From the Department of Pharmacology, University of Tennessee, Memphis, The Health Science Center, Memphis, Tennessee 38163 and the ¶ Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706-1532
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ABSTRACT |
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Binding of epidermal growth factor (EGF) to its
receptor (EGFR) augments the tyrosine kinase activity of the receptor
and autophosphorylation. Exposure of some tissues and cells to EGF also
stimulates adenylyl cyclase activity and results in an increase in
cyclic AMP (cAMP) levels. Because cAMP activates the
cAMP-dependent protein kinase A (PKA), we investigated the
effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc)
stoichiometrically phosphorylated the purified full-length wild type
(WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated
both WT-EGFR as well as a mutant truncated form of EGFR ( Epidermal Growth Factor
(EGF)1 is responsible for a
variety of biological effects ranging from mitogenesis (1) to
influences on glucose metabolism (2). Upon binding of EGF to its
receptor, the epidermal growth factor receptor (EGFR) undergoes
dimerization and displays tyrosine kinase activity. This leads to
autophosphorylation of the EGFR as well as phosphorylation of
intracellular substrates (3). In addition, we have shown that, in the
heart, EGF increases contractility and heart rate by augmenting cAMP
accumulation (4), a result of stimulation of adenylyl cyclase activity
via activation of Gs The EGFR is a 170-kDa glycoprotein with a single transmembrane domain.
Its intracellular domain is susceptible to phosphorylation on various
residues. Thus, the five autophosphorylation sites in the cytosolic
domain of EGFR are located at tyrosine residues 992, 1068, 1086, 1148, and 1173 (10-12). Furthermore, the EGFR is subject to regulation by
other kinases. Hence, protein kinase C (PKC) phosphorylates the EGFR at
Thr-654, thereby decreasing the intrinsic tyrosine kinase activity of
the EGFR (13, 14). Phosphorylation of Ser-1002 by
p34cdc2 (15) and of Ser-1046/1047 on the EGFR by
calmodulin-dependent kinase II (16) is likewise associated
with inhibition of tyrosine kinase activity. The EGFR can also be
phosphorylated on Thr-669 by mitogen-associated protein kinase (MAPK;
Refs. 17 and 18). In addition, in vivo
pp60c-src phosphorylates the EGFR on three
tyrosines (Tyr-845, -891, and -820) that are not the
autophosphorylation sites (19, 20). These novel phosphotyrosines may
provide docking sites for SH2 domain-containing proteins, which would
explain the enhancement of the mitogenic response to EGF observed in
pp60c-src-overexpressing cells (20).
Previous in vitro studies have shown that the EGFR is a
substrate for phosphorylation by PKA (21, 22). However, to date, the
functional significance of the phosphorylation has not been described.
Since we and others have previously shown that EGF can activate
adenylyl cyclase and increase cAMP accumulation in several tissues (4,
23-25), in the present study we have investigated the possibility that
PKA may modulate EGFR tyrosine kinase activity. Our data show that
phosphorylation of the EGFR by PKA on serine residues leads to
decreased tyrosine kinase activity and diminished autophosphorylation
of the EGFR both in vitro and in vivo.
Purification of Wild Type (WT), K721M, and Phosphorylation of EGFR by PKA--
The catalytic subunit of PKA
from bovine heart (PKAc; Sigma) was dissolved in a buffer containing 20 mM MES, pH 6.5, 100 mM NaCl, 100 µM EDTA, 20 mM Phosphoamino Acid Analysis of EGFR--
Bands corresponding to
the 32P-labeled proteins were excised from polyvinylidene
difluoride membranes and subjected to phosphoamino acid analyses as
described previously (8).
