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 Govindarajan, G. Articles by Samarel, A. M. Search for Related Content PubMed PubMed Citation Articles by Govindarajan, G. Articles by Samarel, A. M. Related Collections Mechanism of atherosclerosis/growth factors Cell signalling/signal transduction

(Circulation Research. 2000;87:710.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Focal Adhesion Kinase Is Involved in Angiotensin II–Mediated Protein Synthesis in Cultured Vascular Smooth Muscle Cells

Geetha Govindarajan, Diane M. Eble, Pamela A. Lucchesi, Allen M. Samarel

From The Cardiovascular Institute (G.G., D.M.E., A.M.S), Loyola University Chicago, Maywood, Ill, and Department of Physiology and Biophysics (P.A.L.), University of Alabama at Birmingham, Birmingham, Ala.

Correspondence to Allen M. Samarel, MD, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153. E-mail asamare{at}lumc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—The rate of vascular smooth muscle cell protein synthesis and cellular hypertrophy in response to angiotensin II (Ang II) is dependent on activation of protein tyrosine kinases (PTKs) and both the extracellular signal–regulated kinase (ERK) 1/2 and p70^S6K pathways. One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions. We used an actin depolymerizing agent, cytochalasin D (Cyt-D), and a replication-defective adenovirus encoding FAK-related nonkinase (FRNK), an inhibitor of FAK-dependent signaling, as tools to assess whether FAK was upstream of the ERK1/2 and/or the p70^S6K pathways. Cyt-D reduced basal FAK phosphorylation and blocked Ang II–dependent FAK phosphorylation in a dose-dependent manner. Confocal microscopy indicated that Cyt-D induced actin filament disruption and FAK delocalization from focal adhesions. Cyt-D also reduced Ang II–induced ERK1/2 activation, but p70^S6K activation was relatively unaffected. Cyt-D reduced basal protein synthetic rate and substantially reduced the Ang II–induced increase in protein synthesis. Similarly, FRNK overexpression blocked Ang II–induced FAK phosphorylation and ERK1/2 activation, but not p70^S6K phosphorylation, and markedly inhibited protein synthesis. This is the first report to demonstrate that FAK is a critical component of the signal transduction pathways that mediate Ang II–induced ERK1/2 activation, c-fos induction, and enhanced protein synthesis in vascular smooth muscle cells.

Key Words: FRNK • adenovirus • cytochalasin D • p70S6 kinase • extracellular signal–regulated kinase

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiotensin II (Ang II) plays an important role in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis.^{1 2} Apart from its vasoconstrictor properties, Ang II is a potent hypertrophic growth factor for vascular smooth muscle cells (VSMCs).^{3 4 5} Binding of Ang II to AT₁s activates several signaling pathways implicated in regulating cellular growth.¹

One feature of Ang II–induced VSMC hypertrophy is an increase in protein synthesis. The ERK1/2 family of protein kinases and the ribosomal S6 kinases are key effectors implicated in the regulation of Ang II–induced protein synthesis.^{6 7} ERK1/2 are serine/threonine protein kinases that are activated by the upstream Ras–Raf–mitogen-activated protein kinase/ERK (MEK) cascade. ERK1/2 modulate gene transcription by induction of transcription factors such as c-fos.^{8 9 10} The ribosomal p70 S6 kinase (p70^S6K) and p90 S6 kinase (p90^RSK) families phosphorylate S6 protein, a component of the 40S ribosomal subunit, thereby regulating the protein synthetic machinery.¹¹ p70^S6K is activated by numerous extracellular stimuli. Its activation can be blocked by wortmannin and LY294002, suggesting that it is downstream of phosphatidylinositol-3 kinase.¹² In contrast, p90^RSK is downstream of the ERK1/2 cascade.¹³ The upstream regulators of both of these pathways have not been clearly defined. Several groups have suggested that 1 or more protein tyrosine kinases (PTKs) are involved, as PTK inhibitors such as genistein completely abolish Ang II–induced protein synthesis and block activation of ERK1/2 and the S6 kinases.^{14 15}

One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions.¹⁶ FAK autophosphorylation in response to agonist-induced integrin clustering can lead to its stable association with Src,¹⁷ as well as phosphatidylinositol-3 kinase,¹⁸ which is a well-characterized upstream regulator of p70^S6K. Src can then tyrosine phosphorylate the C-terminal region of FAK, thereby creating an additional binding site for the adapter protein Grb2.¹⁹ Association of Grb2 with the GDP-GTP exchange protein Sos and the small GTP binding protein Ras can activate the ERK1/2 signaling cascade.¹⁰ Thus, FAK may potentially regulate both pathways.

FAK is highly expressed in the medial layer of intact arteries, and high levels of FAK were detected in cultured VSMCs in vitro.²⁰ Okuda et al²¹ have shown that Ang II acutely induces FAK tyrosine phosphorylation in cultured VSMCs. The purpose of this study was to determine whether FAK is indeed an upstream PTK regulating ERK1/2 and p70^S6K activation, as well as enhanced protein synthesis in response to Ang II in VSMCs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Cytochalasin D (Cyt-D) was obtained from Calbiochem. Polyclonal anti-FAK and p70^S6K antibodies were from Upstate Biotechnologies, and anti-PYK2 monoclonal and anti-phosphotyrosine (pTyr) polyclonal antibodies were from Transduction Laboratories. Phosphospecific polyclonal ERK1/2 antibody was from New England Biolabs. c-fos oligodeoxynucleotide probe was from Oncogene Science. Horseradish peroxidase–conjugated goat anti-rabbit IgG was from Bio-Rad. Angiotensin II and protein A and G beads were from Sigma. [³H]Phenylalanine and [³²P]ATP were from Amersham.

