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INTRODUCTION |
The influx of extracellular Ca2+ is a prerequisite for
many cellular functions including cell proliferation and motility. In gastrointestinal smooth muscle, the upstroke of action potential is
principally mediated by Ca2+ influx through
voltage-dependent L-type Ca2+ channels and is
responsible for initiation of contraction. A variety of
neurotransmitters and hormones modulate Ca2+ channel
activity through protein phosphorylation (1). Modulation of
Ca2+ channel activity by serine/threonine kinases
such as cAMP-dependent protein kinase and protein kinase C
has been well established (2, 3), and phosphorylation sites have
been identified on the
subunit of L-type Ca2+ channels
in vascular smooth muscle (4). In addition to their roles in growth and
differentiation, accumulating evidence suggests that tyrosine kinases
are involved in the regulation of smooth muscle contraction. For
instance, activation of both G protein-coupled receptors and growth
factor receptors leads to smooth muscle contraction that is accompanied
by tyrosine phosphorylation of a number of proteins (5, 6). Moreover,
smooth muscle contraction can also be inhibited by structurally
unrelated tyrosine kinase inhibitors (7). The fact that
Ca2+ currents are attenuated by tyrosine kinase inhibitors
and enhanced by growth factors in smooth muscle cells (8) points toward a novel mechanism for tyrosine kinases in the regulation of smooth muscle function.
One of the earlier signaling events associated with activation of G
protein-coupled receptors, particularly Gi protein-coupled receptors, and receptor tyrosine kinases involves activation of c-Src
(9-11). Downstream signaling events of c-Src include the formation of
complexes among Shc, Grb2, and Sos and activation of the Ras and
mitogen-activated protein
(MAP)1 kinase cascade (12).
Focal adhesion kinase (FAK) is a potential substrate for c-Src, and
once phosphorylated, it may provide a docking site for SH2 domains of
the adaptor proteins such as Grb2. In addition, FAK may facilitate
activation of c-Src by displacement of the inhibitory C-terminal
tyrosine phosphorylation (13). Interestingly, activation of G
protein-coupled receptors and receptor tyrosine kinases increases the
activities of c-Src and FAK in smooth muscle (10, 14, 15). The
inhibition of basal Ca2+ currents by tyrosine kinase
inhibitors suggests that there may be constitutively activated tyrosine
kinase(s) that up-regulates Ca2+ channel activity. A
possible candidate is c-Src because of its high levels in smooth muscle
(16). Recent studies in vascular smooth muscle cells indicate that
c-Src may be involved in the regulation of Ca2+ channels
based on the finding that intracellular dialysis of c-Src enhances
Ca2+ currents (17). However, it is not known whether other
downstream signaling molecules may be involved in the regulation of
smooth muscle Ca2+ channels.
In this study, we have examined the kinase activity of c-Src and its
association with FAK in rabbit colonic smooth muscle cells. We have
also evaluated the roles of c-Src and FAK as well as their downstream
components Ras and MAP kinase in the regulation of basal
Ca2+ channel activity and their involvement in
platelet-derived growth factor (PDGF)-induced enhancement of
Ca2+ currents. Our results demonstrate that basal
Ca2+ currents are modulated by c-Src and FAK, but not
Ras/MAP kinase, in differentiated smooth muscle cells. Furthermore,
c-Src and FAK are involved in the functional coupling of PDGF receptors and Ca2+ current enhancement, which is consistent with an
increased phosphorylation of these two kinases leading to the tyrosine
phosphorylation of the Ca2+ channel.