Stoichiometry of EGFR Phosphorylation by PKAc--
This was
achieved by pursuing two approaches. Both approaches involved
decreasing the autophosphorylation of the EGFR. First, we employed the
purified, kinase negative form of the EGFR (EGFR K721M; Ref. 27). This
receptor (10 ng) was phosphorylated as described above. In the second
approach, the tyrosine kinase activity of the wild type EGFR (100 ng)
was inactivated by incubation with 1 mM
N-ethylmaleimide (28) for 15 min in the phosphorylation buffer described above except that vanadate and dithiothreitol were
absent. After addition of dithiothreitol (2 mM) to block further modifications of sulfhydryl groups, PKAc (480 units/ml) and
[ Detection of Tyrosine Phosphorylated Cellular Proteins and Active
EGFR in Cell Lysates--
B82L cells expressing EGFR Serine Phosphorylation of the EGFR by PKA in Intact
Cells--
B82L cells (4 × 105 cells per 35-mm dish)
expressing the K721M form of the EGFR were treated with and without
8-CPT-cAMP for 30 min. The EGFR was then immunoprecipitated from equal
amounts of cell lysates (400 µg of protein) with anti-EGFR antibody
(Transduction Laboratories) as described before (7). Following
separation of proteins in the immunoprecipitate by SDS-PAGE, Western
analysis was performed with anti-phosphoserine antibody (clone A49,
Alexis Corp., 1:250 dilution) according to the manufacturer
instructions. The blot was reprobed with anti-EGFR antibody (Santa
Cruz) to ensure that equal amounts of EGFR were immunoprecipitated.
Tyrosine Kinase Assays--
Purified WT, EGFR Ligand Binding and Internalization of EGFR--
B82L cells
expressing either the WT or Because EGF can increase cAMP accumulation in the heart and other
tissues (4-6, 23-25), we postulated that akin to the negative regulation of the EGFR by protein kinase C (14),
calmodulin-dependent protein kinase II (16) and
p34cdc2 (15), PKA may alter the functional
activity of EGFR. To address this hypothesis, initially, we determined
the effect of pure PKAc on autophosphorylation of the purified WT and
1022-1186)
exclusively on serine residues. Moreover, PKAc also phosphorylated the
cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified
WT as well as EGFR
1022-1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity.
Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog,
8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine
phosphorylation of cellular proteins as well as activation of the
WT-EGFR. Similar effects were also observed in B82L cells transfected
to express the
1022-1186 form of EGFR. Furthermore, activation of
PKAc in intact cells resulted in serine phosphorylation of the EGFR.
The decreased phosphorylation of cellular proteins and diminished
activation of the EGFR in cells treated with the cAMP analog was not
the result of altered binding of EGF to its receptors or changes in
receptor internalization. Therefore, we conclude that PKA
phosphorylates the EGFR on Ser residues and decreases its tyrosine
kinase activity and signal transduction both in vitro and
in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(4-6). Additionally, we have also
demonstrated that EGF-elicited stimulation of adenylyl cyclase activity
requires the tyrosine kinase activity of the EGFR and may involve
phosphorylation of Gs
(7, 8). The cyclic AMP formed in
response to EGF can activate the heterotetrameric
cAMP-dependent protein kinase A (PKA) by binding to its two
regulatory subunits, thereby dissociating them from the catalytic
subunits (9). The catalytic subunit of PKA (PKAc) can then
phosphorylate a variety of intracellular proteins (9).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1022-1186 Forms of
EGFR--
Using the procedures described previously (26) wild type
EGFR was purified from A431 cells. Likewise, using the same method (26), the EGFR K721M and EGFR
1022-1186 were purified from B82L cells transfected to express these forms of the receptor. The final
step in this method involved elution of the EGFR with EGF from an EGFR
antibody affinity column.
-mercaptoethanol, and 50%
ethylene glycol (8000 units/ml). The various forms of purified EGFR
(WT, EGFR
1022-1186, K721M, and EGFRKD (amino acids 644-1186)) were
incubated with 480 units/ml PKAc in 10 µl of phosphorylation buffer
containing 20 mM Hepes (pH 7.4), 5 mM
MgSO4, 2 mM MnCl2, 1 mM
NaF, 1 mM dithiothreitol, 10 µg/ml aprotinin, 20 µg/ml
leupeptin, 10 µM ATP, 50 µM sodium vanadate, and 100 nM EGF (Upstate Biotechnology Inc. or
Intergen Co.) for the indicated times at 30 °C. Reactions were
terminated by addition of 2× Laemmli sample medium and boiling at
100 °C for 5 min. Proteins were separated on 7.5% SDS-PAGE gels.
The samples for autoradiography and phosphoamino acid analyses
contained 5 µCi (6000 Ci/mmol) [
-32P]ATP (NEN Life
Science Products) and were electrophoretically transferred onto
polyvinylidene difluoride membranes (Bio-Rad Laboratories).
