Phosphorylation of p130^Cas by Angiotensin II Is Dependent on c-Src, Intracellular Ca²⁺, and Protein Kinase C

From the Departments of Pathology (P.P.S., M.S.A., M.B.M., K.E.B.) and Medicine (J.B.H.), Emory University School of Medicine, Atlanta, Ga.

Correspondence to Kenneth E. Bernstein, MD, Department of Pathology, Emory University School of Medicine, 1639 Pierce Dr, 7107 WMB, Atlanta, GA 30322. E-mail kbernst{at}emory.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—p130^Cas is a signaling molecule that was initially found to be tyrosine-phosphorylated in v-Crk and v-Src transformed cells. We characterized the regulation of p130^Cas tyrosine phosphorylation in vascular smooth muscle cells by angiotensin II (Ang II). This ligand induced a transient increase in p130^Cas tyrosine phosphorylation, which was sensitive to the actin polymerization inhibitor cytochalasin D and to the intracellular Ca²⁺ chelator BAPTA-AM but not the Ca²⁺ channel blocker verapamil. The Ang II–induced tyrosine phosphorylation of p130^Cas was also dependent on an active Src family tyrosine kinase, since it could be blocked by the Src kinase inhibitors geldanamycin and PP1. Ang II treatment resulted in the ability of p130^Cas to bind at least 11 different phosphate-containing proteins. Analysis of these proteins revealed that protein kinase C and the cell adhesion signaling molecule pp120 formed temporal associations with p130^Cas in response to Ang II. c-Src was found to associate with p130^Cas in a manner that was independent of Ang II treatment. Inhibition of protein kinase C by either calphostin C or phorbol 12-myristate 13-acetate downregulation inhibited the Ang II–induced tyrosine phosphorylation of p130^Cas. These results are the first to demonstrate that the tyrosine phosphorylation of p130^Cas by Ang II is transduced by the Src, intracellular Ca²⁺, protein kinase C signaling pathway.

Key Words: p130^Cas • tyrosine phosphorylation • angiotensin II • vascular smooth muscle cell

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular smooth muscle cells are the principal component of the blood vessel wall and are responsible for maintaining vascular tone. They are also an abundant source of the AT₁ receptor.¹ This G protein–coupled receptor mediates smooth muscle cell contraction through a transient increase in intracellular Ca²⁺ via the production of IP₃. Recent studies indicate that tyrosine phosphorylation is a critical event in many Ang II–mediated signaling events.^{2 3} Known VSMC signaling proteins that are tyrosine-phosphorylated in response to Ang II include paxillin, phospholipase C-1, p120Ras-GAP, Jak2, Stat1, Stat3, Tyk2, and the intracellular tyrosine kinase pp60^c-src.^{4 5 6 7 8}

p130^Cas (a Crk-associated substrate) was initially characterized as a phosphotyrosine-containing protein in v-Crk and v-Src transformed cells.^{9 10} Cloning and expression of the rat p130^Cas cDNA found it to form stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation–dependent manner.¹¹ The protein sequence of p130^Cas suggests that it may serve as an adaptor protein because it contains proline-rich domains, an SH3 domain, and binding motifs for the SH2 domains of v-Crk and v-Src. p130^Cas also appears to be important for integrin-mediated cell adhesion. In response to cell-cell contact, integrin receptors become tyrosine-phosphorylated on the cytoplasmic tail. The end result is cytoskeletal rearrangement.^{12 13} The mechanism(s) of this signal transduction process is not known, but p130^Cas is thought to recruit cytoskeletal signaling molecules such as p125^Fak, paxillin, and tensin to the focal adhesions.^{12 14} However, what seems certain is that tyrosine phosphorylation plays a critical role in this process.

The importance of Ang II–mediated tyrosine phosphorylation in VSMC signal transduction prompted us to investigate the regulation of p130^Cas tyrosine phosphorylation by Ang II. In the present study, we demonstrate that p130^Cas is tyrosine-phosphorylated by Ang II. This phosphorylation is Ca²⁺ dependent and sensitive to cytochalasin D. It also requires active c-Src and PKC signaling molecules. Furthermore, the phosphorylation of p130^Cas appears to have functional consequences in that it can form Ang II–dependent complexes with catalytically active molecules. Western blot analysis identified 3 of the proteins as c-Src, PKC, and the cell adhesion signaling molecule pp120. These results are the first to demonstrate that the tyrosine phosphorylation of p130^Cas by Ang II is transduced by the Src, intracellular Ca²⁺, PKC signaling pathway and suggest that in VSMCs, p130^Cas may serve as a convergence point for 3 different signaling pathways, namely, the serine/threonine PKC pathway, the cell adhesion–mediated pp120 pathway, and the c-Src tyrosine kinase pathway.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
VSMCs were prepared from the aortas of male Sprague-Dawley rats as described.¹⁵ Cells were grown in DMEM+10% FBS at 37°C in a 5% CO₂ humidified atmosphere and used between passages 8 and 22. Dishes (100 mm) at 75% confluence were growth-arrested by incubation in serum-free DMEM for 48 hours before use. Cell culture reagents were obtained from GIBCO/BRL. Inhibitors were purchased from Calbiochem. All other reagents were purchased from Sigma Chemical Co.

Immunoprecipitation
Cells were stimulated as described in the figure legends. To prepare lysates, cells were washed with 2 vol ice-cold PBS containing 1 mmol/L Na₃VO₄ and lysed in 1.0 mL RIPA buffer (20 mmol/L Tris [pH 7.5], 10% glycerol, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 2.5 mmol/L EDTA, 50 mmol/L NaF, 10 mmol/L Na₄P₂O₇, 4 mmol/L benzamidine, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na₃VO₄, and 10 µg/mL aprotinin). Extracts were incubated on ice for 30 minutes and spun at 10 000g for 5 minutes at 4°C. Supernatants were quantified using the D_c protein assay (Bio-Rad). Normalized lysates (300 µg/mL) were immunoprecipitated with 2 µg of antibody and 20 µL of a 50% slurry of Protein A/G Plus agarose beads (Santa Cruz Biotechnology) for 16 hours at 4°C. Immune complexes were washed 3 times with wash buffer (25 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, and 0.1% Triton X-100) and resuspended in sample buffer. Proteins were separated on 8% SDS-PAGE and transferred onto nitrocellulose membranes (Schleicher & Schuell).

Western Blotting
After they were blocked for 1 hour in 5% milk/TBST (wt/vol) at 23°C, nitrocellulose membranes were probed with primary antibody for 2 hours at 23°C in 5% milk/TBST. Blots were washed, and proteins were visualized with enhanced chemiluminescence according to the manufacturer's instructions (Amersham). Films were scanned with an IS-1000 digital imaging system (Alpha Innotech Corp). Statistically significant fold increases are expressed as the mean±SEM. Monoclonal anti-phosphotyrosine (clone PY20), monoclonal anti-p130^Cas, monoclonal anti-pp120, monoclonal anti-PKC, monoclonal anti–PTP-1D (SHP2), and monoclonal anti–PI-3-kinase were from Transduction Laboratories. Monoclonal anti-phosphotyrosine (clone 4G10) was from Upstate Biotechnology. Polyclonal anti-Src (N16) was from Santa Cruz Biotechnology. Monoclonal anti-pp60^c-src (clone GD-11) was a generous gift from Dr Sarah Parsons (University of Virginia). For anti-Tyr(P) Western blotting, a cocktail of PY20/4G10 was used at a final concentration of 1 µg/mL each. For anti-Tyr(P) immunoprecipitation, only PY20 was used.

