©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Angiotensin II Stimulates Tyrosine Phosphorylation of the Focal Adhesion-associated Protein Paxillin in Aortic Smooth Muscle Cells (*)

(Received for publication, July 15, 1994; and in revised form, November 18, 1994)

Isabelle Leduc Sylvain Meloche (§)

From the Centre de Recherche, Hôtel-Dieu de Montréal and the Department of Pharmacology, University of Montreal, Montreal, Quebec H2W 1T8, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(1)-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.


INTRODUCTION

Angiotensin II (AII) (^1)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) . (^2)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(1) and AT(2)(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(1) receptors(6, 7) . Activation of the AT(1) 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(r) 110,000-130,000 and 65,000-75,000 (12, 13, 14, 15, 16) .^2 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.


EXPERIMENTAL PROCEDURES

Materials

AII was purchased from Hukabel Scientific. The AT(1) and AT(2) selective receptor antagonists, losartan and PD123319, were generous gifts from Du Pont Merck, and Parke-Davis, respectively. Human recombinant EGF was from Life Technologies, Inc. Human alpha-thrombin and o-phosphotyrosine were obtained from Sigma. Phorbol 12-myristate 13-acetate was from LC Services Corp. BAPTA-AM was from Calbiochem. Pertussis toxin was obtained from List Biological Laboratories. The PKC inhibitor CGP 41251 and the control compound CGP 42700 were gifts from Ciba-Geigy. The anti-paxillin mAb 165(18, 19) was generously provided by Dr. Christopher Turner (State University of New York, Syracuse). Anti-phosphotyrosine mAbs 4G10 and PY-20 were purchased from Upstate Biotechnology and ICN, respectively.

Cell Culture

Vascular SMC were isolated from the aortas of 12-week-old male Brown-Norway rats as described(20) . Cells were cultured in low-glucose Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 2 mML-glutamine, and antibiotics (50 µg/ml streptomycin and 50 units/ml penicillin) and were used between passages 9 and 14. Quiescent aortic SMC were obtained by incubating 95% confluent cell cultures in serum-free Dulbecco's modified Eagle's medium-F12 (1:1) containing 15 mM Hepes (pH 7.4), 0.1% bovine serum albumin, and 5 µg/ml transferrin for 48 h. Adrenal zona glomerulosa and chromaffin cells were isolated from bovine glands and maintained in culture as previously described(21, 22) . Rat hepatocytes were obtained by a modified collagenase perfusion technique and cultured as described (23) . Rat1 fibroblast cells were grown in minimal essential medium supplemented with 10% calf serum and antibiotics. The cells were made quiescent by incubation in serum-free Dulbecco's modified Eagle's medium-F12 (1:1) for 24 h.

Immunoprecipitations

Quiescent aortic SMC in 60-mm dishes were washed once and stimulated with growth factors as indicated at 37 °C. The cells were then washed twice with ice-cold phosphate-buffered saline and lysed in 0.4 ml of Triton X-100 lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 10M phenylmethylsulfonyl fluoride, 10M leupeptin, 10M pepstatin A, 1% Triton X-100) for 25 min at 4 °C. Lysates were clarified by centrifugation at 13,000 times g for 10 min, and equal amounts of lysate proteins (150-200 µg) were subjected to immunoprecipitation. For immunoprecipitation of paxillin, the lysates were incubated for 2 h at 4 °C with 20 µl of mAb 165 hybridoma supernatant preadsorbed to rabbit anti-mouse immunoglobulin G-coated protein A-Sepharose beads. Phosphotyrosyl proteins were immunoprecipitated by incubation with 20 µl of agarose-coupled PY-20 mAb for 2 h at 4 °C. Immune complexes were washed three times with lysis buffer prior to electrophoresis on 7.5% acrylamide gels and analysis by immunoblotting.

Immunoblot Analysis

After electrophoresis, proteins were electrophoretically transferred to Hybond-C nitrocellulose membranes (Amersham Corp.) in 25 mM Tris, 192 mM glycine and fixed for 15 min in 40% methanol, 7% acetic acid, 3% glycerol. Membranes were blocked in Tris-buffered saline containing 5% non-fat dry milk for 1 h at 37 °C prior to incubation for 2 h at 25 °C with mAb 4G10 (1:5,000) or mAb 165 (1:50) in blocking solution. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Corp.).


RESULTS

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) .^2 We examined whether the cytoskeleton-associated protein paxillin was a constituent of the M(r) 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(r) 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 alpha-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(1) 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(1) 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(1) receptor is also linked to adenylyl cyclase inhibition(27, 28) . To examine the role of G(i)-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.




DISCUSSION

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.^2 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(1) 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(i)-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.


FOOTNOTES

*
This work was supported by grants from the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Scholar of the Medical Research Council of Canada. To whom correspondence should be addressed. Tel.: 514-843-2733; Fax: 514-843-2715.

(^1)
The abbreviations used are: AII, angiotensin II; SMC, smooth muscle cells; EGF, epidermal growth factor; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid tetra-(acetoxymethyl)ester; PKC, protein kinase C; mAb, monoclonal antibody.

(^2)
I. Leduc and S. Meloche, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Christopher Turner for a generous supply of mAb 165, Dr. Ronald Smith (Du Pont Merck) and Dr. Joan Keiser (Parke-Davis) for a supply of losartan and PD123319, respectively, Anna Suter (Ciba-Geigy) for the gifts of CGP 41251 and CGP 42700, Dr. Pierre Haddad for rat hepatocyte cultures, and members of Dr. André De Léan's laboratory for bovine adrenal cells. We also thank Elizabeth Pérès for preparation of the figures and Irène Rémillard for secretarial assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.