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(Circulation Research. 1995;77:1053-1059.)
© 1995 American Heart Association, Inc.

Articles

Angiotensin II Activates pp60^c-src in Vascular Smooth Muscle Cells

Mari Ishida, Mario B. Marrero, Bernhard Schieffer, Takafumi Ishida, Kenneth E. Bernstein, Bradford C. Berk

From the Division of Cardiology (M.I., T.I., B.C.B.), Department of Medicine, University of Washington, Seattle, and the Department of Pathology (M.B.M., B.S., K.E.B.), Emory University, Atlanta, Ga.

Correspondence to Dr Bradford C. Berk, Division of Cardiology, 357710, University of Washington, Seattle, WA 98195. E-mail bcberk@u.washington.edu.

	Abstract

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Abstract The angiotensin II type-1 (AT₁) receptor, a G protein–coupled receptor, lacks intrinsic kinase activity. However, recent data show that angiotensin II (Ang II) stimulates tyrosine phosphorylation of phospholipase C-1 (PLC-1), Stat91 (one of the signal transducers and activators of transcription), and paxillin in vascular smooth muscle cells. The tyrosine kinases responsible for these phosphorylation events are unknown. Src family kinases have been shown to phosphorylate PLC-1 and to be activated by G protein–coupled receptors. We hypothesized that pp60^c-src associates with the AT₁ receptor and is activated after Ang II stimulation of smooth muscle cells. We immunoprecipitated pp60^c-src from Ang II–stimulated vascular smooth muscle cells and measured pp60^c-src activity by autophosphorylation and by phosphorylation of enolase. Both assays demonstrated an approximately threefold increase in pp60^c-src activity within 1 minute. A similar increase in Ang II–stimulated pp60^c-src activity was observed in Chinese hamster ovary cells transfected with the AT₁ receptor but not in untransfected cells. These data are the first to show that pp60^c-src is activated by Ang II. To determine if pp60^c-src associated with the AT₁ receptor, the AT₁ receptor was immunoprecipitated (with two different antibodies), and Western blots were performed with two different anti-pp60^c-src antibodies. No pp60^c-src was detected. In addition, direct interaction between the AT₁ receptor and pp60^c-src could not be demonstrated by using a glutathione S-transferase (GST)-AT₁ fusion protein to bind proteins from cell lysates stimulated by Ang II. In combination with recent findings that anti-pp60^c-src antibodies inhibit Ang II–mediated PLC-1 phosphorylation, our data suggest an important role for pp60^c-src in Ang II signal transduction.

Key Words: angiotensin II • signal transduction • vascular smooth muscle • Src kinase

	Introduction

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Angiotensin II (Ang II) is a critical hormone in vascular homeostasis by virtue of its dual actions as a vasoconstrictor and smooth muscle cell growth factor. Of great interest is the ability of Ang II to stimulate both hyperplasia and hypertrophy of cultured vascular smooth muscle cells, implying activation of several different growth-related signal transduction events.^{1 2 3} These growth-related events are mediated entirely by the AT₁ receptor on the basis of pharmacological effects of specific inhibitors.⁴ The AT₁ receptor has recently been cloned,⁵ and its structure is typical of many G protein–coupled receptors in that it is heptahelical with a long carboxyl cytoplasmic tail. The ability of G protein–coupled receptors to stimulate cell growth in a manner similar to tyrosine kinase–coupled receptors, such as PDGF and EGF receptors, points to the importance of investigating signal transduction events in addition to those mediated by G proteins.

Several findings support the idea that activation of tyrosine kinases and phosphorylation of intracellular substrates are critical events in Ang II–stimulated vascular smooth muscle cell growth. In fact, an increase in tyrosine phosphorylation of several intracellular proteins has been identified as one of the earliest signals stimulated by Ang II.^{6 7 8 9} More recently, specific phosphotyrosine-containing proteins have been identified as substrates for Ang II–stimulated tyrosine kinases, including PLC-1,¹⁰ Stat91 (from the STAT family of transcription factors),^{11 12} and p125^FAK.^{13 14} The recent discovery that STAT proteins are phosphorylated by Ang II¹¹ has implicated the JAK family as important mediators of Ang II signal transduction and suggests that additional similarities exist between the AT₁ receptor, tyrosine kinase–coupled receptors (such as the PDGF and EGF receptors), and cytokine receptors (such as the interleukin and interferon receptors).

A pivotal role for Src family kinases has been postulated for signal transduction events mediated by both G protein–coupled receptors and cytokine receptors. In fact, Src family kinases have been demonstrated to associate with the thrombin receptor¹⁵ and cytokine receptors^{16 17} and to be activated by ligand binding by both dephosphorylation and phosphorylation of critical tyrosine residues.¹⁸ Although the AT₁ receptor has no intrinsic kinase activity, nonreceptor tyrosine kinases are capable of associating with cell surface proteins that lack intrinsic kinase activity and initiating signal transduction in a manner analogous to receptor tyrosine kinases.¹⁹ Three findings suggest that Src kinase may associate with the AT₁ receptor and be important in Ang II signal transduction: (1) PLC-1 has been proposed to be a substrate for Src family kinases.²⁰ By virtue of its Src homology 2 (SH2) and pleckstrin homology domains, PLC-1 may associate with receptors and appropriate kinases.²¹ Since PLC-1 is tyrosine-phosphorylated upon Ang II stimulation,¹⁰ it is possible that PLC-1 is phosphorylated by Src kinase. (2) We recently showed that the AT₁ receptor is constitutively phosphorylated on serine and tyrosine residues^{22 23} and that Src family kinases phosphorylate the carboxyl tail of the AT₁ receptor in vitro.²² (3) Chen et al¹⁵ showed in fibroblasts that both Src and Fyn kinases were activated by the G protein–coupled thrombin receptor, which is similar to the AT₁ receptor in many ways.^{24 25}

On the basis of these findings, we hypothesized that Src kinase associates with the AT₁ receptor and is activated upon stimulation of vascular smooth muscle cells with Ang II. We found that pp60^c-src was rapidly activated by Ang II binding in both cultured vascular smooth muscle cells and CHO cells transfected with the AT₁ receptor. However, pp60^c-src was not associated with the AT₁ receptor, suggesting that pp60^c-src activation occurs via interactions with other molecules involved in signal transduction.