Protein Complex Formation
Cells were scraped in 1.0 mL ice-cold gentle lysis buffer (25 mmol/L Tris [pH 7.5], 10% glycerol, 1% NP-40, 140 mmol/L NaCl, 4 mmol/L benzamidine, 1 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L Na₃VO₄, and 10 µg/mL aprotinin), gently sonicated, and incubated on ice for 1 hour. Samples were spun at 10 000g for 5 minutes at 4°C, and normalized supernatants were precipitated with antibody as described above.

In Vitro Kinase Assays
p130^Cas immunoprecipitates were washed twice with wash buffer followed by 2 washes in kinase reaction buffer (25 mmol/L HEPES [pH 7.6] and 10 mmol/L MgCl₂). The precipitates were resuspended in 50 µL of the same kinase buffer containing 50 mmol/L ATP and 10 µCi [-³²P]ATP (Amersham) and incubated for 25 minutes at 30°C. Additional kinase reactions were performed in the presence of 100 µg/mL GAP p62 (Santa Cruz Biotechnology), a Src family tyrosine kinase substrate. Reactions were terminated by adding SDS sample buffer. Radiolabeled proteins were separated on SDS-PAGE, transferred onto nitrocellulose, and exposed to film.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II Induces Tyrosine Phosphorylation of p130^Cas
To study the regulation of p130^Cas tyrosine phosphorylation in VSMCs, we treated cells with Ang II for varying times. Lysates were immunoprecipitated with anti-p130^Cas mAb and analyzed by Western blotting with anti-Tyr(P) mAbs. Ang II stimulated tyrosine phosphorylation of p130^Cas in a transient manner, with peak phosphorylation occurring at 1 minute (3.91±0.12-fold, n=4) (Figure 1A). Tyrosine phosphorylation remained above basal at 5 minutes (1.94±0.21-fold, n=4) and returned to near basal levels at 20 to 60 minutes. To verify this observation, we again stimulated cells but reversed the addition of antibody so that we immunoprecipitated with anti-Tyr(P) mAb and then Western-blotted with anti-p130^Cas mAb (Figure 1B). p130^Cas was tyrosine-phosphorylated with a time course that was similar to that seen in Figure 1A, demonstrating that both protocols yield nearly identical results.

View larger version (32K): [in this window] [in a new window]   Figure 1. Ang II–induced tyrosine phosphorylation of p130Cas. Quiescent VSMCs were stimulated with 100 nmol/L Ang II for the indicated times. A, Lysates were immunoprecipitated with anti-p130Cas mAb and Western-blotted with anti-Tyr(P) mAbs. The same blot was probed with anti-p130Cas mAb to determine equal loading. Shown is 1 of 4 representative experiments. IP indicates immunoprecipitation. B, Lysates were immunoprecipitated with anti-Tyr(P) mAb and blotted with anti-p130Cas mAb. Also indicated is the IgG heavy chain from the same blot. Shown is 1 of 4 representative experiments. C, Cells were treated for 1 hour either with DMSO (control) or 10 µmol/L losartan and then stimulated with 100 nmol/L Ang II for the indicated times. The anti-p130Cas mAb immunoprecipitates were blotted with anti-Tyr(P) mAb. The same blot was probed with anti-p130Cas mAb to determine equal loading. Shown is 1 of 2 representative experiments.

To define the receptor subtype that mediates the tyrosine phosphorylation of p130^Cas in response to Ang II, VSMCs were pretreated with the AT₁-specific inhibitor losartan. No tyrosine phosphorylation was observed in losartan-treated cells, demonstrating that p130^Cas tyrosine phosphorylation is mediated solely by the AT₁ receptor (Figure 1C). Collectively, these data demonstrate that treatment of VSMCs with Ang II results in AT₁ receptor activation and subsequent p130^Cas tyrosine phosphorylation.

Regulation of p130^Cas Tyrosine Phosphorylation by Cytochalasin D and NaF
Recent studies have shown that the tyrosine phosphorylation of several focal adhesion–associated proteins, including p130^Cas, can be blocked with cytochalasin D.¹⁶ This compound disrupts the formation of focal contacts by inhibiting actin polymerization. However, stimulation of rat liver epithelial cells with Ang II increased the tyrosine phosphorylation of several unidentified proteins in the 115- to 130-kDa range that are insensitive to cytochalasin D pretreatment.¹⁷ To assess the sensitivity of Ang II–induced tyrosine phosphorylation of p130^Cas in VSMCs, quiescent cells were pretreated with cytochalasin D and then stimulated with Ang II. The resulting lysates were immunoprecipitated with anti-Tyr(P) mAb and Western-blotted with anti-p130^Cas mAb. As shown in Figure 2A, the Ang II–induced phosphorylation was inhibited by cytochalasin D in both a dose- and time-dependent manner. These results suggest that in VSMCs, the ability of p130^Cas to interact with the actin filament network is critical for Ang II–induced tyrosine phosphorylation.

View larger version (60K): [in this window] [in a new window]   Figure 2. Regulation of p130Cas tyrosine phosphorylation by cytochalasin D and NaF. A, For the cytochalasin D time course, quiescent VSMCs were pretreated with 3 µmol/L cytochalasin D for the indicated times and stimulated for 1 minute with 100 nmol/L Ang II. For dose-response data, quiescent VSMCs were pretreated for 2 hours with the indicated concentration (Conc.) of cytochalasin D and stimulated for 1 minute with 100 nmol/L Ang II. Lysates were immunoprecipitated with anti-Tyr(P) mAb and blotted with anti-p130Cas mAb. B, Quiescent VSMCs were pretreated with 5 mmol/L NaF for the indicated times and stimulated for 1 minute with saline control (-) or 100 nmol/L Ang II (+). Lysates were immunoprecipitated with anti-Tyr(P) mAb and Western-blotted with anti-p130Cas mAb. IP indicates immunoprecipitation. Shown is 1 of 3 representative experiments for each. Size markers are in kilodaltons.

Several reports have documented increased tyrosine phosphorylation of p130^Cas in response to other G protein–coupled receptor ligands. Carbachol, LPA, vasopressin, bombesin, and endothelin all increased p130^Cas tyrosine phosphorylation in Swiss 3T3 cells.^{18 19 20} Furthermore, the G protein activator GTPS also increased the tyrosine phosphorylation of p125^Fak and paxillin in permeabilized Swiss 3T3 cells.²¹ These results strongly suggest a role for G_ß-dependent tyrosine phosphorylation of p130^Cas. To investigate this role in VSMCs, GTPS was scrape-loaded into quiescent VSMCs. However, both GTPS and sham-scraped control cells were found to have increased p130^Cas tyrosine phosphorylation compared with nonscraped cells (data not shown). As an alternate strategy, we treated cells with the G protein activator NaF. The mechanism of activation is the formation of AlF₄^-, which, in turn, stabilizes the active GTP-protein complex.²² Quiescent VSMCs were pretreated with NaF and stimulated with Ang II, and lysates were prepared. Extracts were immunoprecipitated with anti-Tyr(P) mAb and Western-blotted with anti-p130^Cas mAb (Figure 2B). In the absence of Ang II, 1 minute of NaF induced a 1.83±0.18-fold increase (n=3) in p130^Cas tyrosine phosphorylation compared with the 0-minute controls (lane 3 versus lane 1), thus suggesting a role for G_ß-dependent tyrosine phosphorylation of p130^Cas in VSMCs. We then measured the tyrosine phosphorylation of Cas from cells that were treated with Ang II alone versus those that were treated with NaF and Ang II (lane 2 versus lane 4). Although phosphorylation with NaF and Ang II tended to be greater than that with Ang II alone, statistical analysis of 3 separate experiments failed to show significance between the 2 groups. The same was true for the longer time points of NaF exposure (lanes 5 to 8). The absence of a significant additive effect for NaF and Ang II is consistent with a common signaling mechanism for these 2 activators.

