Down-regulation by Antisense Oligonucleotides Establishes a Role
for the Proline-rich Tyrosine Kinase PYK2 in Angiotensin II-induced
Signaling in Vascular Smooth Muscle*
Petra
Rocic and
Pamela A.
Lucchesi
From the Department of Physiology and Biophysics, University of
Alabama at Birmingham, Birmingham, Alabama 35294-0005
Received for publication, February 22, 2001, and in revised form, March 20, 2001
 |
ABSTRACT |
Abnormal vascular smooth muscle cell (VSMC)
growth plays a key role in the pathogenesis of hypertension and
atherosclerosis. Angiotensin II (Ang II) elicits a hypertrophic growth
response characterized by an increase in protein synthesis in the
absence of DNA synthesis and cell proliferation. Intracellular
signaling mechanisms linking angiotensin type I receptor activation to
protein synthesis in VSMC have not been fully characterized. The
present study investigates the role of the nonreceptor proline-rich
tyrosine kinase 2 (PYK2) in Ang II-induced VSMC protein synthesis and
in the regulation of two signaling pathways that have been implicated in the control of protein synthesis, the extracellular signal-regulated kinase (ERK1/2) and the phosphatidylinositol 3-kinase/Akt
pathways. PYK2 antisense oligonucleotides were used to down-regulate
PYK2 expression in cultured VSMC. An 80% down-regulation in PYK2
expression resulted in an ~80% inhibition of ERK1/2 (3.8 ± 1.3 versus 16.6 ± 1.8), p70S6 kinase (1.03 ± 0.03 versus 3.8 ± 0.5), and Akt activation (3.0 ± 0.8 versus 16.0 ± 1.0) by Ang II. Furthermore,
PYK2 down-regulation resulted in a complete inhibition of Ang
II-induced VSMC protein synthesis. These data conclusively identify
PYK2 as an upstream regulator of both the ERK1/2 and the
phosphatidylinositol 3-kinase/Akt pathways that are involved in Ang
II-induced VSMC protein synthesis.
 |
INTRODUCTION |
Altered VSMC1 growth has
been implicated in the pathogenesis of atherosclerosis, restenosis, and
hypertension. In response to different agonists, VSMC are capable of
both a hyperplastic growth response, characterized by an increase in
cell number, and a hypertrophic growth response, characterized by an
increase in cell size accompanied by an increase in protein synthesis
in the absence of cell division (1, 2). Ang II regulates blood pressure
both acutely via potent vasoconstriction and chronically by its effects
on vascular smooth muscle growth. Ang II induces hypertrophic growth in
cultured VSMC as well as in intact aorta that is characterized by an
increase in protein synthesis (3).
Numerous cellular signaling pathways have been implicated in Ang
II-induced VSMC protein synthesis. These include the nonreceptor tyrosine kinases, c-Src (4), proline-rich tyrosine kinase 2 (PYK2) (5,
6), focal adhesion kinase (FAK) (7), the extracellular signal-regulated
kinase 1/2 (ERK1/2) (8, 9), and phosphatidylinositol 3-kinase
(PI3-kinase) (10, 11). Of these, the ERK1/2 and the PI3-kinase/Akt
pathways are key regulators of cell growth in many cell types (11, 12).
We and others have shown that both pathways are activated in response
to Ang II in VSMC (8, 10, 11). The mechanisms by which ERK1/2 mediate
Ang II-induced protein synthesis have not been fully identified but are
thought to occur at the level of gene expression and the initiation of
protein translation. The activation of PI3-kinase and its downstream
targets, Akt and the ribosomal p70S6 kinase, is critical for protein
synthesis in many cell types, including VSMC (11). For example, p70S6 kinase is thought to be the major in vivo mediator of
ribosomal S6 protein phosphorylation, a necessary step in Ang
II-mediated protein synthesis in VSMC (13). In other cell types, both
ERK1/2 and PI3-kinase have been shown to regulate the association of phosphorylated heat and acid-stable protein 1 (PHAS-1) with the eukaryotic translation initiation factor 4E (eIF4E), a key regulator of
translation initiation (14). We have previously shown that pharmacological inhibition of ERK1/2 and PI3-kinase reduce Ang II-induced protein synthesis (7).
