipitated RGS16 with anti-HA and blotted with
anti-phosphotyrosine or anti-HA to confirm equal RGS16 levels. Whereas
phosphorylation of RGS16 (Y168F) was similar to WT, the level of
phosphorylation of RGS16 (Y177F) was reduced by at least 50% (Fig.
5D), and Y168/Y177F was not phosphorylated (data not shown).
Collectively, although these experiments suggest that Tyr177 may be the site of Src-mediated RGS16
phosphorylation, it also appears that Tyr168 is
phosphorylated in the presence of constitutively active Src in
vivo. As mentioned above, Tyr168 phosphorylation could
be an indirect consequence of Src activation, which is discussed below.
Src-induced RGS16 Tyrosine Phosphorylation Does Not Affect GAP
Activity in Vitro or Localization in Transfected Cells--
In a
previous study, we found that EGFR-mediated tyrosine phosphorylation of
RGS16 increased its GAP activity on G
We hypothesized that because Src-mediated tyrosine phosphorylation did
not appear to affect RGS16 GAP activity, it could play a role in
protein localization. To address this possibility, we expressed
RGS16-GFP in CHO-K1 cells together with lacZ or active Src
and examined the cells by fluorescence microscopy. Transfected RGS16-GFP was distributed throughout the cytoplasm and at the membrane,
consistent with its distribution in fractionation experiments (29), and
co-expression of Src (Y529F) did not grossly alter the expression
pattern of RGS16-GFP (data not shown).
Src-mediated RGS16 Phosphorylation Enhances RGS16 Protein
Stability--
Because phosphorylation was shown to increase stability
of another RGS protein, Sst2 (6), we hypothesized that RGS16 tyrosine phosphorylation could affect RGS16 degradation. To test this
possibility, we expressed RGS16 in CHO-K1 cells in the presence of
either lacZ or Src (Y529F) plasmids. 24 h after
transfection, we added cycloheximide to prevent de novo
protein synthesis and collected cells at various time points to
evaluate RGS16 levels by immunoblotting. The rate of RGS16 degradation
was decreased in the presence of activated Src, suggesting enhanced
protein stability (Fig. 7A).
The half-life of RGS16 in the absence of Src was ~2 h, but in the
presence of active Src, RGS16 levels diminished more slowly over a
period of 4 h (Fig. 7B). To determine whether RGS16
stability in the presence of active Src was related to phosphorylation,
we stimulated cycloheximide-treated cells with pervanadate, and we
assessed the rate of RGS16 degradation by immunoblotting. The half-life of RGS16 in the presence of pervanadate was significantly prolonged, from less than 30 min to greater than 60 min after pervanadate treatment (Fig. 7C). We hypothesize that the variability in
the estimated half-life of RGS16 in our experiments is most likely the
result of differing concentrations of cycloheximide or the presence or
location of epitope tags used in different experiments. To ascertain
whether the enhanced RGS16 stability we observed with Src co-expression
was dependent on RGS16 tyrosine phosphorylation, we expressed the
phosphorylation-resistant RGS16 mutant (Y168F/Y177F) and
assessed RGS16 degradation after pervanadate treatment or in the
presence of co-transfected Src (Y529F) by immunoblotting. In either
case, RGS16 (Y168F/Y177F) showed a similar half-life and did not
undergo phosphorylation (Fig. 7, D-F, and data not shown).
Taken together, these results suggest that Src-mediated RGS16
phosphorylation promotes RGS16 stability in CHO-K1 cells.
To determine whether RGS16 protein turnover was slowed by physiological
Src activation, we analyzed RGS16 levels in BaF3-RGS16 cells stimulated
through CXCR4. We performed pulse-chase analysis of radioactively
labeled RGS16 to exclude the possibility that cycloheximide affected
RGS16 degradation in previous experiments and to measure protein levels
more precisely than by quantitative immunoblotting. Cells were
metabolically labeled with [35S]methionine and then
treated with medium alone or medium containing CXCL12 for various
times. Detergent lysates were then immunoprecipitated with anti-RGS16,
electrophoresed, and visualized by autoradiography. We did not detect
RGS16 in immunoprecipitates of vector-transfected cells (Fig.