Cell Culture
Rat aortic VSMCs were isolated from 10- to 12-week-old Sprague-Dawley rats and cultured on uncoated, plastic dishes, as previously described.²²

Protein Synthesis Measurements
VSMCs were maintained in serum-free medium or stimulated with Ang II (100 nmol/L) for 24 hours. Cells were labeled with [³H]phenylalanine (2 µCi/mL) during the last 6 hours of treatment. Cells were rinsed with PBS and harvested with 10% trichloroacetic acid (TCA) on ice. Samples were centrifuged (25 000g, 5 minutes), washed with TCA, and recentrifuged. Pellets were solubilized in 0.2N NaOH (500 µL, 20 minutes, 60°C). A portion of the sample was analyzed for total protein using a bicinchonic acid protein assay (Pierce Chemical Co). Radioactivity was measured by liquid scintillation spectroscopy. Results are means of triplicate dishes and are expressed as disintegrations per minute (dpm)/µg protein.

Western Blot Analysis
Cell lysates were prepared as previously described.²² Equal amounts of protein (40 µg) were resolved by SDS-PAGE and Western blotting. Primary antibody binding was visualized by enhanced chemiluminescence (Amersham). Band intensity was quantified by laser densitometry.

Immunoprecipitation
Lysates were prepared as previously described.²² Equal amounts of protein (500 µg) were treated overnight with antibodies against phosphotyrosine residues (pTyr) or FAK. Immune complexes were then precipitated using protein A or protein G Sepharose (2 hours; 4°C). Beads were centrifuged, washed, resuspended in sample buffer, and analyzed by SDS-PAGE and Western blotting as described above.

Immunocytochemistry
Cells were fixed, permeabilized, and stained with anti-FAK antibody (to visualize FAK and FAK-related nonkinase [FRNK]) and rhodamine-conjugated phalloidin (to visualize actin filaments).²³ Cells were viewed using a Zeiss model LSM 510 confocal microscope.

Northern Blotting
Total cellular RNA was isolated, size fractionated, and transferred to nylon membranes. c-fos mRNA and 18S rRNA were detected by hybridization to ³²P-labeled oligodeoxynucleotide probes, as previously described.²⁴

Adenoviral Constructs
A replication-defective adenovirus encoding FRNK (Adv-FRNK) was constructed as previously described.²³ A replication-defective adenovirus containing the ß-galactosidase gene, LacZ (Adv-LacZ), was used to control for the nonspecific effects of viral infection. Adenoviruses were amplified and purified using HEK293 cells, as previously described.²³ Viral titers were estimated by absorbance at 260 nm as follows: viral particles per milliliter=A₂₆₀xdilutionx10¹⁰. Preliminary experiments determined that a concentration of 500 particles of Adv-FRNK or Adv-LacZ per cell (ppc) produced readily detectable overexpression of these proteins within 48 hours (Western blot analysis; see Figure 5A) and infected most VSMCs (X-gal staining) (data not shown).

View larger version (79K): [in this window] [in a new window]   Figure 5. FRNK overexpression and its effects on cytoskeletal architecture. A, VSMCs were infected with Adv-FRNK (100 to 1000 ppc). Equal amounts of extracted cell protein (40 µg) were analyzed by SDS-PAGE and Western blotting using an antibody that recognizes both FAK and FRNK. VSMCs were maintained in serum-free medium (B) or were infected with Adv-LacZ (C) or Adv-FRNK (D). Cells were fixed, stained with an antibody that recognizes both FAK and FRNK (green) and with rhodamine-phalloidin (red), and viewed with a confocal microscope. Yellow signals indicate sites of colocalization of FAK/FRNK and F-actin. Std indicates standard.

Data Analysis
Results were expressed as mean±SEM. Data were compared by ANOVA followed by the Dunnet test or Student-Newman-Keuls test using SigmaStat, version 1.0 (Jandel Scientific).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of Protein Kinase Inhibitors on Ang II–Induced Protein Synthesis
Using pharmacological inhibitors, we first examined the relative contribution of the ERK1/2 and p70^S6K pathways to Ang II–mediated increases in protein synthesis. As seen in Figure 1A, Ang II (100 nmol/L) caused a 57±5% increase in [³H]phenylalanine incorporation into total protein. Pretreatment with PD98059 (10 to 30 µmol/L; 30 minutes), a highly specific inhibitor of ERK1/2 activation, resulted in a dose-dependent decrease in Ang II–dependent [³H]phenylalanine incorporation, with a 38% reduction in protein synthesis at the 30 µmol/L concentration. Of note, this concentration of PD98059 completely inhibited Ang II–mediated ERK1/2 phosphorylation, as assessed by Western blotting with phospho-ERK–specific antibody (data not shown). Similarly, pretreatment of cells with rapamycin (1 to 10 nmol/L; 45 minutes), a p70^S6K inhibitor, also significantly reduced the rate of Ang II–induced protein synthesis (Figure 1B). However, rapamycin had no effect on ERK1/2 phosphorylation, as detected by Western blotting (data not shown). Similar results have been obtained by other investigators.^{7 25}

View larger version (24K): [in this window] [in a new window]   Figure 1. Protein kinase inhibitors block Ang II–mediated protein synthesis. VSMCs were treated with Ang II (100 nmol/L, 24 hours) and then labeled with [3H]phenylalanine (2 µCi/mL; 6 hours). Cells were pretreated with either PD98059 (A), rapamycin (B), or genistein or daidzein (C) at the indicated concentrations before Ang II stimulation. Results are expressed as dpm/µg total protein normalized to untreated cultures. *P<0.05 vs control; +P<0.05 vs Ang II.