	Materials and Methods

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Cell Culture
Rat vascular smooth muscle cells were isolated from the aorta of 200- to 250-g male Sprague-Dawley rats and maintained in DMEM supplemented with 10% calf serum as previously described.⁹ Passage-6 to -16 vascular smooth muscle cells at 70% to 80% confluence were growth-arrested by incubation in DMEM supplemented with 0.1% calf serum for 48 hours before use. CHO cells transfected with AT₁ receptor (AT₁R-CHO) were cultured in DME/F-12 HAM media supplemented with 10% fetal calf serum as previously described.²⁶

Preparation of Rat Vascular Smooth Muscle Cell Membranes
Confluent rat vascular smooth muscle cells were washed and scraped off the dish with PBS. Cells were centrifuged for 5 minutes at 1000 rpm. Pellets were resuspended in hypotonic buffer (5 mmol/L HEPES, pH 7.4, 2 mmol/L MgCl₂, 2.5 mmol/L dithiothreitol, 0.1 mmol/L PMSF, and 10 µg/mL leupeptin), dounce homogenized 10 to 30 times, and centrifuged for 20 minutes at 48 000g. The pellets were dissolved in SDS-PAGE sample buffer.

Cell Harvest and Preparation of Lysates
Growth-arrested vascular smooth muscle cells or AT₁R-CHO cells were stimulated with 100 nmol/L Ang II for various times. Cells were lysed in RIPA buffer (20 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L sodium orthovanadate, 1 mmol/L PMSF, 10 µg/mL aprotinin, and 10 µg/mL leupeptin) for pp60^c-src immunoprecipitation or in NP-40 buffer (1% NP-40, 25 mmol/L Tris, pH 7.5, 50 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 137 mmol/L NaCl, 10% glycerol, 1 mmol/L sodium orthovanadate, 1 mmol/L PMSF, 10 µg/mL aprotinin, and 10 µg/mL leupeptin) for the AT₁ receptor immunoprecipitation and in vitro binding of GST fusion protein. Cells were scraped off the dish and centrifuged at 7000 rpm in a microfuge (4°C for 20 minutes), and protein concentrations of the supernatants were determined by DC protein assay (Bio-Rad).

Immunoprecipitation
Lysates containing the same amounts of soluble proteins were precleared against Immunoprecipitin (GIBCO BRL) and incubated overnight at 4°C with rabbit polyclonal anti-rat AT₁R-NH₂ Ab or AT₁R-COOH Ab²⁷ or with mAb327 (Oncogene Science, Inc) at 4°C. Antibody complexes were collected by incubation with protein A–Sepharose CL-4B (Sigma Chemical Co) for AT₁ receptor immunoprecipitation or by incubation with protein G–agarose (GIBCO BRL) for pp60^c-src immunoprecipitation. Precipitates were washed three times in buffer containing 50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 0.1% Triton-X 100, 1 mmol/L PMSF, 10 µg/mL aprotinin, and 10 µg/mL leupeptin and then resuspended in SDS-PAGE sample buffer.

pp60^c-src Immune Complex Kinase Assay
pp60^c-src Immunoprecipitates were washed three times in the buffer described above and twice in kinase reaction buffer (20 mmol/L PIPES, pH 7.0, and 10 mmol/L MnCl₂). The precipitates were then suspended in the kinase reaction buffer (20 mmol/L PIPES, pH 7.0, 10 mmol/L MnCl₂, and 50 µmol/L ATP) with or without 5 µg of acid-denatured (with 25 mmol/L sodium acetate, pH 3.3, 30°C, 5 minutes) rabbit muscle enolase (Sigma). The kinase reaction (final volume, 50 µL) was started by the addition of 10 µCi [-³²P]ATP (specific activity, 3000 mCi/mmol) at 30°C and terminated after 10 minutes by the addition of SDS-PAGE sample buffer. Reactions with enolase contained an additional 30 mmol/L PIPES, pH 7.0, to neutralize the acid from the pretreatment of enolase. Samples were boiled for 5 minutes and subjected to SDS-PAGE.

Construction, Expression, and Purification of GST Fusion Protein
Polymerase chain reaction and the primers 5'-ACTGAATTCACCCTCTGTTCTACGG-3' and 5'-TGGGAATTCGGTCGTAAGCCATTTAG-3' with unique restriction sites were used to amplify the intracellular carboxyl tail of the AT₁ receptor from pCa18b, the cDNA for the rat AT₁ receptor.⁵ The polymerase chain reaction fragment was digested with EcoRI and ligated into EcoRI-digested pGEX-KG to give rise to pGEX-KG-AT₁C (amino acids 297 to 359). The construct, which was inserted in the correct direction, expresses a fusion protein with the intracellular carboxyl tail (amino acids 297 to 359) of the AT₁ receptor linked to GST (GST-AT₁C [amino acids 297 to 359]). BL21 cells (Novagen) were transformed with pGEX-KG-AT₁C (amino acids 297 to 359) and induced with 0.2 mmol/L isopropyl-ß-thiogalactoside at 37°C for 3 hours. Cells were collected by centrifugation and lysed in buffer containing 50 mmol/L Tris, pH 8.0, 5 mmol/L EDTA, 100 mmol/L NaCl, 1 mmol/L PMSF, 10 µg/mL aprotinin, and 10 µg/mL leupeptin plus 1 mg/mL lysozyme and 0.25% sarcosyl and briefly sonicated. The lysate was cleared by centrifugation at 10 000g, and the supernatant was rocked with 50% (vol/vol) glutathione-agarose beads (Sigma) for 1 hour. The beads were collected by centrifugation at 2000g and subsequently washed three times with PBS containing 1% Triton-X, 1 mol/L NaCl, 5 mmol/L EDTA, 1 mmol/L PMSF, and 10 µg/mL leupeptin and once with PBS containing 1 mmol/L PMSF and 10 µg/mL leupeptin. GST-AT₁C (amino acids 297 to 359) fusion protein was stored on beads as a 50% slurry for in vitro binding study at 4°C. The amount of fusion protein was estimated by SDS-PAGE and staining the gel with Coomassie blue.