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EXPERIMENTAL PROCEDURES |
Electrophysiological Recordings--
Single smooth muscle cells
were freshly dispersed from rabbit colonic muscularis mucosae as
described previously (8). Ca2+ currents were recorded using
the whole-cell configuration of the patch-clamp technique (18). All
experiments were performed at room temperature (~25 °C) using an
Axopatch 200A patch-clamp amplifier (Axon Instruments, Inc., Foster
City, CA). Patch pipettes were pulled from thin-walled borosilicate
glass, and the resistance was 3-5 megohms when filled with internal
solution. The internal solution consisted of 100 mM cesium
aspartate, 30 mM CsCl, 2 mM MgCl2,
5 mM HEPES, 5 mM EGTA, 5 mM ATP,
and 0.1 mM GTP (pH 7.2 with CsOH). The cells were
continuously perfused with HEPES-buffered physiological salt solution
(135 mM NaCl, 5.4 mM KCl, 0.33 mM NaH2PO4, 5 mM HEPES, 1 mM MgCl2, 2 mM BaCl2,
and 5.5 mM glucose (pH 7.4 with NaOH)). Antibodies were
applied directly to the cells by means of diffusional exchange during
standard whole-cell patch-clamp recording, and current recordings were
initiated 4 min after rupture of the membrane to allow adequate
intracellular dialysis. Pulse generation and data acquisition were
performed with a PC computer (Deskpro 486/33M, Compaq, Houston, TX)
with pclamp6.0 software (Axon Instruments, Inc.). Currents were
filtered at 1 kHz and normalized with respect to cell capacitance.
Series resistance did not exceed 5 megohms and was not compensated. The
average cell capacitance was 64.6 ± 0.9 pF (n = 128). Currents in the absence and presence of antibody dialysis were
obtained on the same day within the same population of cells.
Immunoprecipitations and Western Blotting--
Following a 5-min
treatment with or without 50 ng/ml PDGF-BB, cell suspensions were
centrifuged, and cell pellets were lysed in a Triton X-100 lysis buffer
(50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02%
sodium azide, 1 mM phenylmethylsulfonyl fluoride, 1 µM aprotinin, 1 µM leupeptin, 1 µM pepstatin A, and 1% Triton X-100). Lysates were
centrifuged at 12,000 rpm at 4 °C for 10 min, and protein contents
of the supernatants were determined. For immunoprecipitation study,
equal amounts of lysate protein (500 µg) were incubated overnight at
4 °C with a monoclonal anti-c-Src antibody. Immune complexes were
recovered with protein A-Sepharose beads. The beads were washed,
resuspended in sample buffer (50 mM Tris-HCl (pH 6.0), 5%
-mercaptoethanol, 2% SDS, 0.1% bromphenol blue, and 10%
glycerol), and boiled at 100 °C for 5 min. Protein samples were
separated by 8% SDS-polyacrylamide gel electrophoresis. After
electrophoresis, proteins were transferred to nitrocellulose membranes.
Membranes were blocked using 2% nonfat dried milk in phosphate-buffered saline (pH 7.2) and incubated overnight with an
anti-Tyr(P) (PY20), monoclonal anti-FAK,
anti-pp60c-src, or anti-c-Src (P416Y) antibody (1 µg/ml each) at 4 °C. Immunoreactive bands were visualized by
chemiluminescence. The L-type Ca2+ channel polyclonal
antibody raised against residues 818-835 of the
1C
subunit was used to detect phosphorylation and interaction of the
Ca2+ channel with c-Src and FAK. Colonic smooth muscle was
homogenized in 300 mM KCl/phosphate-buffered saline with
1% digitonin. For immunoprecipitation, 1 mg of total protein was
precleared with protein A-Sepharose beads for 30 min at 4 °C.
Chemicals--
Collagenase was from Yakult (Tokyo, Japan).
Trypsin was from Sigma. Anti-c-Src and anti-FAK antibodies as well as
the Src kinase substrate peptide were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-mouse IgG, anti-Ras, and anti-Tyr(P) (PY20) antibodies was from Transduction Laboratories (Lexington, KY). Anti-Kv1.5 and anti-
1C antibodies were from Alomone Labs
(Jerusalem, Israel). The anti-c-Src antibody (P416Y) was kindly
provided by Dr. A. P. Laudano. PD 098059 was from Alexis Co. (San
Diego, CA).
Statistical Analysis--
Data are expressed as means ± S.E. Data analysis were performed using Student's t test,
and differences with p < 0.05 were considered
significant.
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RESULTS |
Presence of c-Src and FAK in Rabbit Colonic Smooth Muscle
Cells--
Tyrosine-phosphorylated proteins were identified in freshly
isolated smooth muscle cells of rabbit colonic muscularis mucosae by
immunoblotting with the anti-phosphotyrosine antibody (PY20). Fig.
