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Circulation Research. 2005;97:829-836
Published online before print September 8, 2005, doi: 10.1161/01.RES.0000185322.46009.F5
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(Circulation Research. 2005;97:829.)
© 2005 American Heart Association, Inc.

Molecular Medicine

cAbl Tyrosine Kinase Mediates Reactive Oxygen Species– and Caveolin-Dependent AT1 Receptor Signaling in Vascular Smooth Muscle

Role in Vascular Hypertrophy

Masuko Ushio-Fukai*, Lian Zuo*, Satoshi Ikeda, Taiki Tojo, Nikolay A. Patrushev, R. Wayne Alexander

From the Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga.

Correspondence to Masuko Ushio-Fukai, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, Rm 319, Atlanta, GA 30322. E-mail mfukai{at}emory.edu


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Important output signals of the angiotensin subtype 1 receptor (AT1R) in vascular smooth muscle cells (VSMCs) are mediated by angiotensin II (Ang II)-stimulated transactivation of the epidermal growth factor receptor (EGF-R), which is critical for vascular hypertrophy. Ang II-induced EGF-R transactivation is mediated through cSrc, a proximal target of reactive oxygen species (ROS) derived from NAD(P)H oxidase (NOX) and is dependent on AT1R trafficking through caveolin1 (Cav1)-enriched lipid rafts. Underlying molecular mechanisms are incompletely understood. The nonreceptor tyrosine kinase, proto-oncogene cAbl is a substrate of Src and is a major mediator for ROS-dependent tyrosine phosphorylation of Cav1. We thus hypothesized that cAbl is important for ROS-, cSrc-, and Cav1-dependent growth-related AT1R signal transduction. Here we show that Ang II induces tyrosine phosphorylation of cAbl in rat VSMCs and mouse aorta, and that Ang II promotes association of cAbl with AT1R, both of which are Src-dependent. Pretreatment of rat VSMCs with the NOX inhibitor diphenylene iodonium or the antioxidants N-acetylcysteine or ebselen significantly inhibited Ang II-induced cAbl phosphorylation. Cell fractionation shows that both EGF-Rs and cAbl are found basally in Cav1-enriched membrane fractions. Knockdown of cAbl protein using small interference RNA inhibits Ang II-stimulated: (1) trafficking of AT1R into, and EGF-R out of, Cav1-enriched lipid rafts; (2) EGF-R transactivation; (3) appearance of the transactivated EGF-R and phospho-Cav1 at focal adhesions; and (4) vascular hypertrophy. These studies provide a novel role of cAbl in the spatial and temporal organization of growth-related AT1R signaling in VSMCs and suggest that cAbl may be generally important in signaling of G-protein coupled receptors.


Key Words: cAbl • caveolin-1 • NAD(P)H oxidase • angiotensin II • vascular smooth muscle


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Angiotensin II (Ang II) is important in mediating vascular remodeling and hypertrophy in hypertension. These effects are mediated, in large part, through the G-protein coupled angiotensin subtype 1 receptors (AT1Rs). The growth-related outputs of the AT1R are dependent on the transactivation (tyrosine phosphorylation) of the epidermal growth factor receptor (EGF-R) and its activation of mitogen-activated protein kinases and Akt, which are involved in vascular smooth muscle cell (VSMC) hypertrophy.1,2 Many effects of Ang II are dependent on the AT1R stimulation of reactive oxygen species (ROS) production by NAD(P)H oxidase.2–5 EGF-R transactivation in VSMCs is mediated through the activation of cSrc, a proximal ROS-dependent event.5

Caveolae/lipid rafts are specialized membrane microdomains where multimolecular signaling molecule complexes are compartmentalized, in part, via interacting with caveolin1 (Cav1). We showed that in VSMCs, Ang II stimulation promotes AT1R association with Cav1 and its trafficking into Cav1-enriched lipid rafts, events which in turn are associated contemporaneously with egress of EGF-Rs from their basal location in these fractions.6–8 EGF-R migration from the Cav1-enriched lipid rafts is required for the appearance of transactivated EGF-R at focal adhesions, where they colocalize with vinculin and tyrosine phosphorylated Cav1.7 These results suggest an important role for ROS, cSrc, and phospho-Cav1 in Ang II-induced EGF-R transactivation in AT1R-mediated growth-related signaling events. Underlying organizing molecular mechanisms are incompletely understood.