In Vitro Binding of GST Fusion Protein to Cell Lysates
Cell lysates were mixed with 15 µg of GST-AT₁ fusion protein or GST attached to glutathione-agarose beads for 3 hours at 4°C. The beads were collected by centrifugation, washed four times with cell lysis buffer, and resuspended in SDS-PAGE sample buffer.

For in vitro kinase assay, subsequent washing was done with kinase buffer (20 mmol/L Tris, pH 7.4, 20 mmol/L MgCl₂, 0.1 mmol/L sodium orthovanadate, and 2 mmol/L dithiothreitol) after washing with lysis buffer. Kinase assays were carried out in 30 µL of kinase buffer containing 50 µmol/L ATP and 5 µCi of [-³²P]ATP for 20 minutes at 30°C. The reaction was terminated by adding SDS sample buffer and boiling for 5 minutes, followed by SDS-PAGE.

Western Blot Analysis
Samples subjected to Western blot analysis were separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed. After incubation in blocking solution (GIBCO BRL) overnight, membranes were incubated with primary antibodies for 1 hour at room temperature for anti-AT₁ receptor antibodies, JAK-2 antibody (Santa Cruz), and anti-Src antibody SRC2 (Santa Cruz) or overnight at 4°C for anti-Src antibody mAb327. Excess primary antibody was removed by washing the membranes in PBS containing 0.03% Tween 20. The blots were incubated with appropriate secondary antibodies for 1 hour. The membranes were washed and processed for ECL (Amersham Life Science). In some experiments, membranes were reprobed after stripping in 62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, and 100 mmol/L ß-mercaptoethanol for 30 minutes at 50°C.

	Results

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To explore the role of Src kinase in AT₁ receptor signal transduction by rat vascular smooth muscle cells, we measured pp60^c-src kinase activity in response to Ang II. pp60^c-src was immunoprecipitated from cell lysates treated with Ang II, and pp60^c-src activation was determined by autophosphorylation. pp60^c-src autophosphorylation increased rapidly within 30 seconds after Ang II stimulation, and peak activation was at 1 minute (2.1-fold greater than control, Fig 1). To measure the specific kinase activity of pp60^c-src, phosphorylation of exogenous enolase was determined by an immune complex kinase assay. As shown in Fig 2A and 2B, pp60^c-src activity increased rapidly within 30 seconds in response to Ang II with a maximum 2.7-fold increase 5 minutes after Ang II stimulation. Ang II stimulation also increased the kinase activity of pp60^c-src in AT₁R-CHO cells with a time course similar to that observed in rat vascular smooth muscle cells. In contrast, pp60^c-src was not activated by Ang II in CHO cells not transfected with the AT₁ receptor (Fig 2C). No difference in the amount of pp60^c-src was observed in lysates from control and Ang II–stimulated cells by Western blot analysis with anti-Src antibody mAb327, indicating that the increase in pp60^c-src kinase activity mediated by Ang II was not due to a change in pp60^c-src protein content (data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 1. Ang II activates pp60c-src: autophosphorylation. Growth-arrested vascular smooth muscle cells were treated with Ang II for the indicated times, and cell lysates were prepared with NP-40 buffer for the pp60c-src autophosphorylation assay. The kinase reaction was performed as described in "Materials and Methods." After SDS-PAGE, autophosphorylation was measured by densitometry in the linear range of film development. Results were confirmed by liquid scintillation counting of phosphorylated pp60c-src present in the dried gel. A, Representative autoradiogram of three experiments. B, Densitometric analysis of pp60c-src autophosphorylation. Results were normalized to control (time=0), which was arbitrarily set to 1.0.

View larger version (21K): [in this window] [in a new window]   Figure 2. Ang II activates pp60c-src: specific kinase activity of pp60c-src measured by enolase phosphorylation. Growth-arrested vascular smooth muscle cells or AT1R-CHO cells were treated with Ang II for the indicated times, and cell lysates were prepared with RIPA buffer for pp60c-src immune complex kinase assay using enolase as a substrate. The kinase reaction was performed as described in "Materials and Methods." After SDS-PAGE, phosphorylation of enolase was measured by densitometry in the linear range of film development. Results were confirmed by liquid scintillation counting of phosphorylated enolase present in the dried gel. A, Representative autoradiogram of three experiments in vascular smooth muscle cells. B, Densitometric analysis of enolase phosphorylation from three independent experiments in vascular smooth muscle cells. Results were normalized to control (time=0), which was arbitrarily set to 1.0. C, Densitometric analysis of enolase phosphorylation from two independent experiments in AT1R-CHO cells and control CHO cells.

