(Received for publication, December 22, 1995; and in revised form, March 5, 1996)
From the
Of nine G protein subunits examined, only
and
served as substrates for phosphorylation by
various isoforms of protein kinase C in vitro. A close homolog
of
,
, was not phosphorylated.
Exposure of NIH 3T3 cells that stably express
to
phorbol 12-myristate 13-acetate also resulted in phosphorylation of the
protein. Phosphorylation in vitro occurred near the amino
terminus (probably Ser
), and approximately 1 mol of
phosphate was incorporated per mol of
. Although G
protein heterotrimers containing either
or
were poor substrates for phosphorylation, the
isolated
subunits were phosphorylated equally well in their GDP-
or GTP
S-bound forms. The guanine nucleotide binding properties of
purified
and
were unaltered by
phosphorylation, as was the capacity of
to inhibit
type V adenylyl cyclase. However, phosphorylation of either protein
greatly reduced its affinity for G protein
subunits,
consistent with the newly determined crystal structure of a G protein
heterotrimer. We suggest that protein kinase C regulates
- and
-mediated signaling pathways
by preventing their association with
.
Heterotrimeric guanine nucleotide-binding proteins (G proteins) ()transduce regulatory signals from cell surface receptors
to effectors such as adenylyl cyclases, phosphodiesterases,
phospholipases, and ion channels(1, 2, 3) .
Each G protein oligomer contains a guanine nucleotide-binding
subunit and a high-affinity dimer of
and
subunits. There
are many isoforms of each subunit and thus a very large number of
distinct G protein oligomers. G protein
subunits are commonly
described as members of four subfamilies:
and
(stimulators of adenylyl cyclases);
,
,
,
,
,
,
, and
(functionally diverse group of
pertussis toxin substrates, with the exception of
);
,
,
, and
(activators of phospholipase C-
s); and
and
.
The two members of the
and
subfamily, discovered most
recently, are expressed ubiquitously (4) and share interesting
biochemical characteristics, including relatively slow guanine
nucleotide exchange and hydrolysis(5, 6) . Although
the receptors and effectors that interact with these G proteins have
not yet been identified, overexpression of wild type or mutationally
activated
or
transforms
fibroblasts (7, 8, 9) . Furthermore,
overexpression of constitutively activated
or
stimulates Na
/H
exchange activity(10, 11) . Of interest,
Dhanasekaran et al.(10) showed that this stimulatory
effect of
, but not that of
, is
lost after prolonged exposure of cells to PMA. These results suggest
that
and
transduce similar
regulatory signals related to cell growth or transformation and that
there is further regulation of the
pathway by PKC.
There are other interactions between G protein-regulated pathways
and PKC. Treatment of cells with PMA has a variety of often confusing
effects on their capacity to synthesize cyclic AMP in response to
various activators or inhibitors; certain adenylyl cyclases are
activated following phosphorylation by PKC in
vitro(12, 13) . Activation of phospholipase C by
muscarinic or -adrenergic agonists is blocked by
treatment of astrocytoma cells or hepatocytes, respectively, with
PMA(14, 15) . The inhibitory effects of substance P on
an inward rectifier K
channel appear to be mediated by
a pertussis toxin-insensitive G protein and protein kinase
C(16) . With regard to direct effects of PKC on G protein
subunits, there are descriptions of phosphorylation of G
and G
both in vitro and in
vivo(17, 18, 19, 20) , but the
functional significance of such modification has been unclear. We
describe here the phosphorylation of
by PKC in
vitro and, in addition, in cells exposed to PMA. We further
demonstrate that phosphorylated
and
have reduced affinity for G protein
subunits compared
to the unmodified
subunits. Similar results with
have just been reported by Fields and Casey(21) .
,
, and
were purified as described
previously (5) ;
and
were
purified by the same procedure used for
.
was purified with the Ni-NTA column described for
and then according to Singer et
al.(6) .
C68S was
purified as described (22) and generously provided by Dr. Bruce
Posner (this laboratory).
For preparation of phosphorylated subunits, 2 nmol of
or
were incubated in 4-ml reaction
mixtures with PKC (2 units) and 10 µM ATP (500 cpm/pmol)
for 40 min at 30 °C. Phosphorylated
subunits were purified
(Mono S HR5/5) and processed as described(5) . The
stoichiometry of phosphorylation was approximately 0.5 for
and 1-1.5 for
; protein concentrations
were determined by staining with Amido Black(25) .