The proto-oncogene, nonreceptor tyrosine kinase cAbl is a substrate of Src family kinases.9,10 cAbl is a homologue of the Ablelson murine leukemia virus and fusion mutations are involved in tumorigenesis and leukemia.11 It contains a myristoylation site, SH2 and SH3 domains potentially mediating protein-protein interactions, and a catalytic domain.12 It also has a large, unique C-terminus with several polyproline-rich regions, actin binding domains, and nuclear targeting and export signals. cAbl–/– mice have high intrauterine mortality and are neonatally fragile.13 Nuclear cAbl is involved in apoptosis induced by DNA damage.12 Cytoplasmic cAbl is activated through Src by platelet-derived growth factor or by extracellular matrix proteins and is involved in cytoskeletal remodeling.14 Moreover, cAbl induces c-myc expression and cell growth.10 Thus, cAbl is an important effector of Src-related growth factor-induced mitogenesis and actin cytoskeleton remodeling.15 It is also activated by ROS16–18 and is one of the important mediators of ROS-dependent tyrosine phosphorylation of Cav1.18 cAbl has not been implicated previously in AT1R signaling or for that matter in G-protein coupled signaling generally.

Here, we test the hypothesis that cAbl is important for ROS-, cSrc-, and Cav1-dependent growth-related AT1R signal transduction. We show that Ang II induces tyrosine phosphorylation of cAbl in rat VSMCs and mouse aorta, and that Ang II promotes cAbl association with AT1R, both of which are Src-dependent. Ang II-induced cAbl activation is ROS-dependent. We noted that cAbl has a Cav1 binding consensus sequence and showed that it is localized basally in Cav1-enriched membrane fractions. We provide evidence that cAbl is important for Ang II-induced: (1) trafficking of AT1R into, and EGF-R out of, Cav1-enriched lipid rafts; (2) EGF-R phosphorylation; (3) appearance of the transactivated EGF-R and phospho-Cav1 at focal adhesions; and (4) 3H-leucine incorporation, a surrogate for VSMC hypertrophy. These studies provide evidence for a novel role of cAbl in the spatial and temporal organization of AT1R signaling in VSMCs.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials, cell culture, infection of adenovirus, synthetic small interference RNA (siRNA) and its transfection, Ang II stimulation of the aorta of mice ex vivo and in vivo, immunofluorescence, purification of caveolae fractions, immunoprecipitation and immunoblotting, [3H] leucine incorporation, and statistical analyses are in the Materials and Methods section in the online data supplement available at http://circres.ahajournals.org.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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Ang II Induces Tyrosine Phosphorylation of cAbl in Rat VSMC and Mouse Aorta
Western analysis revealed that cAbl is abundantly expressed in various cells and tissues including VSMCs, endothelial cells, fibroblasts, and aorta as a 145 kDa protein (supplemental Figure I). This molecular mass of cAbl is consistent with that previously reported in other cells and tissues, whereas some cells also include a 110 kDa shorter fragment.19 In rat VSMCs, Ang II (100 nmol/L) induced tyrosine phosphorylation of cAbl that peaked at 5 minutes and remained above baseline for up to 60 minutes (Figure 1A). This response was dose-dependent, with a threshold of 1 nmol/L and a maximum effect occurring at 100 nmol/L (supplemental Figure IIA). Furthermore, the AT1 receptor blocker losartan significantly blocked cAbl phosphorylation (supplemental Figure IIB), suggesting that Ang II-stimulated cAbl phosphorylation is mediated through the AT1 receptor. Similarly, tyrosine phosphorylation of cAbl is also observed in isolated mouse aortas exposed to Ang II (100 nmol/L) for 30 minutes ex vivo (Figure 1B) and in aortas of mice infused with Ang II (0.7 mg · kg–1· d–1) for 24 hours, which did not induce any blood pressure increase (Figure 1C). We confirmed that similar phosphorylation of 145 kDa cAbl by Ang II was obtained with phospho-specific cAbl (pY412) antibody (Cell Signaling) (data not shown).