1A shows several
tyrosine-phosphorylated proteins in unstimulated cells, including bands
at ~60 and 125 kDa. These two bands were identified as c-Src and FAK,
respectively, by stripping the anti-phosphotyrosine blot and reprobing
with specific antibodies (data not shown). The phosphorylation of both
c-Src and FAK was enhanced following treatment of cells with PDGF-BB
(50 ng/ml) for 5 min (Fig. 1A). Immunoprecipitation of the
cell lysates with an anti-c-Src antibody followed by immunoblotting
with PY20 showed that a tyrosine-phosphorylated 125-kDa protein
co-precipitated with c-Src in unstimulated cells, and the association
of the phosphorylated protein with c-Src was enhanced following PDGF-BB
treatment (Fig. 1B).

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Fig. 1.
Anti-phosphotyrosine immunoblot of whole-cell
lysates from rabbit colonic smooth muscle. The Western blots show
tyrosine-phosphorylated proteins in whole-cell lysates (A)
and following immunoprecipitation (I.p.) with the anti-c-Src
antibody (B). Lysates were prepared from unstimulated cells
(Con) and after treatment for 5 min with PDGF-BB (50 ng/ml).
Proteins corresponding to ~60 and 125 kDa were identified as c-Src
and FAK by stripping and reprobing with specific antibodies.
Tyrosyl-phosphorylated proteins were enhanced following treatment with
PDGF. B shows that phosphorylated protein corresponding to
FAK immunoprecipitates with c-Src, and this association is enhanced
following PDGF-BB treatment. The equal staining of mouse IgG heavy
chain indicates equal loading of proteins.
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To further demonstrate that c-Src and FAK co-precipitated, cell lysates
were immunoprecipitated with the anti-c-Src antibody and blotted with
an anti-FAK antibody. Fig. 2A
shows that there was a significant association of c-Src with FAK in
unstimulated cells, which was enhanced following PDGF-BB treatment. The
presence of a c-Src·FAK complex in unstimulated cells suggests that a
constitutively activated c-Src may be present under resting conditions.
This was confirmed by immunoblotting with an anti-c-Src antibody
(P416Y) (Fig. 2B) that recognizes c-Src that is
phosphorylated at Tyr416 and correlates with activated
c-Src (19). This observation is consistent with the previous findings
of a high degree of activated c-Src in resting smooth muscle (16) and
the ability of tyrosine kinase inhibitors to attenuate basal
Ca2+ channel activity (8). In addition, activated c-Src was
also enhanced by PDGF-BB (Fig. 2B).

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Fig. 2.
Immunoprecipitation of FAK with c-Src and
presence of activated c-Src in unstimulated cells. The Western
blots demonstrate that FAK co-precipitates with c-Src. A
shows immunoprecipitation (I.p.) of cell lysates from
unstimulated (Con) and PDGF-BB-treated cells with the
anti-c-Src antibody and immunoblotting with the anti-FAK antibody. Note
the presence of FAK in the unstimulated cells and the enhanced
association of c-Src and FAK following PDGF-BB treatment. B
is an immunoblot of cell lysates with the anti-c-Src antibody raised
against Tyr416 corresponding to activated c-Src. Activated
c-Src was present in unstimulated cells and was markedly enhanced by
PDGF-BB.
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Inhibition of Ca2+ Currents by Src Substrate Peptide
and Anti-c-Src and Anti-FAK Antibodies--
To demonstrate whether
c-Src and FAK may be involved in the regulation of Ca2+
currents, we examined the effects of intracellular dialysis of a c-Src
substrate peptide and anti-c-Src and anti-FAK antibodies on
Ca2+ currents in single smooth muscle cells using the
patch-clamp technique. Ca2+ currents were recorded from a
holding potential of
50 mV using Ba2+ (2 mM)
as the charge carrier and normalized with respect to cell capacitance
(8). The synthetic Src substrate peptide at high concentrations can
result in a significant inhibition of Src kinase activity (20) and has
previously been shown to inhibit Ca2+ currents in vascular
smooth muscle cells (17). Intracellular dialysis of the Src substrate
peptide Cdc2-(6-20)-NH2 (60 µM), derived
from p34cdc2 (21), suppressed the Ca2+ currents
from
6.19 ± 0.65 (n = 6) to
2.52 ± 0.45 (n = 8) pA/pF (p < 0.001),
corresponding to a decrease of 59.3% at the test potential of +10 mV
(Fig. 3, A and B).