The Role of Ca²⁺ in p130^Cas Tyrosine Phosphorylation
Since Ca²⁺ is known to be a critical signaling molecule in VSMCs, we investigated the role of both intracellular and extracellular Ca²⁺ on p130^Cas tyrosine phosphorylation. Quiescent VSMCs were pretreated with BAPTA-AM, a chelator of intracellular Ca²⁺. Since voltage-dependent L-type Ca²⁺ channels are expressed on VSMCs, we also used the Ca²⁺ channel blocker verapamil. Cells were stimulated with Ang II, and lysates were prepared. Extracts were immunoprecipitated with anti-Tyr(P) mAb and then Western-blotted with anti-p130^Cas mAb. The Ang II–induced tyrosine phosphorylation of p130^Cas was completely blocked with BAPTA-AM but not with verapamil (Figure 3A). These results suggest that p130^Cas tyrosine phosphorylation is dependent on intracellular Ca²⁺ but independent of extracellular Ca²⁺ entry. Further supporting the verapamil data was the observation that chelation of extracellular Ca²⁺ by EGTA had no effect on the Ang II–induced tyrosine phosphorylation of p130^Cas (Figure 3B).

View larger version (41K): [in this window] [in a new window]   Figure 3. The role of Ca2+ in p130Cas tyrosine phosphorylation. A, Quiescent VSMCs were pretreated as follows: 10 minutes with DMSO (control), 10 minutes with 50 µmol/L BAPTA-AM, and 0.5 minutes with 10 µmol/L verapamil. Cells were stimulated for 1 minute with saline (-) or 100 nmol/L Ang II (+). Lysates were immunoprecipitated with anti-Tyr(P) mAb and Western-blotted with anti-p130Cas mAb. IP indicates immunoprecipitation. B, Quiescent VSMCs were left untreated or treated for 0.5 minutes with 3 mmol/L EGTA. Cells were stimulated for 1 minute with saline (-) or 100 nmol/L Ang II (+), and the resulting anti-Tyr(P) mAb immunoprecipitates were blotted with anti-p130Cas mAb. C, Quiescent VSMCs were pretreated for 10 minutes with DMSO control (-) or 50 µmol/L BAPTA-AM (+). Cells were then stimulated for 1 minute with either saline control, 100 nmol/L Ang II, 100 nmol/L AVP, or 20 ng/mL PDGF-BB or for 30 minutes with 5 mmol/L NaF. Lysates were immunoprecipitated with anti-Tyr(P) mAb and Western-blotted with anti-p130Cas mAb. Shown is 1 of 3 representative experiments for each.

In contrast to the above result demonstrating a requirement for intracellular Ca²⁺, work in Swiss 3T3 cells has shown that the LPA-induced tyrosine phosphorylation of p130^Cas and other focal adhesion proteins is Ca²⁺ independent.²¹ To confirm our observation, quiescent VSMCs were stimulated with Ang II, AVP, PDGF-BB, or NaF. Extracts were immunoprecipitated with anti-Tyr(P) mAb and then Western-blotted with anti-p130^Cas mAb. All agonists increased the tyrosine phosphorylation of p130^Cas, and all were inhibited by BAPTA-AM (Figure 3C). These results strongly suggest that intracellular Ca²⁺ is critical for p130^Cas tyrosine phosphorylation in VSMCs regardless of whether the ligand activates a G protein–coupled receptor (Ang II, AVP, or NaF) or a tyrosine kinase growth factor receptor (PDGF).

The Role of c-Src in p130^Cas Tyrosine Phosphorylation
We next wanted to define the kinase(s) responsible for the Ang II–induced tyrosine phosphorylation of p130^Cas in VSMCs. c-Src is known to be activated by a broad range of G protein–coupled receptor ligands, including Ang II, platelet-activating factor, LPA, bombesin, and thrombin.^{13 23 24} It is also activated by growth factor receptors.¹³ Others have demonstrated a role for G_ß activation of c-Src.^{25 26} Recent work has also shown that c-Src can be activated by the Ca²⁺-dependent tyrosine kinase Pyk2.²⁷ Last, p130^Cas is known to be hyperphosphorylated in c-Src–transformed cells.¹⁰ The results of our characterization of p130^Cas tyrosine phosphorylation in VSMCs suggested a role for c-Src in that p130^Cas tyrosine phosphorylation was increased by ligands that activated G protein–coupled receptors, growth factor receptors, or activators of G_ß. In addition, all mechanisms of activation were found to be Ca²⁺ dependent.

To access the role of c-Src in the Ang II–induced tyrosine phosphorylation of p130^Cas, we pretreated cells with the Src inhibitor geldanamycin.²⁸ Cells were then stimulated with Ang II for the indicated times, and lysates were prepared. Extracts were immunoprecipitated with anti-p130^Cas mAb and then Western-blotted with anti-Tyr(P) mAbs. Geldanamycin completely blocked the Ang II–dependent phosphorylation of p130^Cas (Figure 4A). The membrane was then blotted with anti-p130^Cas mAb to demonstrate equal precipitation of Cas (Figure 4A). To further demonstrate the requirement of Src in Cas phosphorylation, we pretreated cells with varying doses of the specific Src family kinase inhibitor PP1.²⁹ Cells were then stimulated with Ang II for the indicated times, and lysates were prepared. Extracts were immunoprecipitated with anti-p130^Cas mAb and then Western-blotted with anti-Tyr(P) mAbs. The tyrosine phosphorylation of p130^Cas was eliminated by PP1 in a dose-dependent manner, with maximal inhibition occurring at 20 µmol/L (Figure 4B). To demonstrate that p130^Cas was precipitated equally across all lanes, the blot was subsequently probed with anti-p130^Cas mAb (Figure 4B). To further demonstrate the inhibitory effect of PP1, we pretreated cells with 20 µmol/L PP1 and then stimulated them with Ang II for the indicated times. Lysates were immunoprecipitated with anti-p130^Cas mAb and Western-blotted with anti-Tyr(P) mAbs. The tyrosine phosphorylation of p130^Cas was completely eliminated with 1 minute of Ang II and greatly reduced with 5 minutes of Ang II compared with DMSO controls (Figure 4C). To demonstrate that all lanes were loaded equally, the blot was stripped and probed with anti-p130^Cas mAb (Figure 4C). Taken together, these data indicate that an active Src family tyrosine kinase is critical for Ang II–mediated p130^Cas tyrosine phosphorylation.