8A, lane 1), but we observed a
doublet of ~30 kDa in anti-RGS16 immunoprecipitates. The identity of
the lower band is uncertain, but it has been observed previously in
immunoprecipitates of endogenous RGS16 from rat liver using this
antiserum (29). We hypothesize that the lower molecular weight band
could represent either a proteolytic cleavage product or alternative
start site because it is not recognized by anti-HA antibody (see
below). Treatment with chemokine appeared to decrease the rate of RGS16 degradation over a 3-h period. Interestingly, at the 3-h time point,
RGS16 immunoprecipitates contained several bands of both higher and
lower molecular weight. Because RGS16 has been shown to be an N-end
rule substrate in reticulocyte lysates, signifying proteasome-mediated
degradation, we hypothesized that these bands could represent
multiubiquitinated RGS16 (30). To confirm that RGS16 degradation was
proteasome-dependent in mammalian cells, we performed
pulse-chase in the presence or absence of the proteasome inhibitor
MG132. In this experiment, lysates were immunoprecipitated with agarose
coupled to HA and immunoblotted with anti-RGS16; thus, a single band of
~30 kDa was observed by autoradiography (Fig. 8B) or by
immunoblotting (not shown). With MG132 treatment, the rate of RGS16
degradation appeared to be significantly reduced, comparable with the
rate induced by GPCR stimulation. These results suggest that
GPCR-evoked phosphorylation slows RGS16 turnover and that RGS16
degradation is dependent on the proteasome.
RGS16 Phosphorylation Enhances GAP Activity in Cell
Membranes--
We hypothesized that, over time, induction of RGS16
phosphorylation would lead to a larger pool of available GAP due to
reduced degradation and would thus increase GAP activity in cell
membranes. To explore this possibility, we measured 5-HT-evoked GTPase
activity in HEK 293 membranes expressing a fusion protein between the
5-hydroxytryptamine 1A receptor (5-HT1A) and
G We identified two tyrosine residues in the RGS box conserved in
many mammalian RGS proteins that were potential sites of
phosphorylation. Although our previous work (16) implied a role for
EGFR-mediated phosphorylation of RGS16 on Tyr168 in the
regulation of RGS16 GAP activity, under conditions of reduced tyrosine
phosphatase activity both RGS16 Tyr residues appeared to be
phosphorylated in HEK 293T cells. Therefore, we investigated the
possible role of Tyr177 phosphorylation for RGS16 function.
We found that Src family kinases phosphorylated WT RGS16 but not RGS16
(Y177F), and we observed in vitro phosphorylation of a
peptide with the expected molecular mass of a proteolytic cleavage
product containing Tyr177, suggesting that this residue was
the site of Src-mediated RGS16 phosphorylation. Interestingly, however,
when RGS16 (Y168F) or (Y177F) was expressed with constitutively active
Src in CHO-K1 cells, each mutant was phosphorylated. We hypothesize
that phosphorylation of RGS16 (Y177F) could result from
Tyr168 phosphorylation by a receptor tyrosine kinase
closely related to EGFR (ErbB2) expressed in these cells (25). There is
evidence that Src mediates direct EGFR phosphorylation on tyrosine
residues which augments EGFR activity (32, 33). In addition,
G The importance of Tyr177 for RGS16 function was suggested
by our previous study (16), which showed that mutation of this residue did not affect RGS16 GAP activity in vitro but eliminated
the ability of RGS16 to regulate Gq-coupled MAP kinase
activation or Gi-mediated inhibition of adenylyl cyclase.
We extended these observations in the current work by demonstrating
that Src-induced phosphorylation (putatively on Tyr177) did
not alter the GAP activity of RGS16 toward G CHO-K1 cells. Blockade of endogenous Src activity by
selective inhibitors attenuated RGS16 phosphorylation induced by
pervanadate or receptor stimulation. Furthermore, the rate of RGS16
degradation was reduced in cells expressing active Src or treated with
pervanadate or a G protein-coupled receptor ligand (CXCL12). Induction
of RGS16 tyrosine phosphorylation was associated with increased RGS16 protein levels and enhanced GAP activity in cell membranes. These results suggest that Src mediates RGS16 tyrosine phosphorylation, which
may promote RGS16 stability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit undergoes agonist-evoked GTP binding and dissociation from its
tonic repressor,
. Each G protein component elicits diverse
outcomes including increased enzyme activity or concentration, gene
transcription, or cellular movement. The intrinsic GTPase activity of
the
subunit allows re-formation of an
trimeric complex,
terminating signaling. Regulator of G protein signaling (RGS) proteins,
which are GTPase-activating proteins (GAPs) for G
subunits, may
down-regulate G protein signaling cascades (2-3); however, their
physiological roles have only begun to be elucidated. Some pathological
conditions have been linked to abnormal RGS expression, suggesting that
pharmaceutical modulation of RGS activity may impact the treatment of
diseases associated with abnormal GPCR output (4). How specific RGS
proteins modify discrete GPCRs or G proteins remains an unclear but
intensely studied question (5). For these reasons, it is of vital
importance to understand how individual RGS proteins are regulated.
q (8). By
contrast, ERK2 phosphorylation of RGS-G
-interacting protein enhances
GAP activity toward G
i3 (9, 10). RGS16 is phosphorylated
on Ser194 after epinephrine stimulation of cells expressing
the
2A-adrenergic receptor (11). Mutation of this serine
residue impairs RGS16 GAP activity and its regulation of
epinephrine-stimulated MAP kinase activation.