Genistein, a PTK inhibitor, was then used to determine whether 1 or more PTKs were upstream regulators of these parallel pathways. Pretreatment with genistein (10 to 100 µmol/L; 60 minutes) caused a dose-dependent inhibition of Ang II–induced protein synthesis (Figure 1C). However, daidzein (100 µmol/L, 60 minutes), an inactive analog of genistein, had no effect on Ang II–induced protein synthesis. Herbimycin A (0.1 to 0.5 µmol/L), another PTK inhibitor, produced similar results (data not shown).

Ang II Induces FAK Phosphorylation
We next examined whether Ang II activated FAK. As seen in Figure 2A, VSMCs demonstrated a substantial degree of basal FAK phosphorylation. However, Ang II (100 nmol/L; 2 to 60 minutes) substantially increased FAK phosphorylation in a time- and concentration-dependent manner. FAK activation was maximal at 5 minutes and returned to basal levels by 60 minutes. Ang II–induced FAK phosphorylation was detectable with as little as 10^–12 mol/L and reached a maximum with 10^–7 mol/L of agonist (Figure 2B). These results were obtained by immunoprecipitating cell extracts with anti-pTyr and probing the resulting Western blot with anti-FAK antibody. However, similar results were obtained when the same cell extracts were immunoprecipitated with anti-FAK, and the resulting Western blot was probed with anti-pTyr (data not shown). This time- and dose-dependent response of FAK activation are consistent with previous studies.^{21 22}

View larger version (47K): [in this window] [in a new window]   Figure 2. Ang II stimulates FAK phosphorylation. Cells were stimulated with various Ang II concentrations (10-12 to 10-6 mol/L) for 5 minutes. Cell lysates were immunoprecipitated (IP) with anti-pTyr antibody and analyzed by Western blotting with anti-FAK antibody. Data from 4 or 5 experiments are expressed as fold increase vs unstimulated cells. *P<0.05 vs control. C, VSMCs were pretreated (60 minutes) with genistein (25 to 100 µmol/L) before stimulation with Ang II (5 minutes). Immunoprecipitates were prepared and analyzed as in panels A and B. Std indicates standard.

In light of the effects of genistein on Ang II–induced protein synthesis (Figure 1C), we then examined whether genistein inhibited Ang II–mediated FAK phosphorylation. As seen in Figure 2C, genistein caused a dose-dependent decrease in basal FAK phosphorylation and also significantly inhibited Ang II–mediated FAK phosphorylation at concentrations that also inhibited protein synthesis.

FAK Localization and Ang II–Induced FAK Phosphorylation Are Dependent on the Actin Cytoskeleton
We next examined FAK localization and the effects of cytoskeletal disassembly with Cyt-D on Ang II–mediated FAK phosphorylation. In initial experiments, control VSMCs and VSMCs pretreated with Cyt-D were stained with rhodamine-phalloidin and anti-FAK polyclonal antibody. As seen in Figure 3A, actin stress fibers traversed the length and breadth of untreated, control cells, colocalizing with FAK at focal adhesions. However, pretreatment of VSMCs with Cyt-D (10 µmol/L) resulted in the complete disruption of the actin cytoskeleton and the loss of punctate FAK staining, suggesting delocalization of FAK from focal adhesions (Figure 3D). Lower concentrations of Cyt-D (Figures 3B and 3C) produced less dramatic alterations in FAK localization and stress fiber assembly. However, acute stimulation of VSMCs with Ang II did not result in any obvious differences in FAK localization or cytoskeletal organization in either control or Cyt-D–treated cells (data not shown).

View larger version (55K): [in this window] [in a new window]   Figure 3. Effects of Cyt-D on FAK localization and phosphorylation. VSMCs were maintained in serum-free medium (A) or treated with Cyt-D (0.1, 1.0, or 10 µmol/L; 30 minutes for panels B, C, and D, respectively). Cells were fixed, stained using anti-FAK antibody (green) and rhodamine-phalloidin (red), and viewed under a confocal microscope. Yellow signals represent sites of f-actin and FAK colocalization. E, VSMCs were pretreated with Cyt-D (1 to 10 µmol/L, 30 minutes) and stimulated with Ang II (5 minutes). Cell lysates were immunoprecipitated with anti-pTyr or anti-FAK and immunoblotted with anti-FAK or anti-pTyr. Shown are quantitative results of 4 Western blots in which pTyr immunoprecipitations (IP) were probed with anti-FAK. Results are expressed as fold increase vs unstimulated cells. *P<0.05 vs control; +P<0.05 vs Ang II–treated cells.

Immunoprecipitation studies substantiated these findings. As seen in Figure 3E, dose-dependent disruption of the actin cytoskeleton resulted in a significant decrease in basal FAK phosphorylation. In addition, Ang II–induced FAK phosphorylation was inhibited with as little as 1 µmol/L of Cyt-D and was completely abrogated with 10 µmol/L of the drug. Similar results were obtained using latrunculin A, another cytoskeleton-disrupting agent (data not shown).

