(Received for publication, August 1, 1995; and in revised form, November 20, 1995)
From the
Previous studies from our laboratory have shown that epidermal
growth factor (EGF) stimulates cAMP accumulation in the heart via a
process involving G and the EGF receptor (EGFR)
protein tyrosine kinase activity (Nair, B. G., Parikh, B., Milligan,
G., and Patel, T. B.(1990) J. Biol. Chem. 265,
21317-21322; Nair, B. G., and Patel, T. B.(1993) Biochem.
Pharmacol. 46, 1239-1245). Therefore, studies were performed
to investigate the hypothesis that the EGFR protein tyrosine kinase
phosphorylates G
and activates this protein. Employing
purified EGFR and G
, we have demonstrated that the
EGFR kinase phosphorylates G
in a time-dependent
manner with a stoichiometry of 2 mol of phosphate incorporated/mol of
G
. As determined by phosphoamino acid analysis, the
phosphorylation of G
by the EGFR kinase was
exclusively on tyrosine residues. Interestingly, GDP and guanosine
5`-3-O-(thio)triphosphate (GTP
S) inhibited the
phosphorylation of G
without altering EGFR
autophosphorylation. However, G protein
subunits protected
against GDP- and GTP
S-mediated inhibition of phosphorylation of
G
. In functional studies, phospho-G
demonstrated a greater GTPase activity and also a greater capacity to
bind GTP
S as compared to the nonphosphorylated G
.
Moreover, the phospho-G
augmented adenylyl cyclase
activity in S49 cyc
cell membranes to a greater
extent than its nonphosphorylated counterpart. Therefore, we conclude
that phosphorylation of G
on tyrosine residues by the
EGFR kinase activates this G protein and increases its ability to
stimulate adenylyl cyclase.
Epidermal growth factor (EGF) ()exerts a variety of
biological actions ranging from increased DNA synthesis, hyperplasia,
and increased glucose and fatty acid metabolism, to alterations in
muscular function (see (1) for review). These pleiotropic
actions of EGF are mediated via the activation of several second
messenger systems. For instance, following binding of EGF to its
receptors, the intrinsic protein tyrosine kinase activity of the EGF
receptor is increased, resulting in autophosphorylation of the EGF
receptor as well as of other cellular proteins (reviewed in Refs. 1 and
2). The autophosphorylation of the EGF receptor serves to recruit
proteins containing the Src homology 2 (SH2) domains such as
phospholipase C
(3) , the subsequent phosphorylation of
which increases phosphatidylinositol metabolism, and the generation of
the second messengers inositol 1,4,5-trisphosphate and
diacylglycerol(3) . Likewise, recruitment of the SH2-containing
proteins such as Grb2 and other adaptor proteins to the
phosphotyrosine-containing domains on the EGF receptor also leads to
the activation of serine/threonine phosphorylation cascades such as the
mitogen-activated protein kinase cascade(4) . In addition, EGF
has also been documented to modulate the cAMP second messenger system.
Studies from our laboratory have shown that EGF increases
contractility, beating rate, and cAMP accumulation in the heart (5) by stimulating adenylyl cyclase via a process involving
G
(6, 7) . Moreover, the protein
tyrosine kinase activity of the EGF receptor is important for
EGF-mediated stimulation of cardiac adenylyl cyclase(8) . One
implication of this latter finding is that one, or more, of the
signaling elements involved in stimulation of adenylyl cyclase by the
activated EGF receptor is(are) phosphorylated. Therefore, we have
proposed the hypothesis that EGF phosphorylates G
and
activates this G protein to stimulate adenylyl cyclase activity. This
hypothesis incorporates the requirement for G
and the
EGF receptor protein tyrosine kinase, the two components which our
previous studies have determined to be required for EGF-elicited
stimulation of the effector, adenylyl cyclase(7, 8) .
Among the subunits of heterotrimeric G proteins, G
has been demonstrated to be phosphorylated and inactivated by
protein kinase C(9, 10, 11) . The studies of
Hausdorff et al.(12) have shown that in
vitro, pp60
phosphorylates
G
and that in reconstitution experiments with the
-adrenergic receptor,
-adrenoreceptor agonists such as
isoproterenol augment GTP
S binding to phosphorylated G
to a greater extent than to nonphosphorylated G
.