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  		Figure 1. Ang II stimulates cAbl phosphorylation in rat VSMCs and mouse aorta ex vivo and in vivo. A, Rat VSMCs were stimulated with Ang II (100 nmol/L) for indicated times. B, Thoracic aortae from C57BL/6J mice were stimulated with Ang II (100 nmol/L) for 30 minutes and the vessels were homogenized. C, mice were infused with [Val5]Ang II (0.7 mg · kg–1· d–1) for 24 hours, and aortae were harvested. NC and PC indicate negative control (IP with nonimmune IgG) and positive control (VSMC lysates stimulated with Ang II), respectively. For A, B, and C, cAbl phosphorylation was measured by immunoprecipitation with cAbl antibody and immunoblotting with phosphotyrosine (pTyr) antibody. Immunoblotting with cAbl is shown as a loading control. The graphs represent averaged data (n=3), corrected for total cAbl loading, expressed as change over basal. *P<0.05 for Ang II-induced change vs vehicle alone.

cSrc Mediates Ang II-Induced cAbl Tyrosine Phosphorylation
We examined upstream pathways mediating cAbl tyrosine phosphorylation by Ang II. The Src family kinase inhibitor PP1 blocked Ang II-induced tyrosine phosphorylation of cAbl, but inhibition of phosphatidylinositol-3-kinase by wortmannin or LY294002 or of protein kinase C by GF109203X did not do so (Figure 2A). Ang II-induced cAbl phosphorylation was significantly inhibited by KI-cSrc overexpression, whereas a control virus, Ad.LacZ, had no effect. These results are consistent with the notion that cSrc is an upstream mediator for cAbl activation by Ang II (Figure 2B).



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  		Figure 2. cSrc mediates Ang II-induced cAbl tyrosine phosphorylation. A and B, Rat VSMCs were pretreated with wortmannin (100 nmol/L, 30 minutes), LY242002 (10 µmol/L, 30 minutes), GF109203X (10 µmol/L, 30 minutes), and PP1 (10 µmol/L, 1 hour) (A) or infected with either Ad.KI-Src or Ad.LacZ at 10 MOI (B), and then stimulated with 100 nmol/L Ang II for 5 minutes. Lysates were immunoprecipitated with anti-cAbl antibody and immunoblotted with phosphotyrosine (pTyr) or cAbl antibody. The graphs represent averaged data (n=3), corrected for total cAbl loading, expressed as fold change over basal. *P<0.05 for Ang II-induced change vs vehicle alone; #P<0.05 for Ang II-induced change with inhibitor vs Ang II alone.

Ang II Promotes cSrc-Mediated Association of cAbl With AT1R
To gain insight into the mechanisms involved in the cSrc-dependent AT1R activation of cAbl, rat VSMCs were stimulated with Ang II (100 nmol/L) and lysates were immunoprecipitated with AT1R antibody and immunoblotted with cAbl or cSrc antibody. As shown in Figure 3A, AT1R binds at relatively low levels to cSrc and cAbl basally. Ang II stimulation increased association of cAbl with AT1R, which was detectable at 2 minutes and peaked at 5 minutes, and then continued at a similar level for at least 60 minutes. Reprobing of these blots with anti-phospho-cAbl (pY412) antibody reveals that phospho-cAbl associates with AT1R within 5 minutes, which is cotemporaneous with the Ang II-induced increase in cAbl phosphorylation, raising the possibility that the nonphosphorylated form of cAbl binds initially to the AT1R/cSrc complex within 2 minutes after stimulation. cSrc was also recruited to the AT1R complex within 2 minutes, peaked at 5 minutes, and returned toward baseline within 15 minutes. Ang II-stimulated formation of an AT1R/cSrc/cAbl complex was inhibited by PP1 (Figure 3B). Association of cSrc and cAbl with AT1R was further evaluated by converse experiments: Immunoprecipitating with cSrc (Figure 3C) or cAbl antibody (data not shown) and immunoblotting with AT1R antibody. The results are consistent with a scenario in which Ang II promotes formation of AT1R/cSrc/cAbl complexes in a Src kinase-dependent manner and shows that these interactions may contribute to the cSrc-dependent phosphorylation of cAbl.



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  		Figure 3. Ang II promotes association of cAbl and cSrc with AT1R, which is Src-dependent. A and C, Rat VSMCs were stimulated with 100 nmol/L Ang II. B, Rat VSMCs were pretreated with PP1 (10 µmol/L, 1 hour), and then stimulated with 100 nmol/L Ang II for 5 minutes. Lysates were immunoprecipitated with anti-AT1R (A and B) or cSrc (C) antibody, and immunoblotted with cAbl [cAbl(pY412) for A], cSrc or AT1R antibodies. Blots are representative of 3 separate experiments. The graphs represent averaged data (n=3), corrected for total AT1R loading, expressed as fold change over basal. *P<0.05 for Ang II-induced change vs vehicle alone.

