(Received for publication, July 15, 1994; and in revised form, November 18, 1994)
From the
Treatment of vascular smooth muscle cells (SMC) with angiotensin
II (AII) leads to an increase in the tyrosine phosphorylation of
multiple cellular substrates. Here, we have demonstrated that AII
stimulates tyrosine phosphorylation of the focal adhesion-associated
protein paxillin in rat aortic SMC. AII-induced phosphorylation of
paxillin was detectable within 1 min and was sustained up to 60 min.
Preincubation with the AT-selective antagonist losartan
abolished this response. The stimulatory effect of AII on paxillin
tyrosine phosphorylation was observed only in aortic SMC and not in
other target cells such as adrenal zona glomerulosa cells, chromaffin
cells, or hepatocytes. The effect of AII was dependent on the
activation of phospholipase C. Chelation of intracellular calcium
completely inhibited the ability of AII to stimulate paxillin tyrosine
phosphorylation, while selective inhibition of protein kinase C
partially attenuated the response. In contrast, treatment of the cells
with pertussis toxin had no effect on AII-induced paxillin tyrosine
phosphorylation. These findings identify paxillin as a new substrate
for AII-stimulated tyrosine phosphorylation and suggest a role for
cytoskeleton-associated proteins in the growth response of aortic SMC.
Angiotensin II (AII) ()has been reported to stimulate
the growth of a number of cell types, including 3T3 fibroblasts,
adrenocortical cells, vascular SMC, cardiac myocytes, and cardiac
fibroblasts(1, 2) . In cultured rat aortic SMC, AII
induces cellular hypertrophy, as a result of increased protein
synthesis, but not cell
proliferation(3, 4, 5) . (
)In view
of the potential involvement of AII in the development of
cardiovascular diseases such as hypertension and atherosclerosis, it is
therefore important to define the signaling pathways that mediate the
growth response to the hormone.
AII exerts its physiologic effects
by interacting with two pharmacologically distinct subtypes of
receptors, designated AT and
AT
(6, 7) . Both subtypes of receptor
belong to the superfamily of seven transmembrane domain receptors (8, 9, 10, 11) . Studies using
selective AII receptor antagonists have revealed that most in vitro and in vivo responses to AII are mediated by AT
receptors(6, 7) . Activation of the AT
receptor triggers various G protein-mediated signaling pathways,
including stimulation of phospholipases C and D and inhibition of
adenylyl cyclase(7) . However, the molecular basis for the
growth-promoting effects of AII remains largely unknown.
Recently,
growth factors such as bombesin, vasopressin, endothelin, or AII, which
interact with G protein-coupled receptors, were shown to stimulate the
tyrosine phosphorylation of multiple substrates, including two major
groups of bands migrating with an apparent M 110,000-130,000 and 65,000-75,000 (12, 13, 14, 15, 16) .
One of these prominent tyrosine-phosphorylated proteins was
identified as p125
, a cytosolic tyrosine kinase that
localizes to focal adhesions of cultured cells(17) . In the
present study, we demonstrate that AII stimulates the tyrosine
phosphorylation of another focal adhesion-associated protein, paxillin,
in rat aortic SMC.
Addition of AII to vascular SMC leads to increased tyrosine
phosphorylation of several proteins, including a broad band of apparent
molecular mass 65-75 kDa(16) . We examined
whether the cytoskeleton-associated protein paxillin was a constituent
of the M
65,000-75,000
tyrosine-phosphorylated proteins. Quiescent rat aortic SMC were
stimulated with AII, thrombin, or EGF for 15 min, and lysates of the
cells were incubated with anti-paxillin mAb 165 (18, 19) prior to anti-phosphotyrosine immunoblot
analysis. Fig. 1shows that paxillin contains a relatively low
level of phosphotyrosine in unstimulated aortic SMC and migrates as a
discrete band of M
68,000. All three growth
factors induced a strong increase in the tyrosine phosphorylation of
paxillin, which was accompanied by a characteristic mobility shift of
the protein (Fig. 1A). The effect of AII and thrombin
was consistently stronger than the one produced by EGF. To further
substantiate that AII stimulates tyrosine phosphorylation of paxillin,
lysates of AII-treated aortic SMC were first subjected to
immunoprecipitation with anti-phosphotyrosine mAb PY-20 followed by
immunoblotting with anti-paxillin mAb. As shown in Fig. 1B, AII caused a prominent increase in paxillin
immunoreactivity, which was completely abolished by addition of 1
mM phosphotyrosine to the lysate prior to immunoprecipitation.
