Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is mediated by the Src homology domains

Dominique Trouet, Iris Carton, Diane Hermans, Guy Droogmans, Bernd Nilius, and Jan Eggermont

Laboratory of Physiology, Catholic University of Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We used the whole cell patch-clamp technique in calf pulmonary endothelial (CPAE) cells to investigate the effect of wild-type and mutant c-Src tyrosine kinase on ICl,swell, the swelling-induced Cl- current through volume-regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect ICl,swell. However, transfection of c-Src with a Ser3Cys mutation that introduces a dual acylation signal and targets c-Src to lipid rafts and caveolae strongly repressed hypotonicity-induced ICl,swell in CPAE cells. Kinase activity was dispensable for the inhibition of ICl,swell, since kinase-deficient c-Src Ser3Cys either with an inactivating point mutation in the kinase domain or with the entire kinase domain deleted still suppressed VRAC activity. Again, the Ser3Cys mutation was required to obtain maximal inhibition by the kinase-deleted c-Src. In contrast, the inhibitory effect was completely lost when the Src homology domains 2 and 3 were deleted in c-Src. We therefore conclude that c-Src-mediated inhibition of VRAC requires compartmentalization of c-Src to caveolae and that the Src homology domains 2 and/or 3 are necessary and sufficient for inhibition.

anion channel; caveola; tyrosine kinase; cell volume; volume-regulated anion channel


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN MOST MAMMALIAN CELL TYPES the primary response to cell swelling includes the activation of a Cl- current through volume-regulated anion channels (VRACs). This outwardly rectifying current, termed ICl,swell for swelling-activated Cl- current, is not only involved in cell volume regulation but also participates in electrogenesis, the control of the driving force for Ca2+ influx, and more speculatively in the regulation of intracellular pH and cell proliferation (for reviews, see Refs. 18, 22, and 31).

A major question with respect to VRAC is how cellular volume expansion causes channel activation. In calf pulmonary artery endothelial cells (CPAE), VRAC activation seems to depend on one or more tyrosine phosphorylation steps, since protein tyrosine kinase (PTK) inhibitors can inhibit ICl,swell, triggered either by cell swelling or by alternative isovolumic stimuli such as a decrease in intracellular ionic strength (Gamma i) or intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S; see Refs. 19 and 38). Furthermore, protein tyrosine phosphatase inhibitors potentiate ICl,swell once it is activated by cell swelling (38). In addition to CPAE cells, evidence for PTK-dependent activation has also been reported in cardiac myocytes (30), in human T lymphocytes (14), and human intestinal 407 cells (33). However, nonhydrolyzable ATP analogs can sustain VRAC activation in C6 glioma cells (8) and in N1E115 neuroblastoma cells (3), indicating that at least in these cell types ATP hydrolysis, and consequently phosphorylation, is not required for VRAC activation (3). Currently, it is not clear whether these different observations have biological relevance (e.g., expression of different channel isoforms, alternative cell-specific activation pathways) or whether they are due to methodological or experimental conditions.

Lepple-Wienhues et al. (14) have recently shown that p56lck (or Lck), a lymphocyte-specific PTK belonging to the Src family kinases, mediates the activation of ICl,swell in a human T lymphocyte cell line (14). The vertebrate Src family contains nine PTKs with a conserved domain structure (5, 32). Typically, the catalytic kinase domain is preceded by the proline-binding Src homology domain 3 (SH3) and by the phosphotyrosine- binding Src homology domain 2 (SH2). The kinase domain is followed by a COOH-terminal region containing a conserved, autoinhibitory tyrosine phosphorylation site. Depending on specific stimuli, Src-like kinases adopt either a "closed" inactive or an "open" active conformation. The closed configuration is stabilized by several intramolecular interactions, e.g., between the SH2 domain and the COOH-terminal phosphorylated tyrosine and between the SH3 domain and the linker between the SH2 and the kinase domain (32). Activation is achieved by dephosphorylation of the COOH-terminal tyrosine and/or by binding of proteins containing competing SH2 and/or SH3 domains. Once in the open conformation, Src-like kinases mediate their effect either by tyrosine phosphorylation or by protein-protein interaction via the SH2 and/or SH3 domains.

We have also provided evidence that the VRAC activation cascade is modulated by caveolin-1 (34). Indeed, caveolin-1-deficient Caco-2 cells display little or no ICl,swell upon cell swelling, but transient transfection of caveolin-1 restores the VRAC response (34). Caveolins are 22- to 26-kDa proteins that associate via palmitoyl anchors and an intramembranous hairpin structure with glycosphingolipid- and cholesterol-enriched domains in the plasma membrane (so-called "lipid rafts"; see Ref. 13). The binding of caveolins to lipid rafts generates caveolae, i.e., flask-shaped invaginations of the plasma membrane that have been implicated in endocytosis, signal transduction, and cholesterol transport (1, 16, 23).