ROS Mediate Tyrosine Phosphorylation of cAbl by Ang II
cSrc is an important target of ROS in Ang II signaling.5 Thus, we examined the role of ROS in cAbl phosphorylation by Ang II. Exogenous H2O2 time- (Figure 4A) and dose-dependently (supplemental Figure IIIA) increased cAbl phosphorylation, which was inhibited by PP1 (Figure 4B). Furthermore, pretreatment with various ROS inhibitors (diphenylene iodonium [DPI], an inhibitor of flavin-containing enzymes; N-acetylcysteine [NAC]; and ebselen, a glutathione peroxidase mimetic) caused significant inhibition of tyrosine phosphorylation of cAbl by Ang II (Figure 4C). We also confirmed that polyethylene glycol (PEG)-catalase inhibits Ang II-induced intracellular H2O2 production, as measured by DCF fluorescence (supplemental Figure IIIB), as well as cAbl phosphorylation (Figure 4D), suggesting that Ang II-induced cAbl activation is mediated through H2O2.



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  		Figure 4. Ang II-induced cAbl phosphorylation is ROS-dependent. A, Rat VSMCs were stimulated with 200 µmol/L H2O2. B, Rat VSMCs were pretreated with PP1 (10 µmol/L, 1 hour) and then stimulated with 200 µmol/L H2O2 for 15 minutes. C and D, Rat VSMCs were pretreated with vehicle, DPI (10 µmol/L), NAC (10 mmol/L), or ebselen (40 µmol/L) for 30 to 60 minutes (C) or PEG-catalase (100 U/mL, 24 hour) (D) and then stimulated with 100 nmol/L Ang II for 5 minutes. Lysates were used for the measurement of phosphorylation of cAbl, as described. Values are average of 3 independent experiments. *P<0.05 for Ang II or H2O2-induced change vs vehicle alone.

Knockdown of cAbl Protein by siRNA Inhibits EGF-R Transactivation by Ang II
To determine the functional role of cAbl in Ang II-induced EGF-R transactivation, we designed and tested several siRNA sequences targeting various regions of cAbl (siRNA 1 to 5) (see online supplement Materials and Methods). Transfection of cAbl siRNA in rat VSMCs showed that one of them (cAbl siRNA-1) almost completely knocked down cAbl protein expression without affecting the expression of the cAbl-related protein tyrosine kinase Arg or of {alpha}-tubulin (Figure 5A). We thus used this specific cAbl siRNA for the subsequent functional analysis of cAbl. cAbl siRNA significantly inhibited Ang II-induced transactivation of EGF-R without affecting EGF-induced EGF-R autophosphorylation (Figure 5B), indicating that cAbl is involved in the pathways linking AT1R to EGF-R transactivation.



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  		Figure 5. Effects of cAbl siRNA on EGF-R phosphorylation by Ang II and EGF. A, Rat VSMCs were transfected with scrambled or cAbl siRNAs (15 nmol/L) or transfection reagent alone (control). After 48 hours, lysates were immunoblotted with anti-cAbl, anti-Arg, or {alpha}-tubulin antibody. The results are representative of 3 separate experiments. B, Rat VSMCs transfected with scrambled or cAbl siRNA were stimulated with 100 nmol/L Ang II for 2 minutes (left) or 100 ng/mL EGF for 30 sec (right). Lysates were immunoprecipitated with anti–EGF-R antibody and immunoblotted with phosphotyrosine (pTyr) or EGF-R antibody. The graphs represent averaged data (n=3), corrected for total EGF-R loading, expressed as fold change over basal. *P<0.05 for Ang II- or EGF-induced change vs vehicle alone.