The precise molecular mechanisms that couple AT1 receptor
activation to these distinct signaling pathways have not been fully established. We and others have shown that both PYK2 and the closely related FAK can form signaling complexes with the upstream regulators of the ERK1/2 pathway, Src, Shc, and Grb2, and with p130Cas, an adapter protein implicated in PI3-kinase activation (6, 10, 15).
Govindarajan et al. (7) have demonstrated a role for FAK in
Ang II-mediated VSMC protein synthesis. Little is known about the
interrelationship between PYK2 and FAK signaling in response to Ang II
in VSMC.
In the present study, we examined the role of PYK2 in Ang II-induced
VSMC protein synthesis. We show that down-regulation of PYK2 expression
by antisense oligonucleotides resulted in a significant inhibition of
Ang II-induced protein synthesis that was correlated with inhibition of
ERK1/2, Akt, and p70S6 kinase activation. Moreover, PYK2 antisense
treatment caused a remarkable reduction in Ang II-induced FAK
phosphorylation without any effect on FAK expression. A preliminary
report has appeared (16).
 |
EXPERIMENTAL PROCEDURES |
Materials--
PYK2 antisense oligonucleotides and scrambled
control oligonucleotides were custom-made by BIOGNOSTIK
(Göttingen, Germany). Anti-total PYK2 and anti-total FAK
antibodies were from Transduction Laboratories. Anti-total ERK1/2
antibodies were from Santa Cruz Biotechnology. Anti-phospho
ERK1/2 antibodies were from Promega. Anti-phospho Akt
(pSer-473), anti-phospho p70S6 kinase (pThr-389, pSer-424),
anti-total Akt, and anti-total p70S6 kinase antibodies were from New
England Biolabs. Anti-phospho FAK (pTyr-397 and pTyr-861) was
from BIOSOURCE. [3H]phenylalanine
was from Amersham Pharmacia Biotech. Ang II was from Sigma.
Cell Culture--
VSMC were prepared and cultured as
previously described (6).
PYK2 Antisense Oligonucleotide Incorporation--
VSMC were
grown in 10% CS-DMEM to ~60% confluence. Cells were washed three
times in Opti-MEM medium (Life Technologies, Inc.) 1 h
before antisense treatment. VSMC were treated with PYK2 antisense oligonucleotides (0.75 µM) for 8 h. LipofectAMINE
Plus (10 µg/ml, Life Technologies, Inc.) was used as a transfection
reagent. After 8 h, the medium was replaced with 0.2% CS-DMEM and
left overnight. The next day, 0.2% CS-DMEM was replaced with serum
free-DMEM for at least 1 h prior to treatment with Ang II.
Protein Synthesis Measurements--
VSMC were treated with PYK2
oligonucleotides (antisense and scrambled controls) as described above.
The next day, 0.2% CS-DMEM was replaced with serum free-DMEM for at
least 8 h prior to stimulation with 100 nM Ang II for
24 h. During the last 6 h of Ang II incubation, 1 µCi/ml
[3H]phenylalanine (30 Ci/mmol, Amersham Pharmacia
Biotech) was added to each dish. Total protein measurements were
assessed over the 24-h incubation period. VSMC were rinsed with 1 ml of
ice-cold phosphate-buffered saline, and protein was precipitated by
10% trichloroacetic acid for 30 min on ice. The trichloroacetic
acid-precipitable material was solubilized with 0.2 mM NaOH
for 20 min at 60 °C. A portion of the sample was used to determine
total protein using a bicinchonic acid (Pierce) protein assay, and
[3H]phenylalanine was determined by liquid scintillation
counting. Triplicate dishes were used for each measurement.
Immunoblotting--
Western blot analysis was performed as
described (10).
Data Analysis--
Data are expressed as the mean ± S.E.
for at least n = 3 experiments. One-way repeated
measures analysis of variance (ANOVA) followed by Bonferroni's test
was used for comparisons among multiple groups. Differences among means
were considered significant at p < 0.05. Data were
analyzed using InStat statistical software (Graphpad).
 |
RESULTS |
PYK2 Antisense Oligonucleotides Down-regulate PYK2
Expression--
We first determined the effects of PYK2 antisense
oligonucleotides on PYK2 expression. PYK2 antisense oligonucleotides
decreased PYK2 total protein levels by 80% (0.20 ± 0.06 for
antisense oligonucleotides versus control) as measured by
Western blot analysis. PYK2 antisense treatment had no effect on the
expression of the closely related kinase FAK (1.04 ± 0.04 for
antisense oligonucleotides versus control) as measured by
Western blot analysis with anti-total FAK antibodies (Fig.