-32P]ATP (10 µM) were added to
phosphorylate the EGFR as described above. Following SDS-PAGE (7.5%
acrylamide), the bands corresponding to the EGFR were excised, and
radioactivity associated with the receptor was determined. From the
known specific radioactivity of ATP in the reaction mixture, the moles
of phosphate incorporated into known amount of receptor were determined.
1022-1186 (12)
and REF-52 cells were plated at a density of 2 × 105
cells per 35-mm dish and allowed to grow for 24 h. The cells were
then serum starved overnight and treated with 100 µM
8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP; Sigma) for the indicated times
before addition of 100 nM EGF. Cells were harvested in 2×
Laemmli sample medium, and the samples were boiled at 100 °C for 5 min. An aliquot (20 µl) of the proteins in the Laemmli sample medium
was diluted with water (80 µl) and mixed with 400 µl of 0.1 M sodium phosphate, pH 7.2, to precipitate the SDS.
Standards of bovine serum albumin were similarly treated. The
supernatant from this mixture was utilized to determine protein
concentrations using the Bio-Rad Protein Assay reagent. Equal amounts
of protein (15-45 µg) were then subjected to SDS-PAGE. Western blots
were performed as described previously (8). Either a polyclonal
(Zymed Laboratories Inc.) or a monoclonal (PY-20; ICN
Biomedicals Inc.) anti-phosphotyrosine antibody (both at 1:1000
dilution) was used. To detect activated EGFR, immunoblots of cellular
proteins were performed with anti-active EGFR antibody (Transduction
Laboratories). To ensure that equal amounts of proteins from REF-52
cells were loaded on gels, the blots were reprobed with anti-PKAc-
antibody (Santa Cruz).
1022-1186, or
EGFRKD was preincubated with the indicated amounts of PKAc in
phosphorylation buffer described above for 30 min at 30 °C.
Thereafter, 1 mM Val-5-angiotensin II (Novabiochem) and 1.5 µCi of [
-32P]ATP were added and phosphorylation was
allowed to proceed for 10 min at room temperature in a total volume of
25 µl. The reactions were stopped with 25 µl of 20%
trichloroacetic acid. After centrifugation, the supernatants were
spotted onto P81 Whatman filter disks, rinsed five times for 2 min each
in 75 mM phosphoric acid, dried, and counted for
32P incorporation into Val-5-angiotensin II.
1022-1186 forms of EGFR were plated in
24-well plates (30,000 cells/well) and serum starved overnight. The
cells were washed with 0.5 ml of ice-cold Krebs-Henseleit buffer
modified to contain 20 mM Hepes, pH 7.4, and incubated on
ice for 25 min with the same buffer containing 0.5 mg/ml bovine serum
albumin. Thereafter, 125I-EGF (50 pM) was
added, and incubation was continued on ice for a further 2 h.
Cells were then washed three times with 0.5 ml of ice-cold
Krebs-Henseleit buffer and solubilized in 0.5 ml of 1 N NaOH.
Nonspecific binding was determined in the presence of excess (1 µM), unlabeled EGF. To monitor internalization of the EGFR, the method of Honnegar et al. (29) was pursued.
Essentially, following binding of 125I-EGF to cells as
described above, the cells were incubated at 37 °C for 30 min and
then washed with Krebs-Henseleit buffer as described above. Cell
surface-associated 125I-EGF was removed and counted with
0.5 ml ice-cold 0.5 M acetic acid and 150 mM
NaCl. Thereafter, cells were washed once with Krebs Henseleit buffer
and lysed at 37 °C in 0.5 ml of 1N NaOH to determine the amount of
internalized 125I-EGF.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1022-1186 forms of EGFR; in the
1022-1186 form of EGFR, four of
the five autophosphorylation sites are deleted. Fig.
1A shows that PKAc decreases
the incorporation of 32P label from
[
-32P]ATP into the WT-EGFR, whereas 32P
incorporation into EGFR
1022-1186 remained unchanged in the presence of PKAc. Similar results, i.e. decreases in
32P incorporation, were obtained when the WT-EGFR was
incubated with the PKA holoenzyme activated by 8-CPT-cAMP (not shown).