cAbl Is Involved in Ang II-Stimulated AT1R Trafficking Into, and Egress of EGF-R From, Cav1-Enriched Lipid Rafts
To determine the role of cAbl in AT1R trafficking into Cav1-enriched lipid rafts, which is required for Ang II-induced EGF-R transactivation,7 we isolated Cav1-enriched microdomains.20 In unstimulated rat VSMCs, AT1R was localized in heavy membrane fractions (fractions 10 to 13), but not in the buoyant, light-density Cav1-enriched fractions (fractions 3 to 4) (supplemental Figure IV). Ang II stimulation for 5 minutes promoted migration of AT1R from heavy membrane fractions into Cav1-enriched fractions, contemporaneously with the egress of EGF-R from these membranes (Figure 6). A trafficking of both receptors was significantly inhibited by cAbl siRNA (Figure 6B and 6C). Moreover, cAbl and cSrc were present in both heavy and Cav1-enriched membrane fractions basally (supplemental Figure IV). Ang II stimulation partially reduced the amount of both proteins in the heavy fractions, which was associated with an increase in cAbl protein in Cav1-enriched light membrane fractions (1.9±0.1 fold increase, n=3; Figure 6B) and coimmunoprecipitation of cAbl with Cav1 (data not shown). The amount of phospho-cSrc (pY418) in Cav1-enriched fractions was also increased after Ang II stimulation (data not shown).



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  		Figure 6. Effects of cAbl siRNA on localization of AT1R, cAbl, and EGF-R in Cav1-enriched membrane fractions. Rat VSMCs transfected with scrambled or cAbl siRNA as described above were stimulated with 100 nmol/L Ang II for 5 minutes. A, Caveolae fractions 1 through 8 were immunoblotted for AT1R, cAbl, EGF-R, or Cav1 (A). PC indicates positive control (VSMC cell lysates). B, Equal amounts of Cav1-enriched fraction 3 were immunoblotted with antibodies indicated. C, Graphs represent averaged data (n=3) for the presence of AT1R and EGF-R in Cav1-enriched fraction 3, expressed as % Ang II-induced response (set to 100% in Ang II-treated, scrambled siRNA-transfected cells) for AT1R, and as % control (set to 100% in untreated, scrambled siRNA-transfected cells) for EGF-R. #P<0.05 for Ang II-induced changes in cells transfected with cAbl siRNA vs scrambled siRNA. *P<0.05 for Ang II–induced change vs vehicle alone.

cAbl Is Involved in the Colocalization at Focal Adhesions of Tyrosine Phosphorylated Cav1 and Transactivated EGF-R
Cav1 is tyrosine phosphorylated by cAbl.18 We previously demonstrated that Ang II-induced tyrosine-phosphorylated Cav1 (pY14-Cav1) is localized in focal adhesions together with transactivated EGF-R.7 Thus, we examined the role of cAbl in this response. Immunofluorescence studies showed that, on stimulation with Ang II, phospho-EGF-R colocalized with pY14-Cav1 (Figure 7) and with vinculin, a marker protein of focal adhesions (data not shown). The staining of these proteins is reproducible; more than 90% of cells show the identical patterns. In rat VSMCs transfected with cAbl siRNA, Ang II-stimulated, but not basal, staining of pY14-Cav1 and of activated EGF-R in focal adhesions were markedly reduced. The decrease in Ang II-stimulated pY14-Cav1 level by cAbl siRNA was further confirmed by Western blot analysis (data not shown).



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  		Figure 7. Effects of cAbl siRNA on tyrosine phosphorylated Cav1 and transactivated EGF-R in focal adhesions. Rat VSMCs transfected with scrambled or cAbl siRNA were stimulated with 100 nmol/L Ang II for 5 minutes and double-labeled with rabbit anti-phospho-EGF-R (pY845) antibody and mouse anti-phospho-caveolin (Tyr14) antibody, followed by anti-rabbit fluorescein isothiocyanate-conjugated and anti-mouse Rhodamine Red X-conjugated secondary antibodies. Scale bar=20 µm. All fluorescence images were taken at 5 different fields/well, and the single cell images are representative of 2 different experiments.

cAbl Is Involved in Ang II-Stimulated VSMC Hypertrophy
To gain insights into the functional role of cAbl in AT1R signaling, we examined whether cAbl is involved in Ang II-stimulated VSMC hypertrophy. As shown in Figure 8, cAbl siRNA significantly inhibited Ang II-stimulated [3H]leucine incorporation without affecting basal levels. The trypan blue exclusion test for cell viability indicated that cells transfected with cAbl siRNA were >98% viable. Thus, cAbl plays an important role for Ang II-induced VSMC hypertrophy.



