Src and Pyk2 Mediate G-protein-coupled Receptor Activation of
Epidermal Growth Factor Receptor (EGFR) but Are Not Required for
Coupling to the Mitogen-activated Protein (MAP) Kinase Signaling
Cascade*
Julian
Andreev
,
Maria L.
Galisteo
,
Onno
Kranenburg§,
Susan K.
Logan
,
Ernest S.
Chiu
,
Mitsuhiko
Okigaki
,
Leslie A.
Cary¶,
Wouter H.
Moolenaar§, and
Joseph
Schlessinger
From the
Department of Pharmacology, New York
University School of Medicine, Skirball Institute of Biomolecular
Medicine, New York, NY 10016, the § Division of Cellular
Biochemistry, The Netherlands Cancer Institute, Centre for Biomedical
Genetics, Plesmanlaan 121 1066CX Amsterdam, The Netherlands, and the
¶ Fred Hutchinson Cancer Research
Center, Seattle, Washington 98109
Received for publication, March 14, 2001
 |
ABSTRACT |
The epidermal growth factor receptor (EGFR) and
the non-receptor protein tyrosine kinases Src and Pyk2 have been
implicated in linking a variety of G-protein-coupled receptors (GPCR)
to the mitogen-activated protein (MAP) kinase signaling cascade. In
this report we apply a genetic strategy using cells isolated from
Src-, Pyk2-, or EGFR-deficient mice to explore the roles played
by these protein tyrosine kinases in GPCR-induced activation of EGFR,
Pyk2, and MAP kinase. We show that Src kinases are critical for
activation of Pyk2 in response to GPCR-stimulation and that Pyk2 and
Src are essential for GPCR-induced tyrosine phosphorylation of EGFR. By
contrast, Pyk2, Src, and EGFR are dispensable for GPCR-induced
activation of MAP kinase. Moreover, GPCR-induced MAP kinase activation
is normal in fibroblasts deficient in both Src and Pyk2
(Src
/
Pyk2
/
cells) as well as in fibroblasts deficient in all
three Src kinases expressed in these cells (Src
/
Yes
/
Fyn
/
cells). Finally, experiments are presented demonstrating that, upon
stimulation of GPCR, activated Pyk2 forms a complex with Src, which in
turn phosphorylates EGFR directly. These experiments reveal a role for
Src kinases in Pyk2 activation and a role for Pyk2 and Src in tyrosine
phosphorylation of EGFR following GPCR stimulation. In addition, EGFR,
Src family kinases, and Pyk2 are not required for linking GPCRs with
the MAP kinase signaling cascade.
 |
INTRODUCTION |
The protein tyrosine kinases Src, Pyk2, and epidermal
growth factor receptor
(EGFR)1 have been implicated
as intermediates in signaling networks that couple G-protein-coupled
receptors (GPCRs) with the Ras/MAP kinase signaling cascade (1-4).
Although the mechanisms underlying these pathways are not fully
defined, at least EGFR activation by GPCRs was proposed to be mediated
by intracellular (1, 4) and extracellular (5) processes. Many ligands
acting via GPCRs are known to elicit a mitogenic response in a variety
of cell types; MAP kinases appear to be a critical component of these growth-promoting pathways (6). It has been shown that GPCR-mediated activation of MAP kinases occurs via pertussis toxin-sensitive and -insensitive processes as well as by Ras-dependent and
Ras-independent mechanisms (2, 4, 7, 8). Because many GPCRs couple to
more than one G-protein subtype, their activation will initiate simultaneous stimulation of multiple effector systems. In fibroblasts, however, lysophosphatidic acid (LPA)-induced activation of the MAP
kinase pathway is mediated solely by Gi-regulated Ras
activation (9, 10). Several protein tyrosine kinases have been
implicated as intermediates between Gi and Ras/MAP kinase
activation, including Src, Pyk2, and EGFR (1-4, 11). It has been shown
that EGFR, Pyk2, and Src family kinases are rapidly and
transiently activated by various GPCRs. In PC12 cells, activated Pyk2
binds to Src, association-mediated through binding of the Src homology
2 domain of Src to pY402 of Pyk2 (11). Experiments employing a dominant interfering EGFR mutant or pretreatment of cells with inhibitors of the
protein tyrosine kinase of EGFR suggested that EGFR plays a role in
GPCR-induced mitogenic response (12-14). It has been proposed that Src
kinases play a crucial role in activation of EGF receptors by GPCR
stimulation (14, 15). Moreover, it has been demonstrated that
inhibition of Src kinase activity impairs LPA and
2-adrenergic receptor-mediated activation of MAP
kinase (16).