Ang II–Induced ERK Phosphorylation, but Not p70^S6K Phosphorylation, Is Affected by Cytoskeletal Disruption
Cyt-D was then used to determine whether FAK was upstream of either ERK1/2 or p70^S6K activation or both. As seen in Figure 4A, pretreatment of VSMCs with Cyt-D had no effect on basal ERK1/2 phosphorylation as detected by Western blotting with phosphospecific ERK1/2 antibody. However, Ang II–induced ERK1/2 phosphorylation was completely abrogated at similar Cyt-D concentrations at which FAK phosphorylation was inhibited, suggesting that FAK activation was upstream of ERK1/2. In contrast, p70^S6K phosphorylation, as detected by a band shift on Ang II stimulation, remained unaffected even in the presence of higher doses of Cyt-D than those that inhibited both FAK and ERK1/2 phosphorylation (Figure 4B).

View larger version (36K): [in this window] [in a new window]   Figure 4. Cytoskeletal disruption blocks Ang II–induced ERK1/2 but not p70S6K phosphorylation and inhibits Ang II–induced protein synthesis. A, VSMCs were pretreated (30 minutes) with Cyt-D (1 to 10 µmol/L) and stimulated with Ang II (100 nmol/L; 5 or 15 minutes). Extracted cell proteins (40 µg) were analyzed by Western blotting using phosphospecific ERK1/2 antibody. Results of 4 experiments are expressed as percentage of phospho-ERK2 observed in Ang II–treated cells. *P<0.05 vs Ang II–treated cells. B, Similar cell extracts were analyzed for p70S6K activation by Western blotting. The upward mobility shift is indicative of p70S6K activation. C, Cells were pretreated with Cyt-D (30 minutes, 1 to 10 µmol/L) and stimulated with Ang II (100 nmol/L, 24 hours). Protein synthesis was measured by [3H]phenylalanine incorporation. Quantitative results of 5 experiments are expressed as dpm/µg of total protein normalized to the incorporation rate of untreated cultures. *P<0.05 vs control.

Ang II–Induced Protein Synthesis Is Inhibited by Cytoskeletal Disruption
We then examined whether interfering with FAK phosphorylation with Cyt-D affected protein synthetic rates. As seen in Figure 4C, Cyt-D had only a modest, nonsignificant effect on basal protein synthetic rate. However, Cyt-D produced a dose-dependent decrease in Ang II–induced protein synthesis. Ang II increased protein synthetic rate by only 22% at the highest concentration of Cyt-D tested, as compared with 48% in untreated, control cells. Thus, the ability of Ang II to increase protein synthesis was reduced by cytoskeletal disruption, which is consistent with the effects of Cyt-D on FAK phosphorylation (Figure 3E) and the ERK1/2 signaling cascade (Figure 4A).

Effect of FRNK on FAK Localization and Cytoskeletal Architecture
Although these pharmacological studies provided circumstantial evidence for a role for FAK in regulating Ang II–induced protein synthesis, we sought to more specifically target FAK-dependent signaling using a replication-defective adenovirus encoding FRNK.²⁶ As seen in Figure 5A, 100 to 500 ppc of Adv-FRNK caused the dose-dependent expression of a 41-/43-kDa polypeptide that was recognized by an antibody raised against the C-terminal portion of FAK. Traces of this polypeptide were also observed in uninfected and Adv-LacZ-infected VSMCs. Higher concentrations of virus above 500 particles per cell (ppc) did not further increase FRNK overexpression. Furthermore, FRNK overexpression appeared to have no significant effect on endogenous FAK levels.

We then examined the effect of FRNK overexpression on cytoskeletal architecture. As seen in Figure 5D, FRNK overexpression was inhomogeneous, with some cells nearly completely filled with immunoreactive protein, whereas others displayed much less FAK/FRNK staining. Most VSMCs, however, showed enhanced focal adhesion immunofluorescence, suggesting that FRNK indeed localized to these structures. Despite this inhomogeneity of expression, we noted the complete loss of actin stress fibers in almost all of the Adv-FRNK–infected cells, which suggested that nearly all cells were expressing some level of transgene. The markedly reduced f-actin staining also suggested that FRNK interfered with actin filament assembly. This was not due to a nonspecific effect of adenoviral infection, as both uninfected and ß-galactosidase–overexpressing cells stained normally for actin filaments and FAK (Figures 5B and 5C, respectively).

FRNK Abrogates FAK Phosphorylation
We examined the effect of FRNK overexpression on FAK phosphorylation. As seen in Figures 6A and 6B, basal FAK phosphorylation was reduced by 28±8% by FRNK. FRNK also completely blocked the Ang II–induced increase in FAK phosphorylation, as compared with cells infected with Adv-LacZ. Of note, Adv-LacZ–infected cells displayed somewhat reduced Ang II–induced FAK phosphorylation as compared with uninfected VSMCs (see Figure 2). However, total FAK levels were similar in Adv-FRNK– and Adv-LacZ-infected cells (Figure 6A). Similar immunoprecipitation experiments were then performed to examine the effects of FRNK overexpression on PYK2, the other member of the FAK family of PTKs. As seen in Figure 6C, Ang II–induced PYK2 phosphorylation was also partially blocked by FRNK overexpression, as compared with uninfected cells, or cells infected with Adv-LacZ. Taken together, these results indicate that FRNK indeed interferes with FAK phosphorylation in VSMCs, but there is considerable interaction between FAK, PYK2, and cytoskeleton-dependent signaling in these cells.