View larger version (51K): [in this window] [in a new window]   Figure 4. The role of Src in p130Cas tyrosine phosphorylation. A, Quiescent VSMCs were pretreated for 12 hours with either DMSO (control) or 5 µmol/L geldanamycin (Geld). Cells were then treated with 100 nmol/L Ang II for the indicated times, and the resulting anti-p130Cas mAb immunoprecipitates were blotted with anti-Tyr(P) mAb. The same blot was probed with anti-p130Cas mAb to demonstrate equal loading. Size markers are in kilodaltons. Shown is 1 of 2 representative experiments. IP indicates immunoprecipitation. B, Quiescent VSMCs were pretreated for 30 minutes with increasing amounts of PP1. Cells were then stimulated with 100 nmol/L Ang II for the indicated times, and p130Cas immunoprecipitates were Western-blotted with anti-Tyr(P) mAbs. The same blot was probed with anti-p130Cas mAb to demonstrate equal loading. Size markers are in kilodaltons. Shown is 1 of 3 representative experiments. C, Quiescent VSMCs were pretreated for 30 minutes with DMSO (control) or with 20 µmol/L PP1. Cells were then stimulated with 100 nmol/L Ang II for the indicated times. Lysates were immunoprecipitated with anti-p130Cas mAb and Western-blotted with anti-Tyr(P) mAbs. To demonstrate equal loading, the same membrane was Western-blotted with anti-p130Cas mAb. Size markers are in kilodaltons. Shown is 1 of 2 representative experiments.

p130^Cas Immune Complex In Vitro Kinase Assay
The amino acid sequence of p130^Cas has led some to believe that it may function as an adaptor protein in signal transduction pathways.¹¹ This idea has been supported by studies that demonstrate interactions of p130^Cas with v-Src, v-Crk, and p125^Fak.^{9 10 30} To explore the role of p130^Cas as an adaptor protein in Ang II signal transduction, VSMCs were stimulated with Ang II for varying times, and lysates were prepared. p130^Cas was immunoprecipitated, and the resulting immune complexes were washed and resuspended in kinase buffer containing [-³²P]ATP. After separation on SDS-PAGE and transfer onto nitrocellulose, the proteins were exposed to film (Figure 5A). At least 11 different proteins were found to contain increased ³²P incorporation with 5 minutes of Ang II compared with the 0-minute controls (arrowheads). Since p130^Cas lacks intrinsic kinase activity, the increased ³²P incorporation is the result of catalytically active kinase(s) transferring the phosphate onto substrates. However, what cannot be determined is whether each individual band is a kinase or the substrate of a kinase. The membrane was subsequently blotted with anti-p130^Cas mAb to demonstrate equal loading (Figure 5A). As an alternate strategy to demonstrate that Cas binds active kinases in response to Ang II, we repeated the p130^Cas in vitro kinase assays, but this time the Src family tyrosine kinase substrate GAP p62 was present. Exposure of the film showed an Ang II–dependent increased ³²P incorporation into GAP p62 (Figure 5B). We again demonstrated that Cas was immunoprecipitated equally well by blotting the same membrane with anti-p130^Cas mAb (Figure 5B). Collectively, these studies demonstrate that p130^Cas does act as an adaptor protein, since it binds numerous phosphate-containing proteins and at least one Src family kinase in response to Ang II.

View larger version (28K): [in this window] [in a new window]   Figure 5. p130Cas immune complex in vitro kinase assays. Quiescent VSMCs were stimulated with 100 nmol/L Ang II for the indicated times, and lysates were immunoprecipitated with anti-p130Cas mAb. A, Precipitates were resuspended in kinase buffer containing [-32P]ATP. After SDS-PAGE, radiolabeled proteins were transferred onto nitrocellulose and exposed to film. The membrane was then Western-blotted with anti-p130Cas mAb to demonstrate equal loading across all lanes. Size markers are in kilodaltons. Shown is 1 of 2 representative experiments. IP indicates immunoprecipitation. B, The Src family tyrosine kinase substrate GAP p62 was added to each 32P-containing kinase reaction. After separation on SDS-PAGE and transfer onto nitrocellulose,32P incorporation into GAP p62 was determined by exposing the membrane to film. The membrane was then blotted with anti-p130Cas mAb to demonstrate equal loading across all lanes. Size markers are in kilodaltons. Shown is 1 of 3 representative experiments.

p130^Cas Protein Complex Formation
Our next objective was to begin to identify some of the proteins that were observed in the p130^Cas immune complex assays. To do this, we looked for temporal association of it with other signaling proteins. Quiescent cells were stimulated with Ang II for 0, 1, 5, 20, 40, and 60 minutes. Lysates were immunoprecipitated with anti-p130^Cas mAb, separated by SDS-PAGE, and transferred onto nitrocellulose. Because of our results indicating the importance of a Src family tyrosine kinase in mediating Cas phosphorylation, the blot was probed with anti-Src mAb. c-Src protein was detected across all lanes and did not significantly increase with Ang II treatment as measured in 3 separate experiments (Figure 6A). This result suggests that increased tyrosine phosphorylation of p130^Cas does not increase its association with c-Src. What cannot be determined from this blot is the relative kinase activity of c-Src.

View larger version (48K): [in this window] [in a new window]   Figure 6. p130Cas protein/protein complex formation. A, Quiescent VSMCs were stimulated with 100 nmol/L Ang II for the indicated times, and lysates were prepared. Lysates were immunoprecipitated with anti-p130Cas mAb and Western-blotted as indicated. Size markers are in kilodaltons. Shown is 1 of 3 representative experiments. IP indicates immunoprecipitation. B, Quiescent VSMCs were stimulated with 100 nmol/L Ang II for the indicated times, and lysates were immunoprecipitated as indicated. The precipitates were then blotted as indicated. Shown is 1 of 2 representative experiments for each.

The same blot was also probed with anti-PKC mAb. In VSMCs, PKC is known to mediate proliferation and regulate -actin expression.^{31 32} It is also a member of the Ca²⁺-dependent subfamily of isoforms. Because our data indicated that an intact actin network and intracellular Ca²⁺ were critical for Ang II–induced tyrosine phosphorylation of p130^Cas, we looked for an interaction between p130^Cas and PKC in response to Ang II. At 1 minute, there was significant increased association (1.77±0.08-fold, n=3) of PKC with p130^Cas over the 0-minute controls (Figure 6A). This slowly returned to basal levels by 60 minutes.

The blot was then probed for the cell adhesion signaling molecule pp120. This molecule is a known substrate of c-Src and is tyrosine-phosphorylated in response to growth factor or integrin receptor activation.^{33 34} We have previously shown that Ang II stimulation of VSMCs leads to increased tyrosine phosphorylation of pp120 and that this is c-Src dependent.⁸ An association between p130^Cas and pp120 was observed, but with a temporal pattern that was different from both c-Src and PKC. Peak complex formation (2.07±0.12-fold, n=3) did not occur until 20 minutes after the addition of Ang II (Figure 6A). To demonstrate equal loading, the blot was probed with anti-p130^Cas mAb (Figure 6A).

To demonstrate the specificity of our p130^Cas interactions, the immunoprecipitating anti-p130^Cas mAb was omitted from the protocol in 2 separate experiments. The result was a loss of signal for c-Src, PKC, and pp120 (data not shown). Subsequent Cas immunoprecipitates were probed for 2 additional focal adhesion–associated proteins, phosphatidylinositol-3-kinase and the protein tyrosine phosphatase SHP2. No association was detected with these proteins (data not shown). The SHP2 result, however, is in contrast to a recent report that described an integrin-mediated association of p130^Cas with p59^Fyn and SHP2.³⁵

To confirm the specific Cas interactions, we reversed the order of antibody addition. Stimulated VSMC lysates were immunoprecipitated with either anti-Src, anti-PKC, or anti-pp120 and then blotted with anti-p130^Cas. Blotting of the anti-Src polyclonal antibody immunoprecipitates with anti-p130^Cas again demonstrated that the binding of Src and Cas was largely independent of Ang II treatment (Figure 6B, top, first blot). The levels of precipitated Src were confirmed by blotting with anti-Src mAb (Figure 6B, top, second blot).

Blotting of the anti-PKC immunoprecipitates with anti-p130^Cas again demonstrated an Ang II–dependent association between PKC and Cas (Figure 6B, middle, first blot). Peak association was observed at 5 minutes, as measured in 2 separate experiments. The protocol in Figure 6A demonstrated peak association at 1 minute. Therefore, we conclude that the association between PKC and Cas occurs within 5 minutes of Ang II treatment. Equal precipitation of PKC was demonstrated by blotting the same membrane with anti-PKC mAb (Figure 6B, middle, second blot).