For labeling with either
[S]methionine or
[
P]P
, cells were incubated with
methionine- or phosphate-free Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) for 1 h, followed by incubation with
medium supplemented with [
S]methionine (50
µCi/ml; 3 h) or [
P]P
(0.5
mCi/ml; 2 h). Cells were washed twice with 20 mM NaHepes (pH
7.5) and 150 mM NaCl, harvested, suspended in 500 µl of
hypotonic buffer (20 mM NaHepes (pH 7.5), 1 mM EDTA,
1 mM DTT), frozen, and thawed three times, and centrifuged at
125,000
g at 4 °C for 20 min to prepare cytosolic
and crude membrane fractions. NaF (5 mM) and
-glycerophosphate (10 mM) were included in the lysis
buffer for cells labeled with [
P]P
.
Membrane extracts were prepared with 500 µl of 20 mM NaHepes (pH 7.5), 150 mM NaCl, 1% sodium cholate, 1%
Triton X-100, and 0.5% SDS (RIPA buffer) prior to centrifugation at
125,000
g for 20 min.
For immunoprecipitation, 25
µl of membrane extract was incubated with 2.5 µl of 10% fixed Staphylococcus aureus (Pansorbin; Calbiochem) on ice for 30
min. After centrifugation at 15,000 g for 5 min,
supernatants were incubated overnight at 4 °C with 7.5 µg of
anti-
IgG or control rabbit IgG. Pansorbin (5 µl;
10%) was added for an additional 30 min prior to collection of
immunoprecipitates by centrifugation and suspension in 100 µl of
RIPA buffer. The suspension was layered over 1 ml of RIPA buffer
containing 20% sucrose (w/v) and centrifuged at 15,000
g for 5 min. Pellets were extracted with SDS-PAGE sample buffer,
heated (90 °C; 3 min), and subjected to SDS-PAGE followed by
autoradiography. Gels containing
[
S]methionine-labeled proteins were treated with
EN
HANCE (DuPont NEN).
Figure 1:
Phosphorylation of
G protein subunits by PKC. A, G
subunits
(2.5 pmol) were incubated with 2.5 milliunits of PKC for 20 min. The
products were separated by SDS-PAGE, stained with silver, and subjected
to autoradiography. Upper panel, silver staining of
subunits; lower panel, autoradiography.
,
,
,
,
,
, and
were
purified from Sf9 cells as described under ``Experimental
Procedures.''
and
were
purified from bovine brain and E. coli, respectively. B,
(2.5 pmol) was incubated with 2.5
milliunits of recombinant PKC
for 20 min in the presence or
absence of 5 µM PMA, 10 µg/ml phosphatidylserine, or
125 µM CaCl
as indicated. Proteins were
resolved by SDS-PAGE and subjected to
autoradiography.
We examined NIH 3T3 cells that had been
stably transfected with an expression plasmid encoding to test phosphorylation of the protein in vivo.
Immunoblotting of membranes from these cells (NIH 3T3-G12) demonstrates
significant expression of
(Fig. 2A);
we could not detect the protein in these cells prior to transfection
(using antiserum J169). This antiserum could be used to
immunoprecipitate
from a membrane extract of
[
S]methionine-labeled NIH 3T3-G12 cells (Fig. 2B), and phosphorylated
was
immunoprecipitated from cells labeled with
[
P]P
after exposure to PMA (Fig. 2C). Thus,
appears to be
phosphorylated in vivo after PKC is activated by phorbol
esters.
Figure 2:
Western blotting and immunoprecipitation
of NIH 3T3-G12 cells. A, membranes from NIH 3T3 cells (lane 1) or NIH 3T3-G12 cells (lane 2) (10 µg of
each) were subjected to SDS-PAGE and immunoblotted with antibody J169. B, [S]methionine-labeled NIH 3T3-G12
cell lysate was immunoprecipitated with control rabbit IgG (lane
1) or with J169 IgG (lane 2). The precipitates were
resolved by SDS-PAGE and subjected to autoradiography. C, NIH
3T3-G12 cells were labeled with [
P]P
and treated with vehicle (lane 1) or 5 µM PMA (lane 2) for 20 min. The cell lysates were
immunoprecipitated with J169 IgG. The precipitates were resolved by
SDS-PAGE and subjected to autoradiography. The arrows in A, B, and C indicate the position of
.
The time course and stoichiometry of phosphorylation of
in vitro are shown in Fig. 3A. Since the substrate is over 90% pure (based on
silver staining; Fig. 1A) and other phosphorylated
proteins do not appear in the reaction mixtures (Fig. 1B), we estimated stoichiometry by filtration.