1). Transfection efficiency, as assessed by fluorescein isothiocyanate-labeled antisense oligonucleotides, was
>85% (data not shown).

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Fig. 1.
PYK2 antisense oligonucleotides selectively
reduce PYK2 expression. Lysates from control VSMC
(CTRL, lane 1) or from cells treated with
LipofectAMINE alone (LFN, lane 2), scrambled
control oligonucleotides (S-ODN, lane 3), or PYK2
antisense oligonucleotides (AS-ODN, lane 4), for
8 h, were placed in 0.2% CS-DMEM overnight. Top panel,
representative Western blots using anti-total PYK2 and FAK antibodies.
Bottom panel, cumulative results of n = 6 experiments. *, p < 0.05 versus control;
, p < 0.05 versus scrambled
oligonucleotides. kD, kilodalton.
|
|
PYK2 Antisense Oligonucleotides Inhibit ERK1/2 Activation in
Response to Ang II--
We have previously shown that PYK2 interacts
with Shc and Grb2, upstream activators of the ERK1/2 signaling pathway
(10). To demonstrate that PYK2 is necessary for Ang II-induced ERK1/2 activation, we examined the effects of PYK2 antisense oligonucleotides on ERK1/2 phosphorylation. Down-regulation of PYK2 resulted in a
significant 77% (3.8 ± 1.3-fold increase
versus control for antisense oligonucleotides compared with
16.6 ± 1.8-fold increase versus control for Ang II)
decrease in Ang II-induced ERK1/2 activation as detected by Western
blot analysis with anti-phospho ERK1/2 antibodies. PYK2 antisense
oligonucleotides did not affect ERK1/2 expression as determined
by anti-total ERK1/2 antibodies (Fig. 2)

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Fig. 2.
PYK2 antisense oligonucleotides down-regulate
ERK1/2 activation by Ang II. Lysates from control VSMC
(CTRL, lanes 1 and 2) or from cells
treated with LipofectAMINE alone (LFN, lanes 3 and 4), scrambled control oligonucleotides
(S-ODN, lanes 5 and 6), or PYK2
antisense oligonucleotides (AS-ODN, lanes 7 and
8), for 8 h, were placed in 0.2% CS-DMEM overnight and
then treated with 100 nM Ang II for 5 min as indicated.
Top panel, representative Western blots using anti-total
PYK2, anti-phospho ERK1/2, and anti-total ERK1/2 antibodies.
Bottom panel, cumulative results of n = 6 experiments. *, p < 0.05 versus control; #,
p < 0.05 versus Ang 5; ,
p < 0.05 versus scrambled
oligonucleotides.
|
|
PYK2 Antisense Oligonucleotides Down-regulate Ang II-induced Akt
and p70S6 Kinase Activation--
We have previously demonstrated that
PYK2 associates with the adaptor molecule p130Cas and with PI3-kinase
in response to Ang II (10). Here we sought to determine whether PYK2
down-regulation by PYK2 antisense oligonucleotides would prevent Ang
II-induced activation of Akt and p70S6 kinase, the major downstream
effectors of the PI3-kinase signaling pathway. Here we show that Akt
and p70S6 kinase are activated in response to Ang II. PYK2
down-regulation resulted in an ~80% inhibition (3.00 ± 0.82-fold increase versus control for antisense
oligonucleotides compared with 16.0 ± 1.0-fold increase
versus control for Ang II) in Ang II-induced Akt and a 76%
inhibition of p70S6 kinase activation (1.03 ± 0.03-fold increase
versus control for antisense oligonucleotides compared with
a 3.8 ± 0.5-fold increase versus control for Ang II),
as measured by Western blot analysis with anti-phospho Akt and
anti-phospho p70S6 kinase antibodies, respectively. PYK2 antisense
oligonucleotides did not affect Akt or p70S6 kinase expression as
measured with anti-total Akt and
anti-total p70S6 kinase antibodies (Figs. 3 and
4).

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Fig. 3.