We have employed a genetic strategy to explore the role played by Src,
Pyk2, and EGFR in GPCR-induced activation of EGFR and MAP kinase. Using
cells isolated from Src- or Pyk2-deficient mice we show that Src
kinases are critical for activation of Pyk2 and that Src and Pyk2 are
essential for GPCR-induced activation of EGFR. However, these kinases
are dispensable for GPCR-induced activation of MAP kinase in mouse
embryonic fibroblasts. Moreover, GPCR-induced MAP kinase activation is
normal in fibroblasts deficient in both Src and Pyk2 as well as in
fibroblasts deficient in all three Src kinases expressed in these cells
(Src, Yes, and Fyn). Using fibroblasts isolated from EGFR-deficient
mice, we showed that EGFR activation is dispensable for GPCR-induced
activation of MAP kinase. The experiments described in this report
reveal an intracellular mechanism for activation of Pyk2 and EGFR by Src kinases following GPCR stimulation and demonstrate that EGFR, Src,
and Pyk2 kinases are not required for linking GPCRs with the MAP
kinase signaling cascade.
 |
EXPERIMENTAL PROCEDURES |
Reagents, Antibodies and Plasmids--
LPA, carbachol, and
bradykinin were purchased from Sigma. Recombinant human EGF was from
Intergen. EGFR kinase inhibitors SU1478/009 and SU1517/002 were
provided by Sugen, Inc. Monoclonal anti-Pyk2 antibodies used for
immunoblotting and anti-Ras antibodies were obtained from Transduction
Laboratories. Polyclonal anti-Pyk2 antibodies that were used for
immunoprecipitation were previously described (11). Monoclonal
anti-phosphotyrosine antibodies (4G10) were from Upstate Biotechnology.
Polyclonal anti-EGFR antibodies RK-2 and anti-C antibodies were used
for immunoprecipitation and immunoblotting of EGFR (17). Polyclonal
antibodies against phospho-MAP kinase were from New England BioLabs.
Monoclonal antibodies against Src that were used for
immunoprecipitation were from Calbiochem. The Pyk2 expression vector we
used was previously described (18). The Src expression vector was
obtained from J. Sap, New York University, NY. The EGFR expression
vector was previously described (17). Kinase negative mutant of EGFR
(EGFR-KA) was generated by substituting the codons of Lys-745
with an Ala residue using Stratagene site-directed mutagenesis kit (Stratagene).
Cell Lines and Transfections--
Embryonic fibroblasts derived
from wild type and knockout mice were generated by spontaneous
immortalization. Mouse embryonic fibroblasts and human kidney (293)
cells were grown in Dulbecco's modified Eagle's medium (Cellgro)
supplemented with 10% fetal bovine serum (Life Technologies, Inc.).
Subconfluent 293 cells were transfected using the calcium precipitation
method (19) with 1 µg of DNA/well in a 6-well plate. Pyk2
/
,
Src
/
, and Pyk2
/
Src
/
-deficient fibroblasts were isolated
from Pyk2
/
, Src
/
, and Pyk2
/
Src
/
murine E12.5 embryos
using standard procedures. EGFR
/
mouse embryonic fibroblasts were
obtained from Maria Sibilia and Erwin Wagner, Vienna, Austria.
Src
/
Yes
/
Fyn
/
mouse embryonic fibroblasts were purchased
from ATCC (CRL-2459). Src
/
Fyn
/
cells were obtained from Sheila
Thomas, Boston, MA. Src
/
Yes
/
Fyn
/
mouse embryonic
fibroblasts expressing physiological levels of wild type Src and kinase
negative mutant of Src were generated in Jonathan Cooper's laboratory
(Fred Hutchinson Cancer Research Center, Seattle, WA) by retroviral
infection and selection of low expressing cultures.