When 7 pmol of
was included in the assay, the
maximal incorporation of phosphate was about 3 pmol. Since the
stoichiometry of binding of GTP
S to the
used
here was about 50% (based on the protein assay), we believe that 1 mol
of phosphate is incorporated per mol of
.
(
is not phosphorylated when denatured; data not
shown.) Of interest,
is phosphorylated very poorly
after incubation with a 2-fold excess of
(Fig. 3A); the reaction is almost completely
suppressed when
and
are present at equimolar concentrations (Fig. 3B). Similar results were obtained with
(Fig. 3C). Nonprenylated
subunit complexes have reduced affinity for at least certain
G
subunits(22) ; appropriately, the
complex comprised of
and the nonprenylated Cys
Ser
mutant was a less potent inhibitor
of
phosphorylation (Fig. 3B). Since
did not inhibit the activity of PKC
when a specific substrate peptide from myelin basic protein
(MBP
) was utilized (data not shown), we conclude
that
and
are not substrates for
PKC when associated with
in the G protein heterotrimer.
Figure 3:
Inhibition of phosphorylation of
and
by
. A,
(70 nM) was incubated on ice for 10 min
with or without
(140 nM)
and then phosphorylated with PKC. Aliquots (100 µl) were withdrawn
at the indicated times, filtered, and counted as described under
``Experimental Procedures.'' B,
(50 nM) was incubated with the indicated concentration
of
or
C68S on ice and then phosphorylated
with PKC at 30 °C for 20 min. Aliquots were then filtered and
counted. C,
or
(50
nM) was incubated with the indicated concentration of
on ice for 10 min and then
phosphorylated with PKC for 20 min at 30 °C. Data are expressed as
percent phosphorylation relative to that observed in the absence of
. In A, B, and C, data are the average of duplicate determinations from a
single experiment that is representative of three such
experiments.
Both the GDP-bound and the GTPS-bound forms of
and
are phosphorylated almost equally well by
PKC (Fig. 4A); there was no significant difference in
the time course of phosphorylation of both forms of both proteins (data
not shown). Lounsbury et al.(17) reported that the
GDP-bound form of
was phosphorylated more efficiently
than the GTP
S-activated species. The discrepancy may be explained
by the fact that the
subunits used in this work were purified
from Sf9 cells and thus myristoylated at their amino termini; the
protein used by Lounsbury et al.(17) was synthesized
in Escherichia coli and was not so modified. Myristoylation of
the amino terminus may alter the conformation of this domain, which is
the site of phosphorylation (see below).
Figure 4:
Phosphorylation of the GDP- or
GTPS-bound forms of
or
. A, GDP- or GTP
S-bound
or
(2.5 pmol of each) was phosphorylated with PKC for 20 min at 30
°C. The products were resolved by SDS-PAGE and subjected to
autoradiography. To prepare the GTP
S-bound
subunits,
was incubated with 100 µM GTP
S in
the presence of 10 mM MgSO
at 30 °C for 120
min;
was incubated with 100 µM GTP
S
in the presence of 5 mM EDTA and 3 mM MgCl
at 30 °C for 90 min. Proteins were then gel-filtered into 50
mM NaHepes (pH 8.0), 100 mM NaCl, 3 mM MgCl
, 1 mM EDTA, 2 mM DTT, and 0.7%
CHAPS. The amount of protein was estimated by staining with Amido
Black. B,
-GDP (
) or
-GTP
S (
) (0.5 nM) was incubated
with indicated concentrations of
and
then phosphorylated with PKC and 1 µM [
-
P]ATP (70 cpm/fmol) at 30 °C for
30 min in a total volume of 100 µl. Aliquots were filtered and
counted as described under ``Experimental Procedures.'' Data
shown are the average of duplicate determinations from a single
experiment that is representative of three such
experiments.
Since inhibits
the phosphorylation of
and
, we
assessed the dependence of this effect on
concentration
(using both GDP- and GTP
S-bound forms of
at the
lowest possible concentrations (0.5 nM)) in an attempt to
estimate the affinity of
for the protein (Fig. 4B). Efforts to measure these affinities have
been thwarted in the past by the very high affinity of
-GDP for
and resultant difficulty in detection of an effect of
on
at appropriately low concentrations. However,
phosphorylation of
by PKC offers a very sensitive signal. The
concentrations of
required to
inhibit (by 50%) phosphorylation of
-GDP and
-GTP
S were 0.5 and 50 nM, respectively.
Since the effect of
on
-GDP was still close to stoichiometric, there exists
at least a 100-fold difference in apparent affinity of
for
-GDP and
-GTP
S.