Figure 1:
AII stimulates
paxillin tyrosine phosphorylation in aortic SMC. A, quiescent
rat aortic SMC were incubated for 15 min at 37 °C with either
medium alone (control), 100 nM AII, 1 unit/ml -thrombin,
or 100 ng/ml EGF. Lysates of the cells were then subjected to
immunoprecipitation with anti-paxillin mAb 165 and analyzed by
anti-phosphotyrosine immunoblotting as described under
``Experimental Procedures.'' The positions of paxillin and
IgG heavy chain are indicated by arrows. B, quiescent
aortic SMC were incubated for 15 min at 37 °C with medium alone or
100 nM AII. Cell lysates were then incubated with
agarose-linked anti-phosphotyrosine mAb PY-20 in the absence or
presence of 1 mMo-phosphotyrosine.
Immunoprecipitates were analyzed by immunoblotting with mAb 165. The
position of paxillin is indicated by an arrow.
The stimulatory effect of AII on paxillin tyrosine phosphorylation
was dose dependent with a maximal effect observed at 10 nM (data not shown). Kinetic analysis of paxillin phosphorylation
revealed that AII action was rapid and sustained (Fig. 2). An
increase in phosphorylation was detected 1 min after addition of AII,
reached a maximum at 15 min, and was sustained for up to 60 min. The
increased tyrosine phosphorylation was accompanied by a mobility shift
of the protein to more slowly migrating forms, which was already
apparent after 5 min of stimulation. Incubation with the selective
receptor antagonist losartan but not PD123319 completely prevented this
response, indicating that AII action is mediated through the AT
receptor (data not shown).
Figure 2: Kinetics of AII-induced paxillin tyrosine phosphorylation. Quiescent rat aortic SMC were stimulated with 100 nM AII for the indicated periods of time. Cell lysates were then incubated with mAb 165, and the immunoprecipitates were subsequently analyzed by anti-phosphotyrosine immunoblotting.
A number of observations suggest that tyrosine phosphorylation of focal adhesion-associated proteins may contribute to the signaling pathways that regulate cell growth(24) . To investigate the biological significance of AII-stimulated paxillin phosphorylation and its relationship to cell growth, we examined the state of paxillin tyrosine phosphorylation in different AII target cells. The Rat1 fibroblast cell line, which does not express a significant amount of AII receptors, was used as a negative control. As shown in Fig. 3A, AII only increased tyrosine phosphorylation of paxillin in aortic SMC where it exerts an hypertrophic effect. Immunoblotting experiments showed that paxillin was expressed in all of these cells, albeit at different levels (Fig. 3B).
Figure 3: Effect of AII on paxillin tyrosine phosphorylation in different target cells. The indicated quiescent cells were treated for 15 min with medium alone or 100 nM AII. Lysates of the cells were then subjected to immunoprecipitation with anti-paxillin mAb 165 and analyzed by anti-phosphotyrosine immunoblotting A or by immunoblotting with mAb 165 B. The position of paxillin is indicated by an arrow.
Binding of AII to the AT receptor is known to stimulate the activity of phospholipase C in
various cell types, including vascular SMC(7) . We therefore
investigated the possible involvement of Ca
and PKC
in AII-induced tyrosine phosphorylation of paxillin. To examine the
role of Ca
, quiescent aortic SMC were pretreated with
the membrane-permeable Ca
chelator BAPTA-AM (25) prior to AII stimulation. The concentrations of BAPTA-AM
used are sufficient to suppress AII-mediated Ca
mobilization in these cells (data not shown). Fig. 4A shows that calcium chelation completely prevented the increased
tyrosine phosphorylation of paxillin and the gel mobility shift of the
protein in response to AII. To test the role of PKC activation, we
pretreated the cells with the selective PKC inhibitor CGP
41251(26) . As shown in Fig. 4B, CGP 41251
partially reduced AII stimulation of paxillin tyrosine phosphorylation,
whereas the biologically inactive analog CGP 42700 had no effect. As a
control, the inhibitor CGP 41251 completely blocked phorbol
12-myristate 13-acetate-induced phosphorylation of paxillin (data not
shown). These data indicate that both Ca
mobilization
and PKC activation might be critical for AII-stimulated paxillin
tyrosine phosphorylation.