We next studied the potential interaction between pp60^c-src and the AT₁ receptor. We used two different antibodies in this series of experiments. The first is AT₁R-COOH Ab, which was raised against a GST fusion protein expressing the intracellular carboxy tail (amino acids 306 to 359) of the AT₁ receptor, and the second is AT₁R-NH₂ Ab, which was raised against the extracellular amino terminus (amino acids 15 to 24). Western blot analysis of rat vascular smooth muscle cell membranes with the AT₁R-COOH Ab demonstrated proteins with molecular masses of 48 and 60 kD. These weights are identical to those previously reported for the AT₁ receptor²² and likely represent nascent and mature glycosylated receptors, respectively.²⁸ AT₁R-NH₂ Ab detected a protein with a molecular mass of 48 kD on a similar Western blot (Fig 3). Because the peptide (amino acids 15 to 24) used as an antigen for AT₁R-NH₂ Ab is close to a potential glycosylation site (amino acid 4), this antibody may not recognize the mature glycosylated 60-kD AT₁ receptor, explaining the difference shown in Fig 3. Immunoprecipitation of the AT₁ receptor from rat vascular smooth muscle cells with the AT₁R-COOH Ab identified a protein with a molecular mass of 60 kD (Fig 4A). However, Western blot analysis of the AT₁R-COOH Ab immunoprecipitates with the AT₁R-COOH Ab failed to identify the 48-kD AT₁ receptor, because IgG heavy chain (molecular mass, 46 to 48 kD) was recognized by the secondary antibody. Since the AT₁R-COOH Ab was not affinity-purified, we confirmed that an antibody against GST protein did not precipitate the protein that reacted with the AT₁R-COOH Ab (data not shown). We confirmed that several proteins were coimmunoprecipitated with the AT₁ receptor by silver staining (data not shown). The AT₁ receptor immunoprecipitates were analyzed on Western blots for specific proteins. One of the coimmunoprecipitated proteins was identified as JAK-2, a tyrosine kinase (Fig 4B) as reported previously.¹² However, pp60^c-src was not detected in the AT₁ receptor immunoprecipitates (Fig 4C). Thus, the failure to detect pp60^c-src was unlikely an artifact due to the conditions used for immunoprecipitation.

View larger version (72K): [in this window] [in a new window]   Figure 3. Western blot (WB) analyses of rat vascular smooth muscle cell membranes with two different AT1 receptor antibodies. Rat vascular smooth muscle cell membranes were isolated and prepared for WB analysis as described in "Materials and Methods." Membrane proteins were electrophoresed, and WB analysis was performed with the indicated antibodies (AT1R-COOH and AT1R-NH2).

View larger version (49K): [in this window] [in a new window]   Figure 4. Ang II does not stimulate binding of pp60c-src to the AT1 receptor immunoprecipitated with AT1R-COOH antibody. Growth-arrested vascular smooth muscle cells were treated with Ang II for the indicated times, and cell lysates were prepared for immunoprecipitation of the AT1 receptor with the AT1R-COOH antibody. The immunoprecipitated proteins were electrophoresed, and Western blot (WB) analysis was performed with the indicated antibodies. A, Immunoprecipitation (IP) with AT1R-COOH Ab and WB analysis with AT1R-COOH Ab. Arrowhead indicates the 60-kD immunoreactive protein that is the AT1 receptor. B, IP with AT1R-COOH Ab and WB analysis with JAK-2 antibody. Arrow indicates the 120-kD coimmunoprecipitated JAK-2. C, IP with AT1R-COOH Ab and WB analysis with mAb327 against pp60c-src (Src). Results are representative of three experiments.

It is possible that the AT₁R-COOH Ab may interfere with pp60^c-src binding to the AT₁ receptor, since both AT₁R-COOH Ab and pp60^c-src could interact with the carboxyl tail.²² Therefore, we used two additional methods to confirm the lack of association between the AT₁ receptor and pp60^c-src. First, we used an antibody against the extracellular NH₂ terminus, AT₁R-NH₂ Ab, to immunoprecipitate the AT₁ receptor. As shown in Fig 5A, pp60^c-src was not detected when the AT₁ receptor immunoprecipitates were analyzed on Western blots with an anti-pp60^c-src antibody mAb327, although the AT₁ receptor was immunoprecipitated (Fig 5B). In addition, pp60^c-src was not detected in AT₁ receptor immunoprecipitates when another anti-Src antibody, SRC2, was used (data not shown). No change in pp60^c-src immunoreactive protein was observed in the supernatants after immunoprecipitation of the AT₁ receptor (Fig 5A). Second, to examine whether pp60^c-src binds directly to the carboxyl tail of the AT₁ receptor in vitro, a fusion protein, GST-AT₁C (amino acids 297 to 359), was expressed in bacteria, purified on the glutathione-agarose beads, and incubated with vascular smooth muscle cell lysates. Proteins that associated with the GST-AT₁C (amino acids 297 to 359) fusion protein on beads were subjected to Western blot analysis with anti-pp60^c-src antibody mAb327. Of interest, the presence of a protein kinase in these precipitates could be readily demonstrated. As shown in Fig 6B, addition of [-³²P]ATP to these precipitates resulted in significant phosphorylation of the GST-AT₁C (amino acids 297 to 359) fusion protein. There appeared to be little regulation of this kinase activity by Ang II under the conditions used. As shown in Fig 6A, pp60^c-src was not detected in the coprecipitates with the GST-AT₁C (amino acids 297 to 359) fusion protein as assayed by Western blot with anti-pp60^c-src antibody mAb327. In summary, these results indicate that pp60^c-src does not directly associate with the AT₁ receptor.

View larger version (45K): [in this window] [in a new window]   Figure 5. Ang II does not stimulate binding of pp60c-src to the AT1 receptor immunoprecipitated with AT1R-NH2 antibody. Growth-arrested vascular smooth muscle cells were treated with Ang II for the indicated times, and cell lysates were prepared for immunoprecipitation (IP) of the AT1 receptor with the AT1R-NH2 Ab. A, The AT1 receptor was immunoprecipitated with AT1R-NH2 Ab. The immunoprecipitated proteins and the supernatants remaining after IP were electrophoresed, and Western blot (WB) analysis was performed with mAb327 against pp60c-src (Src). B, The membrane was stripped and reprobed with AT1R-NH2 Ab. Arrows indicate the 48-kD AT1 receptor immediately below the rabbit IgG. Results are representative of three experiments.

