(Received for publication, December 5, 1995)
From the
Angiotensin II is the major effector peptide of the
renin-angiotensin system, and it exerts its physiologic functions via a
G protein-coupled cell surface receptor called AT. We found
that in rat aortic smooth muscle cells, angiotensin II stimulated the
formation of Ras-GTP, Ras-Raf-1 complex formation, and the tyrosine
phosphorylation of two important Ras GTPase-activating proteins (GAPs),
p120 Ras-GAP and p190 Rho-GAP. Electroporation of
anti-pp60
antibody into cultured, adherent
smooth muscle cells blocked the angiotensin II stimulation of Ras-GAP
and Rho-GAP tyrosine phosphorylation. In contrast electroporation of
antibodies against c-Yes or c-Fyn had no effect.
Anti-pp60
antibody also blocked angiotensin
II-stimulated Ras activation and Ras-Raf-1 complex formation. These
data strongly suggest that a G protein-coupled receptor such as the
AT
receptor can activate the Ras protein cascade via the
tyrosine kinase pp60
.
Angiotensin II is the major effector molecule of the renin-angiotensin system. This octapeptide stimulates vascular smooth muscle contraction, elevates vascular resistance, and increases intravascular volume. These effects combine to raise systemic blood pressure(1, 2) . There is also substantial experimental evidence that angiotensin II acts as a growth factor(3) . For instance, cultured vascular smooth muscle cells respond to angiotensin II by expressing early growth response genes such as c-fos, c-jun, and c-myc and by increasing thymidine incorporation(4, 5) . This correlates with in vivo data that infusion of angiotensin II into animals injured with a vascular balloon catheter markedly exacerbates the resulting myoproliferative lesion(6) . A role for angiotensin II in cell growth and tissue remodeling has also been shown in animal models of hypertension, heart failure, and atherosclerosis(7, 8, 9) .
Angiotensin II
exerts its physiologic functions via a high affinity cell surface
receptor now called the AT receptor. This receptor was
first cloned in 1991, and it contains the structural features of a
seven transmembrane, heterotrimeric G protein-associated
receptor(10) . In vascular smooth muscle cells, ligand
activation of the AT
receptor leads to the rapid activation
of phospholipase C and the production of 1,4,5-inositol trisphosphate (11) .
Recently it has become clear that many of the
intracellular signals mediated by the AT receptor are
similar to the signaling pathways activated by receptor tyrosine
kinases. For instance, ligand activation of the AT
receptor
leads to the rapid tyrosine phosphorylation and activation of
phospholipase C-
1 in vascular smooth muscle cells(12) .
This is a critical event for downstream signaling, because inhibition
of tyrosine phosphorylation markedly reduces angiotensin II stimulation
of 1,4,5-inositol trisphosphate production. Separate studies have also
shown that angiotensin II, acting through the AT
receptor,
stimulates JAK2 tyrosine phosphorylation and
activation(13, 14) . Finally, several studies have
indicated that angiotensin II leads to the phosphorylation and
activation of mitogen-activated protein
kinases(15, 16) . The known link between
p21
activity and mitogen-activated protein
kinase stimulation, as well as the central role of p21
in cell growth, prompted us to ask if this molecule is
activated by angiotensin II.
p21 and other
small G proteins are membrane bound guanine nucleotide-binding proteins
that are active when complexed with GTP and inactive when bound to
GDP(17, 18) . The ratio of bound GTP to GDP is
regulated by the rate of nucleotide exchange and by the rate of GTP
hydrolysis. Proteins such as mammalian son-of-sevenless (mSOS)
stimulate the exchange of GTP for bound GDP on
p21
. The deactivation of p21
is regulated by GTPase-activating proteins, called GAPs, (
)which markedly accelerate intrinsic Ras GTPase activity
and lead to Ras-GDP. The best defined GAP is Ras-GAP, a 120-kDa protein
that binds p21
via a Src homology II
domain(19, 20) . A second protein implicated in Ras
deactivation is Rho-GAP, a 190-kDa protein(21) . Although the
exact role of Rho-GAP is not known, it is thought to have a regulatory
role in stress fiber induction and focal adhesion(22) . In
cells stimulated with growth factors the GAPs are phosphorylated on
tyrosine and form a complex that enhances the GTPase activity of
p21
(23) .