12/13-coupled GPCRs
(15). We reported previously that RGS16 undergoes epidermal growth
factor receptor (EGFR)-mediated tyrosine phosphorylation on a conserved
tyrosine residue in the RGS box, Tyr168, which enhances
RGS16 GAP activity in single turnover assays (16). However, when
cellular tyrosine phosphatase activity is inhibited by pervanadate,
RGS16 lacking Tyr168 is also phosphorylated, indicating
that Tyr177 (the only other tyrosine residue in RGS16) is a
possible site of phosphorylation. Moreover, although mutation of
Tyr177 does not affect RGS16 GAP activity in
vitro, the mutant protein is unable to modulate
Gi-coupled adenylyl cyclase inhibition or Gq-coupled MAP kinase activation in cells. These results
suggest that phosphorylation of this residue is of critical importance to the biological activity of RGS16.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1) (Santa Cruz
Biotechnology) or polyclonal anti-RGS16 (CT265, raised against mouse
His6RGS16, the gift of Melvin I. Simon and Ching-Kang Chen,
California Technical Institute (18)). Antigen-antibody complexes were
recovered using sheep anti-rabbit IgG magnetic beads (Dynal) or protein
A-agarose (Invitrogen). Cells transfected with RGS16-V5/His were lysed
in buffer P-Ni (20 mM HEPES, pH 8, 150 mM NaCl,
10 mM
-mercaptoethanol, 15 mM imidazole, 1%
Triton X-100, 1 mM Na3VO4). We
extracted RGS16 from lysates with a 30-µl slurry of Probond resin
(Invitrogen). We washed beads three times prior to addition of Laemmli
buffer and SDS-PAGE. Antibodies against phospho-ERK, ERK1,
5-HT1A receptor, and HA were from Santa Cruz Biotechnology,
anti-V5 from Invitrogen, and anti-phosphotyrosine (4G10) from Upstate
Biotechnology, Inc. The antibody raised against His6RGS4
has been described previously (19, 20). Signal was detected with
SuperSignal enhanced chemiluminescence (Pierce). BaF3 cells were
stimulated with CXCL12 (50 ng/ml, R & D Systems) for 5 min at 37 °C.
Cells were stimulated with goat anti-mouse immunoglobulin G + M (IgG + IgM) (Fab')2 (Jackson ImmunoResearch) essentially as
described (21).
1 bovine serum albumin, 0.1% Triton X-100, 1 mM Na3VO4, 5 µM cold ATP, and 1 µCi [
-32P]ATP (10 mCi/ml, 30 Ci
mmol
1, Amersham Biosciences). For some reactions
containing c-Src or Btk kinase, His6RGS16 or
His6RGS2 (200 ng) was incubated in 40 µl of kinase buffer
(30 mM HEPES, pH 7.4, 5 mM MgCl2, 5 mM MnCl2, and 5 µM ATP)
containing 2 µl of [
-32P]ATP (Amersham Biosciences,
3000 Ci mmol
1) for 1 h at 30 °C. Reactions were
subjected to SDS-PAGE, and gels were dried prior to autoradiography.
The RGS16 band was excised and counted by liquid scintillation
spectrometry. The stoichiometry of phosphorylation was calculated based
on the specific activity of [
-32P]ATP and RGS16
concentration determined by Bradford assay.
i1
(Calbiochem) with [
-32P]GTP (1 µM,
50-100 cpm fmol
1) at 30 °C in buffer containing 50 mM HEPES, pH 8, 5 mM EDTA, 1 mM
dithiothreitol, 0.1% C12E10 (buffer C). The
reaction was chilled on ice before gel filtration on a G-25 Sepharose
column that was equilibrated with buffer C plus bovine serum albumin (0.1 mg ml
1). GTP hydrolysis was initiated by adding G
protein to a tube containing unlabeled GTP (100 µM) and
MgSO4 (10 mM) with or without RGS. Aliquots
were removed at various time points from 10 s prior to the start
of the reaction (arbitrarily set as time 0) to 5 min and quenched in a
chilled, activated charcoal/phosphoric acid slurry. Inorganic phosphate
in supernatants was measured by liquid scintillation spectrometry after
centrifugation at 1500 × g. Net release of
Pi was determined by subtracting the amount at time 0.