Immunoprecipitation and Immunoblotting--
Cells were washed in
ice-cold phosphate-buffered saline and lysed in 50 mM
Hepes, pH 7.2, 150 mM NaCl, 1 mM EDTA, 10%
Glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 40 mM
-glycerophosphate, 10 mM sodium
pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin (lysis buffer). Cells extracts were precleared by centrifugation and loaded on SDS-PAGE (total lysate)
or incubated with antibodies that were cross-linked to protein
A-Sepharose beads in a nutator at 4 °C for 3 h or
overnight. Immunocomplexes were washed in lysis buffer and treated as
described (17).
Ras Activation Assay--
Cells were lysed in a buffer
containing 20 mM Hepes, pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 5 mM MgCl2, and 10%
glycerol. The lysates were incubated with 20 µg of GST-Raf(RBD)
provided by J. Bos, Utrecht, The Netherlands and were washed three
times in lysis buffer. The amount of Ras pulled down was then assessed by immunoblotting with anti-Ras antibodies. In all pull-down
experiments, the lysates were precleared using GST. Analysis of Ras
binding to GST alone was performed in each assay but was not detected, indicating that Ras binds to GST-RBD in RBD- and
stimulus-dependent manners.
 |
RESULTS AND DISCUSSION |
Stimulation of mouse embryonic fibroblasts with LPA, bradykinin,
or carbachol acting on their cognate GPCRs triggers tyrosine phosphorylation of EGFR (Fig. 1,
A). The stimulation of EGFR tyrosine phosphorylation by
these GPCR agonists is similar to the level of EGFR phosphorylation
seen after stimulation with 2 ng/ml of EGF (Fig. 1, A).
Higher concentrations of EGF caused higher levels of EGFR tyrosine
phosphorylation, but the level of MAP kinase activation remained the
same; it was similar to that induced by LPA (2.5 µM)
(Fig. 1, B). It has been proposed that Src kinases play a
pivotal role in EGFR activation in response to GPCR stimulation (2, 4,
15). Because we had previously demonstrated that Pyk2 and Src are
activated by the same stimuli (11), we examined the effects of Src or
Pyk2 deficiencies on LPA-induced activation of EGFR.

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Fig. 1.
LPA, bradykinin, carbachol, and EGF stimulate
tyrosine phosphorylation of EGFR in mouse embryonic fibroblasts.
A, mouse embryonic fibroblasts were starved for
48 h and either left untreated or stimulated with LPA (2.5 µM) for 3 min, Bradykinin (1 µM) for 3 min,
Carbachol (1 mM) for 30 s, or EGF (2 ng/ml) for 3 min.
Cells were lysed, and lysates were subjected to SDS-PAGE after
immunoprecipitation (IP) with anti-EGFR antibodies, and
immunoblotted (IB) with anti-phosphotyrosine antibodies
(pY). The filter was stripped and reblotted with anti-EGFR
antibodies. B, mouse embryonic fibroblasts were starved for
48 h and either left untreated or stimulated with the indicated
concentrations of EGF (ng/ml) or LPA for 3 min, lysed, subjected to
SDS-PAGE directly (Total lysate), and analyzed with
antibodies that recognize the activated form of MAPK (pMAPK). Lysates
were also subjected to immunoprecipitation (IP) with anti-EGFR
antibodies followed by immunoblotting with anti-phosphotyrosine
antibodies (pY). Positions and apparent molecular mass (kDa)
of standard protein markers are indicated on the
right.
|
|
Src and Pyk2 Are Essential for LPA-induced Activation of
EGF--
Src
/
or Pyk2
/
fibroblasts were stimulated with LPA,
and lysates from unstimulated or stimulated cells were subjected to immunoprecipitation with anti-EGFR antibodies followed by
immunoblotting with antibodies against phosphotyrosine (anti-pTyr). The
experiment presented in Fig. 2
demonstrates that both the amplitude and kinetics of LPA-induced
activation of EGFR were reduced in Src
/
or Pyk2
/
fibroblasts.