Figure 4:
Effect of intracellular Ca chelation and PKC inhibition on AII stimulation of paxillin
tyrosine phosphorylation. A, quiescent rat aortic SMC were
pretreated for 30 min at 37 °C in the absence or presence of the
indicated concentrations (µM) of BAPTA-AM. The cells were
then stimulated with medium (control) or 100 nM AII for 15
min. Tyrosine phosphorylation of paxillin was analyzed by
immunoprecipitation with mAb 165 followed by anti-phosphotyrosine
immunoblotting. B, quiescent rat aortic SMC were pretreated
for 30 min at 37 °C in the absence or presence of the selective PKC
inhibitor CGP 41251 (10 µM) or its inactive analog CGP
42700 (10 µM). The cells were then stimulated with 100
nM AII for 15 min. Tyrosine phosphorylation of paxillin was
analyzed as above.
Activation of the AT receptor
is also linked to adenylyl cyclase
inhibition(27, 28) . To examine the role of
G
-mediated pathways in the regulation of paxillin
phosphorylation by AII, quiescent aortic SMC were pretreated with
pertussis toxin prior to stimulation with the hormone. Treatment of the
cells with 100 ng/ml pertussis toxin for 16 h did not affect
AII-induced tyrosine phosphorylation of paxillin (Fig. 5).
Figure 5: Effect of pertussis toxin (PTX) on AII stimulation of paxillin tyrosine phosphorylation. Quiescent rat aortic SMC were pretreated for 16 h at 37 °C with or without 100 ng/ml pertussis toxin. The cells were then stimulated with medium alone (control) or 100 nM AII for 15 min. Tyrosine phosphorylation of paxillin was analyzed by immunoprecipitation with mAb 165 followed by anti-phosphotyrosine immunoblotting.
Protein tyrosine phosphorylation has recently been identified
as an additional signal transduction component of the growth response
to G protein-coupled receptor
agonists(12, 13, 14, 15, 16) .
Although the exact role of this signaling pathway remains to be
established, the observation that tyrosine kinase inhibitors block the
mitogenic response to thrombin (29) , endothelin(30) ,
and bombesin (31) suggest that tyrosine phosphorylation plays
an important role in the growth-promoting effects of these agents.
Similarly, we have recently demonstrated that tyrosine kinase
inhibitors completely abolish the hypertrophic response to AII in rat
aortic SMC. Further determination of the identity and
function of the tyrosine-phosphorylated substrates therefore represents
an important step to address the biological importance of this pathway.
Results from this study identify the cytoskeleton-associated protein paxillin as a target of AII-stimulated protein tyrosine phosphorylation in vascular SMC. Paxillin is a 68-kDa protein that binds to the cytoskeleton protein vinculin and localizes to the focal adhesions of cultured cells(19) . Although the precise physiological function of paxillin is unknown, certain observations suggest that phosphorylation of paxillin may play an important role in the regulation of cell proliferation and differentiation. Paxillin was shown to be heavily phosphorylated on tyrosine residues in Rous sarcoma virus-transformed cells, where normal focal adhesion organization is disrupted(18) . The tyrosine phosphorylation of paxillin is also regulated during chick embryonic development(32) . More recently, paxillin was also identified as a tyrosine-phosphorylated substrate of neuropeptide growth factors (33) . Our observation that AII enhances paxillin tyrosine phosphorylation in aortic SMC but not in target cells that lack a growth response to the hormone is consistent with a role for paxillin in vascular SMC growth. An intriguing possibility is that AII-induced tyrosine phosphorylation of cytoskeleton proteins like paxillin may be critically involved in the migration and increased proliferation of SMC observed following vascular injury.
The mechanism by which AII stimulates the tyrosine
phosphorylation of paxillin was investigated. We first demonstrated
that the action of AII is mediated by AT receptors. This
subtype of receptors is known to be coupled to phospholipase C
activation, leading us to examine the contribution of Ca
and PKC to this response. Results of these experiments indicated
that AII stimulation of paxillin tyrosine phosphorylation was
completely blocked by loading cells with the Ca
chelator BAPTA-AM and was partially attenuated by inhibiting
cellular PKC activity with CGP 41251. These findings are in line with
previous studies by Huckle et al.(12) , showing that
AIIstimulated tyrosine phosphorylation of 66-75-kDa proteins in
WB cells is secondary to, and dependent on, Ca
mobilization. We also demonstrated that AII action is independent
of G
-mediated signals. Thus, AII stimulates paxillin
tyrosine phosphorylation through a pertussis toxin-insensitive
phospholipase C-dependent pathway in aortic SMC. Further work is
required to identify the tyrosine kinase(s) involved in AII action and
to delineate the components of this pathway.