PYK2 antisense oligonucleotides down-regulate
Akt activation by Ang II. Lysates from control VSMC
(CTRL, lanes 1 and 2) or from cells
treated with LipofectAMINE alone (LFN, lanes 3 and 4), scrambled control oligonucleotides
(S-ODN, lanes 5 and 6), or PYK2
antisense oligonucleotides (AS-ODN, lanes 7 and
8), for 8 h, were placed in 0.2% CS-DMEM overnight and
then treated with 100 nM Ang II for 5 min as indicated.
Top panel, representative Western blots using anti-total
PYK2, anti-phospho Akt, and anti-total Akt antibodies. Bottom
panel, cumulative results of n = 6 experiments. *,
p < 0.05 versus control; #,
p < 0.05 versus Ang 5; ,
p < 0.05 versus scrambled
oligonucleotides.
|
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Fig. 4.
PYK2 antisense oligonucleotides down-regulate
p70S6 kinase activation by Ang II. Lysates from control VSMC
(CTRL, lanes 1 and 2) or from cells
treated with LipofectAMINE alone (LFN, lanes 3 and 4), scrambled control oligonucleotides
(S-ODN, lanes 5 and 6), or PYK2
antisense oligonucleotides (AS ODN, lanes 7 and
8), for 8 h, were placed in 0.2% CS-DMEM overnight and
then treated with 100 nM Ang II for 5 min as indicated.
Top panel, representative Western blots using anti-total
PYK2, anti-phospho p70S6 kinase, and anti-total p70S6 kinase
antibodies. Bottom panel, cumulative results of
n = 6 experiments. *, p < 0.05 versus control; #, p < 0.05 versus Ang 5; , p < 0.05 versus scrambled oligonucleotides.
|
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PYK2 Antisense Oligonucleotides Block Ang II-induced VSMC Protein
Synthesis--
To determine whether PYK2 is required for Ang
II-induced VSMC protein synthesis, we measured the effects of PYK2
antisense oligonucleotides on Ang II-induced protein synthesis. Protein synthesis was measured using [3H]phenylalanine
incorporation during the last 6 h of a 24-h Ang II treatment. Ang
II induced a significant increase in VSMC protein synthesis (2.1-fold
increase versus control). Treatment with PYK2 antisense oligonucleotides resulted in a complete inhibition of Ang
II-induced protein synthesis (Fig. 5).
Neither LipofectAMINE alone nor scrambled oligonucleotides had any
significant affect on Ang-II induced protein synthesis.

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Fig. 5.
PYK2 antisense oligonucleotides block Ang
II-induced protein synthesis. VSMC were treated with LipofectAMINE
(LFN), scrambled control oligonucleotides
(S-ODN), or PYK2 antisense oligonucleotides
(AS-ODN) as indicated for 8 h, placed in 0.5% CS-DMEM
overnight, transferred to 0.02% CS-DMEM for at least 6 h, and
then treated with 100 nM Ang II for 24 h.
[3H]phenylalanine was added for the last 6 h of Ang
II treatment. Cumulative data are presented as dpm
[3H]phenylalanine incorporated over total protein. *,
p < 0.05 versus control; #,
p < 0.05 versus Ang 5; ,
p < 0.05 versus scrambled
oligonucleotides.
|
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PYK2 Antisense Oligonucleotides Down-regulate Ang II-induced FAK
Phosphorylation at Tyr-397 and Tyr-861--
Govindarajan et
al. (7) have previously demonstrated a role for FAK in Ang
II-induced protein synthesis. FAK activation results in its
phosphorylation on multiple tyrosine residues including Tyr-397, the
autophosphorylation site, and Tyr-861, a proposed docking site for the
adapter molecules Grb2 and p130Cas. We sought to determine whether FAK
phosphorylation on Tyr-397 and Tyr-861 in response to Ang II is
dependent on PYK2. A decrease in PYK2 protein levels by PYK2 antisense
oligonucleotide treatment resulted in a corresponding decrease in
Tyr-397 and Tyr-861 phosphorylation of FAK, without any effect on FAK
expression (Fig. 6).

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Fig. 6.
PYK2 antisense oligonucleotides decrease FAK
phosphorylation on Tyr-397 and Tyr-861. Lysates from control VSMC
(lanes 1 and 2) or from cells treated with
LipofectAMINE alone (lanes 3 and 4) or PYK2
antisense oligonucleotides (AS ODN, lanes 5 and
6), for 8 h, were placed in 0.2% CS-DMEM overnight and
then treated with 100 nM Ang II for 5 min as indicated.