View larger version (52K): [in this window] [in a new window]   Figure 6. Effects of FRNK overexpression on Ang II–induced FAK and PYK2 phosphorylation. A, VSMCs were infected with Adv-FRNK or Adv-LacZ and then stimulated with Ang II (100 nmol/L, 5 minutes). Cell lysates were immunoprecipitated (IP) with either anti-pTyr or anti-FAK and probed with anti-pTyr or anti-FAK, as in Figure 3. B, Quantitative results of 3 Western blots in which pTyr immunoprecipitations were probed with anti-FAK are depicted. Results are expressed as fold increase vs Adv-LacZ–infected, unstimulated cells. *P<0.05 vs control. C, Uninfected cells or cells infected with Adv-LacZ or Adv-FRNK were stimulated with Ang II. Cell lysates were immunoprecipitated with anti-pTyr and probed with anti-PYK2.

FRNK Overexpression Blocks Ang II–Mediated ERK1/2 Phosphorylation and Protein Synthesis
As expected from the results obtained with Cyt-D (Figure 4), FRNK overexpression markedly suppressed Ang II–induced ERK1/2 phosphorylation but had relatively little effect on p70^S6K activation. As seen in Figure 7A, Ang II–mediated ERK1/2 activation was relatively unaffected by adenoviral infection with Adv-LacZ, as compared with uninfected VSMCs. However, Ang II–stimulated ERK1/2 activation was substantially reduced in Adv-FRNK–infected cells. In contrast, neither Adv-LacZ nor Adv-FRNK infection affected p70^S6K phosphorylation (Figure 7B). Of note, the total amount of p70^S6K was relatively unaffected in either Adv-LacZ– or Adv-FRNK–infected cells, as compared with uninfected, control VSMCs.

View larger version (52K): [in this window] [in a new window]   Figure 7. FRNK overexpression inhibits ERK1/2 phosphorylation and c-fos induction and blocks Ang II–induced protein synthesis. A, VSMCs were maintained in serum-free medium or were infected with Adv-FRNK or Adv-LacZ. Cells were stimulated with Ang II (5 or 15 minutes). Equal amounts of cell protein (40 µg) were subjected to Western blotting, and blots were probed with phospho-ERK1/2 antibodies. Quantitative results of 4 experiments are expressed as percentage of phospho-ERK2 observed in Ang II–treated cells. *P<0.05 vs uninfected, Ang II–stimulated cells. B, Similar cell extracts were analyzed with a phosphospecific p70S6K antibody. Results of 3 experiments are expressed as the percentage of phosphorylated p70S6K in uninfected control cells. C, c-fos mRNA levels were analyzed by Northern blotting in uninfected and in Adv-LacZ– and Adv-FRNK–infected VSMCs under control conditions or after Ang II stimulation (100 nmol/L, 15 and 30 minutes). To ensure equal loading conditions, the blot was also probed with an oligodeoxynucleotide specific for 18S rRNA. D, [3H]Phenylalanine incorporation was analyzed in uninfected and in Adv-LacZ– and Adv-FRNK–infected VSMCs under control conditions or after Ang II treatment (100 nmol/L; 24 hours). Quantitative results of 5 experiments are expressed as fold change in [3H]phenylalanine incorporation relative to uninfected cells. *P<0.05 vs control.

To further substantiate the role of FAK in the ERK1/2 signaling cascade, we examined c-fos induction in FRNK-overexpressing cells. As seen in Figure 7C, Ang II markedly increased c-fos mRNA levels in uninfected and Adv-LacZ–infected VSMCs. However, FRNK overexpression abolished c-fos induction, which correlated with the inhibition of ERK1/2 activation (Figure 7A). Finally, FRNK overexpression had no significant effect on basal rates of protein synthesis but completely abrogated the Ang II–induced increase in [³H]phenylalanine incorporation (Figure 7D). The loss of Ang II responsiveness in Adv-FRNK–infected cells was not due to a nonspecific effect of adenoviral infection, as Ang II stimulated protein synthesis to nearly the same extent in Adv-LacZ–infected and uninfected VSMCs.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is accumulating evidence to indicate that both the ERK1/2 and p70^S6K pathways are regulated by 1 or more upstream PTKs. Indeed, Ang II stimulation in VSMCs leads to the activation of a number of different nonreceptor PTKs, including Src,²⁷ FAK,^{20 21} and PYK2,^{22 28 29} which may all be involved in regulating specific aspects of cellular growth. This study demonstrates for the first time that FAK is a critical component of the signal transduction pathway that may regulate at least 1 of these downstream pathways.

Our results establish the interdependence of FAK and the actin cytoskeleton in Ang II–dependent signal transduction. Disruption of actin stress fibers with Cyt-D resulted in FAK delocalization and dephosphorylation, and inhibition of ERK1/2 phosphorylation and Ang II–mediated protein synthesis. This requirement for actin filaments may be related to their role in maintaining the association of FAK with cell-surface integrins and other components of the cytoskeleton. These components appear necessary for agonist-induced local tyrosine phosphorylation by the signaling molecules that accumulate at focal adhesions.³⁰ It should be pointed out, however, that Cyt-D treatment potently inhibited FAK phosphorylation but only partially blocked Ang II–induced increases in protein synthetic rate. In contrast, FRNK overexpression had a lesser effect on FAK tyrosine phosphorylation but completely abrogated Ang II–induced protein synthesis. The reason for these differences is unclear, but they suggest that phosphorylation-independent determinants on FAK may play a role in regulating protein synthesis in VSMCs.