Interestingly, Src family kinases can be compartmentalized to lipid rafts and caveolae (4). Indeed, all vertebrate Src family kinases, with the exception of c-Src and Blk, contain an NH2-terminal consensus sequence (Gly2-Cys3) for dual acylation, with Gly2 being myristoylated and Cys3 palmitoylated (24). It has been shown for Lck, Fgr, Fyn, and Hck that dual acylation targets these kinases to rafts and caveolae (25, 27, 36). In contrast, c-Src, which contains a Gly2-Ser3 sequence at its NH2 terminus, is only monoacylated (myristoylation), resulting in a low affinity of c-Src for lipid rafts and caveolae (27), although some groups have reported a small association with caveolae (29). Importantly, introduction of a Ser3Cys mutation in c-Src strongly increases its association with rafts and caveolae (27). This is consistent with the observation that dual acylation strongly enhances the caveolar targeting of other proteins such as endothelial nitric oxide synthase and Galpha i-1 (26, 29). Furthermore, it has been shown that caveolin-1 binds c-Src via its caveolin-scaffolding domain, thereby clamping c-Src in the inactive configuration (15).

In view of the link between Src family kinases and caveolae and caveolins and in view of the proposed role of both caveolin and Src family kinases in VRAC activation, we decided to investigate the effect of monoacylated and dually acylated c-Src mutants on ICl,swell in CPAE cells.


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INTRODUCTION
METHODS
RESULTS
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Cells. We used single endothelial cells from an established calf pulmonary artery cell line (CPAE; American Type Culture Collection CCL 209). Cells were grown in DMEM (Life Technologies, GIBCO) containing 20% FCS, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin. CPAE cells were transiently transfected with the bicistronic pCINeo/IRES-green fluorescent protein (GFP) vector (35) containing cDNAs encoding c-Src, c-Src Ser3Cys, c-Src Tyr527Phe, c-Src Ser3Cys/Tyr527Phe, c-Src Ser3Cys/Lys295Arg, c-Src Ser3Cys Delta kinase, c-Src Delta kinase, or c-Src Ser3Cys Delta SH3-SH2. Cells were directly seeded on gelatin-coated coverslips at 5,000 cells/coverslip 24 h before transfection. Transfection of CPAE cells was performed using 1 µg plasmid DNA, 2 µl Lipofectamine, and 7 µl "Plus" reagent (LipofectAMINE PLUS; GIBCO-BRL).

Rat-1 fibroblasts (kindly provided by Dr. P. J. Courtoy, ICP) were used to study the subcellular distribution of c-Src mutants. Rat-1 cells were cultured in DMEM (Life Technologies, GIBCO) supplemented with 20 mM glucose, 4 mM glutamine, 10 mM NaHCO3, 10 mM HEPES, 10 µg/ml streptomycin, 66 µg/ml penicillin, and 10% FCS. Rat-1/BB16 cells were transiently transfected with the bicistronic pCINeo/IRES-GFP vector.

cDNA constructs. A cDNA-encoding chicken c-Src (kindly donated by J. Goris, Biochemistry, K. U. Leuven, Belgium) was subcloned in the bicistronic expression vector pCINeo/IRES-GFP (35). Point mutations were introduced in the c-Src open-reading frame using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The following point mutations were introduced: Ser3Cys (numbering starts with the NH2-terminal Met), Lys295Arg, and Tyr527Phe. c-Src Ser3Cys Delta SH3-SH2 (deletion of amino acids 81-257) was generated by replacing in frame a restriction fragment encoding amino acids 1-257 with a PCR fragment encoding amino acids 1-80, including the Ser3Cys mutation. c-Src Delta kinase and c-Src Ser3Cys Delta kinase (deletion of amino acids 260-533) were generated by replacing a restriction fragment encoding amino acids 258-533 with an oligonucleotide containing a stop codon at position 260 in the c-Src open-reading frame without or with the Ser3Cys mutation. Mutations were verified by nucleotide sequencing using the ALF automated sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden).