70 °C.
o1 fusion protein have been
described previously (22). 30 h post-transfection, cells were
treated overnight with pertussis toxin (PTX, 50 ng/ml, Calbiochem) to eliminate endogenous G
i/o activity. The next day, cells
were scraped and harvested in Tris-buffered saline and centrifuged at
2,000 × g for 10 min at 4 °C. Pellets were
resuspended in ice-cold Tris-HCl/EDTA (TE), pH 7.4, and homogenized
with 30-50 passages through a Dounce homogenizer followed by 15 passages through a 25-gauge needle. Unbroken cells and debris were
pelleted by centrifugation at 4,000 × g for 5 min at
4 °C. The supernatant was centrifuged at 100,000 × g for 30 min at 4 °C to pellet the membrane fraction. Membranes were resuspended in TE buffer to ~2 mg/ml and stored as
aliquots at
80 °C.
-32P]GTP (Amersham Biosciences, 3000 Ci/mmol), and
incubated for 20 min at 30 °C. Reactions were stopped by addition of
ice-cold 10% (w/v) activated charcoal in 50 mM phosphoric
acid followed by centrifugation at 10,000 × g for 20 min at 4 °C. Supernatants containing free
[
-32P]phosphate were analyzed by liquid scintillation
spectrometry. Nonspecific GTPase activity was determined in
simultaneous reactions containing 100 mM GTP.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Detection and phosphorylation of endogenous
RGS16 in MCF-7 cells. A, MCF-7 cells were treated
with medium or doxorubicin (1 µM) overnight prior to
lysis, SDS-PAGE, and immunodetection (ID) with anti-RGS16
antibodies (Ab) (top). Bottom panel
shows an immunoprecipitation of recombinant RGS16 or MCF-7 lysates with
RGS4 or RGS16 antibodies as indicated. Immunoprecipitates were
separated by SDS-PAGE and immunodetected with the same anti-RGS16
antibody used for immunoprecipitation (IP). Ig light chain
(arrow) and RGS16 (arrowhead) bands are
indicated. B, MCF-7 cells were stimulated with medium or EGF
(100 ng/ml) for 5 min at 37 °C prior to lysis and SDS-PAGE. Equal
amounts of protein from cell lysates were blotted with
anti-phosphotyrosine or anti-RGS16 as indicated.
-32P]ATP and measured ATP
incorporation into the protein by autoradiography. RGS16 was
phosphorylated by recombinant p60-Src and Lyn but not by Btk or Abl
kinases, and RGS2 was not phosphorylated by either Src or Btk kinase
(Fig. 2, A and B).
Interestingly, purified Csk kinase was able to phosphorylate RGS16
in vitro; however, this phenomenon was not explored further.
We determined the stoichiometry of phosphorylation by measuring
counts/min of the RGS16 band excised from gels divided by the specific
activity of [
-32P]ATP. Based on this measurement, the
stoichiometry of phosphorylation was 0.6 ± 0.1 mol of phosphate
per mol of RGS16 (mean ± S.E. of 5 independent experiments),
suggesting that Src phosphorylated RGS16 at a single site in
vitro.
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Fig. 2.
In vitro phosphorylation of RGS16
by Src family kinases. A and B, recombinant
RGS16 was incubated with Src, Btk, Abl, Lyn, or Csk kinases and
[ -32P]ATP for 1 h at 30 °C. Reactions were
stopped by the addition of Laemmli buffer before resolution by SDS-PAGE
and autoradiography. Kinase autophosphorylation (Abl, 40 kDa; Csk, 50 kDa; Lyn, 56 kDa) is indicated by arrowheads.
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Fig. 3.
Src-mediated RGS16 phosphorylation in
mammalian cells. A, CHO-K1 cells were transfected with
empty vector or m2 muscarinic receptor plasmids. 24 h
post-transfection, cells were stimulated with either EGF (100 ng/ml) or
carbachol (1 mM) for 5 min at 37 °C. Cells were then
lysed, and equal amounts of protein from lysates were separated by
SDS-PAGE. MAP kinase activity was determined by immunoblotting with
anti-phospho-ERK (top), and ERK levels were assessed with
anti-ERK1 (bottom). ID, immunodetection.
B, CHO-K1 cells were transfected with plasmids encoding
HA-RGS16 together with empty vector or Src (Y529F). RGS16 was
immunoprecipitated (IP) with anti-HA, and immunoprecipitates
were resolved by SDS-PAGE before immunoblotting with
anti-phosphotyrosine (PY) or anti-HA antibodies.