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  		Figure 8. Effects of cAbl siRNA on Ang II-induced vascular hypertrophy. Rat VSMCs transfected with scrambled or cAbl siRNAs as described were used for measurement of [3H]leucine incorporation induced by Ang II (100 nmol/L). The graphs represent averaged data, expressed as fold change over basal. *P<0.05 for Ang II-induced change vs vehicle alone; #P<0.05 for Ang II-induced changes in cells transfected with cAbl siRNA vs scrambled siRNA.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study demonstrates that Ang II activates the cAbl tyrosine kinase in rat VSMCs and mouse aorta and promotes the cSrc-dependent association of cSrc, cAbl, and AT1R. Ang II-induced cAbl tyrosine phosphorylation is mediated through ROS-dependent cSrc activation. Furthermore, cAbl plays an important role for AT1R trafficking into Cav1-enriched lipid rafts, EGF-R transactivation, and tyrosine phosphorylation of Cav1, as well as the colocalization of pEGF-R and pY14Cav1 at focal adhesions. Collectively, these cAbl-dependent responses contribute to regulation of Ang II-induced [3H]-leucine incorporation into rat VSMCs.

cAbl is activated by growth factors9,10,21 and by oxidative stress.16–18 Here, we show that cAbl is expressed in various cells, including VSMCs, endothelial cells, and fibroblasts. Ang II stimulation induces tyrosine phosphorylation of cAbl in rat VSMCs and in the aorta of mice ex vivo and in vivo (independent of pressor effects) (Figure 1). To our knowledge, this demonstration in VSMCs of the phosphorylation of cAbl by AT1R or, more generally, by any G-protein–coupled receptor agonist, is novel. Phosphorylated tyrosine on cAbl may provide binding sites for SH3 domain containing targets of cAbl, including CrkII, Nck, and Grb2,15 thereby activating downstream signaling cascades. The findings in the aorta support the physiological importance of cAbl in Ang II signaling in vivo as inferred by the in vitro cell culture data. Because cAbl is also expressed in endothelial cells and fibroblasts (supplemental Figure I), we cannot eliminate the possible contribution of these cells to total cAbl phosphorylation in mouse aorta. This point requires further investigation.

Although the upstream signaling mechanisms responsible for cAbl activation have not been fully elucidated in other systems, a roles for cSrc and other Src family kinases such as Fyn have been documented.9,10 We demonstrate that both the Src-family kinase inhibitors PP1 and KI-Src almost completely inhibited Ang II-induced phosphorylation of cAbl in rat VSMCs (Figure 2). Thus, cSrc appears to be the major Src family kinase involved here. Activated Src family kinases directly phosphorylate tyrosine residues in the kinase domain of cAbl, which correlates with enhanced activity.9 Phospholipase C{gamma} (PLC{gamma}) is required for cAbl activation by platelet-derived growth factor.22 cSrc is involved in PLC{gamma} activation by Ang II.23 Thus, it is possible that an AT1R/cSrc/PLC{gamma} pathway may contribute to cAbl activation by Ang II in VSMCs.

AT1R slightly associates with cSrc and cAbl basally, and Ang II stimulation rapidly promotes the enhanced recruitment of cSrc and cAbl to a complex with AT1R, which is inhibited by PP1 (Figure 3). Furthermore, non–phospho-cAbl appears to bind initially to AT1R (within 2 minutes), and subsequently phosphorylated cAbl appears in the AT1R complex (Figure 3A). We also found that cAbl siRNA does not affect cSrc binding to the receptor (supplemental Figure V). These results suggest that cSrc is pivotal in Ang II-induced formation of the AT1R/cSrc/cAbl complex, which possibly facilitates cAbl phosphorylation. Our results contrast with a previous report that cSrc does not bind to AT1R in VSMCs.24 This discordance may be attributable to differences in experimental conditions (purified cSrc and AT1R or cell culture conditions, etc) or specificity of AT1R antibodies.

cAbl interacts directly with various receptors, including the Trk nerve growth factor receptor, EphB2, and N-methyl-D-aspartic acid receptors through the SH2 or SH3 domains of cAbl.21,25–27 Of note, another nonreceptor tyrosine kinase (JAK2) binds directly to the AT1R by a mechanism requiring the tyrosine phosphatase SHP-2.28 Also, in transfection experiments, EGF-R associates transiently with AT1R through an SHP-2–dependent mechanism.29 In VSMCs, Ang II stimulation promotes association of SHP-2 with AT1R.28 Thus, it is possible that SHP-2 may function in VSMCs as an AT1R scaffold to form the AT1R/cSrc/cAbl complex. Detailed analysis of cAbl-AT1R association is an objective of future studies.