Solutions. The standard extracellular solution was a modified Krebs solution containing (in mM) 150 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, and 10 HEPES, pH 7.4 with NaOH. The osmolality of this solution, as measured with a vapor pressure osmometer (Wescor 5500; Schlag, Gladbach, Germany), was 320 ± 5 mosmol/kgH2O. At the beginning of the patch-clamp recordings, the Krebs solution was replaced by an isotonic Cs+ solution, containing (in mM) 105 NaCl, 6 CsCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 10 HEPES, and 90 mannitol, pH 7.4 with NaOH (320 ± 5 mosmol/kgH2O). The 25% hypotonic solution (HTS) was obtained by omitting 90 mM mannitol from this solution. The standard pipette solution contained (in mM) 40 CsCl, 100 cesium aspartate, 1 MgCl2, 1.93 CaCl2, 5 EGTA, 4 Na2ATP, and 10 HEPES, pH 7.2 with CsOH (290 mosmol/kgH2O). The concentration of free Ca2+ in this solution was buffered at 100 nM. Activation of VRAC in the absence of an osmotic gradient was achieved by dialyzing the cells with an isosmotic pipette solution that contained 100 µM GTPgamma S (Sigma) or with an isosmotic pipette solution with reduced ionic strength by substituting 70 mM cesium aspartate with 140 mM sucrose.

Electrophysiological recordings. Transfected green fluorescent cells were visualized in a patch-clamp setup, as described previously (35). Currents were monitored with an EPC-7 patch-clamp amplifier (List Electronic, Lambrecht/Pfalz, Germany). Patch electrodes had direct current resistances between 2 and 6 MOmega . An Ag-AgCl wire was used as reference electrode. Whole cell membrane currents were measured using ruptured patches. Currents were sampled at 1-ms intervals and filtered at 1,000 Hz. The following voltage protocol was applied every 15 s from a holding potential of -25 mV: a step to -80 mV for 0.2 s, followed by a step to -100 mV for 0.1 s, and a 1.5-s linear voltage ramp to +100 mV. A step protocol was also used, consisting of 1-s voltage steps from a holding potential of -25 mV to potentials ranging from -100 to +100 mV in 20-mV increments. Experiments were performed at room temperature.

Analysis. Electrophysiological data were acquired with pCLAMP 5.5 (Axon Instruments) and were analyzed with WinASCD (G. Droogmans) and Origin 5.1 (Microcal). Time courses of the whole cell current were obtained by plotting the current at +100 or -100 mV during successive voltage ramps as a function of potential. Current-voltage relations were obtained from the currents measured during the linear voltage ramp. Difference currents (ICl,swell) were calculated by subtracting the basal current under isotonic conditions from the maximal current during hypotonic stimulation. In the experiments with intracellular application of GTPgamma S or reduced ionic strength, the difference current was calculated by subtracting the basal current measured at time 0 from either the peak current reached during transient activation with GTPgamma S or the maximal current at the plateau phase during intracellular perfusion of a pipette solution with reduced ionic strength. Pooled data are given as means ± SE from n cells. Significance between two data sets was tested using the Student's unpaired t-test. One-way ANOVA was used to determine statistical differences of three or more data sets. Differences were considered significant at the level of P < 0.05.

Immunofluorescence. CPAE cells were seeded on gelatin-coated coverslips (7,000 cells/slip) and transfected with pCINeo/IRES-GFP/c-Src S3C. After transfection (48 h), cells were washed three times in PBS and fixed with 3.7% formaldehyde (F-1268; Sigma) in PBS for 10 min at room temperature. After being rinsed with PBS, the cells were permeabilized with 0.2% Triton in PBS for 4 min. The cells were washed with PBS and incubated for 30 min in 10% FCS in PBS. Cells were washed with 1% FCS in PBS and exposed overnight at 4°C to an anti-caveolin-1 polyclonal antibody (diluted 1:400 in PBS containing 1% FCS; Transduction Laboratories C13630) and subsequently for 1 h at room temperature to the secondary tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit IgG (Sigma T-6778; diluted 1:400 in PBS containing 1% FCS). To prevent rapid photobleaching, cells were mounted in Vectashield (J-1000; Vector Laboratories) after washing the coverslips three times in PBS and two times in H2O. Cells were visualized with a Nikon Optiphot-2 microscope using green excitation (510-560 nm) for visualization of TRITC staining and blue excitation (450-490 nm) to identify the GFP signal. Omission of the primary antibody resulted in a very weak or no background staining. As a control, wild-type cells were used.