C, CHO-K1 cells were transfected with empty vector or
RGS16-V5/His. 24 h later, cells were treated with serum-free
medium alone or medium containing PP2 (10 µM) for 20 min
at 37 °C before treatment with pervanadate for 15 min. Cells were
lysed, and RGS16 was affinity-purified by
Ni2+/nitriloacetic acid chromatography. Bead-bound proteins
were separated by SDS-PAGE and detected with anti-V5 and
anti-phosphotyrosine antibodies.
i. Activation of G
i is
associated with increased Src activity (17). We immunoprecipitated
RGS16 using anti-RGS16 antibodies and immunodetected with
anti-phosphotyrosine or anti-HA. Treatment with the CXCR4 ligand CXCL12
induced RGS16 tyrosine phosphorylation, which was blocked by PP2 (Fig.
4A). To test whether
Src-dependent RGS16 tyrosine phosphorylation could occur
independently of GPCR stimulation, we stimulated BaF3/RGS16 cells with
anti-IgM + IgG to cross-link the B-cell receptor, which activates the
endogenous Src family kinase Lyn (21). In these cells, we observed
robust RGS16 tyrosine phosphorylation in response to Ig receptor
cross-linking that was attenuated by PP2 but not by its inactive
homologue PP3 (Fig. 4B). These results suggest that Src
family kinases are required for RGS16 phosphorylation in response to
either B-cell receptor cross-linking or stimulation of CXCR4 receptors
in BaF3 cells.
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Fig. 4.
Src-dependent RGS16
phosphorylation induced by receptor stimulation in BaF3 cells.
A, BaF3 cells stably expressing HA-RGS16 were untreated
or preincubated with PP2 for 20 min (lane 3) before
stimulation with medium alone or CXLC12 (50 ng/ml) for 5 min at
37 °C. RGS16 was immunoprecipitated (IP) with anti-RGS16
antiserum, and immunoprecipitates were separated by SDS-PAGE before
immunoblotting with anti-HA antibody and anti-phosphotyrosine
(PY) as indicated. ID, immunodetection.
B, BaF3-RGS16 cells were stimulated with medium alone
or anti-IgM + IgG in the presence or absence of PP2 or PP3 as indicated
and processed as in B.
-32P]ATP, we affinity-purified His-RGS16
WT or Y177F by nickel chromatography before digestion with EndoLys-C.
We visualized a radioactive band at ~4 kDa in reactions containing
RGS16 WT but not Y177F (Fig. 5B). There was also a
radioactive band at ~10 kDa, which we hypothesize to be a partial
digestion product. Coomassie Blue staining demonstrated a similar
cleavage pattern for both RGS16 WT and Y177F (Fig. 5C), indicating that the C-terminal peptide was not phosphorylated in the
Y177F mutant. This result suggests Src phosphorylates native RGS16 at
residue Tyr177 in vitro.
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Fig. 5.
Src-mediated RGS16 phosphorylation depends on
Tyr177. A, recombinant RGS16 WT or mutants
were incubated with recombinant p60-Src as in Fig. 2.
Reactions were stopped by the addition of Laemmli buffer, and samples
were resolved by SDS-PAGE. Gels were immunoblotted sequentially with
anti-phosphotyrosine (PY) and anti-RGS16 antibodies. RGS16
phosphorylation is indicated by the arrow and Src
autophosphorylation by the arrowhead. B,
His-tagged RGS16 WT or Y177F was incubated with Src kinase (100 ng) and
1 µCi of [ -32P]ATP for 90 min.
Ni2+/nitriloacetic acid beads (20 µl) were added for an
additional 30 min. Beads were then washed twice in detergent-containing
buffer and once in protease incubation buffer. EndoLys-C (500 ng) was
added for an additional 2 h at 37 °C. Digests were separated on
Tricine gels, stained with Coomassie Blue, and exposed to
autoradiography. Arrow indicates full-length RGS16 and the
arrowhead an ~4-kDa peptide generated by EndoLys-C
cleavage that putatively contains Tyr177. C,
Coomassie Blue-stained gel of WT and mutant (mut.) RGS16
proteins in the presence or absence of EndoLys-C. D,
CHO-K1 cells were transfected with HA-RGS16 WT, Y168F, or Y177F
plasmids. 24 h after transfection, cell lysates were processed as
in A, except that anti-HA was used to detect RGS16.
i in single turnover assays. To determine whether Src-mediated RGS16
phosphorylation also affected its catalytic activity, we phosphorylated
RGS16 with Src prior to measuring GTP hydrolysis by G
i1.