View larger version (59K): [in this window] [in a new window]   Figure 6. pp60c-src does not directly bind to the GST-AT1 fusion protein. Growth-arrested vascular smooth muscle cells were left unstimulated (-) or stimulated for 5 minutes with 100 nmol/L Ang II (+). Cell lysates were prepared and incubated with GST alone (lanes 1 and 2) or GST-AT1C (amino acids 297 to 359) fusion protein (lanes 3 and 4) for 3 hours at 4°C. A, The coprecipitated protein and the supernatants remaining after binding of GST-AT1C (amino acids 297 to 359) fusion protein were resolved by SDS-PAGE, and Western blot analysis was performed with mAb327 against pp60c-src (Src). No pp60c-src was detected in the GST-AT1C (amino acids 297 to 359) precipitates with (+) or without (-) Ang II stimulation. pp60c-src was readily detected in the supernatant (arrow). B, The coprecipitated proteins were subjected to in vitro kinase assay as described in "Materials and Methods" and resolved by SDS-PAGE. "Mock" indicates that GST-AT1C (amino acids 297 to 359) fusion protein was subjected to in vitro kinase assay without the addition of cell lysate.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The most important finding of the present study is the demonstration that Ang II activates pp60^c-src in vascular smooth muscle cells, and this is the first study to demonstrate it. Since the AT₁ receptor was cloned, there has been a rapid increase in our understanding of the signal transduction mechanisms responsible for the effects of Ang II. Perhaps most impressive has been the identification of a number of serine/threonine kinases activated by Ang II binding that initiate signal transduction. These include Raf kinase, mitogen-activated protein kinase, protein kinase C, calcium calmodulin–dependent kinase, and S6 kinase.²⁹ Although the AT₁ receptor is a typical G protein–coupled receptor in that it lacks tyrosine kinase activity, many proteins are rapidly tyrosine-phosphorylated when Ang II binds to the AT₁ receptor. This suggests that a receptor-associated tyrosine kinase(s) may be involved in Ang II signaling. Because the AT₁ receptor is itself tyrosine-phosphorylated,²³ binding of SH2-containing proteins, such as pp60^c-src, may be a possible mechanism for activation of the receptor-associated kinase. However, the present study suggests that pp60^c-src does not bind to the AT₁ receptor despite being rapidly activated in cells treated with Ang II. Despite our inability to show a direct association between the AT₁ receptor and pp60^c-src, it is possible that such an association occurs in vivo. For example, the association may be transient and impossible to detect by immunoprecipitation, or a low-affinity interaction, mediated by other cell proteins, may occur. The failure of the AT₁-GST fusion protein to bind pp60^c-src may be explained by improper folding due to the presence of GST or a requirement for other intracellular loops (eg, the third intracellular loop) to maintain the interaction between the AT₁ receptor and pp60^c-src.

A large body of evidence suggests that Src kinase is important in Ang II–stimulated events in vascular smooth muscle cells. First, it has been demonstrated that both p125^FAK and paxillin are tyrosine-phosphorylated rapidly in response to Ang II.^{13 14} p125^FAK has been identified to be important in paxillin phosphorylation.³⁰ Because p125^FAK is itself activated by pp60^c-src-mediated phosphorylation of tyrosines 576 and 577,³¹ it appears plausible that Src kinase is involved in Ang II–mediated p125^FAK and paxillin phosphorylation. Second, -thrombin, which stimulates many of the same events as Ang II in vascular smooth muscle cells,^{1 24 25} has been found to activate Src family kinases in fibroblasts with a time course that is very similar to that shown here for Ang II.¹⁵ In addition, Src kinase has been shown to be involved in signal transduction mediated by other G protein–coupled receptors, including the receptors for lysophosphatidic acid,³² endothelin,³³ and platelet-activating factor receptors.³⁴ Third, we showed previously that the AT₁ receptor was rapidly tyrosine-phosphorylated by Src family kinases in vitro.²² Fourth, we have shown that PLC-1 is rapidly tyrosine-phosphorylated in response to Ang II, with peak phosphorylation at 1 minute and that the phosphorylation and activation of PLC-1 are decreased by the tyrosine kinase inhibitors genistein and tyrphostin.¹⁰ Very recent data from our laboratory show that electroporation of an antibody against pp60^c-src specifically inhibits PLC-1 phosphorylation and activity.³⁵ Thus, it appears that pp60^c-src is responsible for tyrosine phosphorylation of PLC-1 in response to Ang II in vascular smooth muscle cells. Finally, we have recently shown that the receptor-associated kinases of the JAK family bind to the AT₁ receptor and phosphorylate the STAT family of transcription factors.³⁶ Of interest, Stat3-related protein has recently been found to be activated by the Src oncogene tyrosine kinase.³⁷ Thus, it is likely that Src kinase plays an important role in Ang II signal transduction.

The present findings support the concept that the AT₁ receptor acts as a cytokine-like receptor. Early signal transduction events mediated by cytokine receptors include activation of receptor-associated kinases, including JAK, TYK, and the Src family kinases.^{16 17 38 39} The fact that the receptor-associated kinases of the JAK family bind to the AT₁ receptor and phosphorylate the STAT family of transcription factors³⁶ suggests that the long-term effects of Ang II on gene expression may resemble those of tyrosine kinase–coupled receptors, such as the PDGF receptor, and classic cytokine receptors, such as the interleukin and interferon receptors.^{39 40} In the present study, this similarity is extended to the Src family kinases, specifically pp60^c-src. In several cytokine and tyrosine kinase–coupled receptors, pp60^c-src directly associates with the receptors.¹⁶ However, this mechanism of interaction does not pertain to the AT₁ receptor on the basis of the present study. If pp60^c-src does not bind to the AT₁ receptor, how may it be localized to the receptor and be activated? One potential mechanism is suggested by the recent findings that the ß-adrenergic receptor kinase associates with the ß-adrenergic receptor by virtue of G_ß subunits interacting with the pleckstrin homology domain of the ß-adrenergic receptor kinase.⁴¹ Interaction between G_ß subunits, their associated kinases, and kinase substrates may provide the signaling complex that activates and binds pp60^c-src. Thus, future studies should identify the mechanisms by which Src kinase is activated upon binding of Ang II to the AT₁ receptor.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1 receptor = Ang II type-1 receptor AT1R-COOH Ab = AT1 receptor antibody raised against the GST fusion protein of the intracellular carboxyl tail (amino acids 306 to 359) AT1R-NH2 Ab = AT1 receptor antibody raised against the extracellular amino-terminus (amino acids 15 to 24) CHO = Chinese hamster ovary EGF = epidermal growth factor JAK = Janus kinase mAb327 = monoclonal anti-Src antibody p125FAK = p125 focal adhesion kinase PDGF = platelet-derived growth factor PLC-1 = phospholipase C-1 PMSF = phenylmethylsulfonyl fluoride STAT = signal transducers and activators of transcription

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health to Drs Berk, Bernstein, and Marrero and a grant from the Japan Heart Foundation to Dr M. Ishida. Drs Berk and Bernstein are Established Investigators of the American Heart Association. We thank members of the Berk laboratory, especially Drs M. Kusuhara, J. L. Duff, and T. E. Peterson, for their help.