We next examined the time course of LPA-induced activation of EGFR in
fibroblasts derived from mice deficient in both Src and Pyk2. The
experiment presented in Fig. 2, lower right panel shows
higher basal phosphorylation of EGFR in Src
/
Pyk2
/
cells, but
LPA-induced stimulation of tyrosine phosphorylation of EGFR could not
be detected in these fibroblasts. Similar results were obtained with
Src
/
Pyk2
/
fibroblasts that were stimulated with bradykinin or
carbachol (data not shown). In addition, similar results were obtained
with several different primary cultures and with immortalized
Src
/
Pyk2
/
fibroblasts (data not shown). These experiments
demonstrate that Src and Pyk2 are essential for GPCR-induced activation
of EGFR.

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Fig. 2.
Kinetics of LPA-induced tyrosine
phosphorylation of EGFR are altered in Src / , Pyk2 / , and
Pyk2 / Src / fibroblasts. Embryonic fibroblasts derived
from wild type (WT), Src / , Pyk2 / , or
Pyk2 / Src / mutant mice were starved for 48 h and either
left untreated or stimulated with LPA (2.5 µM) or EGF (2 nM) for the indicated times, lysed, and subjected to
SDS-PAGE after immunoprecipitation (IP) with anti-EGFR
antibodies followed by immunoblotting with anti-phosphotyrosine
antibodies (pY). The filters were stripped and reblotted
with anti-EGFR antibodies. All filters were developed simultaneously
for quantitative comparison of the results. Position and size (kDa) of
a standard protein marker is indicated on the right.
|
|
Activation of EGFR Is Not Essential for GPCR-induced Stimulation of
MAP Kinase--
If EGFR plays a critical role in the coupling of GPCRs
to MAP kinase activation (12-14) and Src and Pyk2 are essential for tyrosine phosphorylation of EGFR, one would expect GPCR-induced activation of MAP kinase to be altered in Src
/
or Pyk2
/
fibroblasts and particularly in Src
/
Pyk2
/
fibroblasts. However,
LPA-stimulated MAP kinase activation was readily detected in cells
deficient in either Src (20) or Pyk2 and in cells deficient in both
protein tyrosine kinases (Fig.
3A). In these experiments the
cells were stimulated with LPA for 3 min and analyzed for MAP kinase
activation using antibodies that recognize the phosphorylated
(activated) form of MAP kinase. We next compared the kinetics of
LPA-induced MAP kinase activation in Src
/
, Pyk2
/
, or
Src
/
Pyk2
/
cells with the kinetics of LPA-induced MAP kinase
activation in wild type fibroblasts. Again, no detectable differences
in the kinetics of MAP kinase stimulation were observed (Fig.
3B). Interestingly, LPA-stimulated MAP kinase activation was
also detected in Src
/
Yes
/
Fyn
/
fibroblasts, cells
deficient in all Src family members that are expressed in these cells
(21) (Fig. 3C, left panel). Moreover, LPA-induced
activation of Ras and MAP kinase in Src
/
Yes
/
Fyn
/
fibroblasts was inhibited by pertussis toxin (Fig. 3C,
right panel), indicating that MAP kinase activation in these
cells is mediated by Gi as it is in wild type cells (9,
10). These experiments demonstrate that LPA-induced MAP kinase
activation is normal in fibroblasts deficient in all Src kinases and
that Pyk2 and EGFR activation are not essential for MAP kinase
response.

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Fig. 3.
Activation of Src or Pyk2 is not essential
for LPA-induced MAP kinase activation in fibroblasts.
A, embryonic fibroblasts derived from wild type
(WT), Src / , Pyk2 / , or Pyk2 / Src / mutant mice
were starved for 48 h and either left untreated or stimulated with
LPA (2.5 µM) for 3 min, lysed, subjected to SDS-PAGE, and
immunoblotted with antibodies that recognize the activated form of MAP
kinase (pMAPK). The filter was stripped and reblotted with
anti-ERK1 antibodies as a loading control. All filters were developed
simultaneously for quantitative comparison of the results.