However, we observed no difference in the GAP activity of RGS16 in the
presence or absence of Src (Fig. 6).
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Fig. 6.
Src-induced phosphorylation does not alter
RGS16 GAP activity in vitro. Recombinant RGS16
(10 nM) was incubated with Src kinase or reaction buffer
for 1 h at 30 °C. A single turnover assay measuring GTPase
activity of recombinant G i1 in solution was performed in
the presence of RGS16 (open circles), phosphorylated RGS16
(pRGS16, triangles), or no GAP (closed circles).
An aliquot of the phosphorylation reaction was immunoblotted with
anti-phosphotyrosine to verify RGS16 phosphorylation (data not shown).
Values represent the mean ± S.E. of six independent
experiments.
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Fig. 7.
Phosphorylation affects RGS16 stability.
A, CHO-K1 cells were transfected with RGS16 (no epitope tag)
together with lacZ or Src (Y529F) plasmids. 24 h
post-transfection, cells were treated with cycloheximide (10 µg/ml)
before lysis at the indicated times. Equal volume aliquots from lysates
were separated by SDS-PAGE and immunoblotted with anti-RGS16. The
levels of a more stable protein, ERK1, did not vary substantially over
the indicated period except at 4 h as determined by immunoblotting
with anti-ERK1 (data not shown). ID, immunodetection.
-Gal,
-galactosidase. B, graphic
representation of RGS16 levels (percent of the initial value, mean ± S.E. of 2-4 independent experiments) in the presence or absence of
active Src, as determined by densitometry. C, CHO-K1
cells were transfected with RGS16-V5/His. 24 h post-transfection,
cells were incubated with cycloheximide (15 µg/ml) with or without
pervanadate. Cells were lysed at the indicated times, and lysates were
subjected to SDS-PAGE and immunoblotting with anti-V5 antibodies.
D, cells transfected with RGS16-V5/His (Y168/177F)
together with lacZ or active Src were processed as in
A. E, graphic representation of RGS16
(Y168F/Y177F) levels in the presence of control or active Src plasmids
determined by densitometry. Values represent mean ± S.E. of two
independent experiments. F, same experiment as C,
except that the Y168F/Y177F mutant was transfected.
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Fig. 8.
Alteration in RGS16 degradation by receptor
stimulation and proteasome dependence. A, BaF3 cells
stably transfected with vector (vec.) (lane 1) or
HA-RGS16 (lanes 2-7) were metabolically labeled with
[35S]methionine (150 µCi/ml) for 30 min at 37 °C.
Labeling medium was replaced with medium containing cold methionine in
the presence or absence of CXCL12 for the indicated times. Cells were
lysed in detergent buffer and immunoprecipitated with anti-RGS16
antiserum. After SDS-PAGE, gels were exposed to autoradiography
overnight at 70 °C. B, experiment as in A,
except that labeling medium was replaced by medium containing the
proteasome inhibitor MG132 (10 µM) or not for the
indicated times. Cells were lysed and immunoprecipitated with anti-HA
antibody-coupled agarose.
o1. In this construct, G
contains a pertussis-toxin
(PTX) resistance mutation, enabling PTX treatment of cells prior to
membrane extraction to ablate GTPase activity by endogenous
Gi/Go. We have employed this strategy
previously to measure the GAP activity of co-transfected RGS proteins
in vivo (31). In this case, this method was ideal to
determine the effect of RGS16 phosphorylation induced by pervanadate,
because the activity of the receptor/G protein fusion protein was
unlikely to be directly modulated by tyrosine phosphorylation, in
contrast to activation of downstream signaling components that could be strongly affected by tyrosine kinase activity. We treated cells stably
expressing 5-HT1A/G
o1 and transiently
expressing WT RGS16 with or without pervanadate for 30 min before
preparing membranes and measuring 5-HT-induced GTPase activity. As
expected, GTPase rates were greater in membranes expressing WT RGS16
than in vector-transfected cells (Fig.