Neither the threshold nor the reversal potential was altered by the Src
substrate peptide. Because the Src substrate peptide does not
discriminate between members of the Src family, we studied the effects
of a monoclonal c-Src antibody (anti-c-Src antibody) on
Ca2+ currents to determine the involvement of c-Src in the
modulation of Ca2+ channel activity. In these sets of
experiments, the control Ca2+ current at the test potential
of +10 mV was
6.88 ± 0.70 pA/pF (n = 6),
whereas it was reduced to
2.64 ± 0.54 pA/pF (n = 10; p < 0.0003) by intracellular application of the
anti-c-Src antibody (10 µg/ml), representing an inhibition of 61.6%
(Fig. 4, A and C).
As illustrated in Fig. 4B, inhibition began soon after the onset of the dialysis process and reached its maximum within 4 min. The
steady-state inactivation kinetics of the Ca2+ currents
were not altered by the anti-c-Src antibody (data not shown). To
determine the specificity of the anti-c-Src antibody on
Ca2+ currents, we examined the effects of intracellular
dialysis of an anti-mouse IgG antibody (10 µg/ml) and an anti-Kv1.5
antibody (20 µg/ml). As shown in Fig.
5, the amplitudes of Ca2+
currents were not altered by either antibody.

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Fig. 3.
Effects of Src substrate peptide on
Ca2+ currents. Cells were dialyzed with the c-Src
substrate peptide (60 µM), and Ca2+ currents
were recorded using Ba2+ as a charge carrier. A,
representative traces of Ca2+ currents recorded in the
absence (upper trace; cell capacitance, 68 pF) or presence
(lower trace; cell capacitance, 65 pF) of the Src substrate
peptide. B, effects of the Src substrate peptide on
current-voltage relationship. Currents were obtained by a 600-ms depolarization step from a holding potential of 50 mV and normalized with respect to cell capacitance. Each point represents the mean ± S.E. obtained from six control cells and eight cells dialyzed with
the Src substrate peptide. Current-voltage relationships were measured
4 min following membrane rupture to allow for intracellular dialysis of
the peptide.
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Fig. 4.
Effects of anti-c-Src antibody on
Ca2+currents. Cells were dialyzed with the anti-c-Src
antibody (c-Src-Ab; 10 µg/ml), and Ca2+
currents were recorded using Ba2+ as a charge carrier.
A, representative traces of Ca2+ currents in the
absence (left trace; cell capacitance, 74 pF) or presence
(right trace; cell capacitance, 66 pF) of the anti-c-Src antibody. B, time course of inhibition of Ca2+
currents by the anti-c-Src antibody. Currents were recorded immediately following membrane rupture at the test potential of +10 mV from a
holding potential of 50 mV every 10 s. Currents reached a
plateau within 4 min of membrane rupture. C, effects of the
anti-c-Src antibody on current-voltage relationship. Currents were
obtained by a 600-ms depolarization step from a holding potential of
50 mV and normalized with respect to cell capacitance. Each point represents the mean ± S.E. obtained from 6 control cells and 10 cells dialyzed with the anti-c-Src antibody.
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Fig. 5.
Effects of anti-mouse IgG and anti-Kv1.5
antibodies on Ca2+ currents. Cells were dialyzed with
the anti-mouse IgG (Ig G-Ab; 10 µg/ml) or anti-Kv1.5
(Kv1.5-Ab; 20 µg/ml) antibody, and currents were obtained
by a 600-ms depolarization step from a holding potential of 50 mV and
normalized with respect to cell capacitance. Each point represents the
mean ± S.E. obtained from five to eight cells. Currents in the
absence of intracellular antibodies were obtained on the same day
within the same population of freshly dispersed cells
(Control).
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Since FAK co-immunoprecipitates with c-Src in colonic smooth muscle
cells, we examined whether FAK could modulate Ca2+ channel
activity. Cells were dialyzed with an anti-FAK antibody raised against
residues 748-1053 of human FAK, which includes the region required for
c-Src-induced Grb2 association with FAK (22). Fig.