Western blotting and cell fractionation. Subcellular distribution of c-myc epitope-tagged c-Src mutants was studied in Rat-1 cells transiently transfected with pCINeo/IRES-GFP/c-Src Delta kinase or pCINeo/IRES-GFP/c-Src Ser3Cys Delta kinase. Transfected cells (5 × 106) were harvested by scraping in ice-cold 500 µl lysis buffer (in mM: 25 Tris · HCl, pH 7.2, 50 NaCl, 90 mannitol, and 1 EGTA) supplemented with 1% Triton X-100, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The lysate was adjusted to 45% sucrose and subjected to a sucrose density gradient centrifugation in a Beckman SW50.1 rotor at 250,000 g for 20 h at 4°C. The sucrose gradient was formed by layering 3 ml of 30% sucrose in lysis buffer and 1 ml of 5% sucrose in lysis buffer on top of 1 ml lysate-45% sucrose. After centrifugation, 500-µl fractions were collected from top to bottom (first 250 µl were discarded), and proteins were precipitated with 10% ice-cold TCA. Pellets were washed with acetone, resuspended in Laemmli loading buffer, separated by SDS-PAGE (12.5% polyacrylamide), and transferred by semidry electroblotting to a polyvinylidene difluoride membrane (Immobilon; Millipore). Blots were stained with anti-caveolin-1 monoclonal antibody (C37120; Transduction Laboratories) or anti-c-myc monoclonal antibody (kind gift of Dr. B. De Strooper, Center of Human Genetics, K. U. Leuven, Belgium). Immunoreactive bands were visualized with the Vistra enhanced chemifluorescence detection kit (Amersham Pharmacia Biotech) on a Storm 840 Imager (Molecular Dynamics).


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ABSTRACT
INTRODUCTION
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c-Src Ser3Cys and c-Src Ser3Cys/Tyr527Phe inhibit ICl,swell in CPAE cells. In a first series of experiments, we investigated the effect of transient expression of c-Src- and c-Src-derived mutants on ICl,swell in CPAE cells. Specifically, we compared monoacylated wild-type c-Src with c-Src mutants that are dually acylated due to a Ser3Cys mutation. As shown by Shenoy-Scaria et al. (27), the Ser3Cys mutation generates a dually acylated c-Src isoform with preferential location to lipid rafts and caveolae (27).

When exposed to a 25% HTS, wild-type CPAE cells generate large, outwardly rectifying chloride currents through VRAC. In wild-type CPAE cells, ICl,swell reached a mean current density of 70.3 ± 7.5 pA/pF at +100 mV (n = 16; Fig. 1A). In CPAE cells overexpressing wild-type c-Src, a 25% HTS evoked a similar increase in ICl,swell, with a mean current density of 55 ± 6.8 pA/pF at +100 mV (n = 21; P > 0.05 when compared with wild-type CPAE cells; Fig. 1A). In contrast, transient transfection of CPAE cells with c-Src Ser3Cys resulted in a significant reduction of ICl,swell: 10.5 ± 1.8 pA/pF at +100 mV (n = 15; Fig. 1A). Subsequently, we analyzed the effect of c-Src Tyr527Phe on VRAC. The Tyr527Phe mutation confers constitutive kinase activity to c-Src (11) because it abolishes the inhibitory interaction between the SH2 domain and the phosphorylated Tyr527 and thus destabilizes the closed inactive conformation (32). ICl,swell density was significantly reduced in CPAE cells transfected with c-Src Tyr527Phe (39.3 ± 5.4 pA/pF at +100 mV; n = 25) compared with nontransfected CPAE cells. However, the inhibitory effect of the Tyr527Phe mutation was less pronounced than that of the Ser3Cys mutation (P < 0.05). Interestingly, the Tyr527Phe-mediated downregulation of ICl,swell was further potentiated by the introduction of a dual acylation signal. Indeed, CPAE cells transiently transfected with c-Src Ser3Cys/Tyr527Phe displayed a very small ICl,swell after a 25% HTS with a mean current density of 18.3 ± 3.5 pA/pF at +100 mV (n = 35; Fig. 1A). This corresponds to a significant reduction compared with nontransfected CPAE cells and CPAE cells transfected with wild-type c-Src or c-Src Tyr527Phe. However, it is not significantly different from ICl,swell in CPAE cells transfected with c-Src Ser3Cys.


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Fig. 1.   Inhibition of swelling-activated Cl- current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells by c-Src Ser3Cys and c-Src Ser3Cys/Tyr527Phe is accompanied by a prolonged time for half-maximal activation (t1/2). A: control CPAE cells and CPAE cells transfected with, respectively, c-Src, c-Src Ser3Cys, c-Src Tyr527Phe, or c-Src Ser3Cys/Tyr527Phe, as indicated, were subjected to a 25% hypotonic solution (HTS), and ICl,swell was measured. Mean difference currents (maximal HTS-triggered current - basal current in isotonic medium) at +100 mV are plotted for the different conditions. The mean current densities for the latter 3 conditions are significantly different from those in control and c-Src-transfected CPAE cells. The mean current density for CPAE cells transfected with c-Src Tyr527Phe also differs significantly from those in CPAE cells transfected with c-Src Ser3Cys or c-Src Ser3Cys/Tyr527Phe. B: t1/2 of ICl,swell at +100 mV is plotted for the same 5 conditions. ICl,swell in CPAE cells transfected with c-Src Ser3Cys or c-Src Ser3Cys/Tyr527Phe mutants has a significantly prolonged t1/2 compared with control or c-Src-transfected CPAE cells. In addition, activation of ICl,swell in c-Src Ser3Cys/Tyr527Phe-transfected cells is significantly slower than in c-Src Tyr527Phe cells. Error bars correspond to SE.