The tyrosine kinase pp60
phosphorylates G
on tyrosine residues 37 and 377 (13) with a stoichiometry between 0.4 and 0.9 mol of
phosphate/mol of G
(12) . To date,
phosphorylation of G
by tyrosine kinases other than
pp60
, and in particular receptor
protein tyrosine kinases, has not been reported. Therefore, to address
our aforementioned hypothesis we have investigated whether or not the
EGF receptor protein tyrosine kinase phosphorylates G
and modulates its activity. Our data demonstrate that the
activated EGF receptor (EGFR) stoichiometrically phosphorylates
G
and activates this protein as monitored by its
ability to bind GTP
S and activate adenylyl cyclase. To our
knowledge, this is the first demonstration of phosphorylation of
G
by the EGF receptor with a concomitant increase in
activity of this protein.
Previous data from this laboratory have demonstrated that EGF
stimulates adenylyl cyclase activity in cardiac membranes via
activation of G(6, 7) and that the
EGFR protein tyrosine kinase activity is necessary for this
effect(8) . Therefore, in order to determine whether or not the
EGFR protein tyrosine kinase phosphorylates G
and to
evaluate the functional significance of such a phosphorylation,
experiments were performed with the purified EGFR and pure
G
. Initially, the ability of the EGFR kinase to
phosphorylate G
as a function of time and the
dependence of this phosphorylation on the presence of EGFR and ATP were
determined. As illustrated by the data in Fig. 1, the purified
EGF receptor increased phosphorylation of G
in a
time-dependent manner (Fig. 1A); maximal
phosphorylation of the G
was obtained 60 min after
initiation of the phosphorylation reaction at 25 °C. Moreover, as
demonstrated by the data in Fig. 1B, the
phosphorylation of G
was dependent upon the presence
of the EGFR and ATP. In studies similar to those depicted in Fig. 1, the stoichiometry of phosphorylation of G
by EGFR was determined to be 1.73 ± 0.3 (n = 4) mol of P
incorporated/mol of
G
. As a control, the stoichiometry of
autophosphorylation of the EGF receptor was monitored in parallel and
found to be 4.5 ± 0.5 (n = 5) sites
phosphorylated per mol of the EGFR. While the latter data confirm the
published stoichiometry of the EGFR autophosphorylation(23) ,
the former results indicate that there are two sites on the
G
that are phosphorylated by the EGFR.
Figure 1:
Phosphorylation of G by the EGFR protein tyrosine kinase. Panel A, time
course for phosphorylation of G
by purified EGF
receptor. Purified, recombinant G
was incubated in the
phosphorylation mixture described under ``Materials and
Methods'' in the presence of [
-
P]ATP.
Aliquots (10 µl) were withdrawn at various time intervals and an
equal volume of 2
Laemmli sample buffer was added. EGFR and
G
were separated by SDS-PAGE (12% acrylamide).
Phosphoproteins were visualized by autoradiography. Panel B,
ATP- and EGF receptor-dependent phosphorylation of G
.
Purified, recombinant G
was phosphorylated as
described above in the presence and absence of ATP and EGF receptor.
Phosphoproteins were separated by SDS-PAGE and visualized by
autoradiography.
To determine
whether the phosphorylation of G by the EGF receptor
was on tyrosine residues or involved phosphorylation of serine and
threonine residues also, two approaches were pursued. First,
G
was phosphorylated by the EGFR in the presence of
unlabeled ATP, and following separation of proteins by SDS-PAGE and
transfer onto nitrocellulose, Western analyses with
anti-phosphotyrosine antibody were performed. The data depicted in Fig. 2A demonstrate that both the EGFR and G
are phosphorylated on tyrosine residues, and phosphorylation of
G
is dependent on the presence of ATP and EGFR (see
also Fig. 1B). In additional experiments, phosphoamino
acid analyses of
P-labeled G
and
autophosphorylated EGFR (positive control) demonstrated that the
phosphorylation of both the EGFR and G
was exclusively
on tyrosine residues (Fig. 2B).
Figure 2:
EGF Receptor protein tyrosine kinase
phosphorylates G on tyrosine residues. Panel
A, analysis of phosphoproteins by Western blotting. G
was phosphorylated as described under ``Materials and
Methods.'' Phosphoproteins were separated by SDS-PAGE (12%
acrylamide) and transferred to nitrocellulose. Western analysis was
performed using the Amersham ECL system with a rabbit
anti-phosphotyrosine antibody from Zymed Inc. Panel B,
phosphoamino acid analysis. G
was phosphorylated as
described under ``Materials and Methods'' in the presence of
[
-
P]ATP. Phosphoproteins were separated by
SDS-PAGE and transferred to PVDF membrane. Details of acid hydrolysis
of phosphoproteins and thin layer electrophoresis are described under
``Materials and Methods.'' The phosphoamino acids were
located by autoradiography. The migration of phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) standards are shown.