The biochemical pathway by
which a G protein-coupled receptor such as the AT receptor
regulates p21
activity is not known. These cell
surface receptors lack intrinsic kinase activity and must activate Ras
in a fashion different from that of tyrosine kinase receptors. In this
study we show that angiotensin II stimulates
p21
, leading to p21
-GTP
and Ras-Raf-1 complex formation. Angiotensin II also stimulates the
tyrosine phosphorylation of Ras-GAP and Rho-GAP. The tyrosine kinase
pp60
appears to play a critical early role in
angiotensin II signaling; neutralization of pp60
activity by the electroporation of anti-pp60
antibodies into smooth muscle cells blocked both Ras-GTP
accumulation and Ras-Raf-1 complex formation in response to angiotensin
II. This is the first observation that a G protein-coupled receptor
such as the AT
receptor can activate the Ras protein
cascade via pp60
.
Figure 1:
Ras activation. RASM cells
were labeled with [P]orthophosphate, and
p21
was collected by immunoprecipitation.
Associated GTP and GDP were analyzed by thin-layer chromatography. A, the binding of
P-labeled guanine nucleotides
to p21
under unstimulated conditions (C
) and after 1, 5, and 10 min of exposure to
10
M angiotensin II (A
, A
, and A
) or 100 ng/ml EGF (E
, E
, and E
). B, data
from five separate experiments were quantitated by densitometry. The
results are plotted as the ratio of GTP to the total of GTP plus GDP.
After 1 min, angiotensin II (
) induced a 3-fold increase of
p21
-GTP, whereas EGF (
) induced an
4.5-fold increase.
Activated p21 is known to recruit Raf-1 into a protein
complex(28) . If angiotensin II stimulated Ras-GTP formation,
we then hypothesized that angiotensin II would also induce Ras-Raf-1
complex formation. To measure this, RASM cells were exposed to
angiotensin II, lysed, and immunoprecipitated with anti-p21
antiserum. The immunoprecipitated proteins were probed by Western
blot analysis using anti-Raf-1 antibody. This showed that after
angiotensin II addition, there is a marked increase in Ras-Raf-1
complex formation (Fig. 2). As with Ras-GTP levels, the greatest
Ras-Raf-1 association was present at 1 min, after which levels
declined. We have also measured Ras-Raf-1 complex formation by
stimulating cells with angiotensin II, immunoprecipitating with
anti-Raf-1 antibody, and probing by Western blot analysis using
anti-Ras antibody. This protocol gave identical results to those shown
in Fig. 2.
Figure 2:
Ras-Raf-1 complex formation. RASM cells
were stimulated with angiotensin II for the indicated times. Cell
lysates were immunoprecipitated with monoclonal
anti-p21antibody, separated by
SDS-polyacrylamide gel electrophoresis, and probed by Western blot
analysis with monoclonal anti-Raf-1 antibody.
p21
-Raf-1 complex formation increased
approximately 16-fold 1 min after angiotensin II addition. Molecular
mass markers are indicated in kDa to the right of the
figure.
GAP proteins play an intimate role in the Ras activation-deactivation cycle. Analysis of growth factor signaling has indicated that these proteins serve to limit the time Ras remains in the active Ras-GTP form. To investigate if angiotensin II stimulates the tyrosine phosphorylation of Rho-GAP and Ras-GAP, RASM cells were exposed to angiotensin II, lysed, and immunoprecipitated with an anti-phosphotyrosine antibody. A Western blot of the precipitated proteins was then probed with monoclonal antibodies against either Rho-GAP or Ras-GAP. In unstimulated cells, very little Ras-GAP was phosphorylated on tyrosine (Fig. 3A). However, 1 min after the addition of angiotensin II, Ras-GAP tyrosine phosphorylation increased to about 16-fold greater than at time 0. Even at 10 min, levels of Ras-GAP tyrosine phosphorylation remained elevated. By comparison, Rho-GAP showed less change in tyrosine phosphorylation levels after angiotensin II exposure (Fig. 3B). Densitometry of five experiments showed that on average the tyrosine phosphorylation of Rho-GAP increased 4-fold 1 min after angiotensin II addition. The tyrosine phosphorylation of Rho-GAP was also studied by immunoprecipitation with monoclonal anti-Rho-GAP followed by Western blot analysis using anti-phosphotyrosine. This experiment gave data virtually identical to that of Fig. 3B.