When the anti-pp120 immunoprecipitates were blotted with anti-p130^Cas mAb, we again observed a significant Ang II–dependent association between pp120 and Cas (Figure 6B, bottom, first blot). Similar to the data in Figure 6A, peak association between pp120 and Cas occurred 20 minutes after Ang II treatment. We confirmed the level of precipitated pp120 by blotting the same membrane with anti-pp120 mAb (Figure 6B, bottom, second blot).

Collectively, our protein complex formation data indicate that p130^Cas specifically interacts with PKC and pp120 in an Ang II–dependent manner, whereas Cas specifically binds Src in a manner that is independent of Ang II treatment.

The Role of PKC in p130^Cas Tyrosine Phosphorylation
The Ang II–dependent association between Cas and PKC represented a novel observation in that review of the literature found no reports of the binding of Cas to any PKC isoform. We now wanted to determine whether PKC bound p130^Cas as a consequence of Cas phosphorylation or was required by PKC to mediate Cas tyrosine phosphorylation. To address this issue, we pretreated cells with the PKC inhibitor calphostin C. Cells were then stimulated with Ang II. The resulting lysates were immunoprecipitated with anti-p130^Cas mAb and then Western-blotted with anti-Tyr(P) mAbs. Calphostin C greatly reduced the Ang II–mediated p130^Cas tyrosine phosphorylation compared with DMSO controls (Figure 7A). To demonstrate equal loading, the membrane was Western-blotted with anti-p130^Cas mAb (Figure 7A). These data suggest that PKC activation lies upstream of Cas tyrosine phosphorylation. In a complementary protocol, we downregulated PKC activity by long-term exposure to PMA. p130^Cas immunoprecipitates were again Western-blotted with anti-Tyr(P) mAbs. Inhibition of PKC activity by PMA treatment resulted in decreased p130^Cas tyrosine phosphorylation compared with DMSO controls (Figure 7B). The membrane was then blotted with anti-p130^Cas mAb to demonstrate equal loading (Figure 7B). Although the PMA was not as effective as calphostin C, taken together, the data strongly suggest that PKC activation precedes Cas tyrosine phosphorylation.

View larger version (39K): [in this window] [in a new window]   Figure 7. Role of PKC in p130Cas tyrosine phosphorylation. A, Quiescent VSMCs were pretreated for 15 minutes with DMSO (control) or 1 µmol/L calphostin C. Cells were then stimulated with 100 nmol/L Ang II, and the resulting p130Cas immunoprecipitates were Western-blotted with anti-Tyr(P) mAbs. The same blot was probed with anti-p130Cas mAb. Size markers are in kilodaltons. Shown is 1 of 3 representative experiments. IP indicates immunoprecipitation. B, Quiescent VSMCs were pretreated for 18 hours with DMSO (control) or 1 µmol/L PMA. Cells were then stimulated with 100 nmol/L Ang II, and the resulting p130Cas immunoprecipitates were Western-blotted with anti-Tyr(P) mAbs. The same blot was probed with anti-p130Cas mAb. Size markers are in kilodaltons. Shown is 1 of 2 representative experiments. C, Quiescent VSMCs were pretreated for 1 hour with either DMSO (control) or 100 µmol/L PD90859. Cells were then stimulated with 100 nmol/L Ang II, and the resulting p130Cas immunoprecipitates were Western-blotted with anti-Tyr(P) mAbs. The same blot was probed with anti-p130Cas mAb. Size markers are in kilodaltons. Shown is 1 of 2 representative experiments.

Distal to PKC is MEK activation and subsequent entry into the mitogen-activated protein kinase pathway. Pretreatment of cells with the MEK inhibitor PD90859, at a concentration that was 3- to 4-fold higher than that required to completely block MEK activity,^{36 37} reduced the Ang II–induced p130^Cas tyrosine phosphorylation by only 15±4.3% compared with DMSO controls (Figure 7C). Because the inhibition by BAPTA-AM, PP1, and calphostin C reduced the Ang II–mediated tyrosine phosphorylation of p130^Cas by >90%, we believe that MEK activation is not required for p130^Cas tyrosine phosphorylation. Equal precipitation of Cas was confirmed by blotting the membrane with anti-p130^Cas mAb (Figure 7C).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
p130^Cas was initially characterized as a phosphotyrosine-containing protein in v-Crk and v-Src transformed cells.^{9 10} Cloning and expression of the p130^Cas cDNA found it to form stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation–dependent manner.¹¹ Because Ang II is known to initiate a series a tyrosine phosphorylation signal cascades on binding the AT₁ receptor, we attempted to understand the regulation of p130^Cas tyrosine phosphorylation in VSMCs by Ang II. We found that the phosphorylation is dependent on intracellular Ca²⁺ and an intact actin cytoskeletal network. The requirement of intracellular Ca²⁺ is seen whether activation initiates from G protein–coupled receptors or tyrosine kinase growth factor receptors. This phosphorylation is also Src dependent, since it is blocked by the Src family tyrosine kinase inhibitors geldanamycin and PP1. Our in vitro kinase assays demonstrate that p130^Cas serves as an adaptor protein in Ang II signal transduction, since it binds catalytically active molecules in response to Ang II. Two of these kinases were identified as c-Src and PKC. The integrin-signaling molecule, pp120, also bound Cas in an Ang II–dependent manner. Both c-Src and PKC kinase activities are required for Ang II–mediated p130^Cas tyrosine phosphorylation. The Ang II–mediated association of pp120 and p130^Cas may imply that p130^Cas may serve as a convergence point for 3 different signaling pathways, namely, the serine/threonine PKC pathway, the cell adhesion–mediated pp120 pathway, and the c-Src tyrosine kinase pathway.

Although active c-Src and PKC molecules are required for Ang II–induced tyrosine phosphorylation, it remains possible that other kinase(s) are involved in this mechanism. Two such candidates are the tyrosine kinases Pyk2 and p125^Fak. Recent work has shown that Pyk2 and p125^Fak are activated by Ang II in a Ca²⁺-dependent manner.³⁸ In addition, both kinases bind to Src via a phosphotyrosine interaction with the SH2 domain of Src.^{29 39} Because of the requirement for intracellular Ca²⁺ for the Ang II–mediated Cas phosphorylation in VSMCs, one or both of these molecules may play a role in this signal transduction process. It also remains possible that an additional isoform of PKC may mediate Cas phosphorylation. However, Western blot analysis of Cas immunoprecipitates with an anti-PKC antibody that recognizes all isoforms reveals a single dominant band at 82 kDa (authors' unpublished data, 1998).

Our studies indicate that Src, intracellular Ca²⁺, and PKC are required for Cas phosphorylation. This suggests that Cas phosphorylation is perhaps mediated via the inositol hydrolysis pathway. Specifically, an active Src phosphorylates phospholipase C-1, which, in turn, hydrolyzes phosphatidylinositol diphosphate to the second-messenger molecules IP₃ and DAG. IP₃ binds to its receptors on the endoplasmic reticulum, resulting in a transient increase of intracellular Ca²⁺. DAG, in turn, activates PKC. PKC is classified as a conventional isoform, and its activation requires both Ca²⁺ and DAG. Thus, inhibition of Src, intracellular Ca²⁺, or PKC would presumably block PKC activation and subsequent Cas phosphorylation. This mechanism is supported by the observations that treatment of VSMCs with either PP1, BAPTA-AM, or calphostin C results in little to no Cas phosphorylation by Ang II.