Interestingly, c-Src mutants with the Ser3Cys mutation also slowed down the activation process of VRAC induced by a 25% HTS. The time for half-maximal activation (t1/2) was significantly prolonged in CPAE cells transiently transfected with c-Src Ser3Cys (t1/2 = 101.7 ± 11.5 s; n = 10) or with c-Src Ser3Cys/Tyr527Phe (t1/2 = 115 ± 9.3 s; n = 28) compared with untransfected CPAE cells (t1/2 = 67 ± 10.8 s; n = 15) or with CPAE cells expressing wild-type c-Src (t1/2 = 61.4 ± 5.2 s; n = 21; Fig. 1B). The Tyr527Phe mutation again resulted in an intermediary phenotype: half-maximal activation was reached after 78.7 ± 7.6 s (n = 24), which is significantly faster than the activation process in CPAE cells transiently transfected with c-Src Ser3Cys/Tyr527Phe (Fig. 1B).

c-Src Ser3Cys/Tyr527Phe also represses the isovolumic activation of ICl,swell. We have recently demonstrated in CPAE cells that VRAC can be triggered not only by a hypotonic stimulus but also by reducing Gamma i (19, 37) or by intracellular perfusion with GTPgamma S (20, 38). We were therefore interested to find out whether c-Src Ser3Cys/Tyr527Phe also interfered with the GTPgamma S- or Gamma i-triggered activation of VRAC, which is achieved under isovolumic conditions (37, 38). To this end, we compared the mean current density of ICl,swell in c-Src Ser3Cys/Tyr527Phe-transfected CPAE cells during intracellular perfusion with a pipette solution with reduced Gamma i or containing 100 µM GTPgamma S. Reduced Gamma i generated a manifest ICl,swell in wild-type CPAE cells with a mean current density of 44.6 ± 2.3 pA/pF at +100 mV (n = 4), but it was less potent in eliciting a response in c-Src Ser3Cys/Tyr527Phe-overexpressing CPAE cells (19.3 ± 6.7 pA/pF at +100 mV; n = 11; P < 0.05; Fig. 2). Similarly, intracellular application of 100 µM GTPgamma S induced a typical transient activation of VRAC in wild-type CPAE cells (mean maximal current density of 120.5 ± 26.3 pA/pF at +100 mV; n = 6), but it hardly evoked a response in CPAE cells transfected with c-Src S3C/Y527: mean maximal amplitude of 6.7 ± 3.2 pA/pF at +100 mV (n = 9; Fig. 2). Thus c-Src Ser3Cys/Tyr527Phe represses the swelling induced and the isovolumic activation of ICl,swell in transiently transfected CPAE cells, indicating that the inhibitory effect cannot be attributed to an indirect action on cell swelling.


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Fig. 2.   c-Src Ser3Cys/Tyr527Phe inhibits ICl,swell irrespective of the initial method of volume-regulated anion channel (VRAC) activation. A: time course of the transient activation of the whole cell membrane current (at +100 mV) after breaking into, respectively, a control CPAE cell and a representative c-Src Ser3Cys/Tyr527Phe-transfected CPAE cell with a pipette solution containing 100 µM guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S). Cells were held at -25 mV, and voltage ramps were applied every 15 s. B: time course of the membrane current at +100 mV in a control CPAE cell and in a CPAE cell transfected with c-Src Ser3Cys/Tyr527Phe dialyzed with a pipette solution of reduced ionic strength. As in A, data points were obtained from the voltage ramp, which was applied every 15 s. C: mean current density of ICl,swell in, respectively, control CPAE cells and CPAE cells transfected with c-Src Ser3Cys/Tyr527Phe at +100 mV plotted for the following 2 conditions: activation of ICl,swell by intracellular perfusion with GTPgamma S or by reducing the intracellular ionic strength, respectively. Error bars correspond to SE.