This is in contrast with the results of Rackoff et al. (21)
and Ghosh-Dastidar et al. (22) who showed an increase in
32P incorporation from [
-32P]ATP into the
purified EGFR after addition of PKA. Possible explanations for this
difference are as follows. First, Rackoff et al. (21) incubated the EGFR with PKA for just 30-60 s, which may be too short
to reach steady state phosphorylation of the EGFR on tyrosine and
serine/threonine residues. Second, in the experiments of Rackoff et al. (21) and Ghosh-Dastidar and Fox (22), vanadate, an inhibitor of phosphotyrosine phosphatases was absent. Under these conditions, tyrosine phosphatases, which may co-purify with the EGFR,
would decrease tyrosine phosphorylation of the EGFR, thereby allowing
the net increase in PKA-mediated phosphorylation to be monitored. In
contrast, in our experiments that employed a longer incubation time and
vanadate, the phosphorylation observed with 32P
incorporation would represent the sum of tyrosine and serine/threonine phosphorylation of the EGFR. Therefore, to determine whether PKAc altered tyrosine phosphorylation of the EGFR, experiments similar to
those in Fig. 1A were performed with unlabeled ATP. The
samples were then subjected to SDS-PAGE and Western analyses with
anti-phosphotyrosine antibody. As demonstrated by data in Fig.
1B, incubation of the WT-EGFR with PKAc decreased tyrosine
phosphorylation of the receptor. Similarly, autophosphorylation of the
1022-1186 form of the EGFR was also decreased in the presence of
PKAc. Thus, the decrease in 32P incorporation in WT-EGFR
observed in the presence of PKAc (Fig. 1A) represents a
decrease in autophosphorylation of the receptor. Likewise, because
tyrosine phosphorylation of the one site (Tyr-992) on the
EGFR
1022-1186 is decreased (Fig. 1B), in the presence of
a serine or threonine phosphorylation of this receptor by PKAc, no net
change in 32P incorporation would be evident (Fig.
1A), i.e. phosphorylation of one serine or
threonine residue by PKAc on EGFR
1022-1186 would compensate for the
loss of phosphorylation at Tyr-992.
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Fig. 1.
PKAc decreases tyrosine phosphorylation of WT
and EGFR 1022-1186. Panel A, effect
of PKAc on 32P incorporation into WT (left) and
1022-1186 (right) forms of EGFR. Purified forms of EGFR
(30 ng) were incubated in the presence or absence of PKAc (480 units/ml) for 30 min at 30 °C in the presence of
[
-32P]ATP (10 µM) as described under
"Materials and Methods." The reactions were stopped by addition of
an equal volume of 2× Laemmli sample medium. Phosphoproteins were
separated on a 7.5% SDS-PAGE gel and visualized by autoradiography.
Note that the autoradiograph with WT-EGFR was exposed for 2 h
only, whereas the autoradiograph with EGFR
1022-1186 was exposed
overnight. PKAc autophosphorylation is evident only after long
exposures. Panel B, phosphotyrosine content of WT and
1022-1186 forms of EGFR in the presence and absence of PKAc. The WT
(left) and
1022-1186 (right) EGFR were
phosphorylated by PKAc (480 units/ml) in the presence of unlabeled ATP
(10 µM) as described under "Materials and Methods."
Phosphoproteins were separated by SDS-PAGE (7.5% acrylamide) and
transferred to nitrocellulose. Western blotting was performed with
anti-phosphotyrosine antibody as described under "Materials and
Methods." Representatives of at least three similar experiments are
shown.
To determine whether PKAc phosphorylates serine or threonine residues
on the EGFR, phosphoamino acid analyses were performed. Fig.
2A shows that phosphorylation
of the WT-EGFR occurs predominantly on serine residues, as also found
by Rackoff et al. (21) and Ghosh-Dastidar and Fox (22).
Furthermore, Fig. 2A shows that the appearance of
phosphoserine in the presence of PKAc is accompanied by a decrease in
phosphotyrosine. PKAc also phosphorylates EGFR1022-1186 on serine
residues (Fig. 2B), and this increase in serine
phosphorylation is accompanied by the loss of tyrosine phosphorylation.
These data support the contention that, in experiments performed with EGFR
1022-1186 and [
-32P]ATP, no net difference in
32P incorporation into the receptor is observed in the
presence of PKAc (Fig. 1A, right panel) because
of a gain of serine phosphorylation and concomitant loss of tyrosine
phosphorylation. Because both the WT and EGFR
1022-1186 are
phosphorylated on serine residues by PKAc, the data in Fig. 2
demonstrate that the PKAc phosphorylation site(s) is(are) not located
in the region between amino acids 1022 and 1186. In additional
experiments, we determined whether the purified, cytosolic region of
the EGFR (amino acids 645-1186; EGFRKD) is also phosphorylated by
PKAc. As demonstrated by data in Fig.