Perhaps the most interesting result from these studies was the observation that an active PKC molecule is required for Cas phosphorylation. As shown in Figure 6, we believe that the PKC isoform required for Cas phosphorylation is PKC. Its activation requires both Ca²⁺ and DAG, and previous studies in VSMCs have demonstrated that increased PKC expression leads to (1) increased -actin expression and (2) cellular proliferation.^{31 32} VSMC proliferation is an important event in vascular biology, and abnormal VSMC proliferation has long been associated with vascular disease.⁴⁰ Since p130^Cas tyrosine phosphorylation is coincident with changes in cell morphology,¹⁴ its binding to PKC may suggest that it serves to bridge proliferative and structural responses in vivo. Another possibility is that PKC may activate an intermediary kinase, such as p125^Fak, which, in turn, phosphorylates Cas. One report demonstrated that PKC activity is required for p125^Fak activation.⁴¹ Our results appear to be similar in that PKC activation is required for Cas phosphorylation. A more recent report demonstrated a PKC-dependent tyrosine phosphorylation of p130^Cas in differentiating neuroblastoma cells.⁴² Inhibition of PKC resulted in decreased Cas phosphorylation and a coincident retraction of growth cone filopodia. Whether Cas phosphorylation mediates similar growth responses in VSMCs remains to be investigated. In addition to unidentified kinases, it is also very likely that cytoplasmic protein tyrosine phosphatases will mediate Cas phosphorylation. A recent report identified Cas as a substrate for the cellular phosphatase PTP-PEST.⁴³ Given that p130^Cas binds numerous phosphate-containing proteins in response to Ang II, identifying those that are distal to PKC activation will help to better understand this signal transduction process.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II anti-Tyr(P) = anti-phosphotyrosine AVP = arginine vasopressin DAG = diacylglycerol DMSO = dimethyl sulfoxide IP3 = inositol 1,4,5-trisphosphate LPA = lysophosphatidic acid mAb = monoclonal antibody MEK = extracellular signal–regulated kinase kinase PDGF = platelet-derived growth factor PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate TBST = Tris-buffered saline with Tween 20 VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported by National Institutes of Health grants DK-39777, DK-44280, DK-45215, DK-51445, and HL-47035. Dr Sayeski has been supported by grants T32-DK07298 and F32-HL09678. We wish to thank Drs Daniel Semeniuk and Thanh Doan for their helpful comments. We also thank Shaun Benford for administrative assistance and Elisabeth Seal for tissue culture maintenance.

Received October 15, 1997; accepted April 7, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JAM, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev.. 1993;45:205–251.[Medline] [Order article via Infotrieve]

2. Schieffer B, Bernstein KE, Marrero MB. The role of tyrosine phosphorylation in angiotensin II mediated intracellular signaling and cell growth. J Mol Med.. 1996;74:85–91.[Medline] [Order article via Infotrieve]

3. Bernstein KE, Marrero MB. The importance of tyrosine phosphorylation in angiotensin II signaling. Trends Cardiovasc Med.. 1996;6:179–187.

4. Leduc L, Meloche S. Angiotensin II stimulates tyrosine phosphorylation of the focal adhesion-associated protein paxillin in aortic smooth muscle cells. J Biol Chem.. 1995;270:4401–4404.[Abstract/Free Full Text]

5. Marrero MB, Paxton WG, Duff JL, Berk BC, Bernstein KE. Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-1 in vascular smooth muscle cells. J Biol Chem.. 1994;269:10935–10939.[Abstract/Free Full Text]

6. Scheiffer B, Marrero MB, Paxton WG, Bernstein KE. Angiotensin II controls p21^ras activity via pp60^c-src. J Biol Chem.. 1996;271:10329–10333.[Abstract/Free Full Text]

7. Marrero MB, Scheiffer B, Paxton WG, Heerdt L, Berk BC, Delafontaine P, Bernstein KE. Direct stimulation of Jak/STAT pathway by the angiotensin II AT₁ receptor. Nature.. 1995;375:247–250.[Medline] [Order article via Infotrieve]

8. Marrero MB, Scheiffer B, Paxton WG, Scheiffer E, Bernstein KE. Electroporation of pp60^c-src antibodies inhibits the angiotensin II activation of phospholipase c-1 in rat aortic muscle cells. J Biol Chem.. 1995;270:15734–15738.[Abstract/Free Full Text]

9. Matsuda M, Mayer BJ, Fukui Y, Hanafusa H. Binding of transforming protein, P47^gag-crk, to a broad range of phosphotyrosine-containing proteins. Science.. 1990;248:1537–1539.[Abstract/Free Full Text]

10. Kanner SB, Reynolds AB, Wang HC, Vines RR, Parsons JT. Activation of pp60^c-src tyrosine kinase specific activity in tumor-derived Syrian hamster embryo cells. EMBO J.. 1991;10:1689–1698.[Medline] [Order article via Infotrieve]

11. Sakai R, Iwamatsu A, Hirano N, Ogawa S, Tanaka T, Mano H, Yazaki Y, Hirai H. A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner. EMBO J.. 1994;13:3748–3756.[Medline] [Order article via Infotrieve]

12. Rozengurt E. Convergent signaling in the action of integrins, neuropeptides, growth factors and oncogenes. Cancer Surv.. 1995;24:81–96.[Medline] [Order article via Infotrieve]

13. Erpel T, Courtneidge SA. Src family protein tyrosine kinases and cellular signal transduction pathways. Curr Opin Cell Biol.. 1995;7:176–182.[Medline] [Order article via Infotrieve]

14. Nojima Y, Morino N, Mimura T, Hamasaki K, Furuya H, Sakai R, Sato T, Tachibana K, Morimoto C, Yazaki Y, Hirai H. Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130^Cas, a src homology 3-containing molecule having multiple src homology 2-binding motifs. J Biol Chem.. 1995;270:15398–15402.[Abstract/Free Full Text]

15. Gunther S, Alexander RW, Atkinson WJ, Gimbrone MA. Functional angiotensin II receptors in cultured vascular smooth muscle cells. J Cell Biol.. 1982;92:289–298.[Abstract/Free Full Text]

16. Seufferlein T, Rozengurt E. Sphingosylphosphorylcholine rapidly induces tyrosine phosphorylation of p125^Fak and paxillin, rearrangement of the actin cytoskeleton and focal contact assembly. J Biol Chem.. 1995;270:24343–24351.[Abstract/Free Full Text]

17. Earp HS, Huckle WR, Dawson TL, Li X, Graves LM, Dy R. Angiotensin II activates at least two tyrosine kinases in rat liver epithelial cells. J Biol Chem.. 1995;270:28440–28447.[Abstract/Free Full Text]

18. Gutkind JS, Robbins KC. Activation of transforming G protein coupled receptors induces rapid tyrosine phosphorylation of cellular proteins, including p125^Fak and the p130 v-src substrate. Biochem Biophys Res Commun. 1992;188:155–161.[Medline] [Order article via Infotrieve]

19. Seufferlein T, Rozengurt E. Lysophosphatidic acid stimulates tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130. J Biol Chem.. 1994;269:9345–9351.[Abstract/Free Full Text]

20. Zachary I, Sinnett-Smith J, Rozengurt E. Bombesin, vasopressin, and endothelin stimulation of tyrosine phosphorylation in Swiss 3T3 cells. J Biol Chem.. 1992;267:19031–19034.[Abstract/Free Full Text]