Received June 9, 1995; accepted September 19, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Berk BC, Vekshtein V, Gordon HM, Tsuda T. Angiotensin II–stimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension. 1989;13:305-314. [Abstract/Free Full Text]

2. Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy vs. hyperplasia: autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II. J Clin Invest. 1992;90:456-461.

3. Weber H, Taylor DS, Molloy CJ. Angiotensin II induces delayed mitogenesis and cellular proliferation in rat aortic smooth muscle cells. J Clin Invest. 1994;93:788-798.

4. 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]

5. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991;351:233-236. [Medline] [Order article via Infotrieve]

6. Molloy CJ, Taylor DS, Weber H. Angiotensin II stimulation of rapid protein tyrosine phosphorylation and protein kinase activation in rat aortic smooth muscle cells. J Biol Chem. 1993;268:7338-7345. [Abstract/Free Full Text]

7. Huckle WR, Prokop CA, Dy RC, Herman B, Earp S. Angiotensin II stimulates protein-tyrosine phosphorylation in a calcium-dependent manner. Mol Cell Biol. 1990;10:6290-6298. [Abstract/Free Full Text]

8. Tsuda T, Kawahara Y, Shii K, Koide M, Ishida Y, Yokoyama M. Vasoconstrictor-induced protein-tyrosine phosphorylation in cultured vascular smooth muscle cells. FEBS Lett. 1992;285:44-48.

9. Duff JL, Berk BC, Corson MA. Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1992;188:257-264. [Medline] [Order article via Infotrieve]

10. Marrero MB, Paxton WB, 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]

11. Bhat GJ, Thekkumkara TJ, Thomas WG, Conrad KM, Baker KM. Angiotensin II stimulates sis-inducing factor-like DNA binding activity. J Biol Chem. 1994;269:31443-31449. [Abstract/Free Full Text]

12. Marrero MB, Schieffer 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]

13. Polte TR, Naftilan AJ, Hanks SK. Focal adhesion kinase is abundant in developing blood vessels and elevation of its phosphotyrosine content in vascular smooth muscle cells is a rapid response to angiotensin II. J Cell Biochem. 1994;55:106-119. [Medline] [Order article via Infotrieve]

14. Leduc I, 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]

15. Chen YH, Pouysségur J, Courtneidge SA, Van Obberghen-Schilling E. Activation of Src family kinase activity by the G protein-coupled thrombin receptor in growth-responsive fibroblasts. J Biol Chem. 1994;269:27372-27377. [Abstract/Free Full Text]

16. Minami Y, Kono T, Miyazaki T, Taniguchi T. The IL-2 receptor complex: its structure, function, and target genes. Annu Rev Immunol. 1993;11:245-268. [Medline] [Order article via Infotrieve]

17. Sorio C, Melotti P, Dusi S, Berton G. Interferon-gamma and tumor necrosis factor-alpha enhance p60src expression in human macrophages and myelomonocytic cell lines. FEBS Lett. 1993;327:315-320. [Medline] [Order article via Infotrieve]

18. Cooper JA, B. H. The when and how of Src regulation. Cell. 1993;73:1051-1054. [Medline] [Order article via Infotrieve]

19. Bolen JB. Nonreceptor tyrosine protein kinases. Oncogene. 1993;8:2025-2031. [Medline] [Order article via Infotrieve]

20. Liao F, Shin HS, Rhee SG. In vitro tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2 by src-family protein tyrosine kinases. Biochem Biophys Res Commun. 1993;191:1028-1033. [Medline] [Order article via Infotrieve]

21. Musacchio A, Gibson T, Rice P, Thompson J, Saraste M. The PH domain: a common piece in the structural patchwork of signalling proteins. Trends Biochem Sci. 1993;18:343-348. [Medline] [Order article via Infotrieve]

22. Paxton WG, Marrero MB, Klein JD, Delafontaine P, Berk BC, Bernstein KE. The angiotensin II AT₁ receptor is tyrosine and serine phosphorylated and can serve as a substrate for the Src family of tyrosine kinases. Biochem Biophys Res Commun. 1994;200:260-267. [Medline] [Order article via Infotrieve]

23. Kai H, Griendling KK, Lassegue B, Ollerenshaw JD, Runge MS, Alexander RW. Agonist-induced phosphorylation of the vascular type 1 angiotensin II receptor. Hypertension. 1994;24:523-527. [Abstract/Free Full Text]

24. Berk BC, Taubman MB, Cragoe EJ Jr, Fenton JW 2nd, Griendling KK. Thrombin signal transduction mechanisms in rat vascular smooth muscle cells: calcium and protein kinase C-dependent and -independent pathways. J Biol Chem. 1990;265:17334-17340. [Abstract/Free Full Text]

25. Berk BC, Taubman MB, Griendling KK, Cragoe EJ Jr, Fenton JW, Brock TA. Thrombin-stimulated events in cultured vascular smooth-muscle cells. Biochem J. 1991;274:799-805.

26. Teutsch B, Bihoreau C, Monnot C, Bernstein KE, Murphy TJ, Alexander RW, Corvol P, Clauser E. A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells. Biochem Biophys Res Commun. 1992;187:1381-1388. [Medline] [Order article via Infotrieve]

27. Paxton WG, Runge M, Horaist C, Cohen C, Alexander RW, Bernstein KE. Immunohistochemical localization of rat angiotensin II AT1 receptor. Am J Physiol. 1993;264:F989-F995. [Abstract/Free Full Text]

28. Carson MC, Harper CML, Baukal AJ, Aguilera G, Catt KJ. Physicochemical characterization of photoaffinity-labeled angiotensin II receptors. Mol Endocrinol. 1987;1:147-153. [Abstract/Free Full Text]

29. Duff JL, Berk BC. Angiotensin II-mediated signal transduction events in vascular smooth muscle cells: kinases and phosphatases. Blood Press. 1995;4(suppl 2):55-60.