9A). However, GTPase activity
in cells expressing RGS16 and exposed to pervanadate was nearly
2.5-fold higher than the activity of untreated membranes containing
RGS16. This enhanced GAP activity correlated with a nearly 2-fold
increase in RGS16 protein levels in the membrane as determined by
immunoblotting (inset). By contrast, levels of the fusion
protein (detected with a 5-HT1A receptor antibody) were not
significantly affected by pervanadate treatment. To determine whether
the increase in activity was attributable to the enlarged
pool of RGS16 and to exclude the possibility that pervanadate
stimulated the GTPase activity of the fusion protein directly, we
treated cells expressing the phosphorylation-resistant RGS16 mutant
(Y168F/Y177F) with pervanadate and measured agonist-evoked GTPase
activity. Although GTPase activity in cells expressing the mutant was
higher than in vector-transfected cells, there was no difference in
activity in untreated and pervanadate-stimulated cells. Moreover, the
levels of RGS16 (Y168F/Y177F) did not change with pervanadate
stimulation (Fig. 9B). The fold increase in GTPase activity
between no agonist and the highest 5-HT concentration was significantly
greater for pervanadate-exposed membranes expressing WT RGS16 than for
untreated cells. In contrast, the increase was nearly identical in
membranes expressing the phosphorylation-resistant mutant with or
without pervanadate (p = 0.01, WT
/+ pervanadate) (Fig. 9C). Because the levels of RGS16 (Y168F/Y177F) did not
change appreciably with pervanadate stimulation, increases in RGS16
protein levels due to up-regulated transcription or membrane
translocation over a relatively short time (30 min) seem unlikely. In
summary, induction of RGS16 tyrosine phosphorylation is associated with increased steady-state RGS16 levels and augmented GAP activity in cell
membranes.
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Fig. 9.
Effect of RGS16 stabilization by
phosphorylation on GAP activity in cellular membranes.
A, HEK 293 cells stably expressing a
5-HT1A/G o1 fusion protein were transiently
transfected with lacZ or HA-RGS16 plasmids (5 µg). After
PTX treatment (50 ng/ml overnight) to eradicate activity of endogenous
Gi/Go proteins, cells were treated with
serum-free medium or medium containing pervanadate for 30 min before
harvest and membrane extraction. Membranes were stimulated with the
indicated concentrations of 5-HT for 20 min at 37 °C to measure
agonist-induced GTPase activity. An aliquot of membrane protein (50 µg) was separated by SDS-PAGE and immunoblotted with anti-RGS16
(inset, bottom) and anti-5-HT1A
receptor (inset, top). B, same
experiment as A, except that cells were transfected with
HA-RGS16 (Y168F/Y177F). C, bar graph showing the
fold increase in GTPase activity in unstimulated cells
versus cells treated with 10
3 M
5-HT. Values are mean ± S.E. of 3-4 experiments performed in
triplicate (*, p = 0.01 WT
versus WT+, 2-factor analysis of
variance).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i activation stimulates Src kinase (17) and leads to
EGFR trans-activation (34, 35). Collectively, these studies emphasize
the inter-relatedness of GPCR and RTK pathways, and RGS16
phosphorylation represents yet another potential link between these
signaling cascades.
i in
vitro. Although further studies will be required to determine
whether Src-induced phosphorylation affects G
q GAP
activity or the ability of RGS16 to act as an effector antagonist of
G
q, these results suggest that Tyr177
phosphorylation does not induce a structural change in RGS16 that
affects G protein binding. Extrapolation of the crystal structure of
RGS4 in a complex with G
i1 to RGS16 suggests that
Tyr168, located in helix 8, is in closer proximity to the
G
-binding surface than Tyr177, which is located on helix
9 (Fig. 10A) (36). In fact,
residues that directly contact G
switch I region
(Asp166, Ser167, and Arg170)
surround Tyr168, and NMR analysis of unbound RGS4 suggests
that this region undergoes a conformational change upon G
binding
(37). Therefore, the introduction of negative charge by phosphorylation
could alter this G
-binding surface through electrostatic
interactions. In contrast, Tyr177 faces away from the G
protein interface, in a short helix preceded by a flexible linker,
which might easily facilitate interactions with other proteins (see
below). Therefore, it appears more likely that phosphorylation of
Tyr168 would directly affect GAP activity, in agreement
with our previous results.
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Fig. 10.
Relationship between two conserved
tyrosine residues and the G -binding
interface. A, three-dimensional structure of RGS4
(green) (and by conservation RGS16) bound to
G
i1 (blue) demonstrating the proximity of
Tyr168 (orange) to RGS residues
(yellow) that directly bind switch I Region (red,
forefront) of G
i1. B,
Tyr177 (orange) is located near the N and C
termini (purple) of RGS4 and distant from the RGS residues
(yellow) that directly bind switch I in G
i1.
Images were made using Protein Data Bank file 1AGR and ViewerLite
5.0 from Accelrys.
Because Src-mediated phosphorylation did not alter RGS16 GAP activity in vitro, we explored other possible effects of this modification on RGS16 biological function. We found that co-expression of activated Src or receptor activation of endogenous Src prolonged the half-life of RGS16 in cells. This increased longevity appeared to be related to RGS16 phosphorylation inasmuch as a comparable increase in RGS16 stability was induced by pervanadate treatment. Moreover, the pervanadate-induced reduction in RGS16 degradation was accompanied by a corresponding increase in membrane GAP activity. This result suggests that alterations in RGS16 protein turnover have a direct effect on its activity in cells.