Similarly, interfering with FAK phosphorylation by FRNK overexpression resulted in actin cytoskeletal disassembly. We targeted FAK by overexpressing FRNK, which not only resulted in the inhibition of Ang II–induced FAK phosphorylation but also significantly reduced Ang II–induced ERK1/2 activation and c-fos induction. FRNK, which contains the focal adhesion targeting sequence of FAK but is devoid of the Y-397 autophosphorylation site and kinase domain, appeared to localize in high concentrations within focal adhesions. Although the mechanism whereby FRNK interferes with FAK-dependent signal transduction is not known, it seems reasonable to conclude that FRNK competed with endogenous FAK for specific binding sites present on other focal adhesion components. The displacement of FAK may in turn have resulted in the loss of signals necessary for the maintenance of actin stress fiber assembly and for other downstream signaling events leading to the ERK1/2 cascade.³¹

In contrast, cytoskeletal disruption by Cyt-D and FRNK did not significantly affect Ang II–induced p70^S6K activation. These results indicate that the effects of Cyt-D and FRNK overexpression on ERK1/2 phosphorylation were not just the result of nonspecific effects on cell viability. This was confirmed by the fact that basal rates of protein synthesis were relatively similar in control, Cyt-D–treated, and FRNK-overexpressing cells. It is conceivable that the residual degree of FAK phosphorylation in Cyt-D and FRNK-overexpressing cells was sufficient to maintain agonist-induced downstream signaling to p70^S6K. Also likely, however, is the possibility that p70^S6K activation occurred independently of FAK activation by a different, upstream, PTK. Graves et al¹⁴ previously demonstrated a critical role for a Ca²⁺-sensitive PTK upstream of p70^S6K, but not p90^RSK, in rat liver epithelial cells. They postulated that PYK2, the other member of the FAK family of nonreceptor PTKs, was responsible. Indeed, our previous study²² demonstrated that PYK2 is activated in VSMCs in response to similar concentrations of Ang II and over a similar time period. We found that PYK2 was predominantly localized to the cytoplasm,²² which is quite distinct from the focal adhesion staining of FAK demonstrated in the present report. However, we found that FRNK overexpression partially blocked Ang II–induced PYK2 phosphorylation in VSMCs, suggesting a close interaction between both members of this kinase family and the cytoskeleton. Although it is interesting to speculate that PYK2 and FAK may regulate different components of the signaling pathways involved in Ang II–induced protein synthesis, future studies using more specific antagonists of FAK- and PYK2-dependent signaling will be necessary to confirm this speculation.

	Acknowledgments

 
These studies were supported by NIH Grants RO1 HL34328 (to A.M.S.), HL63711 (to A.M.S.), and HL56046 (to P.A.L.) and by gifts from Mr. and Mrs. James Beck, the Nalco Foundation, and the Ralph and Marian Falk Trust for Medical Research. G.G. is a recipient of an American Heart Association-Midwest Affiliate Predoctoral Fellowship. We thank Alan G. Ferguson for excellent technical assistance.