3A, phosphorylation of the
purified EGFRKD by PKAc resulted in a mobility shift of the EGFRKD on
polyacrylamide gels. These data coupled with the findings with the wild
type and
1022-1186 forms of the EGFR (Figs. 1 and 2) demonstrate
that the serine residue(s) on the EGFR, which is(are) phosphorylated by
PKAc, resides between amino acids 644 and 1022.
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Because the wild type EGFR is phosphorylated both on Tyr as well as Ser residues in the presence of PKAc, and because tyrosine phosphorylation of the receptor decreases in the presence of PKAc (Fig. 2, A and B), the stoichiometry of phosphorylation was difficult to monitor. To circumvent this problem, we employed two approaches. First, phosphorylation of the purified, kinase negative form of the EGFR (EGFR K721M) was monitored. Second, the WT-EGFR was incubated with N-ethylmaleimide to inhibit its tyrosine kinase activity (28). Thereafter, the PKAc-mediated phosphorylation was monitored. As demonstrated in Figs. 3, B and C, autophosphorylation of either the K721M form of the EGFR or the wild type receptor in the presence of N-ethylmaleimide was minimal. Under these conditions, PKAc phosphorylated both the kinase negative EGFR (EGFR K721M, Fig. 3B) and WT-EGFR with a stoichiometry of 0.74 and 0.87 mol of Pi per mol of EGFR, respectively.
Because incubation of the WT and 1022-1186 forms of EGFR in the
presence of PKAc decreased tyrosine phosphorylation of both receptors
(Figs. 1B and 2), it would appear that PKAc-elicited phosphorylation of EGFR on serine residues modulates the tyrosine kinase activity of the EGFR. Therefore, to directly evaluate the functional significance of phosphorylation of EGFR by PKAc, we studied
the influence of PKAc on the tyrosine kinase activity of the WT and
1022-1186 forms of EGFR. Fig. 4
demonstrates that PKAc, in a concentration-dependent
manner, inhibited tyrosine kinase activity of both types of EGFR.
Moreover, as demonstrated in Fig. 4C, PKAc also inhibited
the tyrosine kinase activity of the EGFRKD. Together, the data in Figs.
1-4 demonstrate that the stoichiometric serine phosphorylation of the
EGFR by PKAc is accompanied by a decrease in tyrosine kinase activity
of the receptor. Moreover, the site of serine phosphorylation on the
EGFR is located between residues 644-1022.
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Next we investigated whether or not activation of PKA in
vivo alters EGF-induced tyrosine phosphorylation of cellular
proteins and activation of the EGFR. For this purpose, cells were
incubated with 8-CPT-cAMP, a nonhydrolyzable cAMP analog, that
activates PKA (30). Fig.
5A(i) illustrates
that in REF-52 cells, in the absence of 8-CPT-cAMP, the addition of EGF
resulted in a marked increase in tyrosine phosphorylation of cellular
proteins. The most prominent tyrosine phosphorylation in response to
EGF was observed in proteins of molecular masses of ~180 and ~70
kDa (Fig. 5A(i)). Treatment of REF-52 cells with
8-CPT-cAMP resulted in a marked decrease in EGF-elicited tyrosine
phosphorylation of these proteins (Fig. 5A(i)).
The decrease in tyrosine phosphorylation of cellular proteins in the
presence of 8-CPT-cAMP cannot be attributed to differences in protein
loading because reprobing the same blot with anti-PKAc antibody showed
that the amount of PKAc in each lane was the same (lower
panel of Fig. 5A(i)). Moreover, as assessed with the anti-active EGFR antibody, EGF-elicited activation of the EGFR
in REF-52 cells was also markedly attenuated in the presence of
8-CPT-cAMP (Fig. 5A(ii)); immunoprecipitation of
the EGFR from REF-52 cells treated with 8-CPT-cAMP also showed a
decrease in EGF-elicited autophosphorylation of the immunoprecipitated
EGFR (not shown). Similarly, in B82L cells transfected to express
EGFR1022-1186, activation of PKA by 8-CPT-cAMP for different times
also led to a decrease in tyrosine phosphorylation of proteins (Fig.