Downregulation of VRAC by c-Src Ser3Cys is not mediated by the Src kinase domain but by the SH3-SH2 domains. To investigate whether the dually acylated c-Src inhibited ICl,swell by phosphorylating other proteins, we constructed a kinase-inactive c-Src Ser3Cys mutant by introducing a Lys295Arg mutation in the ATP-binding site of the kinase domain (9). In this series of experiments, ICl,swell reached a mean current density of 100.6 ± 11.6 pA/pF at +100 mV (n = 10; Fig. 3) in wild-type CPAE cells. c-Src Ser3Cys exerted an inhibitory effect on ICl,swell (47.0 ± 11.7 pA/pF at +100 mV; n = 15), albeit somewhat less pronounced than in the previous series of experiments. Importantly, CPAE cells transfected with the kinase-inactive c-Src Ser3Cys/Lys295Arg generated HTS-induced membrane currents of similar amplitude (51.2 ± 4.9 pA/pF at +100 mV; n = 19) as CPAE cells expressing c-Src Ser3Cys (Fig. 3).


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Fig. 3.   Kinase activity is not required for the downregulation of VRAC by c-Src Ser3Cys. Control CPAE cells and CPAE cells transfected with, respectively, c-Src Ser3Cys or c-Src Ser3Cys/ Lys295Arg, as indicated, were subjected to a 25% HTS, and ICl,swell was measured. Mean current densities at +100 mV are plotted for the 3 conditions. A similar degree of downregulation of ICl,swell is observed in both populations of transfected CPAE cells. Error bars correspond to SE.

These results suggested that the c-Src kinase activity is dispensable for the observed inhibition of ICl,swell by c-Src Ser3Cys and, by inference, that the inhibition is mediated by the Src homology domains. This hypothesis was tested by transiently transfecting CPAE cells with c-Src Ser3Cys mutants in which either the entire kinase domain was deleted (c-Src Ser3Cys Delta kinase) or the SH3-SH2 domains were deleted (c-Src Ser3Cys Delta SH3-SH2; Fig. 4). Consistent with the hypothesis, deletion of the SH3-SH2 domains generated a c-Src mutant (c-Src Ser3Cys Delta SH3-SH2) that no longer inhibited ICl,swell (125.5 ± 20.4 pA/pF at +100 mV; n = 8) compared with nontransfected control cells (149.6 ± 20.4 pA/pF at +100 mV; n = 13). Western blotting and immunostaining with a polyclonal c-Src antibody revealed a 40-kDa protein in transiently transfected cells, indicating that the c-Src Ser3Cys Delta SH3-SH2 mutant was expressed (data not shown). In contrast, the c-Src Ser3Cys mutant containing the SH3-SH2 domains but lacking the kinase domain (c-Src Ser3Cys Delta kinase) still exerted a potent inhibition on ICl,swell in transiently transfected CPAE cells: 16.5 ± 4.6 pA/pF at +100 mV (n = 17) vs. 149.6 ± 20.4 pA/pF at +100 mV (n = 13) in control cells (Fig. 4). Moreover, the degree of inhibition by c-Src Delta kinase was significantly affected by the presence of a targeting signal for lipid rafts. Transfection of a c-Src Delta kinase mutant with a wild-type NH2 terminus (Ser at position 3) still inhibited ICl,swell (68.4 ± 12.3 pA/pF at +100 mV; n = 9) compared with control cells, but this inhibition was significantly less than that exerted by c-Src Ser3Cys Delta kinase containing a dual acylation signal (Fig. 4).


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Fig. 4.   Inhibition of ICl,swell is mediated by the c-Src homology 3 (SH3)-homology 2 (SH2) domains. Control CPAE cells and CPAE cells transiently transfected with the c-Src mutants as indicated were subjected to a 25% HTS. Mean current densities of ICl,swell at +100 mV are plotted. Inhibition of ICl,swell requires SH3-SH2 and is potentiated by the presence of a dual acylation signal (Ser3Cys). Error bars correspond to SE.

Cofractionation of c-Src Ser3Cys mutants with caveolin-1-containing light density fractions. A consistent finding in the above-described observations is the critical contribution of the Ser3Cys mutation to the inhibition of ICl,swell by c-Src mutants (Figs. 1 and 4). Because this mutation introduces a dual acylation signal that promotes c-Src targeting to lipid rafts and caveolae (27), this strongly suggests that efficient inhibition requires compartmentalization of c-Src to lipid rafts and/or caveolae. We therefore verified whether the Ser3Cys mutation indeed alters the subcellular distribution of c-Src mutants by examining their cofractionation with caveolin-1 in a sucrose gradient after solubilization of cells with ice-cold Triton X-100. This procedure preserves the lipid composition and hence the low buoyant density of rafts and caveolae (so-called detergent-resistant membranes), which allows their separation in a sucrose gradient (28). c-Myc epitope-tagged c-Src Delta kinase and c-Myc epitope-tagged c-Src Ser3Cys Delta kinase were transiently expressed in Rat-1 cells, and cell lysates were subjected to sucrose density gradient centrifugation. Figure 5 shows that the vast majority of c-Src Delta kinase stays at the bottom of the gradient and that only a minor fraction cofractionates with caveolin-1. In contrast, ~50% of c-Src Ser3Cys Delta kinase floats up in the gradient to the low-density, caveolin-1-containing fractions. Thus the Ser3Cys mutation induces a clear shift in the c-Src Delta kinase distribution toward the detergent-resistant fraction.


