Phosphorylation of p130Cas by Angiotensin II Is Dependent on c-Src, Intracellular Ca2+, and Protein Kinase C
Abstract—p130Cas 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 p130Cas tyrosine phosphorylation in vascular smooth muscle cells by angiotensin II (Ang II). This ligand induced a transient increase in p130Cas 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 p130Cas 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 p130Cas to bind at least 11 different phosphate-containing proteins. Analysis of these proteins revealed that protein kinase Cα and the cell adhesion signaling molecule pp120 formed temporal associations with p130Cas in response to Ang II. c-Src was found to associate with p130Cas 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 p130Cas. These results are the first to demonstrate that the tyrosine phosphorylation of p130Cas by Ang II is transduced by the Src, intracellular Ca2+, protein kinase C signaling pathway.
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-γ1, p120Ras-GAP, Jak2, Stat1, Stat3, Tyk2, and the intracellular tyrosine kinase pp60c-src.4 5 6 7 8
p130Cas (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 p130Cas 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 p130Cas 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. p130Cas 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 p130Cas is thought to recruit cytoskeletal signaling molecules such as p125Fak, 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 p130Cas tyrosine phosphorylation by Ang II. In the present study, we demonstrate that p130Cas 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 p130Cas 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, PKCα, and the cell adhesion signaling molecule pp120. These results are the first to demonstrate that the tyrosine phosphorylation of p130Cas by Ang II is transduced by the Src, intracellular Ca2+, PKC signaling pathway and suggest that in VSMCs, p130Cas 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
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.
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 Dc 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).
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-p130Cas, monoclonal anti-pp120, monoclonal anti-PKCα, 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-pp60c-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
p130Cas 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 [γ-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.
Ang II Induces Tyrosine Phosphorylation of p130Cas
To study the regulation of p130Cas tyrosine phosphorylation in VSMCs, we treated cells with Ang II for varying times. Lysates were immunoprecipitated with anti-p130Cas mAb and analyzed by Western blotting with anti-Tyr(P) mAbs. Ang II stimulated tyrosine phosphorylation of p130Cas 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-p130Cas mAb (Figure 1B⇓). p130Cas was tyrosine-phosphorylated with a time course that was similar to that seen in Figure 1A⇓, demonstrating that both protocols yield nearly identical results.
To define the receptor subtype that mediates the tyrosine phosphorylation of p130Cas 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 p130Cas 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 p130Cas tyrosine phosphorylation.
Regulation of p130Cas Tyrosine Phosphorylation by Cytochalasin D and NaF
Recent studies have shown that the tyrosine phosphorylation of several focal adhesion–associated proteins, including p130Cas, 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 p130Cas 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-p130Cas 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 p130Cas to interact with the actin filament network is critical for Ang II–induced tyrosine phosphorylation.
Several reports have documented increased tyrosine phosphorylation of p130Cas in response to other G protein–coupled receptor ligands. Carbachol, LPA, vasopressin, bombesin, and endothelin all increased p130Cas tyrosine phosphorylation in Swiss 3T3 cells.18 19 20 Furthermore, the G protein activator GTPγS also increased the tyrosine phosphorylation of p125Fak and paxillin in permeabilized Swiss 3T3 cells.21 These results strongly suggest a role for Gαβγ-dependent tyrosine phosphorylation of p130Cas. To investigate this role in VSMCs, GTPγS was scrape-loaded into quiescent VSMCs. However, both GTPγS and sham-scraped control cells were found to have increased p130Cas 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-p130Cas mAb (Figure 2B⇑). In the absence of Ang II, 1 minute of NaF induced a 1.83±0.18-fold increase (n=3) in p130Cas tyrosine phosphorylation compared with the 0-minute controls (lane 3 versus lane 1), thus suggesting a role for Gαβγ-dependent tyrosine phosphorylation of p130Cas 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 p130Cas 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 p130Cas 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-p130Cas mAb. The Ang II–induced tyrosine phosphorylation of p130Cas was completely blocked with BAPTA-AM but not with verapamil (Figure 3A⇓). These results suggest that p130Cas 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 p130Cas (Figure 3B⇓).
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 p130Cas 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-p130Cas mAb. All agonists increased the tyrosine phosphorylation of p130Cas, and all were inhibited by BAPTA-AM (Figure 3C⇑). These results strongly suggest that intracellular Ca2+ is critical for p130Cas 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 p130Cas Tyrosine Phosphorylation
We next wanted to define the kinase(s) responsible for the Ang II–induced tyrosine phosphorylation of p130Cas 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 Gαβγ 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, p130Cas is known to be hyperphosphorylated in c-Src–transformed cells.10 The results of our characterization of p130Cas tyrosine phosphorylation in VSMCs suggested a role for c-Src in that p130Cas tyrosine phosphorylation was increased by ligands that activated G protein–coupled receptors, growth factor receptors, or activators of Gαβγ. 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 p130Cas, 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-p130Cas mAb and then Western-blotted with anti-Tyr(P) mAbs. Geldanamycin completely blocked the Ang II–dependent phosphorylation of p130Cas (Figure 4A⇓). The membrane was then blotted with anti-p130Cas 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-p130Cas mAb and then Western-blotted with anti-Tyr(P) mAbs. The tyrosine phosphorylation of p130Cas was eliminated by PP1 in a dose-dependent manner, with maximal inhibition occurring at 20 μmol/L (Figure 4B⇓). To demonstrate that p130Cas was precipitated equally across all lanes, the blot was subsequently probed with anti-p130Cas 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-p130Cas mAb and Western-blotted with anti-Tyr(P) mAbs. The tyrosine phosphorylation of p130Cas 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-p130Cas mAb (Figure 4C⇓). Taken together, these data indicate that an active Src family tyrosine kinase is critical for Ang II–mediated p130Cas tyrosine phosphorylation.