5B). Thus, 5 min after treatment of cells with 8-CPT-cAMP, a
decrease in EGF-mediated tyrosine phosphorylation of cellular proteins was observed (Fig. 5B). Treatment of these cells with
forskolin, which directly activates adenylyl cyclase and increases cAMP
levels (31), yielded similar results (not shown). To determine whether or not activation of PKA in intact cells phosphorylates the EGFR on
serine residues, the experiment depicted in Fig. 5C was
performed. Essentially, B82L cells transfected to express the
kinase-negative form of the EGFR (EGFR K721M) were treated with
8-CPT-cAMP for 30 min in the absence of EGF. Thereafter, the cells were
lysed, and EGFR was immunoprecipitated. Following separation of
proteins in the immunoprecipitate, Western analysis with
anti-phosphoserine antibody was performed. As shown in Fig.
5C, treatment of cells with 8-CPT-cAMP resulted in serine
phosphorylation of the immunoprecipitated EGFR; the amount of receptor
immunoprecipitated from cells treated with and without 8-CPT-cAMP was
the same (Fig. 5C). The data in Fig. 5 demonstrate that
activation of PKA in intact cells results in serine phosphorylation of
the EGFR, decreased activation of the receptor (Fig.
5A(ii)) and decreased phosphorylation of cellular proteins in response to EGF. Moreover, the data in Fig. 5C
demonstrate that activation of PKA in intact cells can phosphorylate
the EGFR on serine residues in the absence of EGF. Thus, the EGFR does not have to be activated by its ligand for phosphorylation by PKA.
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One possible reason for a decrease in autophosphorylation of EGFR
in intact cells in the presence of the PKA activator 8-CPT-cAMP is that
PKA-elicited phosphorylation of the EGFR may alter binding of EGF to
its receptor or enhance receptor internalization, thereby decreasing
the number of cell surface EGF receptors. To address this possibility,
we monitored the binding of 125I-EGF and internalization of
the ligand in B82L cells expressing EGFR1022-1186 that had been
pretreated with or without 8-CPT-cAMP. These experiments were performed
with 125I-EGF concentrations of 50 pM, the
KD of EGFR for EGF, because any change in
binding affinity would be most pronounced at this concentration of the
ligand. Essentially, our data demonstrated that, in B82L cells
expressing EGFR
1022-1186, the binding of EGF was not altered (Table
I). Likewise, pretreatment of B82L cells
expressing EGFR
1022-1186 with 8-CPT-cAMP did not alter receptor
internalization (Table I). These data indicate that the decrease in
EGF-elicited receptor autophosphorylation observed in cells
preincubated with 8-CPT-cAMP is not the result of a modification in
either EGF binding to its receptors or a change in cell surface EGFR
numbers because of receptor internalization. Thus, the effects of PKA
activation in intact cells reflect the in vitro findings which demonstrate that PKA, by phosphorylating EGFR on serine residues,
decreases its protein-tyrosine kinase activity.
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Regulation of EGFR tyrosine kinase activity by phosphorylation has been described for PKC (13, 14), calmodulin-dependent protein kinase II (16), and p34cdc2 (15). In all of these cases, phosphorylation decreases tyrosine kinase activity. Our data presented here add to the list of protein kinases that regulate EGFR and, for the first time, demonstrate an inhibitory effect of PKA on the early steps of the EGF signaling cascade both in vivo and in vitro. To date, studies of the inhibitory influence of the cAMP-PKA pathway on EGFR signaling have focused mainly on events downstream of Ras, more specifically on MAPK. Thus, it is known that activation of the cAMP-PKA pathway can have inhibitory (32-34), stimulatory (35), or no (36) effects on the activation of MAPK by EGF, depending on the cell type used. This variability is probably because of the various Raf isoforms expressed in the different cell types (37). However, these studies in which intracellular cAMP levels were raised to modulate MAPK activation in response to EGF failed to show an inhibitory effect on WT-EGFR autophosphorylation (32, 33, 38, 39). This is in contrast with our data (Fig. 5) which demonstrate that PKA can interfere with the EGFR signaling cascade at the level of the EGFR itself. One explanation for this discordance may be that the cell lines overexpressing the WT-EGFR, which were used in a number of the studies concerning MAPK activation, may not be suitable for detecting this inhibitory effect of PKA because the amount of EGFR far exceeds the amount of PKA. Indeed, in B82L cells that overexpress the WT-EGFR, we have also not observed any significant decrease in EGF-elicited tyrosine phosphorylation of cellular proteins in the presence of 8-CPT-cAMP (not shown). In any event, our findings, together with those of others (32-34) suggest that, in some cells, PKA can inhibit EGF-mediated activation of MAPK by attenuating both the EGFR kinase as well as interactions of Raf and Ras, and these two mechanisms may act in a mutually reinforcing manner.