In the studies
involving pp60-mediated phosphorylation of
G
, the GDP-bound form of G
was
reported to be a better substrate for the kinase (12) .
Therefore, we investigated the effects of GDP on phosphorylation of
G
by the EGFR kinase. In these experiments,
G
was phosphorylated by the EGFR kinase in the
presence of different GDP concentrations. Following separation of the
proteins by SDS-PAGE and autoradiography, the protein bands
corresponding to EGFR and G
were excised from the gels
and counted for
P content. The data in Fig. 3A
demonstrate that, while the autophosphorylation of the EGF receptor was
not altered by GDP, phosphorylation of G
was inhibited
in a concentration-dependent manner such that, at GDP concentrations in
excess of 3 µM, the phosphorylation of G
was decreased by 50%. Similarly, GTP
S also inhibited by 50% the phosphorylation of G
without altering the
autophosphorylation of the EGFR (Fig. 3A). Thus, in the
presence of guanine nucleotides, the stoichiometry of G
phosphorylation by the EGFR kinase was decreased from 2 mol
P
/mol of G
to 1 mol of P
/mol
of G
(0.7 ± 0.3 mol of P
/mol
G
(n = 3)). Interestingly, in the
absence of GDP or GTP
S, the addition of G protein
subunits did not alter the phosphorylation of G
by the
EGFR kinase (Fig. 3B, cf. lanes 1 and 3). However, in the presence of GDP (10 µM),
subunits protected against the GDP-mediated inhibition of
phosphorylation of G
(Fig. 3B, cf.
lanes 2 and 4), and therefore, the stoichiometry of
G
phosphorylation by the EGFR kinase in the presence
of GDP and
subunits was 2 mol of P
/mol of
G
. Similar protection against inhibition of
phosphorylation of G
by GTP
S was also seen in the
presence of
subunits (not shown). Notably, the
subunits were not phosphorylated by the EGFR kinase (Fig. 3B). The finding that GDP inhibited
phosphorylation of G
(Fig. 3A) is in
stark contrast to the effects of this nucleotide on phosphorylation of
G
by pp60
(12) but
similar to the effects of GDP on phosphorylation of the small molecular
weight G protein by EGFR reported by Hart et al.(24) .
The opposite effects of GDP on phosphorylation of G
by
EGFR and pp60
suggest that the sites on
G
phosphorylated by these two protein tyrosine kinases
may be different. To determine the specificity of phosphorylation of
G
by EGFR kinase, we also investigated the ability of
the EGFR kinase to phosphorylate G
. As compared with
phosphorylation of G
, the EGFR kinase phosphorylated
G
with a very low stoichiometry (0.4 pmol
P
/mole of G
) (data not shown). This
finding is consistent with the report of Hart et al.(24) that EGFR kinase phosphorylated G
and
G
with a very low stoichiometry. Hence, it would
appear that the EGFR kinase selectively phosphorylates
G
.
Figure 3:
GDP and GTPS inhibit the
phosphorylation by EGFR kinase of G
in its monomeric
but not heterotrimeric form. Panel A, inhibition of monomeric
G
phosphorylation in the presence of varying
concentrations of GDP (circles) or GTP
S (squares). Purified G
was phosphorylated by
the EGFR kinase in the absence and presence of guanine nucleotides as
described under ``Materials and Methods.'' After separation
of proteins by SDS-PAGE and location of the G
and EGFR
by autoradiography, the two protein bands were excised and counted for
P content. Data are presented as percent of radioactivity
in the EGFR (open symbols) and G
(filled
symbols) in the absence of guanine nucleotides. Panel B,
GDP does not inhibit phosphorylation of the G
in its
heterotrimeric form. Purified G
mixed with G protein
subunits (G
:
= 1:1) were
phosphorylated in the presence and absence of GDP (10 µM).
Following separation of proteins, the gels were dried and subjected to
autoradiography. Lane 1, G
+ EGFR; lane 2, G
+ EGFR + GDP (10
µM); lane 3, G
+
EGFR; lane 4, G
+ EGFR +
GDP (10 µM). The asterisk indicates the migration
of
subunit as determined by Coomassie staining of the
gel.