30. Turner CE, Schaller MD, Parsons JT. Tyrosine phosphorylation of the focal adhesion kinase pp125FAK during development: relation to paxillin. J Cell Sci. 1993;105:637-645. [Abstract]

31. Calalb MB, Polte TR, Hanks SK. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol. 1995;15:954-963.[Abstract]

32. Jalink K, Eichholtz T, Postma FR, van CEJ, Moolenaar WH. Lysophosphatidic acid induces neuronal shape changes via a novel, receptor-mediated signaling pathway: similarity to thrombin action. Cell Growth Differ. 1993;4:247-255. [Abstract]

33. Simonson MS, Herman WH. Protein kinase C and protein tyrosine kinase activity contribute to mitogenic signaling by endothelin-1. J Biol Chem. 1993;268:9347-9357. [Abstract/Free Full Text]

34. Dhar A, Shukla SD. Electrotransjection of pp60v-src monoclonal antibody inhibits activation of phospholipase C in platelets: a new mechanism for platelet-activating factor responses. J Biol Chem. 1994;269:9123-9127. [Abstract/Free Full Text]

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

36. Marrero MB, Schieffer 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.

37. Yu C-L, Meter DJ, Campbell GS, Larner AC, Carter-Su C, Schwartz J, Jove R. Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science. 1995;269:81-83. [Abstract/Free Full Text]

38. Shuai K, Stark GR, Kerr IM, Darnell JEJ. A single phosphotyrosine residue of Stat91 required for gene activation by interferon-gamma. Science. 1993;261:1744-1746. [Abstract/Free Full Text]

39. Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Thierfelder WE, Kreider B, Silvennoinen O. Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem Sci. 1994;19:222-227. [Medline] [Order article via Infotrieve]

40. Shuai K, Horvath CM, Huang LH, Qureshi SA, Cowburn D, Darnell JEJ. Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions. Cell. 1994;76:821-828. [Medline] [Order article via Infotrieve]

41. Pitcher J, Touhara K, Payne E, Lefkowitz R. Pleckstrin homology domain-mediated membrane association and activation of the ß-adrenergic receptor kinase requires coordinate interaction with G_ß subunits and lipid. J Biol Chem. 1995;270:11707-11710.[Abstract/Free Full Text]

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				Y. Zou, I. Komuro, T. Yamazaki, S. Kudoh, R. Aikawa, W. Zhu, I. Shiojima, Y. Hiroi, K. Tobe, T. Kadowaki, et al. Cell Type–Specific Angiotensin II–Evoked Signal Transduction Pathways : Critical Roles of Gß{gamma} Subunit, Src Family, and Ras in Cardiac Fibroblasts Circ. Res., February 23, 1998; 82(3): 337 - 345. [Abstract] [Full Text] [PDF]

				M. Ishida, T. Ishida, S. M. Thomas, and B. C. Berk Activation of Extracellular Signal–Regulated Kinases (ERK1/2) by Angiotensin II Is Dependent on c-Src in Vascular Smooth Muscle Cells Circ. Res., January 23, 1998; 82(1): 7 - 12. [Abstract] [Full Text] [PDF]

				A. E. Brinson, T. Harding, P. A. Diliberto, Y. He, X. Li, D. Hunter, B. Herman, H. S. Earp, and L. M. Graves Regulation of a Calcium-dependent Tyrosine Kinase in Vascular Smooth Muscle Cells by Angiotensin II and Platelet-derived Growth Factor. DEPENDENCE ON CALCIUM AND THE ACTIN CYTOSKELETON J. Biol. Chem., January 16, 1998; 273(3): 1711 - 1718. [Abstract] [Full Text] [PDF]

				L. M. Luttrell, Y. Daaka, G. J. Della Rocca, and R. J. Lefkowitz G Protein-coupled Receptors Mediate Two Functionally Distinct Pathways of Tyrosine Phosphorylation in Rat 1a Fibroblasts. Shc PHOSPHORYLATION AND RECEPTOR ENDOCYTOSIS CORRELATE WITH ACTIVATION OF Erk KINASES J. Biol. Chem., December 12, 1997; 272(50): 31648 - 31656. [Abstract] [Full Text] [PDF]

				M. van Bilsen Signal transduction revisited: recent developments in angiotensin II signaling in the cardiovascular system Cardiovasc Res, December 1, 1997; 36(3): 310 - 322. [Full Text] [PDF]

				E. Giasson, M. J. Servant, and S. Meloche Cyclic AMP-mediated Inhibition of Angiotensin II-induced Protein Synthesis Is Associated with Suppression of Tyrosine Phosphorylation Signaling in Vascular Smooth Muscle Cells J. Biol. Chem., October 24, 1997; 272(43): 26879 - 26886. [Abstract] [Full Text] [PDF]

				U. Schmitz, M. Ishida, and B. C. Berk Angiotensin II Stimulates Tyrosine Phosphorylation of Phospholipase C-{gamma}–Associated Proteins : Characterization of a c-Src–Dependent 97-kD Protein in Vascular Smooth Muscle Cells Circ. Res., October 19, 1997; 81(4): 550 - 557. [Abstract] [Full Text]

				L. Saward Peter Zahradka Angiotensin II Activates Phosphatidylinositol 3-Kinase in Vascular Smooth Muscle Cells Circ. Res., August 19, 1997; 81(2): 249 - 257. [Abstract] [Full Text]

				J.-i. Abe, M. Takahashi, M. Ishida, J.-D. Lee, and B. C. Berk c-Src Is Required for Oxidative Stress-mediated Activation of Big Mitogen-activated Protein Kinase 1 (BMK1) J. Biol. Chem., August 15, 1997; 272(33): 20389 - 20394. [Abstract] [Full Text] [PDF]