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Fig. 5.   Cofractionation of Ser3Cys mutants with caveolin-1 (cav-1)-containing low-density sucrose fractions. Rat-1 fibroblasts were transiently transfected with c-myc epitope-tagged c-Src Delta kinase (left) or with c-myc epitope-tagged c-Src Ser3Cys Delta kinase (right). After transfection (48 h), nontransfected cells (control) and transfected cells were lysed in a Triton X-100-containing buffer, and the lysate was fractionated in a sucrose density step gradient (5% at top, 45% at bottom). Individual fractions were collected from top to bottom. Fractions 2 (low density) to fraction 10 (high density) were separated on a 12.5% SDS-PAGE and electroblotted. Blots were stained with a monoclonal anti-caveolin-1 antibody (top) or a monoclonal anti-c-myc antibody (bottom) using enhanced chemifluorescence. Histograms show the relative distribution of c-Src Delta kinase (left) or c-Src Ser3Cys Delta kinase (right) across the sucrose gradient. Note that the Ser3Cys mutation induces cofractionation of part of c-Src Delta kinase with the light caveolin-1-containing fractions. Furthermore, the c-Src mutants do not alter the subcellular distribution of caveolin-1 (compare with control blot).

c-Src Ser3Cys does not alter the subcellular localization of endogenous caveolin-1 in CPAE cells. Because v-Src, a constitutively active oncogenic variant of c-Src, downregulates the expression and the plasma membrane location of caveolin-1 and disrupts caveolar structure (12, 21), we investigated whether transient expression of c-Src Ser3Cys altered the subcellular localization of endogenous caveolin-1. Immunofluorescence microscopy on CPAE cells revealed no significant differences in caveolin-1 expression or subcellular localization between wild-type CPAE cells and CPAE cells transiently transfected with c-Src Ser3Cys (Fig. 6). Under both conditions, we detected caveolin-1 signals at the cell periphery (often more concentrated on one edge of the cell). In addition, some caveolin-1 signals resided from the cell interior, as can be expected from the dynamic recycling of caveolin-1 between the plasma membrane and internal organelles (2). The identical caveolin-1 pattern between control and c-Src Ser3Cys-transfected cells virtually excludes the possibility that c-Src Ser3Cys inhibits ICl,swell by altering caveolin-1 expression or its subcellular distribution. A similar conclusion can also be deduced from Figs. 4 and 5: introduction of the Ser3Cys mutation significantly increases ICl,swell inhibition (Fig. 4), yet it does not affect the distribution of caveolin-1 in the sucrose gradient (Fig. 5).


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Fig. 6.   c-Src Ser3Cys does not alter the subcellular localization of endogenous caveolin-1 in CPAE cells. CPAE cells, grown on coverslips, were transfected with pCINeo/IRES-green fluorescent protein (GFP)/c-Src Ser3Cys. Cells were immunostained with a polyclonal primary anti-caveolin-1 antibody and a tetramethylrhodamine isothiocyanate-labeled secondary antibody (A and C). Transfected cells were identified by GFP fluorescence. B: GFP signal of cell shown in A. Subcellular localization of endogenous caveolin-1 in transfected cells (A) was identical compared with untransfected wild-type cells (C). The scale bar corresponds to 40 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we show that c-Src tyrosine kinase represses VRAC activity in vascular endothelial cells (CPAE) if the c-Src protein is targeted to lipid rafts and caveolae by virtue of a dual acylation signal (provided by the Ser3Cys mutation). Furthermore, the inhibitory effect is mediated by the c-Src SH3-SH2 domains, since deletion of these domains abrogated the inhibition. Vice versa, c-Src mutants lacking the kinase domain still exerted an inhibitory effect on ICl,swell.

