Modulation of Voltage-dependent Ca2+ Channels in Rabbit Colonic Smooth Muscle Cells by c-Src and Focal Adhesion Kinase*

Xiang-Qun HuDagger , Namita SinghDagger , Debabrata Mukhopadhyay§, and Hamid I. AkbaraliDagger

From the Divisions of Dagger  Gastroenterology and § Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

There is emerging evidence indicating that smooth muscle contraction and Ca2+ influx through voltage-dependent L-type Ca2+ channels are regulated by tyrosine kinases; however, the specific kinases involved are largely unknown. In rabbit colonic muscularis mucosae cells, tyrosine-phosphorylated proteins of ~60 and 125 kDa were observed in immunoblots using an anti-phosphotyrosine antibody and were identified as c-Src and focal adhesion kinase (FAK) by immunoblotting with specific antibodies. FAK co-immunoprecipitated with c-Src, and the phosphorylation of the c-Src·FAK complex was markedly enhanced by platelet-derived growth factor (PDGF) BB. The presence of activated c-Src in unstimulated cells was identified in cell lysates by immunoblotting with an antibody recognizing the autophosphorylated site (P416Y). In whole-cell patch-clamp studies, intracellular dialysis of a Src substrate peptide and anti-c-Src and anti-FAK antibodies suppressed Ca2+ currents by 60, 62, and 43%, respectively. In contrast, intracellular dialysis of an anti-mouse IgG or anti-Kv1.5 antibody did not inhibit Ca2+ currents. Co-dialysis of anti-c-Src and anti-FAK antibodies inhibited Ca2+ currents (63%) equivalent to dialysis with the anti-c-Src antibody alone. PDGF-BB enhanced Ca2+ currents by 43%, which was abolished by the anti-c-Src and anti-FAK antibodies. Neither the MEK inhibitor PD 098059 nor an anti-Ras antibody inhibited basal Ca2+ currents or PDGF-stimulated Ca2+ currents. The alpha 1C subunit of the L-type Ca2+ channel co-immunoprecipitated with anti-c-Src and anti-phosphotyrosine antibodies, indicating direct association of c-Src kinase with the Ca2+ channel. These data suggest that c-Src and FAK, but not the Ras/mitogen-activated protein kinase cascade, modulate basal Ca2+ channel activity and mediate the PDGF-induced enhancement of L-type Ca2+ currents in differentiated smooth muscle cells.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha  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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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% beta -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 alpha 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-alpha 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

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.

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).

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.

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.

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 alpha 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-alpha 1C, anti-Tyr(P) (PY20), anti-Src, and anti-FAK antibodies and immunoblotted with the anti-alpha 1C antibody. Fig. 8 shows the presence of the alpha 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-alpha 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 alpha 1C subunit (data not shown).


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Fig. 8.   L-type Ca2+ channel (alpha 1C) immunoblot of rabbit colonic smooth muscle. Cell lysates were prepared as described under "Experimental Procedures"; immunoprecipitated (IP) with the anti-alpha 1C (2 µg), anti-phosphotyrosine (PY20), or anti-c-Src (c-Src) antibody; and immunoblotted with the anti-alpha 1C antibody. The tissues were pretreated with PDGF-BB for 5 min prior to homogenization. The alpha  subunit immunoprecipitated with PY20 and c-Src antibodies, indicating tyrosine phosphorylation and association with c-Src kinase following PDGF treatment.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha  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 beta -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 (alpha 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).

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by Grant DK-46367 from NIDDK, National Institutes of Health and Harvard Digestive Disease Center.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: Div. of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2159; Fax: 617-667-2767; E-mail: hakbaral{at}bidmc.harvard.edu.

1 The abbreviations used are: MAP, mitogen-activated protein; FAK, focal adhesion kinase; PDGF, platelet-derived growth factor; pF, picofarads.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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