Figure 5:
Tryptic digestion of .
(2.5 pmol) was phosphorylated with PKC and
[
-
P]ATP as described under
``Experimental Procedures'' and then incubated with 100
µM GDP, 10 mM NaF, 30 µM AlCl
, and 20 mM MgSO
(lanes 2 and 5) or 100 µM GDP alone (lanes 3 and 6) on ice for 10 min. TPCK-treated trypsin (20% of
the
mass) was then added and incubation was
continued at 30 °C for 20 min. The products were resolved by
SDS-PAGE, followed by immunoblotting with antiserum J169 (lanes
1-3) or autoradiography (lanes 4-6). Lanes 1 and 4 show the sample before digestion with
trypsin.
Figure 6:
Time course of GTPS binding to
phosphorylated
or
.
Nonphosphorylated (
) or phosphorylated (
)
(A) or
(B) (100 nM each) was incubated at 30 °C with 5 µM [
S]GTP
S and 10 mM MgSO
(
) or 0.3 mM MgSO
(
) in the presence of 1 mM EDTA.
Aliquots (50 µl) were withdrawn at the indicated times, filtered,
and counted. Data shown are the average of duplicate determinations
from a single experiment that is representative of two such
experiments.
The rate of binding of GTPS to
nonphosphorylated
or
is inhibited
by
(Fig. 7). This reflects the well-known capacity of
to stabilize the GDP-bound form of G protein
subunits.
However, the rate of GTP
S binding to phosphorylated
(Fig. 7A) or phosphorylated
(Fig. 7B) is not inhibited substantially by a
10-fold molar excess of
, and the
modest effects seen could reflect the presence of small amounts of
nonphosphorylated protein in the preparations. The effect of
on GTP
S binding to mock-treated proteins (PKC in the absence of
ATP) was the same as that on the nonphosphorylated proteins (data not
shown).
Figure 7:
Inhibition by of the rate of GTP
S binding to
or
. Phosphorylated (
) or nonphosphorylated
(
)
(A) or
(B) (140 nM) was mixed with the indicated
concentration of
and then incubated
at 30 °C for 60 min with 5 µM [
S]GTP
S and 10 mM MgSO
(
) or 0.3 mM MgSO
(
) in the presence of 1 mM EDTA. After
60 min, aliquots (50 µl) were withdrawn, filtered, and counted. The
amount of GTP
S bound is shown as the percentage bound relative to
that observed in the absence of
. For
nonphosphorylated or phosphorylated
, 100% was 1.0
and 0.8 pmol, respectively. For nonphosphorylated or phosphorylated
, 100% was 1.4 and 1.3 pmol, respectively. Data shown
in A and B are the average of duplicate
determinations from a single experiment that is representative of three
such experiments.
We also examined the interaction of and
by gel filtration. The peaks of both phosphorylated and
nonphosphorylated
were in fractions 38-40 (Fig. 8), corresponding to molecular weights of about 45,000.
Addition of
to nonphosphorylated
shifted the peak of
about three fractions
(35-37), while the migration of phosphorylated
was unchanged after incubation with
. The results shown in Fig. 7and Fig. 8indicate that phosphorylation of
and
interfere with their capacity
to form oligomers with the G protein
subunit complex.
Figure 8:
Superdex 200 gel filtration of
and
.
Nonphosphorylated
(A) or phosphorylated
(B) (250 pmol) was incubated with or
without 750 pmol of
prior to
application to a Superdex 200 gel filtration column. Fractions (20
µl) were subjected to SDS-PAGE and stained with silver. Positions
of molecular weight standards are: void volume (fraction 25),
-globulin (fraction 31), ovalbumin (fraction 39), myoglobin
(fraction 43), and vitamin B
(fraction
52).
Finally, we examined the effect of phosphorylation of on its ability to inhibit the activity of type V adenylyl
cyclase(5) , since this is the only assay available for
interaction of
or
with an effector (Fig. 9). Okadaic acid (1 µM) was included in the
assay to inhibit phosphatases that might be present in the Sf9 cell
membranes utilized as the source of adenylyl cyclase. Phosphorylation
of
had little or no effect on its inhibitory
interactions with adenylyl cyclase.