We reported that production of H2O2 is observed within 1 minute of Ang II stimulation.3 We show that Ang II-induced cAbl tyrosine phosphorylation is significantly inhibited by various ROS inhibitors, including DPI, NAC, ebselen, and PEG-catalase (Figure 4), suggesting an essential role of NAD(P)H oxidase-derived H2O2 in this response. Redox sensitivity of cAbl phosphorylation was further confirmed by the finding that exogenous H2O2 increased cAbl phosphorylation (Figure 4 and supplemental Figure IIIA), which is consistent with other reports.16–18 The relatively slow activation of cAbl phosphorylation by exogenous H2O2 is possibly attributable to barriers to diffusion or to varying antioxidant capacity in different cellular compartments. In contrast, receptor-mediated ROS production may be compartmentalized and may act on preformed, primed AT1R containing cSrc/cAbl complex in appropriate agonist-induced spatial conformations, thereby activating cSrc and cAbl in close proximity to the receptor more rapidly and efficiently. Given that H2O2-induced cAbl phosphorylation is inhibited by PP1 (Figure 4B) and that cSrc is a very proximal target of ROS in Ang II signaling,5 redox-sensitivity of cAbl may be conferred through ROS-dependent activation of cSrc. SHP-2 is a negative regulator of Ang II-induced cSrc activation in VSMCs,30 and phosphatase activity of SHP-2 is inhibited by ROS.31 Thus, evidence supports the notion that Ang II-induced cAbl phosphorylation in VSMCs is mediated through ROS-dependent cSrc activation and may also involve SHP-2.

To explore downstream targets of cAbl in AT1R signaling, we examined whether cAbl is involved in Ang II-induced EGF-R transactivation. Knockdown of endogenous cAbl using siRNA significantly inhibited Ang II-induced EGF-R tyrosine phosphorylation without affecting EGF-mediated EGF-R phosphorylation (Figure 5). Thus, cAbl is involved selectively in the pathways linking AT1R to the EGF-R, but does not appear to be involved in EGF activation of its receptor’s tyrosine kinase. We showed previously, using detergent-free membrane fractionation, that Ang II promotes not only AT1R trafficking into Cav1-enriched/lipid rafts but also egress of EGF-Rs from these microdomains. These events seem to be required for Ang II transactivation of EGF-R and downstream signal generation.7 We noted that cAbl contains 2 Cav1 consensus binding sequences (312YIITEFMTY320 and 423YAFGVLLW430) and found that cAbl is localized basally in Cav1-enriched fractions, and its concentration is enhanced after Ang II stimulation (Figure 6). cAbl siRNA inhibits Ang II-stimulated trafficking of AT1R into, and EGF-R out of, Cav1-enriched lipid rafts. As shown previously, Ang II promotes AT1R association with Cav1,6 and Cav1 siRNA blocks AT1R trafficking into Cav1-enriched fractions.8 We also show that AT1R, cSrc, and cAbl are localized, at least in part, in the heavy membrane fractions basally, and the amounts of these proteins are decreased after Ang II stimulation (supplemental Figure IV) at the time in which AT1R migrates into the lipid rafts fractions (Figure 6). Thus, our current model is consistent with the possibilities that the initial AT1R/cSrc/cAbl interaction and subsequent cSrc-dependent cAbl phosphorylation by Ang II may take place in the heavy membrane fractions and that active AT1R/cSrc/cAbl complexes move into Cav1-enriched fractions. Ang II promotes association of Cav1 with AT1R6 and cAbl (present study), and Cav1 siRNA inhibits Ang II-induced cSrc and cAbl binding to the AT1R, as well as cAbl phosphorylation (unpublished observations, 2005). Thus, Cav1 may function as a scaffold to organize and target AT1R/cSrc/cAbl complexes into the Cav1-enriched/lipid rafts.