In vivo, under basal conditions, prior
to activation of the EGFR, the G would be
predominantly in the GDP bound heterotrimeric form. Therefore, to study
the functional consequences of G
phosphorylation, this
situation was recreated in in vitro studies by phosphorylating
the G
by the EGFR kinase as a heterotrimer (i.e. presence of
and GDP) a condition under which
G
is phosphorylated on two sites (see Fig. 3B). The data in Fig. 4A demonstrate
that the GTPase activity of the phospho-G
was 2-fold
greater than that of its nonphosphorylated counterpart; in these
experiments, controls with
subunits plus EGFR alone were
found to have negligible activity (not shown). This increase in GTPase
activity of the phosphorylated G
also suggests that
the EGFR kinase phosphorylates G
on tyrosine residues
which may be different to those phosphorylated by the
pp60
(13) since phosphorylation of
G
by the latter kinase does not alter its GTPase
activity(12) . As a second approach to assess functional
significance, experiments were also performed to determine if
phosphorylation of G
by EGFR kinase altered the
ability of the protein to bind GTP
S. As demonstrated by the data
in Fig. 4B, phospho-G
bound GTP
S
at a greater rate and to a larger extent as compared to the
nonphosphorylated G
; GTP
S binding to
subunits plus EGFR (control) was negligible (not shown). These data (Fig. 4, A and B) strongly suggested that
phosphorylation of G
in its heterotrimeric form (i.e. in presence of GDP and
subunits) markedly
activated this G protein. Therefore, to determine whether or not the
phosphorylated G
was indeed more effective as an
activator of adenylyl cyclase, experiments were performed employing S49
cyc
cell membranes which do not contain any
endogenous G
(25) . The data from these
experiments demonstrated that upon reconstitution of equal amounts of
nonphosphorylated and phosphorylated G
with
cyc
membranes, the adenylyl cyclase activity was
2-fold greater in the presence of the phospho-G
than
its non-phosphorylated counterpart (Fig. 4C). In
controls performed with
subunits with the EGFR (control),
adenylyl cyclase activity was very low and comparable to the activity
in the absence of any additions (cf. Fig. 4C and (22) ).
Figure 4:
Phosphorylation of G by
EGFR kinase increases its functional activity. G
was
phosphorylated in the presence of GDP and
subunits as
described under ``Materials and Methods.'' Control
(nonphosphorylated) G
was similarly treated except
that the EGFR was not added in the phosphorylation reactions. As
additional control, the EGFR and
were incubated in the
absence of G
. Following the phosphorylation reaction,
GTPase activity, GTP
S binding, and the ability of phosphorylated
and nonphosphorylated G
to stimulate adenylyl cyclase were
monitored. Panel A, GTPase activity of phosphorylated and
nonphosphorylated G
. Following the initial incubation
for 60 min in the phosphorylation reaction with and without EGFR, the
G
(G
) was transferred into the
GTPase reaction mixture, and aliquots were withdrawn at the various
times indicated. A representative experiment of three is shown. Panel B, GTP
S binding to phosphorylated and
non phosphorylated G
. The conditions were similar to
those described for data in Panel A, except that the G
was transferred into GTP
S binding buffer and
aliquots were withdrawn to monitor binding. The precise experimental
conditions are described under ``Materials and Methods.'' A
representative experiment of three is shown. Panel C, ability
of phosphorylated and nonphosphorylated G
to stimulate
adenylyl cyclase activity in S49 cyc
cell membranes.
Following incubation of G
with GDP (10
µM) and
subunits in phosphorylation buffer in
the presence or absence of EGFR, the G
was incubated in the
presence of GTP
S (100 nM) in the binding reaction mixture
for 60 min. Aliquots (1.1 pmol) of G
were then
reconstituted with 10 µg of cyc
cell membranes
and assayed for adenylyl cyclase activity as described under
``Materials and Methods.'' Data are presented as the mean
± S.E. of four determinations.
Since GDP and GTPS inhibit
phosphorylation of G
similarly (Fig. 3), and
because the structures of GDP- and GTP-bound forms of G
subunits are different; see e.g. with transducin
subunit (G
) (26) and
G
(27, 28) , our data (Fig. 3A) would suggest that the tyrosine residue(s)
whose phosphorylation is(are) altered by the guanine nucleotides
is(are) not located in the regions of the molecule that change
conformation upon exchange of GDP for GTP. Indeed the crystal structure
data of the GDP- and GTP-bound forms of G
and
G
(26, 27, 28) indicate that
the GTP-bound form of G
subunits are different from the
GDP-bound form in essentially three regions (switch I, switch II, and
switch III) none of which contains tyrosine residues(26) .