Figure 9:
Inhibition of type V adenylyl cyclase by
phosphorylated . The indicated concentrations of
were mixed with 20 µg of membranes from Sf9 cells
expressing type V adenylyl cyclase in the presence of 50 nM GTP
S-
. Adenylyl cyclase activity was assayed
as described under ``Experimental Procedures.''
subunits were nonphosphorylated
-GDP (
),
nonphosphorylated
-GTP
S (
), phosphorylated
-GDP (
), phosphorylated
-GTP
S (
). The concentrations of
GTP
S-activated
subunits were estimated from
[
S]GTP
S binding. Data shown are the average
of duplicate determinations from a single experiment that is
representative of three such experiments.
We have demonstrated that is
phosphorylated by PKC both in vitro and in vivo; the
homologous subfamily member
is not a substrate.
Among the large number of G protein
subunits tested, the only
other efficient substrate for phosphorylation by various isoforms of
PKC was
. The stoichiometry of phosphorylation of
was equal to that for GTP
S binding and is thus
assumed to be 1.
Phosphorylation of occurs within
the amino-terminal domain that is removed by trypsin selectively from
activated G protein
subunits (Table 1). Examination of
corresponding sites of proteolysis in other G
subunits
indicates that trypsin probably removes the first 49 or 50 residues
from
. There are three serine residues (2, 9, and 38)
and one threonine (7) within the relevant sequence. Although
Ser
and Ser
are both candidates for
phosphorylation by PKC(30) , Ser
is surrounded by
basic residues (RRRSR) and corresponds to one of the phosphorylated
serine residues in
(Ser
; RRSRR). There
is no equivalent of
residues Ser
,
Thr
, or Ser
in
, and there is
no equivalent of
residue Ser
(the other
phosphorylation site) in
. Although these arguments
appear to implicate Ser
in
as the site
of phosphorylation, Ser
cannot be ruled out. Of interest,
both Ser
and Ser
have homologs in
, which is not phosphorylated.
Phosphorylation of
and
does not appear to change
their basic guanine nucleotide binding properties, nor the interactions
of
with type V adenylyl cyclase. However, the
affinity of both
subunits for
is clearly reduced by
phosphorylation, and, reciprocally, their phosphorylation is inhibited
by prior interaction with
. Similar results with
were just reported by Fields and Casey(21) . This effect
suggests that phosphorylation of these proteins could play a role in
desensitization of the relevant signaling pathways if PKC was
stimulated simultaneously. Activation of the G protein causes
dissociation of
from
, and PKC-mediated phosphorylation
would thus be favored. Subsequent inhibition of oligomerization as a
result of phosphorylation of
would presumably attenuate signaling
because of the requirement for
for receptor-mediated
activation of
.
Although the signaling pathway that is
regulated by is unknown, expression of
constitutively activated
activates
Na
/H
exchange in a PKC-dependent
manner(10) . Perhaps
activates certain
isoforms of PKC either directly or indirectly to stimulate
Na
/H
exchange, while PKC attenuates
the activity of
in a classic feedback loop.
The
crystal structure of has been determined in its
GTP
S-, free GDP-, and GDP/
-bound
forms(31, 32, 33) . The conformation of the
amino terminus of the
subunit is a particularly dynamic aspect of
the nucleotide- and
-induced structural changes that have
been observed. The amino terminus is disordered when
is activated
by GTP
S; it forms a compact subdomain with the carboxyl terminus
of
in the free GDP-bound form; it is extended in a long
helix that forms extensive contacts with the
subunit in the
heterotrimer. The serine residue in
(Ser
) that is analogous to Ser
in
and Ser
in
is part of
this interface and is hydrogen-bonded to Lys
in
, consistent with the effect of phosphorylation at
this site on interactions of
with
.
The compact
subdomain formed by the amino and carboxyl termini of in the free GDP-bound state is also of interest. This domain
appears to be stabilized by interactions between arginine residues at
positions 15, 21, and 32 and a sulfate ion contributed by the
crystallization solution. Sulfate ions are capable of binding at sites
that normally interact with phosphate or phosphoserine(34) ,
and the arginine residues involved are close to the sites of
phosphorylation of
and
. It will be
interesting to determine if phosphorylation alters the structure of
this microdomain. The specificity of phosphorylation of G protein
subunits by PKC seems problematic, particularly if phosphorylation
regulates a property as fundamental as
subunit oligomerization.
Although
is apparently phosphorylated following
activation of PKC in hepatocytes or the promyelocytic cell line U937 (19, 20) and phosphorylation in vitro of a
mixture of isoforms of
by PKC was also
described(18) , we were not able to demonstrate phosphorylation
of specific isoforms of
with the preparations of PKC
used in this study. Perhaps phosphorylation of G protein
subunits
is a more general phenomenon than suspected and the appropriate kinases
have not yet been identified.