Received April 26, 2000; revision received August 14, 2000; accepted August 21, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Berk BC, Corson MA. Angiotensin II signal transduction in vascular smooth muscle: role of tyrosine kinases. Circ Res. 1997;80:607–616.[Abstract/Free Full Text]
2. Rosendorff C. Vascular hypertrophy in hypertension: role of the renin-angiotensin system. Mt Sinai J Med. 1998;65:108–117.[Medline] [Order article via Infotrieve]
3. 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]
4. Geisterfer AAT, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 1998;62:749–756.[Abstract/Free Full Text]
5. Holycross BJ, Peach MJ, Owens GK. Angiotensin II stimulates increased protein synthesis, not increased DNA synthesis, in intact rat aortic segments, in vitro. J Vasc Res. 1993;30:80–86.[Medline] [Order article via Infotrieve]
6. Giasson E, Meloche S. Role of p70 S6 kinase in angiotensin II-induced protein synthesis in vascular smooth muscle cells. J Biol Chem. 1995;270:5225–5231.[Abstract/Free Full Text]
7. 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]
8. Taubman MB, Berk BC, Izumo S, Tsuda T, Alexander RW, Nadal-Ginard B. Angiotensin II induces c-fos mRNA in aortic smooth muscle. J Biol Chem. 1989;264:526–530.[Abstract/Free Full Text]
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. Eguchi S, Matsumoto T, Motley ED, Utsunomiya H, Inagami T. Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells. J Biol Chem. 1996;271:14169–14175.[Abstract/Free Full Text]
11. Price DJ, Grove JR, Calvo V, Avruch J, Bierer BE. Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science. 1992;25:973–977.
12. Chung J, Grammer TC, Lemon KP, Kazlauskas A, Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidyl-inositol-3-OH kinase. Nature. 1994;370:71–75.[Medline] [Order article via Infotrieve]
13. Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci U S A. 1993;90:5889–5992.[Abstract/Free Full Text]
14. Graves LM, He Y, Lambert J, Hunter D, Li X, Earp HS. An intracellular calcium signal activated p70 but not p90 ribosomal S6 kinase in liver epithelial cells. J Biol Chem. 1997;272:1920–1928.[Abstract/Free Full Text]
15. Leduc I, Haddad P, Giasson E, Meloche S. Involvement of tyrosine kinase pathway in the growth promoting effects of angiotensin II on aortic smooth muscle cells. Mol Pharmacol. 1995;48:585–592.
16. Schlaepfer DD, Hauck CR, Sieg DJ. Signaling through focal adhesion kinase. Prog Biophys Mol Biol. 1999;71:435–478.[Medline] [Order article via Infotrieve]
17. Schaller MD, Hildebrand JD, Shannon JD, Fox JW, Vines RR, Parsons JT. Autophosphorylation of the focal adhesion kinase, pp125^FAK, directs SH2 binding of pp60^Src. Mol Cell Biol. 1994;14:1680–1688.[Abstract/Free Full Text]
18. Chen HC, Appedu PA, Isoda H, Guan JL. Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. J Biol Chem. 1996;271:26329–26334.[Abstract/Free Full Text]
19. Schlaepfer DD, Hunter T. Evidence for in vivo phosphorylation of the Grb2 SH2 domain binding site on focal adhesion by Src-family of protein tyrosine kinases. Mol Cell Biol. 1996;16:5623–5633.[Abstract]
20. 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]
21. Okuda M, Kawahara Y, Nakayama I, Hoshijima M, Yokoyama M. Angiotensin II transduces its signal to focal adhesions via angiotensin II type I receptors in vascular smooth muscle cells. FEBS Lett. 1995;368:343–347.[Medline] [Order article via Infotrieve]
22. Sabri A, Govindarajan G, Griffin T, Byron KL, Samarel AM, Lucchesi PA. Calcium and protein kinase C dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle. Circ Res. 1998;83:841–851.[Abstract/Free Full Text]
23. Eble DM, Strait JB, Govindarajan G, Lou J, Byron KL, Samarel AM. Endothelin induced cardiac myocyte hypertrophy: role for focal adhesion kinase. Am J Physiol Heart Circ Physiol. 2000;278:H1695–H1707.[Abstract/Free Full Text]
24. Samarel AM, Engelmann GL. Contractile activity modulates myosin heavy chain-ß expression in neonatal rat heart cells. Am J Physiol Heart Circ Physiol. 1991;261:H1067–H1077.[Abstract/Free Full Text]
25. Sadoshima J, Izumo S. Rapamycin selectively inhibits angiotensin II-induced increase in protein synthesis in cardiac myocytes in vitro. Circ Res. 1995;77:1040–1052.[Abstract/Free Full Text]
26. Richardson A, Parsons JT. A mechanism for the regulation of the adhesion-associated protein tyrosine kinase pp125^FAK. Nature. 1996;380:538–540.[Medline] [Order article via Infotrieve]
27. Ishida M, Marrero MB, Schieffer B, Ishida T, 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]
28. Brinson AE, Harding T, Diliberto PA, He Y, Li X, Hunter D, Herman B, Earp HS, Graves LM. Regulation of a calcium dependent tyrosine kinase in vascular smooth muscle cells by angiotensin II and platelet derived growth factor. J Biol Chem. 1998;273:1711–1718.[Abstract/Free Full Text]
29. Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo PD, Akiyama SK, Yamada KM. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol. 1995;131:791–805.[Abstract/Free Full Text]
30. Ishida M, Ishida T, Thomas SM, Berk BC. Activation of extracellular signal-regulated kinases (ERK1/2) by angiotensin II is dependent on c-Src in vascular smooth muscle cells. Circ Res. 1998;82:7–12.[Abstract/Free Full Text]
31. 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]

This article has been cited by other articles:

				M. F. Walsh, D. R. Ampasala, J. Hatfield, R. Vander Heide, S. Suer, A. K. Rishi, and M. D. Basson Transforming Growth Factor-{beta} Stimulates Intestinal Epithelial Focal Adhesion Kinase Synthesis via Smad- and p38-Dependent Mechanisms Am. J. Pathol., August 1, 2008; 173(2): 385 - 399. [Abstract] [Full Text] [PDF]

				H. Louis, A. Kakou, V. Regnault, C. Labat, A. Bressenot, J. Gao-Li, H. Gardner, S. N. Thornton, P. Challande, Z. Li, et al. Role of {alpha}1beta1-integrin in arterial stiffness and angiotensin-induced arterial wall hypertrophy in mice Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2597 - H2604. [Abstract] [Full Text] [PDF]

				S. Albinsson and P. Hellstrand Integration of signal pathways for stretch-dependent growth and differentiation in vascular smooth muscle Am J Physiol Cell Physiol, August 1, 2007; 293(2): C772 - C782. [Abstract] [Full Text] [PDF]

				L. Jin, M. J. Kern, C. A. Otey, B. R. Wamhoff, and A. V. Somlyo Angiotensin II, Focal Adhesion Kinase, and PRX1 Enhance Smooth Muscle Expression of Lipoma Preferred Partner and its Newly Identified Binding Partner Palladin to Promote Cell Migration Circ. Res., March 30, 2007; 100(6): 817 - 825. [Abstract] [Full Text] [PDF]

				M. Takeji, T. Moriyama, S. Oseto, N. Kawada, M. Hori, E. Imai, and T. Miwa Smooth Muscle {alpha}-Actin Deficiency in Myofibroblasts Leads to Enhanced Renal Tissue Fibrosis J. Biol. Chem., December 29, 2006; 281(52): 40193 - 40200. [Abstract] [Full Text] [PDF]