Figure 3: Tyrosine phosphorylation of Ras-GAP and Rho-GAP. RASM cells were stimulated with angiotensin II for 0 or 1 min. Cell lysates were then immunoprecipitated with anti-phosphotyrosine antibody, separated by SDS-polyacrylamide gel electrophoresis, and probed by Western blot analysis with anti-Ras-GAP (A) and anti-Rho-GAP antibody (B). Ras-GAP and Rho-GAP protein bands (insets) were quantitated by densitometry; the results are expressed as an increase in arbitrary units (mean ± S.D., n = 5 for each figure). Angiotensin II induces the rapid tyrosine phosphorylation of Ras-GAP and Rho-GAP.
Figure 4:
Inhibition of GAP phosphorylation with
anti-pp60 antibody. A, RASM cells
were electroporated with either Hanks' balanced salt solution (E), anti-pp60
antiserum (ES), rabbit IgG (EI), or BSA (EB).
Angiotensin II was added for 0 or 1 min. Cell lysates were analyzed for
the tyrosine phosphorylation of Ras-GAP or Rho-GAP as described in the
legend to Fig. 3. Anti-pp60
blocks the
angiotensin II-stimulated tyrosine phosphorylation of Ras-GAP and
Rho-GAP. B, RASM cells were electroporated with either
Hanks' balanced salt solution (E),
anti-pp60
antibody preabsorbed with an excess
of Src peptide (EA), or anti-pp60
antiserum that was sham absorbed (ES). Cells were
then treated with angiotensin II for 0, 1, or 5 min. Ras-GAP and
Rho-GAP tyrosine phosphorylation was measured as described in the
legend to Fig. 3. Antibody preabsorbed with Src peptide no
longer inhibited GAP tyrosine phosphorylation. C, RASM cells
were electroporated with either anti-pp60
antibody (ES), anti-c-Yes antibody (EY),
or anti-c-Fyn antibody (EF). Cells were treated with
angiotensin II for 0 or 1 min, and GAP tyrosine phosphorylation was
measured. Anti-c-Yes and anti-c-Fyn antibodies do not inhibit
angiotensin II stimulation of GAP tyrosine
phosphorylation.
We also studied the role of pp60 in Ras-GTP formation. When cells were electroporated in the
presence of vehicle, they responded to angiotensin II or to EGF with
the rapid conversion of Ras-GDP to Ras-GTP, very similar to
nonelectroporated cells (Fig. 5A, E). When
cells were electroporated with either rabbit IgG or BSA, these cells
responded to angiotensin II or EGF in a fashion indistinguishable from
cells electroporated with vehicle alone (Fig. 5A, EI and EB). In contrast, when cells were
electroporated with rabbit anti-pp60
antiserum, Ras activation in response to angiotensin II was
abolished (Fig. 5A, ES). This was not a toxic
effect because these cells remained fully capable of activating Ras in
response to EGF.
Figure 5:
Inhibition of Ras activation with
anti-pp60 antibody. A, RASM cells
were electroporated with either Hanks' balanced salt solution (E), rabbit IgG (EI), BSA (EB), or
anti-pp60
antiserum (ES). Cells were
either harvested unstimulated (C
) or treated for 1
min with either EGF (E
) or angiotensin II (A
). Ras-associated nucleotides were isolated and
analyzed as described in Fig. 1. The bar graph plots the ratio
of GTP compared with the total of GTP plus GDP. The data are given as
mean ± S.D. (n = 3) for each time point.
Anti-pp60
antibody inhibits the angiotensin
II-stimulated increase of p21
-GTP but has no
effect on the EGF stimulation of p21
-GTP. B, RASM cells were electroporated as described for A.
Angiotensin II was added for 0 or 1 min, and Ras-Raf-1 complex
formation was measured as described in Fig. 2.
Anti-pp60
antibody inhibits the angiotensin
II-stimulated formation of this complex.
We also used the electroporation technique to
investigate the role of pp60 on angiotensin
II-mediated formation of a Ras-Raf-1 complex. Consistent with the above
data, anti-pp60
antibody completely blocks
Ras-Raf-1 complex formation (Fig. 5B, ES). No
effect on angiotensin II-mediated complex formation was observed with
either rabbit IgG or BSA (Fig. 5B, EI and EB). These data strongly suggest that pp60
or a highly related enzyme plays a critical role in
angiotensin II-mediated activation of the Ras signaling pathway.