				J. Chung, A.-G. Gao, and W. A. Frazier Thrombspondin Acts via Integrin-associated Protein to Activate the Platelet Integrin alpha IIbbeta 3 J. Biol. Chem., June 6, 1997; 272(23): 14740 - 14746. [Abstract] [Full Text] [PDF]

				B. C. Berk and M. A. Corson Angiotensin II Signal Transduction in Vascular Smooth Muscle : Role of Tyrosine Kinases Circ. Res., May 19, 1997; 80(5): 607 - 616. [Abstract] [Full Text]

				M. S. Ali, B. Schieffer, P. Delafontaine, K. E. Bernstein, B. N. Ling, and M. B. Marrero Angiotensin II Stimulates Tyrosine Phosphorylation and Activation of Insulin Receptor Substrate 1 and Protein-tyrosine Phosphatase 1D in Vascular Smooth Muscle Cells J. Biol. Chem., May 9, 1997; 272(19): 12373 - 12379. [Abstract] [Full Text] [PDF]

				M. Hernandez-Presa, C. Bustos, M. Ortego, J. Tunon, G. Renedo, M. Ruiz-Ortega, and J. Egido Angiotensin-Converting Enzyme Inhibition Prevents Arterial Nuclear Factor-{kappa}B Activation, Monocyte Chemoattractant Protein-1 Expression, and Macrophage Infiltration in a Rabbit Model of Early Accelerated Atherosclerosis Circulation, March 18, 1997; 95(6): 1532 - 1541. [Abstract] [Full Text]

				L. M. Luttrell, G. J. Della Rocca, T. van Biesen, D. K. Luttrell, and R. J. Lefkowitz Gbeta gamma Subunits Mediate Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor. A SCAFFOLD FOR G PROTEIN-COUPLED RECEPTOR-MEDIATED Ras ACTIVATION J. Biol. Chem., February 14, 1997; 272(7): 4637 - 4644. [Abstract] [Full Text] [PDF]

				K. K. Griendling, M. Ushio-Fukai, B. Lassegue, and R. W. Alexander Angiotensin II Signaling in Vascular Smooth Muscle: New Concepts Hypertension, January 1, 1997; 29(1): 366 - 370. [Abstract] [Full Text] [PDF]

				Y. Zou, I. Komuro, T. Yamazaki, R. Aikawa, S. Kudoh, I. Shiojima, Y. Hiroi, T. Mizuno, and Y. Yazaki Protein Kinase C, but Not Tyrosine Kinases or Ras, Plays a Critical Role in Angiotensin II-induced Activation of Raf-1 Kinase and Extracellular Signal-regulated Protein Kinases in Cardiac Myocytes J. Biol. Chem., December 27, 1996; 271(52): 33592 - 33597. [Abstract] [Full Text] [PDF]

				D.-F. Liao, J. L. Duff, G. Daum, S. L. Pelech, and B. C. Berk Angiotensin II Stimulates MAP Kinase Kinase Kinase Activity in Vascular Smooth Muscle Cells: Role of Raf Circ. Res., November 1, 1996; 79(5): 1007 - 1014. [Abstract] [Full Text]

				L. M. Luttrell, B. E. Hawes, T. van Biesen, D. K. Luttrell, T. J. Lansing, and R. J. Lefkowitz Role of c-Src Tyrosine Kinase in G Protein-coupled Receptorand Gbeta gamma Subunit-mediated Activation of Mitogen-activated Protein Kinases J. Biol. Chem., August 9, 1996; 271(32): 19443 - 19450. [Abstract] [Full Text] [PDF]

				B. Schieffer, W. G. Paxton, Q. Chai, M. B. Marrero, and K. E. Bernstein Angiotensin II Controls p21[IMAGE] Activity via pp60[IMAGE] J. Biol. Chem., April 26, 1996; 271(17): 10329 - 10333. [Abstract] [Full Text] [PDF]

				B. Schieffer, W. G. Paxton, M. B. Marrero, and K. E. Bernstein Importance of Tyrosine Phosphorylation in Angiotensin II Type 1 Receptor Signaling Hypertension, March 1, 1996; 27(3): 476 - 480. [Abstract] [Full Text]

				L. Guillemot, A. Levy, Z. J. Zhao, G. Bereziat, and B. Rothhut The Protein-tyrosine Phosphatase SHP-2 Is Required during Angiotensin II-mediated Activation of Cyclin D1 Promoter in CHO-AT1A Cells J. Biol. Chem., August 18, 2000; 275(34): 26349 - 26358. [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]

				P. P. Sayeski, M. S. Ali, S. J. Frank, and K. E. Bernstein The Angiotensin II-dependent Nuclear Translocation of Stat1 Is Mediated by the Jak2 Protein Motif 231YRFRR J. Biol. Chem., March 23, 2001; 276(13): 10556 - 10563. [Abstract] [Full Text] [PDF]

				A. Bonacchi, P. Romagnani, R. G. Romanelli, E. Efsen, F. Annunziato, L. Lasagni, M. Francalanci, M. Serio, G. Laffi, M. Pinzani, et al. Signal Transduction by the Chemokine Receptor CXCR3. ACTIVATION OF Ras/ERK, Src, AND PHOSPHATIDYLINOSITOL 3-KINASE/Akt CONTROLS CELL MIGRATION AND PROLIFERATION IN HUMAN VASCULAR PERICYTES J. Biol. Chem., March 23, 2001; 276(13): 9945 - 9954. [Abstract] [Full Text] [PDF]

				P. Rocic and P. A. Lucchesi Down-regulation by Antisense Oligonucleotides Establishes a Role for the Proline-rich Tyrosine Kinase PYK2 in Angiotensin II-induced Signaling in Vascular Smooth Muscle J. Biol. Chem., June 8, 2001; 276(24): 21902 - 21906. [Abstract] [Full Text] [PDF]

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