6A shows that dialysis of the
anti-FAK antibody (7.3 µg/ml) resulted in a decrease of 43.1% in the
peak amplitude of Ca2+ currents (from
7.31 ± 1.31 (n = 6) to
4.16 ± 0.43 (n = 6)
pA/pF (p < 0.05)). To test whether the action of c-Src
and FAK on Ca2+ currents was additive, the cells were
co-dialyzed with anti-c-Src and anti-FAK antibodies. Application of
both antibodies to the cells reduced the peak currents by 62.9% (from
8.91 ± 0.70 (n = 4) to
3.31 ± 0.29 (n = 4) pA/pF (p < 0.05)) (Fig.
6B). This inhibition is comparable to that obtained with the
anti-c-Src antibody alone, suggesting that c-Src and FAK may modulate
Ca2+ currents through a complex of c-Src and FAK, rather
than acting separately.

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Fig. 6.
Effects of dialysis of anti-FAK antibody and
co-dialysis of c-Src and FAK antibodies on Ca2+
currents. Cells were dialyzed with the anti-FAK antibody (FAK-Ab; 7.3 µg/ml; A) or co-dialyzed with the
anti-c-Src antibody (c-Src-Ab; 10 µg/ml) and the anti-FAK
antibody (FAK-Ab; 7.3 µg/ml; B). A:
panel a, trace showing the effects of the anti-FAK antibody on Ca2+ currents. The cell capacitance is 80 pF in control
cell and 70 pF in cells dialyzed with the anti-FAK antibody.
Panel b, bar graph summarizing the effects of the anti-FAK
antibody on Ca2+ currents obtained at +10 mV. Data are
expressed as means ± S.E. of six control cells and six cells
dialyzed with the anti-FAK antibody. *, p < 0.05 versus control. B: panel a, trace
showing the effects of the anti-c-Src and anti-FAK antibodies on
Ca2+ currents. The cell capacitance is 75 pF in control
cell and 69 pF in cells dialyzed with the anti-c-Src and anti-FAK
antibodies. Panel b, bar graph summarizing the effects of
the anti-c-Src and anti-FAK antibodies on Ca2+ currents
obtained at +10 mV. Data are expressed as means ± S.E. of four
control cells and four cells dialyzed with the anti-c-Src and anti-FAK
antibodies. *, p < 0.05 versus
control.
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PDGF-induced Enhancement of Ca2+ Currents Is Mediated
by c-Src and FAK--
Previously, we have shown that Ca2+
currents are enhanced by epidermal growth factor (8). In the present
study, Ca2+ currents were also enhanced by PDGF-BB (Fig.
7A). Perfusion of PDGF-BB (50 ng/ml) resulted in a 42.8 ± 4.2% (n = 11;
p < 0.001) increase in peak currents. The onset of
this enhancement was rapid and reached a maximum at ~2 min. It is
well known that c-Src and FAK are involved in the regulation of
PDGF-mediated responses (23). We therefore investigated the potential
roles of c-Src and FAK in PDGF-BB-mediated enhancement of
Ca2+ currents. Similar to the observation described above,
dialysis of the cells with the anti-c-Src antibody (10 µg/ml), but
not the anti-mouse IgG antibody (10 mg/ml), resulted in a reduction of
the basal Ca2+ currents. PDGF-BB-mediated increases in
Ca2+ currents were abolished by the anti-c-Src antibody,
whereas they remained unchanged in the presence of the anti-mouse IgG
antibody (Fig. 7B). Moreover, dialysis with the anti-FAK
antibody also suppressed the basal Ca2+ currents and
blocked PDGF-BB-mediated enhancement of Ca2+ currents (Fig.
7B).

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Fig. 7.
Enhancement of Ca2+ currents by
PDGF-BB: effects of anti-mouse IgG, anti-c-Src, and anti-FAK
antibodies. A, effects of PDGF-BB on current-voltage
relationship. Control currents were recorded 2 min prior to perfusion
with PDGF-BB (50 ng/ml). The peak amplitude of currents was normalized
with respect to cell capacitance (n = 11).