21. Seckl MJ, Morii N, Narumiya S, Rozengurt E. Guanosine 5'-3-O-(thio)triphosphate stimulates tyrosine phosphorylation of p125^FAK and paxillin in permeabilized Swiss 3T3 cells. J Biol Chem.. 1995;270:6984–6990.[Abstract/Free Full Text]

22. Blackmore PF, Bocckino SB, Waynick LE, Exton JH. Role of guanine nucleotide-binding regulatory protein in the hydrolysis of hepatocyte phosphatidylinositol 4,5-bisphosphate by calcium mobilizing hormones and the control of cell calcium: studies utilizing aluminum fluoride. J Biol Chem.. 1985;260:14477–14488.[Abstract/Free Full Text]

23. Ishida M, Marrero MB, Schieffer B, Bernstein KE, Berk BC. Angiotensin II activates pp60^c-src in vascular smooth muscle cells. Circ Res.. 1995;77:1053–1059.[Abstract/Free Full Text]

24. Dhar A, Shukla SD. Electrotransjection of pp60^v-src monoclonal antibody inhibits activation of phospholipase C in platelets. J Biol Chem.. 1994;269:9123–9127.[Abstract/Free Full Text]

25. Li S, Couet J, Lisanti MP. Src tyrosine kinases, G_a subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin. J Biol Chem.. 1996;271:29182–29190.[Abstract/Free Full Text]

26. Luttrell LM, Hawes BE, van Biesen T, Luttrell DK, Lansing TJ, Lefkowitz RJ. Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbg subunit-mediated activation of mitogen-activated protein kinases. J Biol Chem.. 1996;271:19443–19450.[Abstract/Free Full Text]

27. Dikic I, Tokiwa G, Lev S, Courtneidge SA, Schlessinger J. A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation. Nature.. 1996;383:547–550.[Medline] [Order article via Infotrieve]

28. Yamaki H, Nakajima M, Seimiya H, Saya H, Sugita M, Tsuruo T. Inhibition of the association with nuclear matrix of pRB, p70, and p40 proteins along with specific suppression of c-myc expression by geldanamycin, an inhibitor of Src tyrosine kinase. J Antibiot (Tokyo).. 1995;48:1021–1026.[Medline] [Order article via Infotrieve]

29. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. J Biol Chem.. 1996;271:695–701.[Abstract/Free Full Text]

30. Harte MT, Hildebrand JD, Burnham MR, Bouton AH, Parsons JT. p130^Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase. J Biol Chem.. 1996;271:13649–13655.[Abstract/Free Full Text]

31. Leszczynski D, Joenvaara S, Foegh ML. Protein kinase C- regulates proliferation but not apoptosis in rat coronary vascular smooth muscle cells. Life Sci.. 1996;58:599–606.[Medline] [Order article via Infotrieve]

32. Haller H, Lindschau C, Quass P, Distler A, Luft FC. Differentiation of vascular smooth muscle cells and the regulation of protein kinase C-. Circ Res.. 1995;76:21–29.[Abstract/Free Full Text]

33. Reynolds AB, Herbert L, Cleveland JL, Berg ST, Gaut JR. p120, a novel substrate of protein tyrosine kinase receptors and of pp60^v-src, is related to cadherin-binding factors beta-catenin, plakoglobin and armadillo. Oncogene.. 1992;7:2439–2445.[Medline] [Order article via Infotrieve]

34. Kim L, Wong TW. The cytoplasmic tyrosine kinase FER is associated with the catenin-like substrate pp120 and is activated by growth factors. Mol Cell Biol.. 1995;15:4553–4561.[Abstract]

35. Manie SN, Astier A, Haghayeghi N, Canty T, Druker BJ, Hirai H, Freedman AS. Regulation of integrin-mediated p130^Cas tyrosine phosphorylation in human B cells. J Biol Chem.. 1997;272:15636–15641.[Abstract/Free Full Text]

36. Cook SJ, Beltman J, Cadwallader KA, McMahon M, McCormick F. Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-related kinase-dependent and Ca⁺²-dependent signal pathways in rat-1 cells. J Biol Chem. 1997;272:13309–13319.[Abstract/Free Full Text]

37. Servant MJ, Giasson E, Meloche S. Inhibition of growth factor-induced protein synthesis by a selective MEK inhibitor in aortic smooth muscle cells. J Biol Chem.. 1996;271:16047–16052.[Abstract/Free Full Text]

38. Li X, Earp HS. Paxillin is tyrosine-phosphorylated by and preferentially associates with the calcium-dependent tyrosine kinase in rat liver epithelial cells. J Biol Chem.. 1997;272:14341–14348.[Abstract/Free Full Text]

39. Schlaepfer DD, Hunter T. Focal adhesion kinase overexpression enhances Ras-dependent integrin signaling to ERK2/mitogen-activated protein kinase through interactions with and activation of c-Src. J Biol Chem.. 1997;272:13189–13195.[Abstract/Free Full Text]

40. Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascular disease. Circ Res.. 1986;58:427–444.[Abstract/Free Full Text]

41. Vuori K, Ruoslahti E. Activation of protein kinase C precedes ₅ß₁ integrin-mediated cell spreading on fibronectin. J Biol Chem.. 1993;268:21459–21462.[Abstract/Free Full Text]

42. Fagerstrom S, Pahlman S, Nanberg E. Protein kinase C-dependent tyrosine phosphorylation of p130^Cas in differentiating neuroblastoma cells. J Biol Chem.. 1998;273:2336–2343.[Abstract/Free Full Text]

43. Garton AJ, Flint AJ, Tonks NK. Identification of p130^Cas as a substrate for the cytosolic protein tyrosine phosphatase PTP-PEST. Mol Cell Biol.. 1996;16:6408–6418.[Abstract]

This article has been cited by other articles:

				M. D. Godeny and P. P. Sayeski ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKC{zeta}-controlled ERK1/2 activity within the nucleus Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1297 - C1307. [Abstract] [Full Text] [PDF]

				M. D. Godeny and P. P. Sayeski ERK1/2 regulates ANG II-dependent cell proliferation via cytoplasmic activation of RSK2 and nuclear activation of elk1 Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1308 - C1317. [Abstract] [Full Text] [PDF]

				T.-J. Park, K. Boyd, and T. Curran Cardiovascular and Craniofacial Defects in Crk-Null Mice. Mol. Cell. Biol., August 1, 2006; 26(16): 6272 - 6282. [Abstract] [Full Text] [PDF]

				T. A. Wallace, D. VonDerLinden, K. He, S. J. Frank, and P. P. Sayeski Microarray analyses identify JAK2 tyrosine kinase as a key mediator of ligand-independent gene expression Am J Physiol Cell Physiol, October 1, 2004; 287(4): C981 - C991. [Abstract] [Full Text] [PDF]

				E. M. Sandberg and P. P. Sayeski Jak2 Tyrosine Kinase Mediates Oxidative Stress-induced Apoptosis in Vascular Smooth Muscle Cells J. Biol. Chem., August 13, 2004; 279(33): 34547 - 34552. [Abstract] [Full Text] [PDF]

				M. Kyaw, M. Yoshizumi, K. Tsuchiya, S. Kagami, Y. Izawa, Y. Fujita, N. Ali, Y. Kanematsu, K. Toida, K. Ishimura, et al. Src and Cas Are Essentially but Differentially Involved in Angiotensin II-Stimulated Migration of Vascular Smooth Muscle Cells via Extracellular Signal-Regulated Kinase 1/2 and c-Jun NH2-Terminal Kinase Activation Mol. Pharmacol., April 1, 2004; 65(4): 832 - 841. [Abstract] [Full Text]