The Ser3Cys mutation, which creates a dual acylation signal in the c-Src NH2 terminus, maximalized the inhibition of ICl,swell by c-Src. Because dually acylated proteins are preferentially targeted to lipid rafts and/or caveolae, this observation indicates that these plasma membrane compartments play a crucial role in the expression of VRAC activity. However, it should be stressed that lipid rafts as such (i.e., sphingolipid- and cholesterol-enriched membrane domains) are not sufficient for VRAC activity. Indeed, Triton X-100 solubilization of caveolin-1-deficient Caco-2 cells followed by sucrose density centrifugation revealed the presence of low-density lipid rafts (17). Yet, VRAC could only be efficiently activated in Caco-2 cells after transient transfection with caveolin-1 (34). Together, these data suggest that a crucial component of the VRAC cascade (either the channel itself, an essential regulatory protein, or both) is confined to caveolae, i.e., caveolin-1-containing lipid rafts.

How can we explain the downregulation of VRAC by c-Src targeted to caveolae? One possibility would be that the expression of dually acylated c-Src disrupts caveolar structure and/or composition. However, we could not detect alterations in caveolin-1 expression or in its subcellular distribution in cells transfected with c-Src mutants that clearly exerted an inhibitory effect on VRAC. This observation excludes caveolar disruption as the causative factor. Taking into account that inhibition requires the presence in c-Src of the Src homology domains, which are bona fide protein-protein interaction domains, we envisage two plausible hypotheses. One hypothesis postulates a crucial step in the VRAC activation cascade that is mediated by a caveolar protein containing phosphotyrosine(s) and/or a proline-rich domain. In this scenario, binding of this (as yet unidentified) protein to the c-Src SH2 and/or SH3 domain would result in its sequestration, thereby preventing it from participating in the normal VRAC activation cascade. Such an inhibition by sequestration would resemble the process of "squelching" whereby overexpression of a transcription factor reduces transcriptional activity by titrating out other essential transcription factors (6). The molecular identity of the c-Src interaction partner(s) remains enigmatic, but any candidate should fulfill at least two requirements as follows: 1) it must localize to caveolae and 2) it must be able to interact with SH2 and/or SH3 domains, i.e., it must possess phosphotyrosines, proline-rich domains, or both. Interestingly, Liu et al. (16) have shown that some of the dually acylated Src family kinases, such as Yes, Fyn, Lck, and Lyn, are concentrated in caveolae obtained from lung endothelial cells. In view of the postulated role for tyrosine kinases in VRAC activation in endothelial cells (38) and in view of the requirement for Lck to activate VRAC in lymphocytes (14), it will be interesting to address the role of these dually acylated Src family kinases in the endothelial VRAC cascade.

Alternatively, inhibition of VRAC by caveolar-targeted c-Src can be explained by invoking the caveolar recruitment of an inhibitory protein that normally resides in the cytoplasm. Translocation of this inhibitor to caveolae would depend on its interaction with the SH2 and/or SH3 domain of c-Src. This mechanism bears some resemblance to the Cbp-mediated recruitment of Csk kinase to lipid rafts (10). Cbp is a lipid raft-located phosphoprotein that in its tyrosine-phosphorylated state provides a binding site for Csk, which normally resides in the cytoplasm. After translocation to rafts, Csk phosphorylates the autoinhibitory tyrosine in the COOH terminus of Src family kinases, thereby inactivating the raft-associated Src family kinases (7). Whether overexpression of dually acylated c-Src also induces caveolar translocation of Csk (or of another inhibitory protein) remains to be shown.

We therefore conclude that inhibition of VRAC by c-Src requires compartmentalization of c-Src to caveolae. Furthermore, because SH2 and SH3 are necessary and sufficient to confer inhibition, we suggest that c-Src is incorporated in a caveolar protein-protein interaction network that negatively interferes with the VRAC activation cascade.


    ACKNOWLEDGEMENTS

We thank J. Goris (Laboratory of Biochemistry, K. U. Leuven, Belgium) for the gift of c-Src cDNA and B. De Strooper (Center of Human Genetics, K. K. Leuven, Belgium) for the anti-c-myc antibodies. The technical help of J. Prenen, A. Florizoone, M. Crabbé, and H. Van Weijenbergh is greatly appreciated. D. Trouet is a Research Assistant of the Flemish Fund of Scientific Research [Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen].


    FOOTNOTES

This research was supported by grants from the Interuniversitaire Attractiepolen (IUAP P4/23) FWO-Vlaanderen (FWO G.0214.99), Geconcerteerde Onderzoeksactie (GOA 99/07), and the `Jean en Alphone Forton' foundation (R7115 B0 to B. Nilius).

Address for reprint requests and other correspondence: J. Eggermont, Laboratory of Physiology, Campus Gasthuisberg, B-3000 Leuven, Belgium (E-mail Jan.Eggermont{at}med.kuleuven.ac.be).

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.

Received 13 April 2000; accepted in final form 12 February 2001.


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