Representative Western blots using anti-total PYK2, anti-phospho
Y397FAK, anti-phospho Y861FAK, and anti-total FAK antibodies are
shown.
|
|
 |
DISCUSSION |
Angiotensin II is a potent mediator of VSMC hypertrophy. The
intracellular signaling components that link AT1 receptors
to VSMC growth involve a complex network of protein-protein
interactions and kinase cascades, but little is known about the
intracellular signaling intermediates that link the AT1
receptor to these pathways. There is increasing evidence that
non-receptor tyrosine kinases are the upstream regulators of signaling
pathways important in the regulation of cellular growth. Here, using
antisense oligonucleotide strategies, we established PYK2 as a proximal
signaling intermediate that links the AT1 receptor
activation to the activation of ERK1/2 and the activation of Akt and
p70S6 kinase, downstream effectors of PI3-kinase signaling. Inhibition
of these pathways by PYK2 down-regulation resulted in a complete
inhibition of Ang II-induced VSMC protein synthesis.
We chose an antisense strategy to down-regulate PYK2 expression rather
than overexpression of a dominant negative PYK2 mutant because we were
concerned that protein overexpression could alter protein-protein
interactions that are largely governed by protein concentration and
localization (17). Treatment with PYK2 antisense oligonucleotides, but
not scrambled oligonucleotides, lead to a significant down-regulation
of PYK2 in VSMC. This effect was specific, because the expression of
FAK and downstream signaling molecules was unaltered.
PYK2 has been shown to be an upstream regulator of a variety of
cellular signaling pathways, including Src and multiple members of the
mitogen-activated protein kinase family (18). We and others have
previously shown that Ang II induced complex formation between PYK2 and
the upstream regulators of the ERK1/2 pathway, Src, Shc, and Grb2, in
VSMC (6, 10), suggesting a role for PYK2 in the regulation of ERK1/2
activation in response to Ang II. Using PYK2 antisense oligonucleotides
in this study, we conclusively showed that PYK2 is required for Ang
II-induced ERK1/2 activation (Fig. 2).
Ang II has been shown to activate PI3-kinase (11), p70S6 kinase
(19), and Akt (20) in VSMC. We have previously demonstrated a
Ca2+-dependent complex formation between PYK2,
the adaptor molecule p130Cas, and PI3-kinase in response to Ang II
(10). These data are suggestive of PYK2-dependent
activation of the PI3-kinase signaling pathway. We here demonstrated
that PYK2 is required for activation of the PI3-kinase signaling
pathway by Ang II, because PYK2 down-regulation significantly blocked
Ang II-induced phosphorylation of both Akt and p70S6
kinase (Figs. 3 and 4). On the other hand, Eguchi et al.
(19) reported that Ca2+-dependent
transactivation of the epidermal growth factor receptor was involved in
Ang II-induced Akt and p70S6 kinase. Therefore it is possible that the
Ca2+-sensitive PYK2 may play a role in Ang II-induced
epidermal growth factor receptor transactivation to mediate
PI3-kinase signaling.
Recent evidence suggests that ERK1/2 and PI3-kinase play a crucial role
in Ang II-induced VSMC hypertrophy (7, 13, 21, 22). These pathways
appear to be independent in smooth muscle, because inhibitors of
PI3-kinase do not block Ang II-induced ERK1/2 activation (10), and
inhibitors of ERK1/2 signaling have no effect on Ang II-induced
activation of Akt (19). Because PYK2 is necessary for the activation of
these pathways, we reasoned that PYK2 down-regulation would prevent Ang
II-induced protein synthesis. As shown in Fig. 5, pretreatment with
PYK2 antisense oligonucleotides completely blocked protein synthesis in
response to Ang II. These results suggest that PYK2 links the
AT1 receptor to divergent signaling pathways that control
VSMC protein synthesis.
The regulation of translation initiation is the rate-limiting step for
protein synthesis. Both Akt and ERK1/2 are thought to regulate this
step via phosphorylation of the eukaryotic initiation factor
eIF4E/PHAS-1 complex. eIF4E mediates the initiation phase of mRNA
translation, the rate-limiting step for protein synthesis (23). The
availability of eIF4E is regulated by PHAS-1; when phosphorylated,
PHAS-1 disassociates from eIF4E, allowing the factor to participate in
translation initiation (14). Both proteins are regulated via
phosphorylation by ERK1/2 and PI3-kinase pathways (14, 23). Thus, the
ability of PYK2 antisense oligonucleotides to prevent Ang
II-induced protein synthesis may be due, in part, to decreased
phosphorylation of the eIF4E/PHAS-1 complex by ERK1/2 and PI3-kinase
pathways. Future studies will elucidate the exact mechanisms involved
in the regulation of translation initiation by PYK2.