However, on G
, tyrosine residues (Tyr-176 and -239)
are located proximal to switch I and switch III regions and it is
plausible that phosphorylation of one or both of these residues by the
EGF receptor increases the rate of GTP
S binding that is observed (Fig. 4B). Likewise since tyrosine residues (Tyr-325,
-344, and -346) are also located in the proximity of adenylyl cyclase
interacting regions on G
(see e.g.(26) ), it is tempting to speculate that phosphorylation
of one of these residues increases the interaction of G
with adenylyl cyclase, thereby augmenting activity as observed in Fig. 4C. Interestingly, phosphorylation of the
heterotrimeric form of G
by the EGF receptor was not
altered by either GDP (Fig. 3B) or GTP
S (not
shown) suggesting that the tyrosine residues on G
that
are phoshorylated reside in region(s) whose conformation is stabilized
by the
subunits. Additional studies which will identify the
sites on G
that are phosphorylated by the EGF receptor
will provide more mechanistic information in the light of the crystal
structure of G
and G
. Presently, from
the data presented in Fig. 3, we can conclude that guanine
nucleotide binding (either GDP or GTP) inhibits the phosphorylation of
G
by the EGF receptor and that
subunits
protect against such inhibition of phosphorylation.
Several
laboratories have reported the phosphorylation of subunits of G
proteins by different kinases as a potential regulatory event in signal
transduction. However, functional significance of the phosphorylation
has been demonstrated in only a few of the studies. Thus it is clear
that phosphorylation of G
by protein kinase C
decreases the activity of this G
protein(9, 10, 11) . On the other hand, the
functional consequence of phosphorylation of G
by
protein kinase C (29) remains to be elucidated. Similarly,
although the
subunits of transducin(30) , G
,
and G
(31) have been shown to be phosphorylated by
the insulin receptor protein tyrosine kinase, the functional
consequences of these phosphorylations remain unknown. It should be
noted that G
is not phosphorylated to any significant
extent by either protein kinase C (29) or the insulin receptor
protein tyrosine kinase(31) . In this respect, our findings for
the first time, demonstrate the phosphorylation of G
by a receptor protein tyrosine kinase and provide information
concerning the alteration in G
function due to these
phosphorylations. Most importantly, the data presented here provide a
tenable mechanism for EGF-mediated stimulation of adenylyl cyclase
activity. Notably, however, this may not be the only mechanism involved
in EGF-elicited stimulation of adenylyl cyclase activity but may
represent one of two different, but not mutually exclusive, manners by
which EGF may augment adenylyl cyclase activity. Hence, recently we
have shown that a juxtamembrane 13-amino acid sequence in the EGFR can
activate G
and thereby stimulate adenylyl cyclase activity (22) . This finding is consistent with the hypothesis that upon
binding to its receptors, EGF stimulates autophosphorylation of the
receptors which results in a change in the conformation of the
cytosolic domain of the EGF receptor from a compact to an extended form (32) and thereby, allows the juxtamembrane region to interact
with and stimulate G
. However, in addition to this mode of
activation of G
, simultaneous phosphorylation of
G
by the EGF receptor protein tyrosine kinase could,
in a mutually reinforcing manner, amplify the signal from EGFR to
G
and ultimately to adenylyl cyclase, since the
phospho-G
is a better activator of this effector (Fig. 4C). This latter possibility, i.e. amplification of signaling by combinatorial effects of
phosphorylation and interaction of the cytosolic, juxtamembrane, region
of the EGF receptor with G
and its implications on adenylyl
cyclase activity remains to be experimentally tested.
In conclusion,
we have presented experimental evidence to demonstrate that EGF
receptor protein tyrosine kinase can phosphorylate G on tyrosine residues and stimulate the functional activity of
this G protein. To our knowledge, this is the first demonstration that
a receptor protein tyrosine kinase can phosphorylate and activate
G
. Moreover, these data provide mechanistic insights into
EGF-elicited stimulation of adenylyl cyclase activity. Presently, the
identity of the tyrosine residues on G
that are
phosphorylated by EGFR kinase remain to be elucidated. However,
stoichiometry analyses indicate that at least 2 tyrosine residues are
phosphorylated. Whether or not the phosphorylation of one of these 2
tyrosine residues alters the GTPase and GTP
S binding activities
preferentially or phosphorylation of both tyrosines is required to
observe the functional changes is not known. These questions and the
possibility that there may be a hierarchy in the phosphorylation of the
two tyrosines on G
by the EGFR kinase forms the
subject of our future investigations.