				B. Callaghan, S.D. Koh, and K.D. Keef Muscarinic M2 Receptor Stimulation of Cav1.2b Requires Phosphatidylinositol 3-Kinase, Protein Kinase C, and c-Src Circ. Res., March 19, 2004; 94(5): 626 - 633. [Abstract] [Full Text] [PDF]

				E. M. Sandberg, X. Ma, D. VonDerLinden, M. D. Godeny, and P. P. Sayeski Jak2 Tyrosine Kinase Mediates Angiotensin II-dependent Inactivation of ERK2 via Induction of Mitogen-activated Protein Kinase Phosphatase 1 J. Biol. Chem., January 16, 2004; 279(3): 1956 - 1967. [Abstract] [Full Text] [PDF]

				H. Kodama, K. Fukuda, E. Takahashi, S. Tahara, Y. Tomita, M. Ieda, K. Kimura, K. M. Owada, K. Vuori, and S. Ogawa Selective Involvement of p130Cas/Crk/Pyk2/c-Src in Endothelin-1-Induced JNK Activation Hypertension, June 1, 2003; 41(6): 1372 - 1379. [Abstract] [Full Text] [PDF]

				C. Berry, R. Touyz, A. F. Dominiczak, R. C. Webb, and D. G. Johns Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2337 - H2365. [Abstract] [Full Text] [PDF]

				L. R. James, A. Ingram, H. Ly, K. Thai, L. Cai, and J. W. Scholey Angiotensin II activates the GFAT promoter in mesangial cells Am J Physiol Renal Physiol, July 1, 2001; 281(1): F151 - F162. [Abstract] [Full Text] [PDF]

				P. Rocic, G. Govindarajan, A. Sabri, and P. A. Lucchesi A role for PYK2 in regulation of ERK1/2 MAP kinases and PI 3-kinase by ANG II in vascular smooth muscle Am J Physiol Cell Physiol, January 1, 2001; 280(1): C90 - C99. [Abstract] [Full Text] [PDF]

				R. M. Touyz and E. L. Schiffrin Signal Transduction Mechanisms Mediating the Physiological and Pathophysiological Actions of Angiotensin II in Vascular Smooth Muscle Cells Pharmacol. Rev., December 1, 2000; 52(4): 639 - 672. [Abstract] [Full Text] [PDF]

				J. L. Hall, C. M. Matter, X. Wang, and G. H. Gibbons Hyperglycemia Inhibits Vascular Smooth Muscle Cell Apoptosis Through a Protein Kinase C-Dependent Pathway Circ. Res., September 29, 2000; 87(7): 574 - 580. [Abstract] [Full Text] [PDF]

				J. Haendeler, M. Ishida, L. Hunyady, and B. C. Berk The Third Cytoplasmic Loop of the Angiotensin II Type 1 Receptor Exerts Differential Effects on Extracellular Signal-Regulated Kinase (ERK1/ERK2) and Apoptosis via Ras- and Rap1-Dependent Pathways Circ. Res., April 14, 2000; 86(7): 729 - 736. [Abstract] [Full Text] [PDF]

				H. Tang, Z. J. Zhao, E. J. Landon, and T. Inagami Regulation of Calcium-sensitive Tyrosine Kinase Pyk2 by Angiotensin II in Endothelial Cells. ROLES OF Yes TYROSINE KINASE AND TYROSINE PHOSPHATASE SHP-2 J. Biol. Chem., March 17, 2000; 275(12): 8389 - 8396. [Abstract] [Full Text] [PDF]

				S. Kim and H. Iwao Molecular and Cellular Mechanisms of Angiotensin II-Mediated Cardiovascular and Renal Diseases Pharmacol. Rev., March 1, 2000; 52(1): 11 - 34. [Abstract] [Full Text] [PDF]

				P. P. Sayeski, M. S. Ali, A. Safavi, M. Lyles, S.-O. Kim, S. J. Frank, and K. E. Bernstein A Catalytically Active Jak2 Is Required for the Angiotensin II-dependent Activation of Fyn J. Biol. Chem., November 12, 1999; 274(46): 33131 - 33142. [Abstract] [Full Text] [PDF]

				M. Okuda, M. Takahashi, J. Suero, C. E. Murry, O. Traub, H. Kawakatsu, and B. C. Berk Shear Stress Stimulation of p130cas Tyrosine Phosphorylation Requires Calcium-dependent c-Src Activation J. Biol. Chem., September 17, 1999; 274(38): 26803 - 26809. [Abstract] [Full Text] [PDF]

				Z. Zhang, L. Hernandez-Lagunas, W. C. Horne, and R. Baron Cytoskeleton-dependent Tyrosine Phosphorylation of the p130Cas Family Member HEF1 Downstream of the G Protein-coupled Calcitonin Receptor. CALCITONIN INDUCES THE ASSOCIATION OF HEF1, PAXILLIN, AND FOCAL ADHESION KINASE J. Biol. Chem., August 27, 1999; 274(35): 25093 - 25098. [Abstract] [Full Text] [PDF]

				P. P. Sayeski, M. S. Ali, K. Hawks, S. J. Frank, and K. E. Bernstein The Angiotensin II–Dependent Association of Jak2 and c-Src Requires the N-Terminus of Jak2 and the SH2 Domain of c-Src Circ. Res., June 11, 1999; 84(11): 1332 - 1338. [Abstract] [Full Text] [PDF]

				J. Sadoshima Versatility of the Angiotensin II Type 1 Receptor Circ. Res., June 29, 1998; 82(12): 1352 - 1355. [Full Text] [PDF]

				Z. Zhang, R. Baron, and W. C. Horne Integrin Engagement, the Actin Cytoskeleton, and c-Src Are Required for the Calcitonin-induced Tyrosine Phosphorylation of Paxillin and HEF1, but Not for Calcitonin-induced Erk1/2 Phosphorylation J. Biol. Chem., November 17, 2000; 275(47): 37219 - 37223. [Abstract] [Full Text] [PDF]

				T. Ohmori, Y. Yatomi, H. Okamoto, Y. Miura, G. Rile, K. Satoh, and Y. Ozaki Gi-mediated Cas Tyrosine Phosphorylation in Vascular Endothelial Cells Stimulated with Sphingosine 1-Phosphate. POSSIBLE INVOLVEMENT IN CELL MOTILITY ENHANCEMENT IN COOPERATION WITH Rho-MEDIATED PATHWAYS J. Biol. Chem., February 9, 2001; 276(7): 5274 - 5280. [Abstract] [Full Text] [PDF]

				K. Suzuki-Inoue, Y. Ozaki, M. Kainoh, Y. Shin, Y. Wu, Y. Yatomi, T. Ohmori, T. Tanaka, K. Satoh, and T. Morita Rhodocytin Induces Platelet Aggregation by Interacting with Glycoprotein Ia/IIa (GPIa/IIa, Integrin alpha 2beta 1). INVOLVEMENT OF GPIa/IIa-ASSOCIATED Src AND PROTEIN TYROSINE PHOSPHORYLATION J. Biol. Chem., January 5, 2001; 276(2): 1643 - 1652. [Abstract] [Full Text] [PDF]

				K. C. Chung, J. Y. Sung, W. Ahn, H. Rhim, T. H. Oh, M. G. Lee, and Y. S. Ahn Intracellular Calcium Mobilization Induces Immediate Early Gene pip92 via Src and Mitogen-activated Protein Kinase in Immortalized Hippocampal Cells J. Biol. Chem., January 12, 2001; 276(3): 2132 - 2138. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sayeski, P. P. Articles by Bernstein, K. E. Search for Related Content PubMed PubMed Citation Articles by Sayeski, P. P. Articles by Bernstein, K. E.