				M. Brancaccio, E. Hirsch, A. Notte, G. Selvetella, G. Lembo, and G. Tarone Integrin signalling: The tug-of-war in heart hypertrophy Cardiovasc Res, June 1, 2006; 70(3): 422 - 433. [Abstract] [Full Text] [PDF]

				L. H. Romer, K. G. Birukov, and J. G.N. Garcia Focal Adhesions: Paradigm for a Signaling Nexus Circ. Res., March 17, 2006; 98(5): 606 - 616. [Abstract] [Full Text] [PDF]

				T. A. Hornberger, D. D. Armstrong, T. J. Koh, T. J. Burkholder, and K. A. Esser Intracellular signaling specificity in response to uniaxial vs. multiaxial stretch: implications for mechanotransduction Am J Physiol Cell Physiol, January 1, 2005; 288(1): C185 - C194. [Abstract] [Full Text] [PDF]

				R. Arya, V. Kedar, J. R. Hwang, H. McDonough, H.-H. Li, J. Taylor, and C. Patterson Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy J. Cell Biol., December 20, 2004; 167(6): 1147 - 1159. [Abstract] [Full Text] [PDF]

				P. Rocic, H. Jo, and P. A. Lucchesi A role for PYK2 in ANG II-dependent regulation of the PHAS-1-eIF4E complex by multiple signaling cascades in vascular smooth muscle Am J Physiol Cell Physiol, December 1, 2003; 285(6): C1437 - C1444. [Abstract] [Full Text] [PDF]

				H. K. Avraham, T.-H. Lee, Y. Koh, T.-A. Kim, S. Jiang, M. Sussman, A. M. Samarel, and S. Avraham Vascular Endothelial Growth Factor Regulates Focal Adhesion Assembly in Human Brain Microvascular Endothelial Cells through Activation of the Focal Adhesion Kinase and Related Adhesion Focal Tyrosine Kinase J. Biol. Chem., September 19, 2003; 278(38): 36661 - 36668. [Abstract] [Full Text] [PDF]

				L. J. Sundberg, L. M. Galante, H. M. Bill, C. P. Mack, and J. M. Taylor An Endogenous Inhibitor of Focal Adhesion Kinase Blocks Rac1/JNK but Not Ras/ERK-dependent Signaling in Vascular Smooth Muscle Cells J. Biol. Chem., August 8, 2003; 278(32): 29783 - 29791. [Abstract] [Full Text] [PDF]

				S. Nakao, T. Kuwano, T. Ishibashi, M. Kuwano, and M. Ono Synergistic Effect of TNF-{alpha} in Soluble VCAM-1-Induced Angiogenesis Through {alpha}4 Integrins J. Immunol., June 1, 2003; 170(11): 5704 - 5711. [Abstract] [Full Text] [PDF]

				G. D. Frank, M. Mifune, T. Inagami, M. Ohba, T. Sasaki, S. Higashiyama, P. J. Dempsey, and S. Eguchi Distinct Mechanisms of Receptor and Nonreceptor Tyrosine Kinase Activation by Reactive Oxygen Species in Vascular Smooth Muscle Cells: Role of Metalloprotease and Protein Kinase C-{delta} Mol. Cell. Biol., March 1, 2003; 23(5): 1581 - 1589. [Abstract] [Full Text] [PDF]

				S. Bhattacharya, N. Sen, M. T. Yiming, R. Patel, K. Parthasarathi, S. Quadri, A. C. Issekutz, and J. Bhattacharya High Tidal Volume Ventilation Induces Proinflammatory Signaling in Rat Lung Endothelium Am. J. Respir. Cell Mol. Biol., February 1, 2003; 28(2): 218 - 224. [Abstract] [Full Text] [PDF]

				E. A. Goncharova, A. J. Ammit, C. Irani, R. G. Carroll, A. J. Eszterhas, R. A. Panettieri, and V. P. Krymskaya PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells Am J Physiol Lung Cell Mol Physiol, August 1, 2002; 283(2): L354 - L363. [Abstract] [Full Text] [PDF]

				A. L. Bayer, M. C. Heidkamp, N. Patel, M. J. Porter, S. J. Engman, and A. M. Samarel PYK2 expression and phosphorylation increases in pressure overload-induced left ventricular hypertrophy Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H695 - H706. [Abstract] [Full Text] [PDF]

				M. C. Heidkamp, A. L. Bayer, J. A. Kalina, D. M. Eble, and A. M. Samarel GFP-FRNK Disrupts Focal Adhesions and Induces Anoikis in Neonatal Rat Ventricular Myocytes Circ. Res., June 28, 2002; 90(12): 1282 - 1289. [Abstract] [Full Text] [PDF]

				Y. Matsumoto, K. Tanaka, G. Hirata, M. Hanada, S. Matsuda, T. Shuto, and Y. Iwamoto Possible Involvement of the Vascular Endothelial Growth Factor-Flt-1-Focal Adhesion Kinase Pathway in Chemotaxis and the Cell Proliferation of Osteoclast Precursor Cells in Arthritic Joints J. Immunol., June 1, 2002; 168(11): 5824 - 5831. [Abstract] [Full Text] [PDF]

				P. Rocic, T. M. Griffin, C. N. McRae, and P. A. Lucchesi Altered PYK2 phosphorylation by ANG II in hypertensive vascular smooth muscle Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H457 - H465. [Abstract] [Full Text] [PDF]