B, bar graph showing the effects of the anti-mouse IgG
(IgG-Ab), anti-c-Src (c-Src-Ab), anti-FAK
(FAK-Ab), anti-Ras (Ras-Ab) antibodies and the
MEK inhibitor PD098059 on PDGF-BB-induced enhancement of
Ca2+ currents. Cells were dialyzed with the anti-mouse IgG
(10 µg/ml), anti-c-Src (10 µg/ml), anti-FAK (7.3 µg/ml), or
anti-Ras (5 µg/ml) antibody, and Ca2+ currents were
recorded before and 2 min after bath application of PDGF-BB (50 ng/ml)
and normalized with respect to cell capacitance. The MEK inhibitor PD
098059 (30 µM) was applied to the bath, and Ca2+ currents were recorded before and after application of
PDGF-BB. Data are expressed as means ± S.E. obtained from five to
six cells. *, p < 0.05 versus cells not
treated with PDGF-BB.
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Effects of Anti-Ras Antibody and PD 098059 on Ca2+
Currents--
Since activation of c-Src and FAK stimulates the Ras/MAP
kinase cascade (12, 13), and the anti-FAK antibody used in this study
might block the binding of Grb2 to FAK, we investigated the roles of
the Ras/MAP kinase pathway in the regulation of Ca2+
currents using an anti-Ras antibody and the MEK inhibitor PD 098059 (24). As shown in Fig. 7, intracellular dialysis of the anti-Ras
antibody (5 µg/ml) did not depress the basal Ca2+ current
or PDGF-BB-stimulated Ca2+ currents. In the presence of the
anti-Ras antibody, PDGF-BB enhanced Ca2+ currents by
39.5 ± 6.7% (n = 5). Furthermore, perfusing the
cells with 30 µM PD 098059 also did not inhibit basal
Ca2+ currents or PDGF-mediated enhancement (37 ± 8.1%, n = 5).
Interaction of c-Src with
1C Ca2+
Channel--
To determine whether c-Src or FAK directly associates
with the Ca2+ channel, rabbit colonic tissue was treated
with PDGF-BB and homogenized in 1% digitonin. The samples were
immunoprecipitated with cardiac anti-
1C, anti-Tyr(P)
(PY20), anti-Src, and anti-FAK antibodies and immunoblotted with the
anti-
1C antibody. Fig. 8
shows the presence of the
1C L-type Ca2+
channel (first lane), corresponding to a band of ~210 kDa.
Bands of similar size were also observed following immunoprecipitation with anti-phosphotyrosine (PY20) (second lane) and
anti-c-Src (third lane) antibodies and immunoblotting with
the anti-
1C antibody, suggesting that the
Ca2+ channel is phosphorylated by tyrosine kinases and
associates with c-Src kinase. However, the anti-FAK antibody failed to
immunoprecipitate the
1C subunit (data not shown).

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Fig. 8.
L-type Ca2+ channel
( 1C) immunoblot of rabbit colonic smooth muscle.
Cell lysates were prepared as described under "Experimental Procedures"; immunoprecipitated (IP) with the
anti- 1C (2 µg), anti-phosphotyrosine (PY20), or
anti-c-Src (c-Src) antibody; and immunoblotted with the
anti- 1C antibody. The tissues were pretreated with
PDGF-BB for 5 min prior to homogenization. The subunit immunoprecipitated with PY20 and c-Src antibodies, indicating tyrosine
phosphorylation and association with c-Src kinase following PDGF
treatment.
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DISCUSSION |
In this study, we have examined the roles of the non-receptor
tyrosine kinases c-Src and FAK in the regulation of Ca2+
channel activity in differentiated smooth muscle cells of rabbit colon.
The results demonstrate the presence of activated c-Src, which
interacts with FAK to form a c-Src·FAK complex, in unstimulated smooth muscle cells. The phosphorylation of the c-Src·FAK complex is
enhanced by PDGF-BB. Furthermore, we provide evidence that Ca2+ channels are constitutively modulated by c-Src, and
enhanced phosphorylation of the c-Src·FAK complex by PDGF-BB
correlates with enhanced Ca2+ currents. This study also
shows that c-Src directly associates with the
subunit of the
Ca2+ channels in smooth muscle. The modulation of
Ca2+ channel activity by c-Src and FAK implicates an
important role for these two kinases in regulating excitability of
smooth muscle cells.