In the present study, we show that PYK2 is involved in Ang II-induced
FAK activation. A recent report, using overexpression of the C-terminal
domain of FAK (FRNK), demonstrated that FAK is necessary for ERK1/2
activation and the induction of protein synthesis by Ang II (7). FRNK,
however, inhibited Ang II-induced protein synthesis only partially,
which may be related to its inability to inhibit Ang II-induced
activation of p70S6 kinase (7). Graves et al. (24) also
reported that p70S6 kinase is activated independently of FAK by an
upstream, Ca2+-sensitive tyrosine kinase. To link the
inhibition of ERK1/2, Akt, and p70S6 kinase phosphorylation observed in
this study to specific down-regulation of PYK2, we examined the effects
of PYK2 antisense oligonucleotides on FAK expression. Under conditions where PYK2 was significantly down-regulated, PYK2 antisense
oligonucleotides had no effect on FAK expression (Fig. 1). We then
determined whether PYK2 was involved in Ang II-induced FAK
phosphorylation. Using phospho-specific antibodies, we showed that PYK2
down-regulation led to a significant decrease in Ang II-induced FAK
phosphorylation at Tyr-397 and Tyr-861 (Fig. 6). Tyr-397 is an
autophosphorylation site that can also be phosphorylated by Src in
response to integrin engagement (25, 26). Tyr-861 has recently been
shown to be a major site that is phosphorylated by Src (27). These data suggest that PYK2 may be at least one of the upstream regulators of FAK
activation by Ang II.
The mechanisms by which PYK2 links AT1 receptor activation
to FAK phosphorylation remain to be elucidated. One possible mechanism is that the adapter protein p130Cas assembles both PYK2 and FAK in
response to Ang II. The proline-rich motifs of both PYK2 and FAK are
thought to bind to the SH3 domains of the large adapter protein
p130Cas. We have previously shown that PYK2, p130Cas, and PI3-kinase
form a signaling complex in VSMC in response to Ang II (10), and others
have shown an interaction between FAK and p130Cas in other cell types
(28). Therefore, it is tempting to speculate that this adaptor protein
is a necessary link between PYK2 and FAK activation. On the other hand,
the PYK2-Src complex that forms in response to Ang II activation (6)
may directly activate FAK. Furthermore, PYK2 can localize into focal
adhesions in response to G-protein-coupled receptor and protein kinase
C activation, resulting in PYK2 binding to the focal adhesion
proteins paxillin and p130Cas (29).
In summary, our data establish a requirement for PYK2 in the activation
of two signaling pathways implicated in the regulation of VSMC growth,
the ERK1/2 and the PI3-kinase/Akt pathways, in response to Ang II.
Furthermore, we establish a requirement for PYK2 in the initiation of
Ang II-induced VSMC protein synthesis, the major hallmark of VSMC
hypertrophy. Thus, PYK2 may represent an important molecular target for
molecular and pharmacological strategies to minimize VSMC growth
in vivo.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL56046 and HL63318 (to P. A. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: University of Alabama
at Birmingham, Dept. of Physiology and Biophysics, MCLM-986, 1530 3rd
Ave. S., Birmingham, AL 35294-0005.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101684200
 |
ABBREVIATIONS |
The abbreviations used are:
VSMC, vascular smooth muscle cell(s);
Ang II, angiotensin II;
PYK2, proline-rich tyrosine kinase 2;
FAK, focal adhesion kinase;
ERK1/2, extracellular signal-regulated kinase 1/2;
PI3-kinase, phosphatidylinositol 3-kinase;
p70S6, 70-kDa ribosomal S6 kinase;
PHAS-1, phosphorylated heat and acid stable protein 1;
eIF4E, eukaryotic initiation factor 4E;
p130Cas, 130-kDa Crk-associated
substrate;
p, phospho;
CS, calf serum;
DMEM, Dulbecco's modified
Eagle's medium.
 |
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