B, embryonic fibroblasts derived from Src / , Pyk2 / ,
or Pyk2 / Src / mutant mice were starved for 48 h and either
left untreated or stimulated with EGF (2 nM) or LPA (2.5 µM) for the indicated times, lysed, subjected to
SDS-PAGE, and immunoblotted with antibodies that recognize the
activated form of MAP kinase (pMAPK). The same filters were
reblotted with anti-ERK1 antibodies. All filters were developed
simultaneously for quantitative comparison of the results.
C, LPA-induced pertussis toxin-sensitive activation of Ras
and MAP kinase in Src / Yes / Fyn / fibroblasts. Mouse embryonic
fibroblasts derived from wild type mice (WT) or
Src / Yes / Fyn / mice were cultured in serum-free medium
overnight in the absence or presence of 100 ng/ml pertussis toxin.
Cells were then stimulated with LPA (2.5 µM) for 3 min,
and Ras activation was assessed by GST-RBD pull-down from cell lysates
as described under "Experimental Procedures." MAPK ac- tivation was analyzed by immunoblotting of total lysates using
anti-phospho-MAPK antibodies. All filters were developed simultaneously
for quantitative comparison of the results. Positions and sizes (kDa)
of standard protein markers are indicated on the
right.
|
|
To address the role of EGFR in LPA-induced MAPK activation more
directly, we compared LPA-stimulation of MAP kinase in wild type
fibroblasts to fibroblasts derived from EGFR
/
mice. In this
experiment, wild type or EGFR
/
fibroblasts were stimulated with LPA
for different times. Lysates from unstimulated or stimulated cells were
analyzed for MAP kinase activation with antibodies that recognize
activated, phosphorylated MAP kinase. These experiments showed no
difference in the kinetics or amplitude of LPA-induced MAP kinase
activation of wild type and EGFR
/
fibroblasts at the indicated time
points (Fig. 4A) and up to 90 min of LPA stimulation (data not shown). Surprisingly, pretreatment of
wild type fibroblasts with two different inhibitors that block the
tyrosine kinase activity of EGFR but not Src did not reduce EGF or
LPA-induced MAP kinase activation (Fig. 4B). It is
noteworthy that the tyrosine kinase inhibitors that were used in this
experiment also block the tyrosine kinase activity of erbB2 and erbB4
(data not shown), demonstrating that erbB2 and erbB4 do not compensate
for the loss of EGFR and that these receptors are not involved in this
process. Taken together, these experiments demonstrate that activation
of EGFR is not essential for GPCR-induced MAP kinase activation.

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Fig. 4.
EGFR activation is not essential for
LPA-induced MAP kinase activation in fibroblasts. A,
time course of LPA-induced MAP kinase activation in wild type or
EGFR / fibroblasts. Mouse embryonic fibroblasts derived from wild
type (WT) or EGFR / mutant mice were starved for 48 h and either left untreated or stimulated with LPA (2.5 µM) for the indicated times, lysed, subjected to
SDS-PAGE, and immunoblotted with antibodies that recognize the
activated form of MAP kinase (pMAPK). The filters were
stripped and reblotted with anti-ERK1 antibodies. B,
inhibitors of EGFR do not influence EGF- or LPA-induced MAP
kinase activation in wild type fibroblasts. Mouse embryonic fibroblasts
isolated from wild type mice were starved for 48 h and either left
untreated or pre-treated with EGFR tyrosine kinase inhibitors
SU1478/009 or SU1517/002 (1 µM) for 20 min, stimulated
with EGF (2 nM) or LPA (2.5 µM) for 3 min,
lysed, subjected to SDS-PAGE directly (Total lysate), and
analyzed with antibodies that recognize the activated form of MAP
kinase (pMAPK). Lysates were also subjected to
immunoprecipitation (IP) with anti-EGFR or anti-Src
antibodies and analyzed with anti-phosphotyrosine antibodies
(pY). The filters were stripped and reblotted with
anti-ERK1, anti-EGFR, or anti-Src antibodies, respectively. Positions
and sizes (kDa) of standard protein markers are indicated on the
right.
|
|
We have previously shown that stimulation of PC12 cells with LPA leads
to tyrosine phosphorylation of Pyk2, which in turn recruits Src via its
Src homology 2 domain (11). The experiment presented in Fig.