p130Cas Immune Complex In Vitro Kinase Assay
The amino acid sequence of p130Cas 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 p130Cas with v-Src, v-Crk, and p125Fak.9 10 30 To explore the role of p130Cas as an adaptor protein in Ang II signal transduction, VSMCs were stimulated with Ang II for varying times, and lysates were prepared. p130Cas was immunoprecipitated, and the resulting immune complexes were washed and resuspended in kinase buffer containing [γ-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 p130Cas 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-p130Cas 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 p130Cas 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-p130Cas mAb (Figure 5B⇓). Collectively, these studies demonstrate that p130Cas 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.
p130Cas Protein Complex Formation
Our next objective was to begin to identify some of the proteins that were observed in the p130Cas 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-p130Cas 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 p130Cas does not increase its association with c-Src. What cannot be determined from this blot is the relative kinase activity of c-Src.
The same blot was also probed with anti-PKCα mAb. In VSMCs, PKCα is known to mediate proliferation and regulate α-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 p130Cas, we looked for an interaction between p130Cas and PKCα in response to Ang II. At 1 minute, there was significant increased association (1.77±0.08-fold, n=3) of PKCα with p130Cas 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 p130Cas and pp120 was observed, but with a temporal pattern that was different from both c-Src and PKCα. 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-p130Cas mAb (Figure 6A⇑).
To demonstrate the specificity of our p130Cas interactions, the immunoprecipitating anti-p130Cas mAb was omitted from the protocol in 2 separate experiments. The result was a loss of signal for c-Src, PKCα, 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 p130Cas with p59Fyn 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-PKCα, or anti-pp120 and then blotted with anti-p130Cas. Blotting of the anti-Src polyclonal antibody immunoprecipitates with anti-p130Cas 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-PKCα immunoprecipitates with anti-p130Cas again demonstrated an Ang II–dependent association between PKCα 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 PKCα and Cas occurs within 5 minutes of Ang II treatment. Equal precipitation of PKCα was demonstrated by blotting the same membrane with anti-PKCα mAb (Figure 6B⇑, middle, second blot).
When the anti-pp120 immunoprecipitates were blotted with anti-p130Cas 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 p130Cas specifically interacts with PKCα 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 PKCα in p130Cas Tyrosine Phosphorylation
The Ang II–dependent association between Cas and PKCα 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 PKCα bound p130Cas as a consequence of Cas phosphorylation or was required by PKCα 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-p130Cas mAb and then Western-blotted with anti-Tyr(P) mAbs. Calphostin C greatly reduced the Ang II–mediated p130Cas tyrosine phosphorylation compared with DMSO controls (Figure 7A⇓). To demonstrate equal loading, the membrane was Western-blotted with anti-p130Cas 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. p130Cas immunoprecipitates were again Western-blotted with anti-Tyr(P) mAbs. Inhibition of PKC activity by PMA treatment resulted in decreased p130Cas tyrosine phosphorylation compared with DMSO controls (Figure 7B⇓). The membrane was then blotted with anti-p130Cas 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.
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 p130Cas 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 p130Cas by >90%, we believe that MEK activation is not required for p130Cas tyrosine phosphorylation. Equal precipitation of Cas was confirmed by blotting the membrane with anti-p130Cas mAb (Figure 7C⇑).
p130Cas was initially characterized as a phosphotyrosine-containing protein in v-Crk and v-Src transformed cells.9 10 Cloning and expression of the p130Cas 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 p130Cas 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 p130Cas 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 PKCα. The integrin-signaling molecule, pp120, also bound Cas in an Ang II–dependent manner. Both c-Src and PKCα kinase activities are required for Ang II–mediated p130Cas tyrosine phosphorylation. The Ang II–mediated association of pp120 and p130Cas may imply that p130Cas 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 p125Fak. Recent work has shown that Pyk2 and p125Fak 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-γ1, 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. PKCα 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 PKCα 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 PKCα. Its activation requires both Ca2+ and DAG, and previous studies in VSMCs have demonstrated that increased PKCα expression leads to (1) increased α-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 p130Cas tyrosine phosphorylation is coincident with changes in cell morphology,14 its binding to PKCα may suggest that it serves to bridge proliferative and structural responses in vivo. Another possibility is that PKCα may activate an intermediary kinase, such as p125Fak, which, in turn, phosphorylates Cas. One report demonstrated that PKC activity is required for p125Fak 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 p130Cas 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 p130Cas 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|
|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|
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.
- © 1998 American Heart Association, Inc.
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