The AT receptor is a seven transmembrane receptor
responsible for virtually all of the physiologic actions of angiotensin
II(1, 2) . Whereas the process of angiotensin
II-mediated smooth muscle contraction has been intensely studied, the
intracellular signals associated with angiotensin II-mediated cell
growth are less understood. Previous investigators have established
that angiotensin II activates the mitogen-activated protein kinase
cascade(15, 16) . Here we show that angiotensin II
stimulates the activation of p21
(Ras). This was first
established by directly verifying the conversion of Ras-GDP to Ras-GTP
and by demonstrating Ras-Raf-1 association. Thus, angiotensin II
appears to use the Ras pathway to activate mitogen-activated protein
kinase in a fashion analogous to growth factors such as EGF. That said,
there must be differences in the signaling initiated by angiotensin II
and by classic growth factors because angiotensin II is a less potent
growth factor than EGF or similar molecules. In analyzing the Ras
activation in response to angiotensin II, we noted two differences from
the Ras activation in response to EGF. The magnitude of angiotensin
II-induced Ras-GTP formation was less, and Ras was more rapidly
inactivated. A major intracellular mechanism used to regulate Ras
activity is the action of Ras-GTPase proteins or GAPs. In response to
angiotensin II, there is a rapid and marked tyrosine phosphorylation of
Ras-GAP. In association with Rho-GAP, this molecule converts the active
Ras molecule back to the Ras-GDP form(17) . Thus, we
hypothesize that the rapid activation of the Ras-GAP system by tyrosine
phosphorylation acts to modulate the stimulatory potential of
angiotensin II.
Another obvious difference between angiotensin II
signaling and that of classic growth factors is that the AT receptor lacks intrinsic kinase domains(10) . Thus, the
AT
receptor must recruit an intracellular kinase to
initiate any type of kinase cascade. Recent experimental evidence
indicates that pp60
is activated by
angiotensin II(29) . In vascular smooth muscle cells it appears
to play a major role in angiotensin II-mediated phosphorylation of
phospholipase C-
1 and 1,4,5-inositol trisphosphate
generation(25) . In response to growth factors, Chang and
colleagues (30) showed that pp60
exerts a signaling effect upstream of Ras. To investigate
the role of pp60
in stimulation of Ras, we
established an electroporation protocol to insert anti-pp60c-src
antibodies into cells to examine if this blocks angiotensin II-mediated
Ras activation. This technique uses an electrode that allows
electroporation of cultured cells while still attached to a tissue
culture dish; it is efficient and gentle in that cells need not be
trypsinized either before or after electroporation (25) .
Anti-pp60 antibody blocked the angiotensin
II-mediated conversion of Ras-GDP to the active Ras-GTP form. This
appeared to be a specific effect for angiotensin II because EGF
activated Ras in the presence of the antibody. These data clearly
support a role for pp60
upstream of
angiotensin II-mediated Ras activation. Whether pp60
directly activates a nucleotide exchange protein complex or
whether it activates Ras in some indirect fashion remains unknown.
Potential indirect mechanisms include the release of intracellular
Ca
and the tyrosine phosphorylation of linkers such
as SHC and IRS-1(31, 32) . Experiments with the
anti-pp60
antibody also show that Src plays a
role in Ras-GAP and Rho-GAP tyrosine phosphorylation. As with the
activation of Ras, the precise mechanism by which Src leads to the
tyrosine phosphorylation of GAPs is unknown. Both Ras-GAP and Rho-GAP
have been shown to be substrates of Src family
kinases(19, 33) .
Thus, in conclusion, angiotensin
II activates the Ras pathway in vascular smooth muscle cells. This is
consistent with its known role as a growth factor for these cells. In
response to angiotensin II, there is the rapid tyrosine phosphorylation
of GAP proteins which serve to regulate the active Ras complex.
Critical to angiotensin II-mediated activation of the Ras pathway is
the intracellular kinase pp60. This enzyme
plays a role upstream of p21
activation. It also leads to
the tyrosine phosphorylation of GAPs perhaps through direct
pp60
kinase activity.