5A shows that LPA stimulation
leads to complex formation between endogenous Src and Pyk2 in
fibroblasts. Moreover, in accordance with the data reported previously
for different cell types (9, 14, 22), Src and EGFR form a complex in LPA-stimulated fibroblasts, as shown in Fig. 5A. However, we
have detected association between EGFR and Src in lysates from
unstimulated Pyk2
/
fibroblasts. It is possible that Src forms a
complex with EGFR in unstimulated Pyk2
/
fibroblasts because of the
higher basal activity of EGFR in Pyk2
/
cells. To further analyze
the nature of the association between Src-Pyk2 complex and EGFR, 293 cells were transfected with expression vectors that direct the synthesis of EGFR and EGFR-KA (a kinase negative EGFR point mutant at
Lys-745) together with expression vectors for Src or Pyk2. The
experiment presented in Fig. 5B shows that, in transfected 293 cells, the kinase negative mutant of EGFR is
tyrosine-phosphorylated by Src but not by Pyk2.

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Fig. 5.
LPA-induced Pyk2-Src complex mediates EGFR
phosphorylation. A, mouse embryonic fibroblasts
derived from wild type (WT) or Pyk2 / mutant mice were
starved for 48 h and either left untreated or stimulated with LPA
(2.5 µM) for the indicated times, lysed, subjected to
SDS-PAGE after immunoprecipitation (IP) with anti-Src
antibodies, and immunoblotted with anti-Pyk2 or anti-EGFR antibodies.
B, EGFR is phosphorylated by Src and not by Pyk2. Lysates
from 293 cells transfected with the expression vector for EGFR alone
(EGFR-WT), the kinase negative mutant of EGFR (EGFR-KA) and
Src, the kinase negative mutant of EGFR (EGFR-KA) and Pyk2, or with
kinase negative mutant of EGFR (EGFR-KA) alone were
subjected to immunoprecipitation (IP) with anti-EGFR
antibodies and immunoblotting with anti-phosphotyrosine antibodies
(pY). The filter was stripped and reblotted with anti-EGFR
antibodies. Position and size (kDa) of a standard protein marker is
indicated on the right.
|
|
Src Kinases Are Critical for GPCR-induced Stimulation of
Pyk2--
We have next tested whether LPA-induced activation of Pyk2
is dependent upon Src kinases. In this experiment wild type or Src
/
Yes
/
Fyn
/
fibroblasts were stimulated with LPA, and
lysates from stimulated or unstimulated cells were subjected to
immunoprecipitation with anti-Pyk2 antibodies followed by
immunoblotting with anti-pTyr antibodies. The experiment presented in
Fig. 6 shows that LPA failed to stimulate
Pyk2 activation in Src
/
Yes
/
Fyn
/
fibroblasts, demonstrating
that Src kinases are crucial for LPA-induced activation of Pyk2.
Similar results were obtained with Src
/
Yes
/
Fyn
/
fibroblasts
stimulated with bradykinin, carbachol, or angiotensin II (data not
shown). However, LPA-induced activation of Pyk2 was normal in
Src
/
fibroblasts, indicating that either Yes or Fyn are able to
compensate for the loss of Src in these cells. Indeed, stimulation of
Src
/
Fyn
/
fibroblasts with LPA resulted in normal activation of
Pyk2, confirming that Yes is capable in mediating LPA-induced Pyk2
activation. Moreover, LPA-induced Pyk2 activation is completely
restored in Src
/
Yes
/
Fyn
/
fibroblasts by ectopic expression
of Src but not by a kinase negative Src mutant demonstrating that
expression of a single active Src kinase is sufficient for linking LPA
stimulation with Pyk2 activation.

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Fig. 6.