Cav-1 is a major tyrosine phosphorylation substrate of cAbl.18 We showed that Ang II-induced egress of EGF-R from Cav-1 enriched lipid rafts in VSMCs is required for the appearance of transactivated EGF-R at focal adhesions, where it colocalizes with pY14-Cav1.7 cAbl siRNA inhibits Ang II-induced colocalization of phosphor–EGF-Rs and pY14-Cav1 at focal adhesions. Western analysis confirmed that Ang II-induced tyrosine phosphorylation of Cav1 is inhibited by cAbl siRNA. Cav1 is tyrosine phosphorylated by various growth factors, including EGF32 and VEGF.33,34 Phospho-Cav1 interacts with the scaffold Grb735 and Csk, a negative regulator for Src,36 suggesting that pY14-Cav1 functions in organizing phosphotyrosine-binding molecules involved in growth factor-mediated signaling. Lewis et al14 reported that cAbl is involved in signal transduction activated by integrins. Thus, cAbl-dependent localization of transactivated EGF-R and pY14-Cav1 at focal adhesions, a major site of tyrosine kinase signaling,37 indicates an important role of cAbl in growth-related AT1R signaling in VSMCs. The site(s) at which the Cav1 and EGF-R phosphorylation events initially occur are not known, but they could be at the focal adhesions. This point requires further investigation.

Proposed functional roles for cAbl include oncogenesis, apoptosis during DNA damage, actin cytoskeleton regulation,9,12,15 and growth factor-induced DNA synthesis.10 The present study reveals a new function of cAbl in AT1R signaling in VSMCs. We show that cAbl siRNA significantly inhibits Ang II-stimulated increase in [3H]leucine incorporation, indicating that this kinase is involved in VSMC hypertrophy. The partial inhibition of cAbl siRNA may be attributable to the facts that Ang II-induced vascular hypertrophy is mediated through both ROS-dependent Akt and ROS-independent ERK1/2 pathways and that cAbl siRNA inhibits Akt, but not ERK1/2, phosphorylation by Ang II (unpublished observation, 2005). The mechanisms by which cAbl siRNA inhibits Ang II-induced VSMC hypertrophy may be, at least in part, related to its inhibition of phosphorylation of EGF-R and Cav1 in focal adhesions, as transactivated EGF-R38 and Cav139 are important in Ang II-induced increases in protein synthesis in VSMCs.

As noted, cAbl is expressed within both the cytosol and nucleus. The inhibitory effect of cAbl siRNA on early signaling events activated by Ang II may reflect the role of cytoplasmic cAbl in AT1R-induced vascular hypertrophy. We cannot exclude possible contributions of nuclear cAbl in this response, which is the subject of future investigation.

Imatinib (STI571, Glivec), a relatively selective inhibitor of the Bcr-Abl tyrosine kinase, inhibits Ang II infusion-induced hypertrophy in rat mesenteric arteries in vivo,40 as well as diabetes-associated atherosclerosis in aorta of apolipoprotein E–/– mice.41 Atherosclerosis in this model is highly dependent on AT1R.42 Thus, these results inferentially associating cAbl with Ang II/AT1R signaling are supported by our results that cAbl is a major mediator of Ang II responses in VSMCs.

In summary, we demonstrated that cAbl is activated by Ang II in rat VSMCs and mouse aortae and that Ang II stimulation promotes association of cAbl with AT1R in a cSrc-dependent manner. Furthermore, Ang II-induced cAbl activation is ROS-dependent. We also found that cAbl is involved in Ang II-stimulated: (1) AT1R trafficking into Cav1-enriched fractions; (2) egress of EGF-R from these microdomains; (3) appearance of transactivated EGF-R and phosphorylated Cav1 at focal adhesions; and (4) VSMC hypertrophy. These findings suggest a novel role of cAbl in the spatial and temporal organization of ROS-sensitive AT1R signaling in VSMCs.


*    Acknowledgments
 
This work was supported by National Institutes of Health grant HL60728 (R.W.A. and M.U.-F.) and an American Heart Association National Scientist Development Grant 0130175N and an American Heart Association Grant-in-Aid 0555308B (to M.U.-F.). Immunofluorescence analysis was supported by National Institutes of Health grants PO1 HL058000 and PO1 HL075209.


*    Footnotes
 
*Both authors contributed equally to this work. Back

Original received May 2, 2005; resubmission received August 8, 2005; revised resubmission received August 25, 2005; accepted August 29, 2005.


*    References
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