A significant finding of this study is that PDGF-BB-induced enhancement
of Ca2+ channel activity was blocked by anti-c-Src and
anti-FAK antibodies. These data provide the first evidence for a direct
involvement of c-Src and FAK in the regulation of Ca2+
currents in differentiated cells by PDGF. The activities of c-Src and
FAK are elevated by PDGF (9, 14), and c-Src is required for PDGF
mitogenic signaling (25, 26). We confirmed these findings in colonic
smooth muscle cells by showing that phosphorylation of these two
kinases is enhanced by PDGF-BB, which is consistent with the
observations in the electrophysiological study. Two binding sites for
c-Src have been identified in the
-type PDGF receptors, Tyr579 and Tyr581 (23). However, an absence of
both SH2 and SH3 domains in FAK excludes the possibility of a direct
interaction of FAK with receptor tyrosine kinases (27), and c-Src is
suggested as an intermediate between receptor tyrosine kinases and FAK
(28). Our finding that PDGF-BB enhances the association of c-Src with
FAK supports such a role for c-Src.
The formation of a c-Src·FAK complex is crucial for cell adhesion and
integrin signaling (12) and may also be required for modulating
Ca2+ channels. While FAK does not directly associate with
the Ca2+ channel, its involvement is of particular
relevance to smooth muscle contraction since one of the potential
targets for FAK are the cytoskeletal proteins talin and paxillin, which
are phosphorylated following activation of G protein-coupled receptors
and receptor tyrosine kinases (6, 14, 15, 29). The membrane-associated dense plaques of smooth muscle are structurally similar to the focal
adhesion sites of cultured cells in that both contain the cytoskeletal
proteins. FAK autophosphorylation at Tyr397 promotes the
binding of FAK to the SH2 domain of Src family kinases (27, 30, 31).
Subsequent phosphorylation of FAK in the kinase domain at
Tyr576 and Tyr577 enhances the activity of FAK
(32). By forming such a complex, c-Src activity is also up-regulated
(13) and leads to modulation of the smooth muscle Ca2+
channel. A recent study in HEK 293 cells also suggests that formation of the c-Src·FAK complex is essential for coupling FAK to the Ras
signaling pathway (33). In rat glomerular mesangial cells, PDGF was
shown to enhance voltage-independent Ca2+ channels through
Ras (34). The failure of the anti-Ras antibody and the MEK inhibitor PD
098059 to suppress basal Ca2+ currents or to alter
PDGF-induced enhancement of Ca2+ currents suggests that the
Ras/MAP kinase cascade, the downstream signaling components of c-Src
and FAK, is not involved in the regulation of
voltage-dependent Ca2+ channels.
In transfected HEK 293 cells, v-Src associates with the human delayed
rectifier-type K+ channel Kv1.5, and tyrosine
phosphorylation of the channels is accompanied by an inhibition of
K+ currents (35). Furthermore, the association of c-Src
with N-methyl-D-aspartate channels has been
observed in rat central neurons (36). Our studies show similar
modulation of smooth muscle L-type Ca2+ currents by c-Src.
The L-type Ca2+ channel is tyrosine-phosphorylated and
associates with c-Src following PDGF treatment (Fig. 8). The full
cDNA sequence of the L-type Ca2+ channel
(
1C subunit) from rat vascular smooth muscle reveals a
potential phosphorylation site of tyrosine kinases, which is located at
residues 1869-1876 (RLSEEVEY) of an alternately spliced region
(residues 1854-1921) (4). It is known that the SH3 domain interacts
with proline-rich sequences (RPLPXXP) in the target protein
(37), and RPLPRYIP, a sequence similar to the SH3 domain-binding motif,
is present in the rat aorta Ca2+ channel (4).
We thank Drs. J. T. LaMont and A. Mercurio for critical reading of the manuscript. We also thank Drs.
W. R. Giles and A. P. Laudano for providing anti-Kv1.5 and
anti-c-Src (P416Y) antibodies, respectively.