In contrast to our findings, another protein with an RGS domain, GRK2,
was shown to undergo Src-mediated phosphorylation, which
promoted GRK2 proteolysis by the proteasome pathway (38). However, GRK2 phosphorylation appeared to occur on tyrosine residues (Tyr13, Tyr86, and Tyr92) distinct
from the two phosphorylated residues in RGS16. Only two of three
tyrosines in GRK2 that are targets of Src phosphorylation are located
within the GRK2 RGS domain (on helix 4) (39). In addition, increased
GRK2 degradation required recruitment of an additional protein,
-arrestin, as well as GRK2 kinase activity, because a
kinase-inactive GRK2 mutant underwent phosphorylation in cells
co-expressing active Src but did not exhibit altered proteolysis. Thus,
the mechanism whereby Src phosphorylation promotes stabilization of
RGS16 is likely to be distinct from its effect on GRK2 turnover.
It is perhaps not surprising that a regulatory protein such as RGS16 is
relatively unstable in order to allow greater flexibility in local RGS
concentration at the site of G protein action before and after
stimulation of receptors. In addition to Sst2, which undergoes
pheromone-induced phosphorylation that stabilizes the protein (6),
another example of this type of regulation involves RGS7. Tumor
necrosis factor-induced RGS7 phosphorylation, which is dependent
on p38 MAP kinase, reduces RGS7 degradation by the proteasome (40).
Like RGS7, RGS16 is an N-end rule substrate, which specifies
ubiquitination and proteasome-dependent degradation (30).
We found that RGS16 degradation was also likely to be mediated by the
proteasome in mammalian cells because the proteasome inhibitor slowed
the rate of RGS16 turnover. This process appears to be mediated by an
Arg-transferase, which couples an Arg residue to
Cys2 in RGS16. It is conceivable that phosphorylation on
Tyr177 alters access of this enzyme to the RGS16 N
terminus, which was disordered in the crystal structure (of RGS4) but
appears to be in close proximity to Tyr177 in C-terminal
helix 9 (Fig. 10B). Alternatively, RGS16 phosphorylation could promote binding to another protein that shields it from degradation. Although RGS16 Tyr177 is not contained within
a consensus sequence predicted to bind protein tyrosine binding domains
(NPXpY, where X is any amino acid and pY is
phosphotyrosine), broader ligand binding specificities for
protein-tyrosine binding motifs have been demonstrated recently (41).
Another possibility is that RGS16 tyrosine phosphorylation could alter
its subcellular localization, which might protect it from the
proteasome. An example of this process is Abl-mediated IB
phosphorylation, which leads to nuclear accumulation of I
B
and
enhanced stability (42). In preliminary studies we failed to observe a
gross change in the localization of a RGS16-GFP fusion protein in
CHO-K1 cells co-transfected with active Src. One possibility is that
phosphorylation could induce or prevent RGS16 localization in lipid
rafts, which might also affect access to the proteasome. More detailed
experiments utilizing markers for organelles or cytoskeletal elements
and better visualization methods (EM, confocal microscopy) as well as
localization of endogenous RGS16 may be necessary to determine
differences in RGS16 locale after phosphorylation.
Because Src activity is evoked by numerous stimuli, including, among
others, cytokines (43), extracellular matrix proteins (44), steroids
(45), ion channels, and integrins (46), Src-mediated RGS16
phosphorylation and the resultant stabilization of RGS16 levels may
represent a mechanism by which diverse cell surface receptors
down-regulate GPCR output in a complex cellular milieu. Our findings
provide a framework to study the effects of tyrosine phosphorylation on
RGS16 function and their generalization to closely related RGS proteins.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: NIAID, National Institutes of Health, 12441 Parklawn Dr., Rockville, MD 20852. Tel.: 301-435-8875; E-mail: kdruey@nih.gov.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M210371200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
RGS, regulator of G protein signaling;
GAP, GTPase-activating protein;
HA, hemagglutinin epitope of influenza
virus;
MAP kinase, mitogen-activated protein kinase;
EGF, epidermal
growth factor;
EGFR, epidermal growth factor receptor;
PTX, pertussis
toxin;
5-HT, 5-hydroxytryptamine;
WT, wild type;
ERK, extracellular
signal-regulated kinase;
AMP-PNP, adenosine
5'-(,
-imino)triphosphate;
GFP, green fluorescent protein;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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