Interestingly, the EGFR is not the only receptor tyrosine kinase target for regulation by PKA. Hence, pretreatment of cells with cAMP-elevating agents increases serine/threonine phosphorylation of the insulin receptor and decreases its insulin-dependent tyrosine kinase activity by 50% (40). This decrease in insulin receptor tyrosine kinase activity by cAMP-elevating agents has been confirmed in in vitro experiments which demonstrated that PKA phosphorylates the insulin receptor and decreases its tyrosine kinase activity (41). These findings coupled with our observations would suggest that PKA may play a more generalized role in regulation of receptor protein-tyrosine kinase signaling.
Although, at present, the identity of the serine residue(s) on
EGFR which is(are) phosphorylated remains unknown, there are some sites
that can be discarded. Thus, because PKA phosphorylates and modulates
the activity of the WT-EGFR, EGFR 1022-1186, and cytosolic domain
(EGFRKD) of the receptor similarly, it would appear that the
functionally important serine residue(s) which is(are) phosphorylated
and modulates EGFR kinase activity must reside in the cytosolic region,
i.e. between amino acids 644 and 1022. Within this region of
the EGFR, serine residues 671, 967, 971, and 1002 have been shown to be
phosphorylated (15, 42). Among these, however, only the phosphorylation
of Ser-1002 by p34cdc2 has been shown to
decrease EGFR tyrosine kinase activity (15). The functional
significance of phosphorylation of Ser-671, -967, and -971 remains to
be determined (42). Notably, however, none of these sites including
Ser-1002 conforms to the PKA consensus sequence. Nevertheless, the
identity of the precise serine residues on EGFR that are phosphorylated
by PKA and that alter EGFR kinase activity forms the subject of future investigations.
In conclusion, we have demonstrated that PKA can phosphorylate EGFR on
serine residue(s) and that this phosphorylation is accompanied by a
decrease in EGFR protein-tyrosine kinase activity both in
vitro and in vivo. In view of our previous findings
that EGFR kinase activity is required to stimulate adenylyl cyclase activity (7) and increase intracellular cAMP levels (5), the findings
presented here would suggest that activation of PKA can, in a feedback
regulatory manner, attenuate EGFR tyrosine kinase activity.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Gordon N. Gill, University of California, San Diego, for providing us with the purified cytosolic region of the EGFR (EGFRKD).
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FOOTNOTES |
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* This work was supported by Grants HL 48308 (to T. B. P.), GM 53271 (to P. J. B.) from the National Institutes of Health and a grant-in-aid from the American Heart Association, Tennessee Affiliate (to H. M. P.).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.
Recipient of a Postdoctoral Fellowship from the American Heart
Association, Tennessee Affiliate.
§ Contributed equally to this work.
To whom correspondence should be addressed: Dept. of
Pharmacology, University of Tennessee, Memphis, The Health Science
Center, 874 Union Ave., Memphis, TN 38163. Tel.: 901-448-6006; Fax:
901-448-7300; E-mail: tpatel{at}physio1.utmem.edu.
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ABBREVIATIONS |
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The abbreviations used are:
EGF, epidermal
growth factor;
EGFR, EGF receptor;
WT, wild type;
EGFR1022-1186, EGFR in which all amino acids after 1021 are deleted;
EGFRKD, cytosolic
region (amino acids 644-1186) of the EGFR in which the extracellular
and transmembrane domains are deleted;
PKA, cAMP-dependent
protein kinase;
PKAc, catalytic subunit of PKA;
MAPK, mitogen-activated
protein kinase;
PKC, protein kinase C;
8-CPT-cAMP, 8-(4-chlorophenylthio)-cAMP;
MES, 4-morpholineethanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis.
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