LPA-induced stimulation of Pyk2 is dependent
upon Src kinases. Mouse embryonic fibroblasts derived from
wild type (WT), Src / Yes / Fyn /
(SYF / ), Src / Fyn / (SF / ), or
Src / mutant mice or SYF / cells overexpressing wild type
(SYF / SrcWT) or kinase-dead (SYF / SrcKD)
Src were starved for 48 h and either left untreated or stimulated
with LPA (2.5 µM) for 3 min, lysed, subjected to SDS-PAGE
after immunoprecipitation (IP) with anti-Pyk2 antibodies,
and immunoblotted with anti-phosphotyrosine antibodies (pY). The blot
was stripped and reblotted with anti-Pyk2 antibodies. Position and size
(kDa) of a standard protein marker is indicated on the
right.
|
|
 |
CONCLUSIONS |
Previous studies performed in PC12 cells using dominant negative
mutants of Pyk2 (11) and studies performed in Rat-1 fibroblasts using
dominant interfering mutants of EGFR or Src (12, 23) demonstrated that
EGFR, Src, and Pyk2 play a role in linking GPCR activation with the MAP
kinase signaling pathway. Experiments performed in embryonic
fibroblasts derived from EGFR
/
, Src
/
, Pyk2
/
, or
Src
/
Pyk2
/
mice demonstrate that both Src and Pyk2 are essential
for GPCR-induced tyrosine phosphorylation of EGFR. However, together
with EGFR, they are dispensable for coupling to the MAP kinase
signaling cascade. Moreover, GPCR-induced MAP kinase stimulation is
normal in fibroblasts deficient in Src, Yes, and Fyn. Taken together,
these studies show that different signaling networks may couple GPCRs
with the Ras/MAP kinase signaling pathway in different cell types. In
fibroblasts, different experimental strategies have produced
conflicting results as to the role played by EGFR, Src, and Pyk2 in
GPCR-induced MAP kinase activation. Experiments with dominant
interfering EGFR and Src mutants could be misleading because vast
overexpression of mutant proteins necessary for inhibition of MAP
kinase may interfere with the action of other proteins involved in
linking GPCR activation with the MAP kinase signaling cascade. Certain
EGFR kinase inhibitors are certainly not sufficiently specific, and
most likely block the action of protein tyrosine kinases other than
EGFR. In addition, studies using a genetic approach are not always easy
to interpret; deficiency in a protein tyrosine kinase may be
compensated for by another member of the same family of enzymes. We
have addressed this issue for Src kinases by using fibroblasts
deficient in all Src kinases expressed in these cells. Our results
clearly demonstrate that GPCR-induced activation of MAP kinase is
normal even in Src
/
Yes
/
Fyn
/
fibroblasts. However, a single
Src kinase is sufficient for mediating LPA-induced stimulation of Pyk2.
Taken together, these studies suggest that establishment of
intracellular links between signaling proteins cannot be based upon the
sole use of inhibitors or expression of dominant negative mutant
proteins. Genetic tools should be used for establishing links between
signaling pathways, for testing hypotheses, and confirming conclusions
drawn from experiments based upon indirect approaches. Finally, an
alternative and intriguing possibility is that the link between GPCR
activation and the MAP kinase signaling cascade is mediated by several
parallel signaling pathways. Inhibition of one of the pathways by a
drug or mutation may lead to a bypass or rerouting of the information
flow via alternative pathway(s) for coupling GPCR-activation with the
MAP kinase signaling cascade.
 |
ACKNOWLEDGEMENTS |
We thank M. Sibilia and E. Wagner for
providing EGFR
/
fibroblasts, and S. Thomas for Src
/
Fyn
/
fibroblasts. W. M. acknowledges the technical assistance of I. Verlaan.
 |
FOOTNOTES |
*
W. M. acknowledges the Dutch Cancer Society for its
support. J. C. and L. C. acknowledge the National Institutes of
Health for their support.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: Dept. of
Pharmacology and Skirball Institute, NYU School of Medicine, 550 First Ave., New York, NY 10016. Tel.: 212-263-7111; Fax: 212-263-7133; E-mail: schlej01@popmail.med.nyu.edu.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M102307200
 |
ABBREVIATIONS |
The abbreviations used are:
EGFR, epidermal
growth factor receptor;
GPCR, G-protein-coupled receptor;
MAP, mitogen-activated protein;
LPA, lysophosphatidic acid;
EGF, epidermal
growth factor;
PAGE, polyacrylamide gel electrophoresis;
GST, glutathione S-transferase;
RBD, Ras binding domain;
MAPK, MAP kinase.
 |
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